Plant Pedia

Glossary in Plant Biotechnology (Part 2)

2012-01-30T19:56:00.000-08:00

Genetic engineering (GE): the manipulation of the genetic material of an organism using recombinant DNA technology.

Genetic enhancement: the broadening of the genetic base of a species using breeding and/or GE methods.

Genome-wide selection (GWS) or genomic selection (GS): works at the whole genome level without the need for the identification of a subset of markers associated with the traits as in the case of MAS, MABC and MARS. GWS relies on the fact that the genomic regions containing the same rare haplotypes are usually identical by descent, harboring the same QTL allele, and thereby these markers or marker haplotypes, which are in close LD with QTL, can be used for selection.

Genomic estimated breeding values (GEBVs): estimates of the breeding values of genotyped individuals (breeding population), calculated based on marker effects derived from the genotyping and phenotyping data obtained from trained individuals (training population).

Intergovernmental Panel on Climate Change (IPCC): scientific and intergovernmental organization, developed as a collaborative effort of the UN Environment Programme and the World Meteorological Organization. The aim of the IPCC is to review the scientific, technical and
socioeconomic impacts of climate change.

Marker-assisted backcrossing (MABC): marker-aided foreground selection to introgress precisely the donor segment into the elite breeding line accompanied by marker-assisted background selection to ensure the maximum recovery of recurrent parent genome.

Marker-assisted recurrent selection (MARS): marker-aided population improvement scheme relying on the recovery of superior or ideal genotypes, which are generally made up of various genomic fragments harboring smaller effectQTLs. The isolation of suchanideal genotypeis not possible in simple biparental mapping populations.

Molecular breeding (MB): theprocessofgeneticimprovementthrough the deployment of molecular tools such as DNA markers in traditional breeding. MB enhances genetic gain by increasing the selection efficiency coupled with the reduced length of breeding cycles.

Next-generation sequencing (NGS) technologies: high-throughput sequencing technologies such as Roche/454 (http://www. 454.com/), Solexa/Illumina (http://www.illumina.com/) and AB-SOLiD (http://www.appliedbiosystems.com/), which provide reduced cost per data point. NGS techniques are ideal for resequencing genomes, but currently these are being used for de novo whole-genome sequencing in many crops.

Nitrogen use efficiency (NUE): expressed in terms of grain yield per unit of available soil nitrogen. NUE can be divided into two components: uptake efficiency (to take nitrogen from the soils) and usage efficiency (to convert the nitrogen uptake into protein).

Quantitative trait loci (QTLs): genomic regions associated with complex quantitative traits governed by several large effect as well as smaller effect genes. Special statistical software is needed to identify the locations and effects associated with these regions.

RNA chaperones: class of proteins required for the proper folding of RNA or for resolving incorrectly folded RNA structures.

RNAi: the natural mechanism of silencing the expression of genes with the help of RNA molecules such as miRNA and siRNA. RNAi facilitates the rapid identification of gene functions.

Targeted gene replacement: the in vitro modification of a cloned DNA fragment and subsequent introduction into the host cell through homologous recombination or gene targeting.

Training population: one of the components of a GS scheme. Genotyping and phenotyping data are recorded for ‘model’ or ‘trained’ individuals, which are subsequently used to calculate the GEBVs of individuals.

Transgenic or genetically modified organism: contains a foreign gene that has been introduced into its genome by GE.

Wild relatives: wild species, particularly those closest to domesticated plants that might harbor lots of novel variations not available in the cultivated germplasm pool.

Glossary in Plant Biotechnology (Part 1)

2012-01-30T19:42:00.000-08:00

Allele mining: the identification and isolation of novel allelic variants associated with the phenotype of interest that exists within large germplasm collections.

Aquaporins: integral proteins of the cell membrane that are required for regulating the movement of water in and out of the cell while excluding ions and metabolites.

Association mapping or linkage disequilibrium (LD) mapping: mapping methods that rely on the historical recombination and nonrandom association of alleles or LD that persists in random mating populations. Association genetics facilitates the identification of marker–trait association based on whole-genome or candidate gene-based LD analysis.

Best linear unbiased prediction: statistical method to calculate random effects in linear mixed models that is widely used for estimating breeding values in plant breeding.

Candidate genes: genes that might be related to the trait of interest. Candidates genes can be identified through either map-based methods such as QTL analysis (positional candidate genes) or functional genomics approaches such as transcriptomics and expression genetics (functional candidate genes).

Cis-genics: GE approach that relies on the identification and transfer of natural indigenous genes or cis-genes, isolated from the same species of the plant or other sexually compatible species.

Cold shock proteins (CSPs): family of proteins that are induced by a decrease in temperature. During cold shocks, most of the cellular protein synthesis processes slow down; however, the number of protein synthesis processes operating in CSPs increases to a maximum during the ‘acclimation’.

Constitutive expression: the continuous expression of genes (i.e. no control over expression).

C-repeat binding factors (CBFs): also known as dehydration responsive element binding factors. They are activated by cold stress and have a conserved ‘CCGAC’ core sequence, which is found in the promoter of many cold-inducible genes.

Food insecurity: the lack of access to an adequate food supply, leading to a deficient food supply at the household level and malnourishment at the individual level.

Gene pyramiding: the process of introducing desirable genes into a single genotype from different donor sources. This is also known as gene stacking.

Biological effects of essential oils – A review

2011-08-24T03:51:00.000-07:00

F. Bakkalia, b, S. Averbecka, D. Averbecka, Corresponding Author Contact Information, E-mail The Corresponding Author and M. Idaomarb

aInstitut Curie-Section de Recherche, UMR2027 CNRS/IC, LCR V28 CEA, Bât. 110, Centre Universitaire, 91405 Orsay cedex, France

bUniversité Abdelmalek Essâadi, Faculté des Sciences, Laboratoire de Biologie et Santé, BP 2121, Tétouan, Morocco

Abstract

Since the middle ages, essential oils have been widely used for bactericidal, virucidal, fungicidal, antiparasitical, insecticidal, medicinal and cosmetic applications, especially nowadays in pharmaceutical, sanitary, cosmetic, agricultural and food industries. Because of the mode of extraction, mostly by distillation from aromatic plants, they contain a variety of volatile molecules such as terpenes and terpenoids, phenol-derived aromatic components and aliphatic components. In vitro physicochemical assays characterise most of them as antioxidants. However, recent work shows that in eukaryotic cells, essential oils can act as prooxidants affecting inner cell membranes and organelles such as mitochondria. Depending on type and concentration, they exhibit cytotoxic effects on living cells but are usually non-genotoxic. In some cases, changes in intracellular redox potential and mitochondrial dysfunction induced by essential oils can be associated with their capacity to exert antigenotoxic effects. These findings suggest that, at least in part, the encountered beneficial effects of essential oils are due to prooxidant effects on the cellular level.

Keywords: Essential oil; Cytotoxicity; Genotoxicity; Antigenotoxicity; Prooxidant activity

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The Synthesis of Starch

2011-08-18T20:18:00.000-07:00

Following the uptake of carbon into the amyloplast, starch synthesis proceeds variously via (i) plastidial phosphoglucomutase and plastidial ADPglucose pyrophosphorylase, (ii) only via plastidial ADPglucose or (iii) via no intermediate steps prior to the polymerising reactions of starch synthases and branching enzymes (Smith et al., 1997). The exact route depends on the nature of the imported carbon source (see Figure 25.3). The first reaction of plastidial starch metabolism both in the potato tuber (Tauberger et al., 2000) and in the pea embryo (Hill and Smith, 1991) is the interconversion of glucose-6- and glucose-1-phosphate catalysed by plastidial phosphoglucomutase. Compelling evidence for the involvement of this enzyme in pea starch synthesis was provided by studies on the rug3 mutant which revealed that this locus encodes a plastidial phosphoglucomutase and that mutation at this locus results in a severe depletion of starch levels in pea embryos (Harrisson et al., 1998). In addition the depleted starch accumulation in transgenic potato plants exhibiting reduced levels of phosphoglucomutase (Tauberger et al., 2000) and the reduced gravitropic response of roots of the TC7 mutant of Arabidopsis (also deficient in this enzyme; Kiss et al., 1996) highlight its involvement in starch synthesis in other species. Moreover, transgenic potato plants with a reduction in the cytosolic isoform of phosphoglucomutase also exhibited reduced levels of starch, most probably due to a reduction in the available glucose 6-phosphate for uptake into the plastid (Fernie et al., 2001b).

The next reaction on the path to starch synthesis, which is catalysed by plastidial AGPase, has received much attention for a number of years. This reaction is often considered to be the first committed step of starch synthesis. AGPase utilises ATP and produces pyrophosphate, which is then hydrolysed by pyrophosphatase to yield 2Pi. The hydrolysis of PPi serves to remove the ADPglucose pyrophosphorylase reaction away from equilibrium. A cDNA encoding a soluble inorganic pyrophosphatase has been cloned from potato (Du Jardin et al., 1995), however, a functional assessment of the in vivo role of this protein is yet to be performed. Some evidence of a role for a pyrophosphatase activity was provided in experiments in which potato tuber discs were treated with fluoride (Viola and Davies, 1991), however, caution is required while interpreting these data as fluoride is a relatively promiscuous inhibitor. In many species including pea embryos, soybean cell suspension cultures and cauliflower buds, AGPase appears to be located exclusively in the plastid (MacDonald and ap Rees, 1983; Journet and Douce, 1985; Smith, 1988) and this isoform thus plays an important role in mediating the flux of carbon to starch. On removal or severe reduction of the AGPase activity in Arabidopsis or potato the level of starch was found to be dramatically reduced in all tissues (Lin et al., 1988a, 1988b; Müller-Röber et al., 1992). In direct contrast, when a non-regulated bacterial AGPase was expressed in various plant tissues the starch levels were dramatically increased (Stark et al., 1991). Plant AGPases are multisubunit proteins and expression studies in which the potato tuber enzyme way expressed in E. coli revealed that maximal activity can only be achieved on expression of both the large and the small subunit (Iglesias et al., 1993). Moreover, they are also allosterically regulated, being activated by 3-PGA and inhibited by Pi (Preiss, 1988; Sowokinos and Preiss 1992; Ballicora et al., 1995), and there is a clear evidence that changes in these metabolites are involved in the regulation of starch synthesis within leaves allowing the coordination of carbon assimilation, sucrose synthesis and starch synthesis (Stitt, 1997). There is also increasing evidence of a strong correlation between the 3PGA and ADPglucose levels and the rate of starch synthesis in potato tubers under a wide range of conditions (Geigenberger et al., 1997; 1998a). It is worth noting that using mutagenesis, 3PGA insensitive forms of the plant enzyme have also been created (Greene et al., 1996). However, since there are also several reports in the literature that the plant plastidial AGPase is clearly regulated, at least in vitro, by redox status (Fu et al., 1998; Ballicora et al., 2000), it is probable that this should also be taken into account in the design of future strategies intent on increasing starch content.

Whilst there is a wealth of information on the regulation of the plastidial isoform of AGPase detailed knowledge of the cytosolic isoform is very much limited. Several import studies provide evidence that the ADPglucose produced in the cytosol can be taken up by the plastid (Pozueta-Romera et al., 1991a, 1991b; Tetlow et al., 1994; Möhlmann et al., 1997), a process most probably mediated by the Brittle 1 protein (Sullivan et al., 1991). From these studies and from characterisation of maize Brittle 1 mutants which accumulated ADPglucose to 13 times the level found in wild-type plants (Shannon et al., 1996) it would seem that the Brittle 1 gene encodes for an amyloplastidial ADPglucose transporter. Despite these findings the physiological significance of cytosolic ADPglucose production remains unclear for a range of species. Thus, calculations of Denyer et al. (1996) demonstrate that the AGPase activity of the plastid is insufficient to account for measured rates of starch synthesis in barley endosperm, suggesting that at least some of the ADPglucose required for this process is provided by cytosolic production.

The recent purification of an ADPglucose pyrophosphatase from a range of plant species (Rodriguez et al., 2000) complicates matters further. This enzyme is believed to be co-localised with AGPase and to compete with starch synthase thus markedly blocking starch synthesis. Moreover, in studies in E. coli it was found that when this protein was reduced by insertional mutagenesis the level of glycogen marginally increased, suggesting this protein could play an important role in the regulation of carbohydrate storage (Moreno-Bruna et al., 2001). Furthermore, recent studies of 14-3-3 proteins within starch granules of Arabidopsis chloroplasts (Sehnke et al., 2001) indicate that these proteins may also be involved in the regulation of starch metabolism. However, to date no substantial data have been reported to characterise the physiological role of these proteins in plants and it is therefore unclear how important these proteins are in the regulation of starch metabolism. Despite this note of caution, it is clear that such regulatory genes could represent important future strategies. This is especially true when it is considered that the modification of pathway enzymes often has less than the desired effect.

Whilst the involvement of the above enzymes in starch biosynthesis are strictly species dependent, the starch polymerising activities are ever present and responsible for the formation of the two different macromolecular forms of starch, amylose and amylopectin. Starch synthases catalyse the transfer of the glucosyl moiety from ADPglucose to the non-linear end of an αα-1,4 glucan. The various starch synthases are able to extend 1, 4-glucans in both amylose and amylopectin. At least four different classes of starch synthases exist, designated as GBSS (granule-bound starch synthase), SSI, SSII and SSIII, which vary greatly in molecular weight, need for primers, substrate affinities and antigenic properties (for a review see Sivak and Preiss, 1998). It seems likely that most plant species contain the four different classes of starch synthase; however, the extent to which they contribute in vivo probably differs considerably between species (Denyer et al., 2001). Starch branching enzymes (SBE) are responsible for the formation of αα-1,6 branch points within amylopectin. Although there are more than two isoforms present in most plant species, all isoforms can be separated into two classes—most simply designated as A and B forms (Burton et al., 1995). The precise mechanism by which this is achieved is unknown, however it is thought to involve cleavage of a linear αα-1,4 linked glucose chain and reattachment of the chain to form an αα-1,6 linkage (Kossmann and Lloyd, 2000). The combined action of starch synthases and branching enzymes play an important role in determining the structure of starch which will be described in detail below. Other enzymes of starch synthesis and degradation are less well understood. Disproportionating enzyme (D-enzyme) is able to synthesise αα-1,4 glucans from maltose and has been suggested to be a candidate as a source of the malto-oligosaccharide primers required for starch synthesis. However, several lines of evidence suggest that this is unlikely to play a major role in starch synthesis in vivo. The maltose present in plant tissues is almost exclusively derived from starch (Kossmann and Lloyd, 2000) and transgenic plants exhibiting reduced D-enzyme expression had no effect on starch content (Takaha et al., 1998). Recent studies on an Arabidopsis mutant deficient in D-enzyme reveal a minor decrease in starch under certain conditions. However, they indicate that this enzyme primarily plays a role in the removal of malto-oligosaccharides during starch degradation (Critchley et al., 2001).

A further protein with a possible role in starch synthesis is the R1 protein. This protein has long been thought to be involved in the phosphorylation of starch since starch isolated from transgenic potato plants in which the R1 protein was reduced by antisense repression displayed only 10&percnt; of the phosphate content in wild-type potatoes (Lorbeth et al., 1998). Consistent with this proposal the enzymatic function of the protein was recently proved to be a starch water dikinase (Ritte et al., 2002).

To fully comprehend factors that determine starch biosynthesis knowledge of both synthetic and degradative functions is required. Currently, understanding of the roles of the starch degradative enzymes is relatively rudimentary. However, on the basis of several recent studies it has been proposed that several of the enzymes once regarded as operating exclusively in starch degradation also have a role in the synthesis of starch. The proposed catabolic and anabolic roles of debranching enzymes (isoamylases and pullanases), starch phosphorylase and αα- and ββ-amylases will be discussed below and their role in starch structure will be covered in greater detail in the later sections.

The endosperm from the Sugary-1 (Su-1) mutant of maize contains a second type of branched glucan other than amylopectin that is known as phytoglycogen. This mutant was shown to be deficient in an isoform of debranching enzyme (Pan and Nelson, 1984). This has since been confirmed when the Su-1 gene was cloned and found to encode an isoamylase-type enzyme (James et al., 1995). A similar phytoglycogen-accumulating Su-1 mutant has also been found in rice which exhibits changes in the activities of several enzymes of starch metabolism. The most dramatic of these by far was a 90&percnt; reduction in total debranching enzyme activity (Nakamara et al., 1996). That this reduction was specific to pullulanase was demonstrated immunologically, and pullulanase activity was found to correlate closely to phytoglycogen accumulation across rice lines producing different concentrations of soluble sugars (Nakamura et al., 1997). The sta-7 mutant of the monocellular green algae Chlamydomonas rheinhardtii has been found to contain no starch but a small amount of phytoglycogen (Mouille et al., 1996). Studies of this mutant revealed that the only activity of starch metabolism missing was that of a debranching enzyme. In combination these data present compelling evidence of a role of debranching enzymes in starch synthesis. However, the exact mechanism for this remains controversial. In the last few years two models have been proposed for amylopectin synthesis (summarised in Figure 25.4). In the model of Ball et al. (1996) glucans are synthesised within amylopectin until they reach a certain regular length which allows branching enzymes to act on them. Branching enzymes subsequently produce an uncrystalline glycogen-like polysaccharide (preamylopectin) on the outside of the linear chains. Debranching enzymes are then proposed to trim back the preamylopectin to leave amylopectin and in the process regenerate primer molecules to trigger a further cycle of synthesis and degradation. The second model of Zeeman et al. (1998a) suggests that starch is made exclusively by starch synthases and SBE and phytoglycogen is made as a byproduct of this process . They argue that phytoglycogen is subsequently degraded by a suite of enzymes including debranching enzymes and the products of this degradation can be used to support starch synthesis. Whichever of these models is correct it is clear that debranching enzymes play an important, albeit perhaps indirect, role in the process of starch biosynthesis.

The role of starch phosphorylase is less clear, since it catalyses a reversible reaction whereby glucose 1-phosphate is liberated from or incorporated into the non-reducing end of a glucan chain. Previously, based on the assumption that there was only a small amount of glucose 1-phosphate in the amyloplast, it was thought that the degradative reaction was favoured in vivo (Preiss and Levi, 1980; Steup, 1988). Plants contain both plastidial and cytosolic isoforms of this enzyme. On germination of pea embryos the plastidial isoform decreases 10-fold in activity whilst the cytosolic isoform remains unchanged (van Berkel et al., 1991). These data seem to preclude a major degradative role for the plastidial isoform and suggest that it is possible that the cytosolic isoform gains access to the starch as the amyloplastid membrane degrades. However, when the cytosolic isoform or either of the plastidial isoforms of this enzyme in potato is reduced by antisense repression no changes are observed in the rate of starch degradation (Duwenig et al., 1997a).

The degradation of starch by αα- and ββ-amylases is perhaps better characterised. Alpha-amylases are endoamylolytic, being able to break αα-1,4 bonds in amylose and amylopectin. These enzymes have been studied extensively, particularly in cereal endosperm, and are very varied with respect to both degradative ability and the extent of post-translational modification that they are subjected to (see Kossmann and Lloyd, 2000). Furthermore, in many species they appear to be gene families of αα-amylases with at least 10 in rice (Huang et al., 1990) and five in potato (Gausing and Kreiberg, 1989). It is thought that this great diversity may reflect the different roles for these enzymes in different tissues. Alpha-amylases appear to play a role in starch mobilisation during seed germination especially in cereals. In these crop plants it is thought that only hydrolytic enzymes have a role in the degradation of starch since large amounts of maltose and glucose accumulate and these substances are not known to be produced by the other degradative enzymes (Beck and Ziegler, 1989). Although the effects of αα-amylase during cereal seed germination has been well characterised for other plants its role is less clear. Studies on an Arabidopsis mutant with a reduced capacity to degrade leaf starch demonstrated that several by αα-amylases were extra-plastidial (Zeeman et al., 1998b). However, in this mutant it was one of the plastidial located αα-amylases that had a greatly reduced activity indicating that this isoform is responsible for starch degradation in Arabidopsis leaves. The role of αα-amylases in potato is currently unknown. However, in vitro studies of an αα-amylase purified from the potato starch granule revealed that at least one potato isoform is able, and correctly located, to degrade starch (Witt and Sauter, 1996).

In contrast to the αα-amylases, the ββ-amylases are exoamylolytic and liberate maltose residues progressively from the non-reducing ends of amylose and amylopectin until they react an αα-1,6 branch point. Their role in starch degradation is unclear as many isoforms have been found to be located in the vacuole. However, their activity increases during germination of seeds of mustard, maize and rice (Okomoto and Akazawa, 1980; Subbaramaiah and Sharma, 1989; Wang et al., 1997) and also on cold sweetening in potatoes (Hill et al., 1996; Nielsen et al., 1997). Since both these processes are associated with a time of active starch breakdown, it follows that there is at least some role for ββ-amylases during starch mobilisation.

The above studies have largely focussed on pathways as individual linear entities, however metabolism is in fact highly branched and better represented as a network since many pathways are linked by common metabolites and co-factors. In plant systems most prominent amongst such molecules are pyrophosphate and the adenylate and uridinylate pools. In addition other important factors that influence the regulation of carbohydrate metabolism should be considered before we begin discussing biotechnological strategies in earnest.

Pyrophosphate is an essential co-factor in starch storing organs since following SuSy-dependent sucrose cleavage, sucrose mobilisation requires the reaction catalysed by the pyrophosphate-dependent UDPglucose pyrophosphorylase (Zrenner et al., 1993). Since one molecule of pyrophosphate is required for each sucrose cleaved by the SuSy-dependent pathway and given that, in many heterotrophic cells the majority of the incoming sucrose is converted to starch, there are only two possible sources for the necessary pyrophosphate to fuel this reaction. It is either recycled across the amyloplast membrane from the starch biosynthetic pathway or it is produced by a cycling process involving pyrophosphate: fructose 6-phosphate, 1-phosphotransferase (PFP) or the tonoplast pyrophosphatase (Stitt, 1998). Correlative evidence for the former proposal includes (i) the observation that pyrophosphatase activity does not increase during the developmental switch from elongating stolon to growing tuber (Appeldoorn et al., 1999), (ii) the description of a pyrophosphate transporter in chloroplasts (Lunn and Douce, 1993) and (iii) measurements of pyrophosphate contents and the rates of sucrose degradation and starch synthesis in a range of transgenic lines exhibiting altered levels of pyrophosphate (Farre et al., 2000). Furthermore, analysis of metabolite levels in transgenic lines exhibiting reduced levels of PFP suggest that this enzyme is operating in the glycolytic direction and thus consuming pyrophosphate in vivo (Hajirezaei et al., 1994) and, therefore, is incapable of supporting sucrose degradation. Moreover, there was no difference in the rate of sucrose degradation (or resynthesis) in heterotrophic transgenic tobacco cells exhibiting increased levels of fructose 2,6-bisphosphate and therefore elevated in vivo activity of PFP (Fernie et al., 2001c). Taken together these data indicate that it is unlikely that PFP supplies pyrophosphate for sucrose degradation and it seems possible that the pyrophosphate level provides an important link between the catabolic reactions of the cytosol and the anabolic reactions of the plastid.

Direct evidence to support this model is however unfortunately lacking. When pyrophosphate levels were depressed by the expression of a bacterial pyrophosphatase, initial experiments revealed an inhibition of sucrose breakdown and a reduction in starch accumulation (Jelitto et al., 1992), whereas subsequent experimentation carried out with plants at a different developmental stage showed the exact opposite (Geigenberger et al., 1998b). A further discrepancy in the behaviour of plants exhibiting low levels of pyrophosphate is that they have been variously reported to be characterised by accelerated (Farre et al., 2001a) and delayed (Hajirezaei and Sonneewald, 1999) sprouting. However, different independent transgenic lines were used in these two studies and it is possible that different pyrophosphate levels trigger different effects. Despite these discrepancies, the combination of results to date suggest that pyrophosphate is clearly capable of effecting a variety of processes and, therefore, has a central, if somewhat enigmatic, role in the regulation of the sucrose to starch transition in heterotrophic tissues.

Studies on the effects of increasing the levels of adenylates (Tjaden et al., 1998; Loef et al., 2000; Geigenberger et al., 2001) and uridinylates (Loef et al., 2001) on biosynthesis, although limited to date, have revealed that these compounds are also important in the regulation of the starch biosynthetic pathway, with increases in cellular ATP (Loef et al., 2001) or of ATP supply to the plastid (Tjaden et al., 1998; Geigenberger et al., 2001) stimulating starch synthesis. Similarly, feeding potato tuber slices with precursors of uridinylate synthesis resulted in increase of uridinylates which stimulated starch synthesis and resulted in an increased partitioning of carbon towards starch.

In addition to their role as intermediates in metabolic pathways recently much attention has focused on the potential role of sugars as regulatory signals (Koch et al., 1990; Purcell et al., 1998). Driven by compelling evidence from the yeast system the primary candidate for a signal is glucose acting via sensing mechanisms involving either hexokinase or SNF1. In potato tubers the expression of invertase in the cytosol resulted in an accumulation of glucose and a resultant shift in partitioning from starch towards glycolysis (Trethewey et al., 1998). A similar metabolic shift was observed following the incubation of wild-type tuber discs in high-glucose concentrations (Geiger et al., 1998). However, the expression of a bacterial sucrose phosphorylase and the supertransformation of invertase expressing tubers with a bacterial glucokinase (Trethewey et al., 1998; Trethewey et al., 2001) displayed essentially the same metabolic phenotype despite the fact that they were not characterised by an increased glucose concentration. Furthermore, modulation in the activities of either isoform of potato tuber hexokinase had no major impact on either tuber morphology or on metabolism (Veramendi et al., 1999; 2001) thereby strongly arguing against a role for glucose-dependent signalling processes in the tuber.

Thus, the subject of sugar sensing remains highly controversial and there is reasonable evidence that sugar carriers or other factors at the plasma membrane may play a role in the regulation of heteotrophic metabolism (Sonnewald et al., 1997; Lalonde et al., 1999; Fernie et al., 2000, 2001a; Roitsch et al., 2000). Firstly, potato tubers expressing invertase in the apoplast are characterised by an increased rate of cell division and display a different metabolism to those expressing the invertase at a cytosolic location (Sonnewald et al., 1997) despite the fact that the hexoses released at both sites are able to enter metabolism (Fernie et al., 2000). Secondly, the rate of sucrose degradation and starch synthesis is stimulated when the unmetabolisable sucrose analogue palatinose is supplied to isolated tuber discs despite the fact that the uptake of palatinose into the tuber parenchyma is negligible (Fernie et al., 2001a). Whilst these studies allow us to speculate that these responses are mediated by a factor(s) localised in the plasma membrane (Lalonde et al., 1999), a lot of work is needed using a variety of approaches before the precise nature and role of sugar sensing in heterotrophic tissues can be defined and usefully manipulated. They do, however, remain an attractive alternative approach for metabolic manipulation since if such regulatory processes can be harnessed by the metabolic engineer as an opportunity to orchestrate rather than merely modify metabolism may arise.

Uptake of Carbon into Amyloplasts

2011-08-18T20:09:00.000-07:00

The form in which carbon crosses the amyloplast membrane and enters into starch biosynthesis has been the subject of considerable debate. Categorical evidence that carbon enters potato tuber, Chenopodium rubrum, maize endosperm, wheat endosperm and tobacco amyloplasts in the form of hexose monophosphates (or nucleosides), rather than triose phosphates was provided by determination of the degree of randomisation of radiolabel in glucose units isolated from starch following incubation of the various tissues with glucose labelled at the C1 or C6 positions (Keeling et al., 1988; Viola et al., 1991; Hatzfeld and Stitt, 1990; Fernie et al., 2001c). These data are in agreement with the observation that potato tubers lack plastidial fructose 1, 6-bisphosphatase activity (Entwistle and ap Rees, 1990) and the failure to find expression of plastidial FBPase in tubers (Kossmann et al., 1992).

Although it is clear that triose-phosphates are not the substrate taken up by amyloplasts to support starch synthesis there has been considerable debate as to whether glucose 1-phosphate (Naeem et al., 1997; Tetlow et al., 1994; Tyson and ap Rees, 1988) or glucose 6-phosphate (Schott et al., 1995; Wischmann et al., 1999) is the preferred substrate for uptake. More recently, particularly in cereals, the uptake of cytosolically produced ADPglucose has also been much discussed (Pozeuta-Romera et al., 1991a, 1991b; ap Rees, 1995). The necessary pathways to support starch synthesis presuming uptake of one of these three substrates are presented in Figure 25.3. The results of recent transgenic and immuno-localisation experiments have indicated that the substrate for uptake is most probably species specific with clear evidence of the predominant route of uptake in the developing tuber in the form of glucose-6-phosphate, whereas in barley, wheat, oat and possibly maize, the predominant form of uptake is as ADPglucose (Denyer et al., 1996; Thorbjornsen et al., 1996; Shannon et al., 1998).

The cloning of a hexose monophosphate transporter from potato and the finding that the cauliflower homologue is highly specific for glucose 6-phosphate provides strong support for the first theory (Kammerer et al., 1998). Furthermore, when this observation is taken together with in vivo evidence that transgenic potato lines, in which the activity of the plastidial isoform of phosphoglucomutase was reduced by antisense inhibition, were characterised by a large reduction in starch content (Tauberger et al., 2000), then there are compelling grounds for asserting that glucose 6-phosphate is the major form in which tuber amyloplasts import carbon from the cytosol. Since these antisense plants were not starchless we cannot, however, exclude the possibility that glucose 1-phosphate makes some contribution to the flux to starch. Nor should we, in light of recent findings of extra-plastidial isoforms of ADPglucose pyrophosphorylase (Beckles et al., 2001), overlook the possibility of production of ADPglucose by a cytosolically localised enzyme and its subsequent transport in to the plastid to supplement starch synthesis.

However, this seems unlikely since following non-aqueous fractionation of potato tuber tissue AGPase activity always co-localised with pyrophosphatase activity which is known to be located exclusively in the plastid (Farre et al., 2001b) and expression of a bacterial AGPase in the cytosol of potato tubers did not result in an altered starch content (Stark et al., 1991). Furthermore, results from recent comprehensive studies in which the ratio of ADPglucose to UDPglucose was determined in a wide range of species suggest that the presence of a cytoplasmic AGPase isoform is limited to Graminaceous endosperms and is not a general feature of starch-storing organs (Beckles et al., 1991). This rationale behind these measurements is that the metabolite ratio is expected to be high in organs in which UDPglucose and ADPglucose are both mainly produced in the cytosol since the reactions of AGPase and UGPase will be coupled and close to equilibrium.

The results from this study are in direct contrast to earlier immunolocalisation studies using antisera against AGPase which suggested that there was an extraplastidiary isoform of AGPase in tomato fruit (Chen et al., 1998), but not in maize endosperm (Miller and Chourey, 1995; Brangeon et al., 1997). However, it is possible that the immunogold-labelling patterns seen in these studies do not accurately reflect the in vivo situation. Further studies by Beckles and Smith (2001) indicated that this is indeed probably the case since the proportion of the total activity of AGPase that was confined to the plastid was similar to that of the total activity of enzymes known to be confined to the plastid. When samples of plastid and total homogenate fractions were subjected to immunoblotting with an antisera raised against AGPase, most or all of the protein detected was plastidial.

The utilisation of UDPglucose as a substrate for starch synthesis has not been discussed here as it is unlikely to be a major route of starch synthesis in plants for several reasons. Firstly, unlike certain glycogen synthases that can efficiently utilise UDPglucose as a substrate, plant starch synthases are either specific for ADPglucose, or have affinities for this nucleoside that are far in excess of those for UDPglucose (Smith, 1990). Furthermore, since UGPase appears to be absent from the plastid (Entwistle and ap Rees, 1988) there is no route other than through starch synthases by which UDPglucose can support starch synthesis. Secondly, as described above the reduction of ADPglucose production by decreasing the AGPase activity in a wide range of species by approaches of mutagenesis or transgenesis reduces starch accumulation (Tsuai and Nelson 1966; Lin et al., 1988a, 1988b; Smith et al., 1989; Müller-Rober et al., 1992). Additionally, as described above genetic manipulation of SuSy which produces UDPglucose had no effect on starch synthesis.

Plant Science Scholarship -Master of Science (MSc) programme in Plant Biotechnology (Wageningen University)

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Wageningen University's Master of Science (MSc) programme in Plant Biotechnology caters to several types of bachelor students, who wish to continue their studies at Master of Science (MSc) level. Besides our own BSc students in Plant Sciences and BSc students in Biotechnology and Biology from our own and other Dutch universities, it also aims at Dutch students who hold a bachelor of professional education's degree in e.g. (Botanical) Laboratory Research, Biotechnology or Agri- and Horticulture. How ever the majority of Plant Biotechnology master students comes from abroad.

There are three specialisations.

Functional Plant Genomics

Genomics profoundly affects plant molecular biology and genetics. Genomic information on Arabidopsis and rice has revolutionised insight into plant genomics. By using array technology, gene expression can be studied to improve our understanding of the complexity of the plant transcriptome and interactions between genes and gene products.

Plants for Human and Animal Health

Plants are increasingly being used as a safe and inexpensive alternative for the production of valuable proteins for food supplements and pharmaceuticals. This specialisation provides a fundamental understanding of how plants can be exploited for the production of foreign proteins and metabolites. In addition biomedical aspects, including immunology and food allergy, and also nutritional genomics and plant metabolomics can be studied.

Molecular Plant Breeding and Pathology

Molecular approaches to analyse and change qualitative and quantitative traits in cultivated plants are highly effective to improve yield and quality of food and renewable resources, disease resistance and abiotic stress tolerance. Molecular plant breeding focuses on the application of molecular markers and genomics to explore natural variation and on the development of transgene technologies to expand genetic variation. Molecular plant pathology aims at understanding and exploitation of plant-insect, plant-pathogen and crop-weed interactions and the development of new technologies for integrated plant health management. These technologies include improved molecular detection of pathogens and transgene technologies to introduce resistance genes into crops.

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Production of Hexose Phosphates in the Cytosol

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The circular pathway operating between sucrose and hexose phosphates is well established and has been intensively investigated (Figures 25.1 and 25.2). In the last decade a battery of transgenic plants expressing sense or antisense constructs targeted against most of the genes involved in this pathway (and ectopic expression of proteins specifically directed at many of the metabolic intermediates) have been generated. Moreover, in the last 2 years the genes for plastidial and cytosolic phosphoglucomutase (Tauberger et al., 2000; Fernie et al., 2001b) and sucrose phosphate phosphatase (Lunn et al., 2000) have been cloned suggesting that the jigsaw puzzle of genes is now complete.

Sucrose delivered to the tuber can be cleaved in one of three ways (i) as described above in the apoplast by acid invertase or in the cytosol by either (ii) alkaline invertase or (iii) sucrose synthase (SuSy). As indicated in Figures 25.1 and 25.2 the primary route of sucrose cleavage mirrors the mechanism of unloading with invertase activities being high during the early stages of tuber initiation whilst SuSy dominates in the developing tuber (Appeldorn et al., 1999), whereas the opposite is true for the developing tomato fruit (Damon et al., 1988; Robinson et al., 1988; DemnitzKing et al., 1997). Cereal seeds tend to have high apoplasmic invertase activities to facilitate unloading, however, as mentioned above these are not a prerequisite for unloading hence it is most likely that both pathways have a role to play in sucrose mobilisation within this sink organ (Schmalstig and Hitz, 1987).

The products of sucrose cleavage enter into metabolism by the concerted action of fructokinase and UDPglucose pyrophosphorylase (Zrenner et al., 1993) or fructokinase and hexokinase (Smith et al., 1993; Veramendi et al., 1999, 2002) in the case of the SuSy and invertase pathways, respectively. The hexose phosphates produced by these reactions are equilibrated by the action of cytosolic isoforms of phosphoglucose isomerase and phosphoglucomutase (Fernie et al., 2001b). Sucrose mobilisation has been subject to intense investigation in all crop species, however, perhaps the greatest scrutiny has been applied to the potato.

Experiments of Fu and Park (1995a) (Fu et al., a,b) revealed that potato contains two differentially expressed classes of genes encoding for SuSy in potato which they named SuS3 and SuS4. Both classes were shown to contain 13 introns, including a particularly long leader intron, and their coding regions were found to be 87&percnt; identical at the nucleotide level. Using GUS fused to the 5′ flanking sequences they found that SuS3 is strongly expressed in vascular tissues of leaves, stems, roots and tubers—implying a possible role in energy provision in phloem cells: whereas SuS4 genes are strongly expressed in sink tissues such as root tips, basal tissues of the shoot and potato tubers. The SuS4 genes correspond to the cDNA for the T-type isoform first cloned by Salanoubat and Belliard (1987) who proposed that this isoform plays a dominant role in the metabolism of sucrose within the tuber. The T-type isoform was subject to antisense inhibition using the 35S promoter (Franck et al., 1980), and a reduction in SuSy activity was only found in the tubers (Zrenner et al., 1993). In tubers a reduction in activity of up to 95&percnt; resulted in a reduction in starch and storage protein content of mature tubers but surprisingly no changes in the level of sucrose. There was, however, a significant increase in the level of hexoses which was in keeping with an observed 40-fold increase in the invertase activity of these lines. The exact reason for this compensatory increase is unknown but it serves to provide further evidence of the flexibility inherent within plant metabolism. Such metabolic flexibility appears to be a central feature of plant metabolism and probably accounts for the absence of major metabolic effects of many transgenic manipulations (e.g., Burrell et al., 1994; Hajirezaei et al., 1994; Fernie et al., 2001c). Thus, when taken together the results of the studies of Zrenner and co-workers strongly indicate that SuSy plays a significant role in determining potato tuber sink strength—at least when activities are reduced. The fact that the enzyme is likely to operate close to equilibrium in vivo (Geigenberger and Stitt, 1993), probably explains the failure to increase sink strength via overexpression strategies (Howard et al., 2001). Further transgenic studies confirmed the pre-eminance of the SuSy route of sucrose cleavage within the developing tuber. Potato plants repressed in the activities of hexokinases (Veramendi et al., 2002, 1999) or of acid invertase (Zrenner et al., 1996) exhibiting very little difference from wild-type tubers implying that these enzymes do not play such a crucial role in metabolism during this stage of the tuber life cycle.

Although the net flux in the tuber is one of sucrose degradation, the rate of sucrose (re)synthesis within this and other tissues is considerable (Wendler et al., 1991; Geigenberger et al., 1997; 1999a; Fernie et al., 2001c; Nguyen-Quoc and Foyer, 2001). This process can also proceed via two different pathways: the reverse of the SuSy degradative pathway or the reactions catalysed by sucrose phosphate synthase and sucrose phosphate phosphatase (Geigenberger and Stitt, 1993). There is clear evidence from feeding experiments with labelled sugars that both pathways contribute to sucrose (re)synthesis within the developing tuber (Geigenberger and Stitt, 1993). It is thought that the combined operation of these pathways with the degradative pathway allows the cell to respond sensitively to both variations in sucrose supply and the cellular demand of carbon for biosynthetic processes (Hatzfeld and Stitt, 1990). The importance of SuSy is demonstrated following antisense repression of this enzyme which resulted in a reduced starch content, coupled to a reduced tuber dry weight and a reduction in storage proteins. Furthermore, when SuSy is bypassed either by introduction of a yeast invertase (Sonnewald et al., 1997; Trethewey et al., 1998) or a bacterial sucrose phosphorylase (Trethewey et al., 2001) there are similar dramatic repercussions on metabolism.

In contrast, sucrose synthesis via sucrose phosphate synthase is relatively well defined (Geigenberger et al., 1997). Although the reaction catalysed by sucrose phosphate synthase is not irreversible, the efficient removal of its product by sucrose phosphate phosphatase means that sucrose phosphate synthase is the first commited reaction in the sucrose synthetic pathway. In photosynthetic tissue sucrose phosphate synthase has been found to be regulated at a variety of levels including allosteric activation by glucose 6-phosphate and inhibition by inorganic phosphate (which allows sucrose synthesis to proceed at times when substrate is plentiful) and deactivated by protein phosphorylation (Reimholz et al., 1994). There is now a growing body of correlative evidence that the potato tuber enzyme is regulated in an analogous manner to the leaf enzyme (Geigenberger et al., 1994, 1999a; Fernie et al., 2001b). The complex regulation of this enzyme suggests that it plays an important metabolic role, a view that is supported by antisense studies which reveal only a minor influence on starch metabolism but a major role in sucrose synthesis in response to water stress (Geigenberger et al., 1997).

In sharp contrast to the lack of changes observed following antisense repression of the majority of enzymes involved in sucrose degradation in the potato are the severe changes seen following repression of the same enzmyes in tomato. With tomato plants altered in invertase (Dickinson et al., 1991; Klann et al., 1996), SuSy (D'Aoust et al., 1999) and hexokinase (Dai et al., 1999) activities display both dramatic phenotypes and marked shifts in metabolism. Transgenic tomatoes expressing a yeast invertase in their apoplast were severely repressed in their growth (Dickinson et al., 1991), however, this transgene was under the control of the tissue-constitutive 35S promoter, and it is most likely that this phenotype results from a block in photoassimilate export. Interestingly, when the expression of one of the tomatoes' six endogenous invertase genes, TIV1, was reduced constitutively by antisense repression of the fruit size was also reduced (Klann et al., 1996) and the precise role of this or any of the other tomato invertases remains to be elucidated. In contrast, the role of SuSy in tomato fruit is well defined and analogous to that in the potato tuber, it seems to be an important determinant of sink strength as indicated by the decreased fruit setting and sucrose unloading capacity of plants exhibiting reduced expression of the enzmye (D'Aoust et al., 1999). Marked phenotypic changes were also observed on the overexpression of an Arabidopsis hexokinase gene in transgenic tomato plants which displayed dramatic growth reduction and early senescence changes which were not seen in the potato (Veramendi et al., 1999). However, it is likely that many of these changes result from the dramatic reduction of leaf photosynthesis in these plants (Dai et al., 1999). In addition to these phenotypic changes, changes in the sugar content and its subsequent mobilisation via metabolism were observed in all the above examples and also in situations wherein the activities of sucrose phosphate synthase (Nguyen-Quoc et al., 1999) were altered transgenically and various invertase activities were altered by conventional plant breeding (Chetelat et al., 1995; Fridmann et al., 2000; Husain et al., 2001). One clear reason for the differences observed on manipulating certain enzyme activities in tomato as opposed to potato is that the tomato fruit goes through large developmental changes whereas the potato tuber does not. This is also reflected in their metabolism. For example starch accumulation in potato proceeds linearly with time (Moorby and Milthorpe, 1975) but is merely transient in tomato fruit (Schaeffer and Petroikova, 1997).

The phenotypes observed in maize seeds on the modification of pathways of sucrose mobilisation are equally severe. We have described the minature1 mutant of maize deficient in cell wall invertase above. Maize further contains two isoforms of SuSy SH1 and SUS1 encoded by the Shrunken1 (Sh1) and Sucrose synthase I (Sus1) loci, respectively. Although both genes are expressed in the developing endosperm, SH1 contributes the majority of the total SuSy enzyme activity. Seeds of the shrunken1 mutant have a mild starch deficiency and degenerate storage cell whereas those of the sus1 mutant shows no phenotypic change (Chourey et al., 1998). Furthermore, distinct phenotypes have been observed on studies of transgenic carrot plants wherein the activities of either invertase or SuSys were altered in the taproots. When invertase activity is severely reduced the carrot plants fail to develop roots suggesting a crucial role for this enzyme in the regulation of carbon portioning (Tang et al., 1999). Reduction of the SuSy activity of carrot roots results in decreased root growth. The transgenic plants also displayed a reduced sucrose utilisation but an increase in starch and cellulose accumulation which was interpreted to suggest that SuSy played an important developmental role within the carrot plant (Tang and Sturm, 1999).

When taken together these examples highlight that plant species vary dramatically with respect to the importance of various routes of both sucrose delivery and utilisation and that successful manipulation at a certain metabolic loci in one species does not imply that such a manipulation will be possible in other species.

It is also important to note that hexose phosphates have a crucial mediatory role in the sucrose to starch transition; they also serve as precursor substrates for glycolysis and oxidative pentose phosphate pathway. However, these pathways have also been the subject of many transgenic studies in heterotrophic tissues and this subject area has been extensively reviewed elsewhere in recent years (Stitt and Sonnewald, 1995; Neuhaus and Emes, 2000; Given, 1999).

Pathways of Heterotrophic Carbohydrate Metabolism

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The Supply of Sucrose

In photosynthetic and gluconeogenic tissues sucrose is predominantly exported from cells, most probably by facilitated diffusion, and subsequently taken up by the phloem complex through a specific sucrose/H+ co-transport mechanism (Riesmeier et al., 1994; Frommer and Sonnewald, 1995). Once in the phloem complex, sucrose is transported to cells of heterotrophic ‘sink’ organs. Sucrose obtained through translocation can enter a cell via the symplasm (Figure 25.1) or the apoplasm (Figure 25.2) and in many species the nature of the predominantly used route is hotly debated. Several studies using assymetrically labelled sucrose suggest that carbon obtained by heterotrophic cells moves primarily through the symplasmic route and is not cleaved to glucose and fructose during transport. It seems likely that cells of many species receive most of their sucrose by such a route (Patrick, 1990; Tegeder et al., 1999; Lalonde et al., 1999).

However, in certain tissues it is clear that sucrose must be supplied through the apoplasm. This is certainly the case in developing seeds in which protoplasmic connections between maternal and embryonic tissue simply do not exist. Thus, studies on the pathway of uptake of sucrose from the apoplast revealed that there is not a single route of uptake. Hydrolysis of sucrose precedes uptake by developing seeds of maize, sorghum and pearl-millet, whereas in wheat, rye and barley sucrose appears to be transferred without cleavage (Thorne, 1985; Weschke et al., 2000). However, even in species where apoplasmic hydrolysis of sucrose occurs this does not seem to be a prerequisite for uptake since the invertase-resistant sucrose analogue 1-fluorosucrose is taken up by maize seeds at similar rates to that of sucrose (Schmalstig and Hitz, 1987). Studies on the minature-1 mutant of maize, deficient in apoplasmic invertase activity, revealed that seeds were only one fifth the normal weight (Miller and Chourey, 1992), suggesting that apoplasmic hydrolysis of sucrose may play an important role in the maintenance of source to sink sucrose gradients.

The pathway of phloem unloading in the tuber has been the subject of much debate. It has been clear for some time that plasmodesmatal connections between the phloem and the surrounding parenchyma cells exist in the tuber (Oparka and Prior, 1987) and that plasmolysis of growing tubers has an inhibitory effect on the flux of sucrose into the tuber (Oparka and Wright, 1988) suggesting that unloading occurs via a symplastic mechanism. However, isolated tuber discs display a substantial capacity to take up sucrose supplied to the surrounding media (Geigenberger et al., 2000; Fernie et al., 2001a). Furthermore transgenic expression of a yeast-derived invertase in the apoplast under the control of the tuber specific B33-patatin promoter significantly altered tuber yield (Sonnewald et al., 1997) indicating the importance of apoplastic sucrose.

Recent studies using a combination of confocal microscopy, autoradiography and biochemical analyses have provided definitive evidence that unloading in the potato tuber is predominantly apoplastic during stolon elongation and becomes primarily symplastic during the initial phases of tuberisation (Viola et al., 2001). This is in direct contrast to the situation observed in the developing tomato fruit in which sucrose unloading is predominantly symplasmic during early, starch accumulating, stages of development (Damon et al., 1988; Ruan and Patrick, 1995) and apoplasmic during later, hexose accumulating, stages (Patrick et al., 1990; Ruan and Patrick, 1995).

Given that sucrose unloading is essentially symplastic in the developing potato tuber the impact on tuber morphology following expression of a heterologous invertase in the apoplast at this developmental stage (Sonnewald et al., 1997; Hajirezaei et al., 2000) is intriguing. However, despite the large morphological changes apoplastic expression of invertase had no effect on the levels of cellular metabolites (Hajirezaei et al., 2000) and the role of sugars in the apoplastic space of the tubers remains unsolved. Interestingly, when the rate of glucose consumption in these transgenic lines was increased by the cytosolic expression of a bacterial glucokinase the total hexose content of the tuber was reduced implying that apoplastic hexose is somehow able to enter cytosolic metabolism (Fernie et al., 2000). Comparison of transgenic plants exhibiting apoplastic with those exhibiting cytosolic expression of the invertase reveals that a completely different phenotype is produced depending upon the compartment to which the enzyme is targeted. This observation together with results from biochemical studies, suggests that the route of entry of hexoses into metabolism differ according to whether they are generated in the cytosol or the apoplast.

For sugars synthesised in the apoplast this could imply either an endocytotic-like mechanism of transport to the vacuole and subsequent release to the cytosol or delivery into the cytosol by a specific hexose transporter in the plasma membrane which have a signalling capacity (Lalonde et al., 1999; Fernie et al., 2000). It is clear that an apoplasmic unloading mechanism needs the presence of one or both types (monosaccharide and sucrose) of transporter at the plasma membrane. Tables 25.1 and 25.2 list the currently sequenced monosaccharide and sucrose transporters of agronomically important plant species and of Arabidopsis. Since these transporters have been the subject of two excellent recent reviews (Lalonde et al., 1999; Lemoine, 2000) and to our knowledge there has been very little success, from a biotechnological standpoint, in the genetic manipulation of these transporters we will not discuss them in detail here. It is however worth pointing out that although the transport mechanism of the much studied potato sucrose proton transporter SUT1 has been characterised by expression in Xenopus oocytes (Boorer et al., 1996) its precise role in planta is yet to be fully elucidated. Since this is one of the best characterised transporters it, therefore, follows that much work is required before the factors controlling the intracellular movement of sugars within the heterotrophic cell can be fully resolved.

Carbohydrate Metabolism in Plants

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The yield from crop plants has been subject to constant improvement through conventional breeding and refinements in agricultural practice for many decades. In the case of the potato, which will form the major focus of this chapter, the harvest index (ratio of dry weight of harvestable organs to the dry weight of the entire plant) has been increased from 0.09 in wild species up to 0.81 in modern cultivars (Inouhe and Tanaka, 1978), with smaller, yet dramatic, improvements found in other crop species (Ellen, 1993; Hay, 1995).

Although such improvements have proved both dramatic and revolutionary they were also time consuming and slow. The emergence of molecular-assisted breeding (see Chapter 6) and plant transformation technologies (see Chapters 8 and 9) offers the possibility of manipulating metabolism using a more rapid, targeted approach. Indeed since the advent and widespread adoption of transgenesis approaches some 15 years ago gave rise to the discipline of molecular plant physiology, much information has been obtained concerning the potential to manipulate plant metabolism. In this chapter we intend to review the many previous studies of genetic manipulation of heterotrophic carbohydrate metabolism in plants.

It is clear that the successful manipulation of plant metabolism requires detailed understanding of the underlying factors that regulate it. For this reason we intend to describe the current understanding of the central pathways of carbohydrate metabolism. Some stress will be put on the sucrose to starch transition since this pathway has received great attention over the past few years. In the case of the potato (Solanum tuberosum) tuber all the genes believed to be directly involved in the sucrose to starch transition have been cloned, the final gaps being filled within the last couple of years (Veramendi et al., 1999; Tauberger et al., 2000; Fernie et al., 2002; Veramendi et al., 2002).

Furthermore, an impressive variety of transgenic lines have been generated where the activities of most of the individual genes have been modulated, alone or in combination. In addition, a large range of transgenic potato lines has been created where deregulated alternatives to endogeneous enzymes have been introduced. These studies have allowed the confirmation of many longstanding hypotheses which were previously based upon indirect methodologies all of which will be summarised within this chapter. In addition, this chapter will also cover the synthesis of fructans and the introduction of novel carbohydrates into plants and will review recent advances in understanding and influencing structural properties of starch. As stated above we intend to split this review into two major sections. The first of these is a description of the pathways operating in various agronomically important plants following the path of carbohydrate from sucrose transported from source tissues to its assimilation into storage carbohydrate in sink tissues.

The metabolism of the major forms of carbohydrate: sucrose, starch and fructans will be covered in some detail. The second section provides a review of strategies taken to manipiulate these pathways for commercial gain with particular prominence given to alteration of starch functionality and to increasing end product accumulation of starch and sucrose and finally, the manipulation of minor and novel sugars are reviewed.

Benefits of Virus-Resistant Crops

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Viral-resistance in crop plants is engineered based on the premise that the host plants express genes that interfere with the essential functions of the virus thereby upsetting the balance of related components. Coat-protein mediated pathogen-derived resistance is the commonly used method to introduce resistance in crop plants to viruses. In this approach, plants are transformed with a specific virus coat protein gene which interfere with critical processes such as replication, post-transcriptional gene expression, virion coating and uncoating and intercellular transport (Beachy et al., 1990; Kaniewski and Lawson, 1998). Constitutive expression of the coat protein gene confers protection against infection with the virus from which the gene is derived and possibly against infection from other related viruses (Di et al., 1996).

Commercially available virus-resistant crops include papaya, summer squash and potato. Virus-resistant papaya and squash have been available since 1998. Virus-resistant trait was stacked with Bt to broaden the range of protection against pest populations in potato and was discussed in insect-resistant plants section.

Adoption of virus-resistant papaya has been rapid since its introduction (53&percnt; in 2000). It is expected that virus-resistant papaya will be planted on almost 90&percnt; of the acreage in the next few years. On the other hand, biotechnology-derived summer squash was planted on less than 10&percnt; of the total acreage in the United States in 2000. Lack of resistance to important pathogenic viruses coupled with the availability of virus-resistance trait in only few varieties is cited to be the reasons for low adoption.

Biotechnology-derived virus-resistant crops are particularly valuable as management options that limit viral infestations to prevent serious yield losses are limited. Since viral infestations cannot be controlled by chemical means, conventional way to manage viruses is to manage their transmission by controlling insects. Preventing the spread of virus by controlling insect vectors is not effective for two reasons: virus transmission through insects is almost instantaneous which render insecticide applications futile and secondary hosts that harbour the viruses do not exhibit symptoms. Another widely used management technique to control viruses is use of resistant varieties in crops such as squash. Natural resistance may not be available to combat viruses in crops such as papaya. However, both these methods are not completely effective in preventing viral infestations.

Papaya industry in the United States concentrated mainly in Hawaii was on the brink of extinction in 1990s due to the epidemic infestations of papaya ringspot virus (PRSV). PRSV is the most important disease of papayas. The PRSV is transmitted by aphids and cannot be eradicated as secondary hosts harbour the virus without exhibiting any symptoms. Hawaiian farmers had no choice other than destroying the infested plants to contain the disease.

Viruses that limit summer squash production in the United States are zucchini mosaic virus (ZMV), watermelon mosaic virus 2 (WMV), cucumber mosaic virus (CMV) and papaya ringspot virus (PRSV). All these viruses are transmitted by aphids and affect a range of plants making it difficult and impossible to prevent virus infestations. Foliar applications of highly refined petroleum oil are widely used to serve as a barrier between aphid and the plant to prevent virus transmission. However, frequent applications are needed to ensure season-long protection.
Virus-resistant plants enable growers to reduce the use of pesticide by eliminating the need to spray insecticides to control the insects that transmit viral diseases, or herbicides to kill the weeds that harbour those insects. As a result, overall pesticide use and crop production costs have been reduced. An indirect benefit of virus-resistant crops is they do not serve as reservoirs for viruses unlike their conventional counterparts. As a result, further spread of virus to susceptible plants by vectors is prevented.

Papaya

Virus-resistant papaya is an exemplary example that demonstrated the promise biotechnology holds. It literally saved an industry that could disappear. A recent survey by USDA suggested that papaya yields increased by 33&percnt; in 2000 compared to 1998, which is a direct consequence of using PSRV-resistant plants (USDA-NASS, 2001b).

Squash

Biotechnology-derived virus protection in squash translated to increased number of harvests and increased yield per harvest. Evidence suggests that virus-resistant squash produces greater marketable yields of high quality fruit, particularly in production areas where high virus incidence limits the growing season both in terms of number of plantings and number of harvests per planting (Fuchs et al., 1998; Schultheis and Walters, 1998). However, virus-resistant squash has not reduced insecticide use as chemical applications that control aphids also control white flies and will be made to biotechnology-derived squash also.

Benefits of Insect-Resistant Bt Crops

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Insect-resistant Bt crops offered positive benefits to growers in two ways: by reducing insecticide costs and increasing yields. Since Bt crops eliminate the need for insecticide applications, major impact of insect-resistant crops has been the reduction in insecticide use targeted for key pest control. Insect-resistant crops express toxic proteins during much of the crop season as a result of which supplemental insecticide applications are not needed for pest control. Unlike herbicide-tolerant crops, insect-resistant crops increased crop yields due to enhanced levels of insect control. Overall, direct grower benefits from insect-resistant Bt corn, cotton, potato and sweet corn were reported to be $112 million in 1999 in the United States (EPA, 2000). By 2001, net income of US growers was reported to increase by $228 million from Bt corn and cotton plantings alone (Gianessi et al., 2002).

Corn

A primary benefit of insect-resistant corn has been the opportunity to control a pest that previously escaped control and reduced yields. Though modest, adoption of Bt corn led to reductions in insecticide use. Since the introduction of Bt corn, acreage sprayed with insecticides for ECB control has been reduced resulting in over one million fewer acres treated for ECB (Carpenter and Gianessi, 2001). Only a minor acreage, about 5&percnt;, is treated for ECB control in the United States, which is the reason for the modest reduction in insecticide use due to Bt corn (Phipps and Park, 2002). Additionally, the insecticides used against the ECB are also used to control other insect pests to which Bt trait does not provide resistance and would still be applied regardless of ECB.

Yield gain and economic benefit from Bt-corn fluctuates based on variability in ECB infestation levels. While yield increased and resulting economic benefits were lower in low infestation years, Bt-corn delivered a significant economic benefit when ECB outbreaks occurred (Alstad et al., 1997). As a result, net returns have been higher for Bt corn in spite of seed premium and technology fees (Fernandez-Cornejo and McBride, 2000). On an average, yield advantage from Bt-corn ranged from 4&percnt; to 8&percnt;, depending on the levels of ECB infestation (Marra et al., 1998).

A significant benefit of Bt-corn is decreased secondary pathogen infestations (e.g. ear rot) due to reduction in entryways left by ECB (Alstad, 1997). Fusarium ear rot is the most common ear rot disease in the corn belt; it has been found in nearly every corn field at harvest. The severity of this disease is usually low, but it can reduce yield and quality. Symptoms of Fusarium ear rot are often highly correlated with ear damage by ECB. The primary importance of this disease is its association with mycotoxins, particularly the fumonisins. Fumonisins are a group of mycotoxins that can be fatal to livestock and are probable human carcinogens (Munkvold and Desjardin, 1997). The importance of fumonisins in human health is still a subject of debate, but there is evidence that they have some impact on cancer incidence (Marasas, 1995). Multi-year studies showed that kernel feeding by insects, extent of ear rot infestation and fumonisin levels in Bt corn were significantly lower than conventional corn (Munkvold et al., 1999). Volunteer corn in the following season has been reduced, as ears dropped due to ECB infestation are less with Bt corn (Alstad, 1997).

Depending on the prevalence of ECB populations, Bt-corn influences the local ECB population (Alstad, 1997; Andow and Hutchison, 1998). It is possible that planting non-Bt-corn near Bt-corn could suppress ECB populations in non-Bt corn and this localised benefit is called the halo effect. Similar effects may be noted with other insect-resistant crops.

Cotton

Insect-resistant Bt cotton has provided a tool to cotton growers to control the most damaging pests. Insect-resistant cotton resulted in highest per acre grower benefits and largest reduction in insecticide use among all the insect-resistant crops. In states such as Alabama, growers used the least amount of insecticides on cotton since the 1940s (Smith, 1997). A 1999 estimate by the EPA (2000) suggested a reduction of 1.6 million pounds of insecticide active ingredient use and 7.5 million acre treatments due to Bt cotton. Based on the USDA pesticide use data, growers in six major cotton-growing states reduced insecticide use by 16&percnt; and insecticide applications by 25&percnt; in 2000 compared to 1995 (Carpenter and Gianessi, 2002). A similar estimate by Fernandez-Cornejo and McBride (2000) also showed that Bt cotton growers applied 2.5 fewer insecticide applications per acre. Though not as dramatic as reductions in insecticide use, insect-resistant cotton led to reduced yield losses as a result of which yield advantage has been realised in many cotton-growing states (Fernandez-Cornejo and McBride, 2000). The overall effect of reduction in insecticide use and gains in yields has been higher net return to cotton growers, despite the technology fee. Grower benefits have increased from 16 million in 1996 to 44 million in 1999 due to Bt cotton (EPA, 2000).

By targeting specific insects through the naturally occurring protein in the plant, Bt cotton reduces the need for and use of chemical insecticides. By eliminating chemical sprays, the beneficial insects that naturally inhabit agricultural fields are maintained and can even provide a secondary level of pest control. This is the reason why Bt cotton adoption is high in areas where boll weevil eradication programmes are in effect as insurance against unchecked bollworm and budworm populations due to elimination of natural predators with the use of malathion.

Evidence states that insect-resistant crops impact local ecosystems favourably. Beneficial insect-feeding bird populations have been reported to be higher in numbers in Bt cotton fields compared to conventional fields (Edge et al., 2001).

A major worry concerning the success of Bt crops, especially cotton, is the potential vulnerability to eventual adaptation by insect pests to Bt toxin. Large-scale deployment of Bt crops will impose selection pressure for pre-existing Bt-resistant insects to increase their numbers resulting in the loss of viability of this environmentally sound pest control practice. Several resistance management strategies have been proposed to slow the evolution of insect adaptation to Bt genes such as refuges, intense field monitoring of insect-resistant plants for potential escapes and alternate control strategies.

To slow the adaptation of insects to Bt toxin, the EPA has mandated that cotton growers should plant at least 4&percnt; of their biotechnology-derived crop with conventional cotton varieties and this refuge cannot be treated with any insecticides. The advantage of planting refuges is that they will harbour susceptible insects and thus retard the evolution of insect resistance against the Bt gene. Gould et al., (1997) predicted that Bt cotton could remain efficacious for 10 years with 4&percnt; refuge.

Potato

Due to low adoption rates, insecticide use reductions in potato are not as dramatic as in cotton. Based on 4&percnt; market share of Bt potato, insecticide use reduction from Bt potato has been reported to be 89 000 less acre treatments with corresponding grower benefit of $9.30 (EPA, 2000) to $11.50 (Gianessi et al., 2002) per acre. Insect-resistant Bt potato has not yet made a significant impact on overall yield.

An indirect benefit of insect-resistant crops, potato and cotton in particular, is the worker safety the technology affords. Insecticides routinely used for pest control in cotton and potato such as organophosphates, carbamates and synthetic pyrethroids are known to cause adverse health effects in workers. Insect-resistant Bt cotton eliminates the need for the use of the above chemicals as a result of which occupational risk is minimised.

Sweet Corn

A notable impact of Bt sweet corn is the reduction in number of insecticide applications. Based on sweet corn acreage planted with Bt varieties in 1999, EPA (2000) reported that reduction in insecticide applications have been 4.3 per acre or a total of 127 000 acre applications in the United States. Reduction in insecticide applications on a per acre basis has been the highest in Bt sweet corn compared to any biotechnology-derived crop. Benefits from improved pest control and reduced application costs have been $5.40 per acre (EPA, 2000). An added benefit to insect-resistant sweet corn is the reduction in yield loss caused by feeding damage of fall armyworm and corn earworm. Season long protection offered by Bt sweet corn resulted in significantly higher marketable yield than conventional varieties (Stegelin, 2000). Overall, once the market penetration of Bt sweet corn increases, growers are expected to note significant reductions in overall insecticide use and enhanced returns.

Insect-Resistant Crops

2011-08-02T03:34:00.000-07:00

Insect-resistant crops were developed to contain genes from Bacillus thuringiensis (Bt) that encode for proteins toxic to insects. Bt is a soil bacterium, which produces crystalline proteins (referred to as Cry proteins) that are toxic, to select insect orders such as lepidoptera, diptera and coleopera (Swadener, 1994). When insects ingest the protein produced by Bt, the toxin binds to specific receptors on the mid gut epithelial cells. As a result, the cell membrane develops pores, which affects the insects' ability to regulate osmotic pressure. The function of insects' digestive system is thus disrupted, leading to starvation and eventual death.

The Cry proteins have been used as insecticides since 1961, mainly in organic crop production. The commercial Bt products are available in powder formulation, containing a mixture of dried spores and toxin crystals. They represent about 1&percnt; of the total agrochemical market across the world.

There are four insect-resistant Bt crops that have been approved for commercial production in the United States to date. They are field corn, cotton, potato and sweet corn. Field corn, cotton and potato were commercialised in 1996 while sweet corn was commercially introduced in 1998.

Insect-resistant field corn was developed to express Cry1A(b) and Cry1A(c) proteins that confer protection against European corn borer (ECB), south-western corn borer, fall armyworm, corn earworms and stalk borers. Insect-resistant Bt corn has no activity on other corn pests such as aphids, spider mites, cut worms and soil insects such as rootworms, wireworms and seed corn maggots. European corn borer is the primary target pest for Bt corn. Since its introduction, adoption of Bt corn steadily increased until 1999 (26&percnt;) but remained flat at 19&percnt; in 2000 (Table 61.2). Low corn prices and low pest pressure in the past two growing seasons were suspected to be the reasons for the slight decline in adoption rate in 2000
Insect-resistant cotton acreage steadily increased since its introduction and was planted on about 28&percnt; of the total cotton acreage in 2000 (Table 61.2). Bt cotton adoption rate is higher in certain states in the United States such as Alabama, where losses from bollworm/budworm infestations are huge. Target pests for Bt cotton are tobacco budworm, cotton bollworm and pink bollworm. While cotton bollworm and tobacco budworm infest south-east and mid-south production areas, pink bollworm is prevalent in western states of the United States.

The Colorado potato beetle-resistant potatoes developed to express Cry proteins (CryIIIA) from Bacillus thuringiensis var tenebrionis were marketed as NewLeafpotatoes in 1996. NewLeafPlus with resistance to the potato leafroll virus; and NewLeafY with additional resistance to potato virus Y were developed by stacking the virus-resistance traits with Bt and were introduced in 1999. Combined adoption of all three types of Bt potatoes has been limited and planted acreage never exceeded greater than 4&percnt; in the United States (Table 61.2). The low adoption is attributed to marketing concerns.

Insect-resistant Bt sweet corn [Cry1A(b)] tolerant to lepidopteran pests such as corn earworm and fall armyworm was commercialised in 1998. Adoption of Bt corn has been very low thus far due to the reluctance of fresh corn marketers' to purchase biotechnology-derived produce.

Use of insecticides has been the most commonly used method for insect control since 1930s in the United States. Insecticides have routinely been used in an integrated approach along with cultural practices such as crop rotation, tillage, and insect-resistant crop varieties. Major limitations to the use of insecticides are resurgence of primary and secondary pests and development of insect resistance to insecticides.

Resurgence occurs when insects normally killed by insecticides return in larger numbers. When insecticides remove target insects and their natural enemies, opportunity exists for the temporarily removed pests to reproduce before their natural enemies return. Spider mites, for example, caused havoc when DDT and other insecticides killed their predators.

The problem of insect resistance to conventional insecticides is already a serious issue, estimated to contribute about 25&percnt; of the pest control expenditure in the United States. Insect-resistance results in diminished utility of insecticides and places tremendous selection pressure on few products, which could further aggravate the problem. Resistance is the result of selection, where few insects in the population with genes of specific resistance mechanisms survive the insecticide sprays and multiply, thereby increasing the proportion of resistant insects in the population. The Colorado potato beetle is considered to be the most resistant pest in North America since it has developed resistance to every group of insecticides that growers have used against it. Other gaps in insect management using conventional tactics specific to each crop for target Bt pests are discussed below. Biotechnology-derived insect-resistant crops bridge the gaps in conventional insect management tactics with no need for chemical sprays to control target pests.

Corn

European corn borer damage results in poor ear development, broken stalks and broken ears, and ultimately yield losses due to larval feeding on kernels, leaves and conductive tissue. Its feeding on stalks and kernels increases the incidence of secondary infestations of stalk-rot fungi and mycotoxin-producing fungi, respectively. European corn borers also carry spores of secondary pathogens such as ear rot fungi from the leaves to the developing kernels and thereby increase the incidence of kernel rot and symptomless infections.

European corn borer is a difficult pest to control for two reasons. First, European corn borer levels are difficult to predict and vary greatly from year to year. As a result, growers are usually reluctant to incur costs on scouting to determine the feasibility and profitability of insecticide applications. Second, ECB control is complicated due to the feeding and survival behaviour of the insect. Corn borer larvae feed in leaf whorls after hatching and eventually move into the stalks to pupate inside the stem burrows thereby avoiding insecticide applications. Insecticides need to be applied during the two to three days period between egg hatching and their burrowing in the stems. Thus, carefully timed insecticide applications are the key for the successful control of ECB.

European corn borer control with the conventional insect-tolerant varieties and available insecticide options is only marginal to good. Consequent yield losses from ECB have been as high as 300 million bushels per year accounting to monetary losses of up to one billion dollars in the United States (Mason et al., 1996).

Cotton

Chemical control costs for cotton bollworm, tobacco budworm and pink bollworm amount to about 60&percnt; to 70&percnt; of the total pesticide costs to US cotton growers. Cotton insect management is very intensive; more than 90&percnt; of the entire cotton acreage is treated with insecticides and use of about ten insecticide applications was not uncommon in one season. More insecticides are applied to cotton than in any other crop in the United States (Gianessi and Marcelli, 1997). A limitation to cotton insect management using conventional insecticides has been the development of resistance in insects to pyrethroids, organophosphates and carbamates.

Potato

Roughly two-thirds of the total potato insecticides are targeted to control two insect pests, Colorado potato beetle (CPB) and green peach aphids. Colorado potato beetle is the most devastating and is particularly difficult to control as it has developed resistance to a broad range of insecticides such as arsenicals, organochlorines, organophosphates, carbamates and synthetic pyrethroids. Sprays of Bt are not widely used due to lack of effectiveness on early instars, lack of residual activity and stringent requirements on application timing (Whalon and Ferro, 1998).

The green peach aphid serves as a vector in transmitting potato leaf roll virus (PLRV). Potato leaf roll virus causes net necrosis in tubers, thereby lowering the marketable value of the crop. Since no chemical control options are available for virus control, the only way to limit virus infestations is to control insect vectors that transmit the virus.

Sweet Corn

Similar to ECB, the internal feeding habit of corn earworm and fall armyworm on sweet corn makes them only susceptible to pesticide applications during a narrow window of time when they migrate to newly developing ears. Once the larvae enter the ears, they are virtually impervious to chemical sprays. Most sweet corn growers consider 2&percnt; to be the maximum tolerable damage level for fall armyworm or corn earworm. As a result, it is not uncommon for sweet corn growers in states such as Florida to make up to 12 insecticide applications throughout silking stage to control these pests. Insect-resistant sweet corn varieties and biological methods have been used to no avail.

Map of Plant Protein Interactions

2011-08-01T00:21:00.000-07:00

An international team of scientists has described their mapping and early analyses of thousands of protein-to-protein interactions within the cells of Arabidopsis thaliana -- a variety of mustard plant that is to plant biology what the lab mouse is to human biology.

"With this one study we managed to double the plant protein-interaction data that are available to scientists," says Salk Institute plant biologist Joseph Ecker, a professor in the Plant Molecular and Cellular Biology Laboratory. "These data along with data from future 'interactome' mapping studies like this one should enable biologists to make agricultural plants more resistant to drought and diseases, more nutritious, and generally more useful to mankind."

The four-year project was funded by an $8 million National Science Foundation grant, and was headed by Marc Vidal, Pascal Braun, David Hill and colleagues at the Dana Farber Cancer Institute in Boston; and Ecker at the Salk Institute. "It was a natural collaboration," says Vidal, "because Joe and his colleagues at the Salk Institute had already sequenced the Arabidopsis genome and had cloned many of the protein-coding genes, whereas on our side at the Dana Farber Institute we had experience in making these protein interaction maps for other organisms such as yeast."

In the initial stages of the project, members of Ecker's lab led by research technician Mary Galli converted most of their accumulated library of Arabidopsis protein-coding gene clones into a form useful for protein-interaction tests. "For this project, over 10,000 'open reading frame' clones were converted and sequence verified in preparation for protein-interaction screening," says Galli.

Vidal, Braun, Hill and their colleagues systematically ran these open reading frames through a high quality protein-interaction screening process, based on a test known as the yeast two-hybrid screen. Out of more than forty million possible pair combinations, they found a total of 6,205 Arabidopsis protein- protein interactions, involving 2,774 individual proteins. The researchers confirmed the high quality of these data, for example by showing their overlap with protein interaction datafrom past studies.

The new map of 6,205 protein partnerings represents only about two percent of the full protein- protein "interactome" for Arabidopsis, since the screening test covered only a third of all Arabidopsis proteins, and wasn't sensitive enough to detect many weaker protein interactions. "There will be larger maps after this one," says Ecker.

Even as a preliminary step, though, the new map is clearly useful. The researchers were able to sort the protein interaction pairs they found into functional groups, revealing networks and "communities" of proteins that work together. "There had been very little information, for example, on how plant hormone signaling pathways communicate with one another," says Ecker. "But in this study we were able to find a number of intriguing links between these pathways."

A further analysis of their map provided new insight into plant evolution. Ecker and colleagues Arabidopsis genome data, reported a decade ago, had revealed that plants randomly duplicate their genes to a much greater extent than animals do. These gene duplication events apparently give plants some of the genetic versatility they need to stay adapted to shifting environments. In this study, the researchers found 1900 pairs of their mapped proteins that appeared to be the products of ancient gene-duplication events.

Using advanced genomic dating techniques, the researchers were able to gauge the span of time since each of these gene-duplication events -- the longest span being 700 million years -- and compare it with the changes in the two proteins' interaction partners. "This provides a measure of how evolution has rewired the functions of these proteins," says Vidal. "Our large, high-quality dataset and the naturally high frequency of these gene duplications in Arabidopsis allowed us to make such an analysis for the first time."

The researchers found evidence that the Arabidopsis protein partnerships tend to change quickly after the duplication event, then more slowly as the duplicated gene settles into its new function and is held there by evolutionary pressure. "Even though the divergence of these proteins' amino-acid sequences may continue, the divergence in terms of their respective partners slows drastically after a rapid initial change, which we hadn't expected to see," Vidal says.

In the July 29 issue of Science researchers from the Arabidopsis interactome mapping study reported yet another demonstration of the usefulness of their approach. Led by Jeffery L. Dangl of the University of North Carolina at Chapel Hill, they examined Arabidopsis protein interactions with the bacterium Pseudomonas syringae (Psy) and a fungus-like microbe called Hyaloperonospora arabidopsidis (Hpa). "Even though these two pathogens are separated by about a billion years of evolution, it turns out that the 'effector' proteins they use to subvert Arabidopsis cells during infection are both targeted against the same set of highly connected Arabidopsis proteins," says Ecker. "We looked at some of these targeted Arabidopsis proteins and found evidence that they serve as 'hubs' or control points for the plant immune system and related systems."

Ecker and his colleagues hope that these studies mark the start of a period of rapid advancement in understanding plant biology, and in putting that knowledge to use for human benefit. "This starts to give us a big, systems-level picture of how Arabidopsis works, and much of that systems-level picture is going to be relevant to -- and guide further research on -- other plant species, including those used in human agriculture and even pharmaceuticals,"Ecker says.

The "Arabidopsis Interactome Mapping Consortium" consists of over 20 national and international laboratories that contribute to this study with support from a number of funding agencies including the National Science Foundation and the National Institutes of Health.

Herbicide Tolerant Crops

2011-07-21T16:37:00.000-07:00

Herbicide-tolerant crops have been the most widely used application of agricultural biotechnology in the United States. Currently, crops have been modified to be tolerant to three herbicides: bromoxynil, glyphosate and glufosinate. Bromoxynil controls broadleaf weeds only while glyphosate and glufosinate are broad-spectrum with activity on both grass and broadleaf weeds.

Herbicide-tolerance results from three mechanisms: metabolic detoxification, resistance at the site of action and prevention of the herbicide from reaching the site of action. Bromoxynil-tolerant crops were developed by introducing a gene that encodes for bromoxynil-specific nitrilase from a soil bacterium, Klebseilla ozaenae (Stalker et al., 1996). Crop tolerance to bromoxynil results from metabolic detoxification. While introduction of the glyphosate-insensitive EPSPS from Agrobacetrium sp. strain CP4 into crops by genetic modification techniques was successful in conferring glyphosate tolerance (Padgette et al., 1996), glufosinate tolerance was achieved through the use of bar gene isolated from another soil bacteria, Streptomyces hygroscopicus. The bar gene encodes for an enzyme, phosphinothricin acetyl transferase, which detoxifies the herbicide glufosinate (Vasil, 1996).

Commercialised herbicide-tolerant crops to date include bromoxynil-tolerant cotton, glyphosate-tolerant soybean, cotton, corn, sugarbeet and canola, and glufosinate-tolerant corn and canola (Table 61.1). Bromoxynil-tolerant cotton was introduced in 1995, while glyphosate-tolerant soybean, cotton, corn, sugarbeet and canola have been available in the United States since 1996, 1997, 1998, 1999 and 1999, respectively. Glufosinate-tolerant corn and canola were commercialised in 1997 and 1999, respectively.
The adoption of bromoxynil-tolerant cotton has been low in the United States (Table 61.1) due to several reasons. Although bromoxynil provides effective control of problem weeds such as morning glory and cocklebur, it is weak on sickle pod, which is a key weed species in several cotton growing states and has no activity on grass weeds. Marketability of bromoxynil-tolerant cotton is further limited, as the herbicide-tolerance trait has not been stacked with the insect-resistance trait.

On the other hand, the commercial adoption of glyphosate-tolerant soybean, cotton and canola has been the most rapid cases of technology diffusion in the history of agriculture. In 2001, glyphosate-tolerant soybean, cotton and canola were planted on approximately 68, 70 and 50&percnt; of the total planted acreage, respectively (Table 61.1). Herbicide-tolerant (glyphosate and glufosinate tolerant included) corn was planted on only 7&percnt; of the total acreage in 2001. Lack of approval for biotechnology-derived glyphosate-tolerant corn imports into the European Union and non-availability of herbicide-tolerance trait in popular varieties adapted for various corn growing regions have hindered the adoption of herbicide-tolerant corn thus far. Although glyphosate-tolerant sugarbeet has been available for commercial planting since 1999, adoption has been zero due to issues related to marketing.

A traditional weed control program in conventional crops involves the use of several herbicides, targeted to a specific weed or groups of weeds. Herbicides are typically applied either as preplant incorporated (PPI) treatments prior to planting, pre-emergence (PRE) applications at planting or before crop emergence, post-emergence (POST) applications after the crop has emerged or a combination of PRE followed by POST applications. Several constraints limit the success of PPI and PRE herbicide applications. Preplant incorporated and PRE treatments involve guesswork as herbicide applications are made anticipating the weed species that may emerge. The efficacy of soil-applied PRE herbicides is highly dependent on rainfall, with poor weed control under extremely low or high rainfall conditions. As a result, there is an increasing trend towards total POST herbicide programmes. Herbicide-tolerant crops, on the other hand, facilitate the use of POST herbicides, such as glyphosate and glufosinate, wherein herbicides are selected based on emerged weed species in the field within the limits of crop and weed growth stages.

Conventional herbicides pose carryover concerns resulting in planting restrictions as many of them have long soil persistence periods. For example, herbicide labels suggest that crops such as alfalfa, dry beans, cabbage, lima beans, muskmelon, onions, peas, peppers and pumpkins should not be planted for 18 months following the application of a premix of atrazine/rimsulfuron/nicosulfuron (trade name: Basis Gold) in corn. Similarly, 26 months should elapse after imazethapyr application when planting potato, and 40 months for tomato, watermelon, squash and pumpkin following imazethapyr application in soybean (Pest Management Recommendations for Field Crops, 2000). Herbicide-tolerant crops resolve this problem because herbicides used in biotechnology-derived crops, such as glyphosate and glufosinate, have no residual activity and thus no carryover potential.

Crop injury from herbicide applications is a major concern in crop production. The potential for crop injury is generally greater with certain conventional herbicides in crops such as cotton and soybean. For instance, herbicides, such as acifluorfen and 2,4-DB, can cause substantial injury to conventional soybean leading to yield losses (Kapusta et al., 1986; Wax et al., 1973). Weed control is compromised if herbicide rates are decreased to lessen crop injury. Herbicide-tolerant crops offer growers remarkable flexibility because crop injury is practically non-existent.

Herbicide-tolerant crops add flexibility to weed management as they offer programmes that are less restricted by crop growth stage, weed species, weed size, tank-mix partners and adjuvant type. Herbicides used in conjunction with herbicide-tolerant crops can be applied at later crop growth stages compared to conventional herbicides, and the maximum height at which they are effective on weed species is greater than that of currently used herbicides. For example, glyphosate can be used up to the 4-leaf stage on cotton, 6-leaf stage on canola and up to flowering on soybean. These application windows are much wider than those with conventional herbicides. Labels instruct that maximum height up to which glyphosate applications can be made for the control of foxtail and fall panicum, two problem weeds in corn, is 6 inches in contrast to 3 inches with the premix of conventional herbicides atrazine/rimsulfuron/nicosulfuron (Curran et al., 1999).

Herbicides used in herbicide-tolerant crops are broad-spectrum and non-selective in activity. As a result, control of both annual and perennial broadleaf and grass weeds can be obtained with one herbicide application alone and with no need for a tank-mix partner in most situations. This is in contrast to intense weed management programmes used in crops such as cotton, which on average receive three herbicide applications consisting of three active ingredients in combination with one to three cultivations. This simplicity in weed control coupled with no crop injury is the reason cited most often by growers for the adoption of herbicide-tolerant crops.

Perennial weeds are a major issue in crop production as they are difficult weeds to control. The difficulty arises due to their propagation behaviour that includes both vegetative and reproductive methods. Many of the currently available conventional herbicides are not effective on perennial weeds. Though herbicides such as clopyralid are effective, high cost limit their use. Herbicide-tolerant crops provide a viable choice for perennial weed management as herbicides such as glyphosate provide excellent perennial weed control in addition to control of other weeds and are cost-effective.

Benefits of Commercialised Biotechnology-Derived Crops in the United States

2011-07-13T15:42:00.000-07:00

Agricultural biotechnology has been a significant step forward inpest management in the United States. The range of its applications has been extensive and is expanding rapidly. The principal commercialised applications thus far include herbicide tolerance, insect resistance and virus resistance.

Biotechnology-derived crops were first introduced for commercial production in the United States in the mid 1990s. In spite of the dichotomy of opinion regarding biotechnology-derived crops, adoption has been dramatically rapid in the United States since their introduction. The United States is the principal country that planted most of the biotechnology-derived crop acreage (68&percnt; of the global) followed by Argentina (22&percnt;), Canada (6&percnt;) and China (3&percnt;) in 2001 (James, 2001). In 2001, biotechnology-derived crops were planted on 88 million acres of US crop acreage, up by 18&percnt; compared to 2000 (James, 2001). James (2001) noted that adoption of biotechnology-derived crops has been the highest ever for new agricultural technologies and attributed this to grower satisfaction and significant benefits. Current trends suggest that in the next few years almost all acreage of the major crops grown in North America will be biotechnology-derived.

A conflicting aspect of agricultural biotechnology is the amount of public debate and furore it has generated. Opposing opinions regarding biotechnology-derived crops centre on different perceptions regarding their benefits, environmental and ecological safety, implications on human health and ethics. An understanding of the benefits of agricultural biotechnology for pest management is pivotal to judge the merit of the technology and to resolve the public discussion.

This chapter examines the importance of pest management in crop production and details the commercially available biotechnology-derived traits and their need in the context of available pest management options in conventional crops. The discussion is mainly focused on the actual and potential benefits of this innovation for crops commercialised so far in the United States. Economic advantage to growers is the ultimate key factor, which determines the adoption and success of biotechnology-derived crops. Economic benefits normally result from reduced input costs or increased yields or both.

Importance of Pests in Agriculture

Pest populations have been and will continue to be the major constraints to crop production in the United States. Based on a 1988–1990 estimate, the impact of weeds, insects and pathogens on the production value of eight major crops grown in North America was 11.4, 10.2 and 9.6&percnt;, respectively, amounting to a total of $23 billion (Oerke et al., 1994). A recent estimate suggests that impact of weeds alone on US economy exceeds $20 billion annually (Bridges, 1994).

Pest management in crops is a dynamic activity that evolves as new technologies are developed. Growers have relied on a variety of tactics such as manual methods, cultural practices, biological control, quarantine and pest-resistant cultivars to combat pests thus far. Use of chemicals replaced manual and cultural methods in the 1940s, after which crop productivity increased dramatically in the United States.

Weeds are a constant and major challenge to farmers worldwide. About 72&percnt; of the pesticides used in the United States are herbicides, 21&percnt; are insecticides and 7&percnt; are fungicides (Duke, 1998), which emphasises the importance of weeds as crop pests. Control of weeds is critical as they compete with crops for nutrients, water, sunlight and space resulting in significant yield and quality losses. Season-long weed infestations can result in severe yield losses depending on the competing weed species and their density. For example, corn yields were reduced 10&percnt; by giant foxtail, 11&percnt; by common lambsquarters, 18&percnt; by velvetleaf and 22&percnt; by common cocklebur at a density of two per foot of row (Beckett et al., 1988). Additionally, weeds increase the cost of agricultural production, reduce land use and human efficiency, and act as hosts for insects and pathogens thereby increasing their control costs. As a result, almost all of the acreage of major crops in the United States is treated with herbicides to avoid yield loss.

Insects infest crop plants for the most basic reasons: to obtain food or protection for overwintering and oviposition or to complete their life cycle. The direct impact of insects result from their feeding on plant parts, which leads to reduction in crop productivity and quality. Losses due to insects each year in the United States were estimated to be 13&percnt; or $33 billion (USBC, 1998). The concentrated large acreages of a single crop in successive years (monoculture) have led to a general increase in insect pest populations in the United States. Monocultures lead to unstable agroecosystems due to increased abundance of food supply, decreased competition, low diversity of insect pests and increased ease of locating food supply.

Pathogens that infect plants fall under diverse groups such as viruses, bacteria, fungi, algae, protozoans and nematodes. These pathogens cause harmful physiological and metabolic effects in crop plants thereby resulting in significant yield losses. For example, estimated crop losses due to diseases in the United States are over 10&percnt; (El-Zik and Frisbie, 1991). Annual crop losses to all plant pathogens total an approximate $33 billion in the United States (USBC, 1998).

Crop loss estimates due to various pests are often misleading as they represent average loss over a wide area of production. In reality, losses are usually much higher on individual farms. Thus, crop losses definitely justify research to explore new methods such as modern biotechnology to manage pest populations.

The following discussion centres on why growers have adopted biotechnology-derived crops. It outlines the shortcomings in conventional pest management and suggests how biotechnology-derived crops offer solutions to these problems. Finally, the benefits derived from the technology for specific crops are highlighted.

Application of Proven Agronomic Biotechnologies to Ornamental Plants

2011-07-10T02:20:00.000-07:00

In 1996, interdisciplinary scientists ushered in a new age of collaboration between molecular biologists and plant breeders with the introduction of herbicide- and insect-tolerant transgenic plants on a commercial agronomic scale. In the past 6–7 years, herbicide and insect tolerance traits have been utilised in many different agronomic and vegetable crops, and now make up a significant percentage of the acreage planted yearly in the United States for corn, soybeans and cotton. Although there have been no published reports on the production of herbicide- or insect-tolerant ornamental crops, research is currently being conducted on turfgrass, and a select number of significant floriculture crops to engineer glyphosate resistance. The transfer of glyphosate resistance into creeping bentgrass is an obvious example of how proven agronomic traits can be used to make weed control in municipal and highly managed turf environments more efficient. It is very likely that any number of horticultural crops could be engineered with herbicide resistance, but the trait will probably only be commercially viable in crops grown in the field or planted in the ground in large public areas, such as vegetable crops, turfgrass, bedding plants and nursery crops. The use of herbicide resistance as a selectable marker in tissue culture, and subsequently as a ‘stacked’ trait on top of other introduced traits may influence the appearance of this technology in future crops grown for ornamental purposes as well.

From a broad perspective, it appears that the future for application of biotechnology to ornamental crops is promising. Many technical advancements have been made that have provided ‘proof of concept’ of their commercial utility in ornamental crops, and this work will likely supply the next generation of flower breeders with many novel traits. As progress is made in sequencing new plant genomes and as functional genomic tools become more widely used in crops of lesser economic value, scientists working to apply biotechnology to ornamental crops will likely find themselves with more novel traits to work on than they have people in their labs. There is no question that the main technical limitations that exist for applying biotechnology to ornamental crops lie mainly in the area of development of transformation systems for the large number of plant species used in the industry. For this reason, it will be imperative for breeders to introgress commercially viable traits into breeding stocks, whether their goal is to reproduce their crops by sexual or asexual means.

The real potential of biotechnology in ornamental crops is probably not going to be determined by any major difficulties encountered in the technical realm—it appears that there will be many genes and promoters that will have commercial viability in the ornamental plant industry. The factors that will determine how the ornamental plant industry accepts and utilises biotechnology will be more influenced by economic and regulatory issues. It is obvious that in terms of regulatory hurdles, ornamental crops have a particular advantage over food crops because they are non-edible. Since consumers do not physically ingest these products, it is likely that much public relations benefit can be gained in terms of consumer perception of genetically engineered crops by providing them an ornamental plant that is extremely novel and desirable. The introduction of new and novel plants has been the basis of progress in the ornamental plant industry since its inception, so it should be no surprise if that happens.

It is also logical to think that since there may be fewer regulatory hurdles for ornamental plants, the cost of introducing a new biotechnological ornamental crop would be reduced. However, the current regulatory restrictions that have been developed around the introduction of genetically engineered food crops will have to be revisited when regulatory packages for new ornamental crops are submitted to federal agencies. While it may be feasible to submit regulatory packages for each individual cultivar of genetically engineered corn or soybean, it will be cost prohibitive for any company to acquire all of the required regulatory data necessary for the hundreds of individual engineered cultivars that could be developed and potentially released by any particular breeding company. Since an individual ornamental crop has much less commercial value than any individual agronomic crop and a much shorter market life expectancy, the costs of gaining regulatory approval from federal agencies will have far more impact on the economic decisions made by those parties interested in engineering these crops. With the current royalty structures and profit margins in the ornamental plant industry, combined with a comparatively low market volume, it would take a company several years to recur the initial cost of their biotechnology investment, even when they capture large percentages of a given market share. In addition, many cut flowers and vegetative propagules used in the ornamental plant industry are produced in Africa, Central and South America and Europe, and are imported into the United States and all throughout Europe and Asia. With the current global status of genetically engineered plants in most of these regions being more restrictive than in the United States, any segment of the industry depending on import or export of products outside the United States will have far more complications in the physical movement of genetically engineered ornamental plants. These types of scenarios ultimately restrict the application of plant biotechnology to ornamental crops far more than any limitations at the technical end.

Phytohormone Synthesis and Perception

2011-07-10T02:13:00.000-07:00

Flower colour and scent of ornamental crops are important candidate target traits for genetic manipulation with biotechnology tools because they are the obvious reasons that these crops are grown. However, there has been much research attention given to the manipulation of phytohormone synthesis and perception in ornamental crops due to the wide range of physiological processes in which they are involved. Currently, several issues in crop production and post-harvest handling in the ornamental horticulture industry reflect a need to be able to understand physiological processes that are normally controlled by plant hormones. For example, significant effort and money is spent applying chemicals to plants to control the synthesis of gibberellic acid (GA) and subsequent plant height during crop production. In many potted flowering crops and most bedding plant crops, as many as three to five applications of growth-regulating compounds may be used during a crop cycle. This adds a significant amount of production cost for chemicals and labour to apply and handle them, while being a negative factor environmentally. In addition, many of the newly introduced ornamental species that have not been in commercial production for a long period of time are receiving particular attention in conventional breeding and selection for dwarf plants because their natural habit does not fit into the mass-market operation scheme requiring compact plants that fit into transport vessels. It is apparent that the manipulation of endogenous GA concentration in plants through genetic engineering has the potential to produce ornamental and flowering plants with a diverse array of reduced-height phenotypes.

Considerable research over the past two decades has been conducted to isolate GA biosynthesis mutants (for review, see Hedden and Proebsting, 1999) and to elucidate the GA biosynthetic pathway (for review, see Hedden and Kamiya, 1997; Hedden and Phillips, 2000). The initial cloning and characterisation of gibberellin 20-oxidase genes from Arabidopsis (Phillips et al., 1995) and spinach (Wu et al., 1996) provided some of the first practical molecular tools needed to develop plants with altered endogenous GA levels. In Arabidopsis thaliana, GA 20-oxidase catalyses consecutive steps late in GA biosynthesis, and is encoded by three genes with differential patterns of expression (Phillips et al., 1995; Coles et al., 1999). Over-expression of GA 20-oxidase in Arabidopsis leads to increased production of active GA (GA4) and subsequent stem elongation, while expression of antisense GA 20-oxidase leads to reduced levels of active GAs and reduced rates of stem elongation (Coles et al., 1999). Heterologous over-expression of a pumpkin GA 20-oxidase gene driven by constitutive promoters in both transgenic Solanum dulcamara (Curtis et al., 2000) and lettuce (Lactuca sativa ‘Vanguard’) (Niki et al., 2001) also resulted in the production of dwarf plants. In both cases, endogenous concentrations of active forms of GA, such as GA1 and GA4 were reduced in transgenic dwarf plants, while concentrations of various inactive forms of GA such as GA25 were increased, indicating a diversion of the normal pathway of GA biosynthesis towards the production of inactive forms of GA.

More recently, additional tools for use in engineering dwarf plants have been developed as a result of the cloning and characterisation of genes involved in the regulation of GA degradation. The first steps in the degradation of biologically active GA appear to involve hydroxylation reactions that are similar to the final steps of GA synthesis. Similarly to the GA3-oxidases, which hydroxylate the 3-position of GA precursors to form active GAs (Chiang et al., 1995; Talon et al., 1990), GA-2 oxidases hydroxylate the 2-position of active C19-GAs as a first step to render them inactive (Sakamoto et al., 2001; Martin et al., 1999; Thomas et al., 1999). Two additional GA2-oxidases (AtGAox7 and AtGAox8) have recently been isolated from Arabidopsis that hydroxylate C20-GA precursors, but not active C19GAs (Schomburg et al., 2003in press). When over-expressed in transgenic Arabidopsis and tobacco, these genes result in plants with decreased levels of active GAs and a range of corresponding dwarf phenotypes. Transgenic CaMV35S::AtGAox7 and AtGAox8 petunias have a range of dwarf phenotypes, suggesting that the control of endogenous GA levels may be applicable to important floriculture crops as well (Figure 45.1). It is interesting to note that the dwarf phenotype can be reversed by treatment of plants with exogenous GA (Figure 45.2). This is very important for the application of dwarfing technology to the ornamental plant industry, because producers of vegetative propagules may actually want to temporarily increase shoot elongation at various points during cutting production, then have the plants resume a dwarf phenotype after propagation.Breeder selection for the appropriate plant height among transgenic lines will be critical for applying these technologies to any given crop, and breeders should be able to select any particular plant height they desire. Traditional horticultural experiments similar to those used in any breeding programme with a focus directed towards plant height control should be relatively straightforward, and are well established in the industry.

It is likely that dwarfing technologies will find utility in many areas of ornamental plant production. There should be ample room in the industry for potted plants that fit in transit vessels easily, annual bedding plants that do not grow out of the confines of a small garden spot, and shrubs and trees that require less pruning. One extremely important application for dwarfing technology will likely be observed in plant species used in the turfgrass industry. Slower growing lawn grasses that require significantly less labour input for maintenance have been a dream of suburban homeowners and turfgrass maintenance experts for many years. This concept is being developed by commercial scientists at the Scotts Company, with a great deal of progress being made in producing dwarf creeping bentgrass (Agrostis sp.). In high-management areas, these ‘low mow’ grasses have a great deal of potential for saving money on labour costs and equipment maintenance. In addition, slow-growing grasses could help reduce lawn mower pollution and use of fossil fuels, and may use less water and fertilisers. It is likely that these ‘low-mow’ turfgrasses will be important for homeowners and municipal or roadside situations where there is a low amount of foot traffic. It is not likely that these grasses will be used to any great extent on golf courses or sports turf facilities. Due to the high amount of wear and tear that is experienced under these conditions,they may not grow fast enough to cover the damage inflicted by constant use.

Another aspect of hormonal control of plant morphology currently being pursued in ornamental crops concerns the engineering of plants with delayed leaf senescence. Senescence is the final developmental process in the lifecycle of a leaf through which macromolecules (e.g. chlorophyll, proteins, nucleic acids) of leaf cells are metabolised to basic components and transported to the growing shoot and reproductive organs of the plant (Matile et al., 1996; Noodén et al., 1997). Natural leaf senescence in many plants is characterised by lower leaf yellowing or chlorosis as nutrients and other components of the cells are degraded (especially chlorophyll). In horticultural terms, leaf chlorosis can cause a decreased aesthetic appearance of ornamental plants and thus a decrease in the salability of those plants.

One way to prevent leaf senescence is through the manipulation of cytokinins. Cytokinins are an important class of phytohormones that influence numerous aspects of plant growth and development, and have been shown to delay and, in some cases, reverse the leaf senescence process (Gan and Amasino, 1996). The limitation of the use of cytokinins in the prevention of senescence has most often been related to difficulties of controlling temporal and spatial delivery of cytokinins. Exogenously applied cytokinins may not enter the cells, nor be transported to the area needed, nor quickly become conjugated or metabolised to non-active forms (Klee and Lanahan, 1995; Gan and Amasino, 1996; Kaminek et al., 1997). Researchers have recently turned to the manipulation and engineering of endogenous cytokinin biosynthesis in order to solve these problems.

After many years of research on cytokinins, the mechanisms of biosynthesis and perception are just now being elucidated. One enzyme, isopentenyl transferase (IPT), has received much research attention because of its known involvement in cytokinin synthesis. IPT enzyme activity has been isolated from many organisms including plants (Nishinari and Syono, 1980; Blackwell and Horgan, 1994), Dictyostelium discoideum (Taya et al., 1978; Ihara et al., 1984), and Rhodococcus fasciens (Crespi et al., 1992). Although it is well known that there is IPT activity in plants, the genes that encode IPT have only recently been isolated and cloned from plants (Kakimoto, 2001; Takei et al., 2001). As a result, experiments focused on the manipulation of endogenous cytokinin synthesis in transgenic plants with these genes have been lacking. In contrast, a gene from Agrobacterium tumefaciens that encodes the IPT enzyme has been available for use in transgenic plant research for a number of years. IPT is encoded on the Ti (tumour inducing) plasmid of Agrobacterium tumefaciens (Akiyoshi et al., 1984; Barry et al., 1984). It catalyses the condensation of dimethylallylpyrophosphate (DMAPP) and 5′AMP to form isopentenyladenosine 5′-phostphate ([9R-5′P]iP), which is then quickly converted to different cytokinins. This is presumed to be the rate-limiting step in cytokinin biosynthesis since expression of this one gene can cause an over-production of cytokinins (Medford et al., 1989; Klee and Lanahan, 1995; Gan and Amasino, 1996). Attempts have been made to use this gene under the control of various promoters, for example, modified CaMV 35S (Faiss et al., 1997), Cu2+-inducible (McKenzie et al., 1998), or heat shock inducible (Smart et al., 1991) to stop leaf senescence. However, the results of such experiments were often complicated by abnormal growth patterns, or the possibility that the induction treatment (i.e. high temperature or CuSO4 treatment) was causing the observed phenotype (Smart et al., 1991; Buchanan-Wollaston, 1997).

In an attempt to overcome the problems associated with past work on transgenic plants overproducing cytokinins, Gan and Amasino (1995) developed a genetic construct that utilised the highly senescence-specific promoter from SAG 12 (PSAG-12) to drive IPT expression. This construct had three important features due to PSAG-12 specificity: temporal regulation, spatial regulation and quantitative regulation (Gan and Amasino, 1995, 1996). When leaf senescence is triggered, the transcription of IPT is activated by PSAG-12, leading to the production of functional IPT enzyme. The enzyme then catalyses cytokinin production, which in turn delays senescence. Without senescence signals, the PSAG-12 promoter attenuates IPT transcription and subsequent enzyme production, thus providing autoregulatory control of cytokinin synthesis. The PSAG-12-IPT construct was transformed into tobacco. The transformed plants displayed a normal growth habit except that senescence was inhibited and there was a significant increase in flower number, an increased biomass due to the presence of lower leaves, and increased seed yield (Gan and Amasino, 1995). Since then, transgenic PSAG-12-IPT lettuce plants (McCabe et al., 2001) Nicotiana alata (Schroeder et al., 2001) and petunias (Clark et al., in press) with similar delayed leaf senescence phenotypes have been produced (Figure 45.3). The PSAG-12 promoter has also been used to drive the expression of the KNOTTED-1 mutant gene from maize in transgenic tobacco plants to confer a delayed leaf senescence phenotype (Ori et al., 1999), thus extending the utility of delayed leaf senescence technologies. Transgenic PSAG-12:KNOTTED-1 and PSAG-13-IPT petunias both have delayed leaf senescence after nutrition stress induced at the onset of flowering. It is likely that horticultural performance studies will show that breeder selection of transgenic plants under a wide range of selection criteria will be essential for providing the growers with the methods required to produce these plants effectively on a commercial scale.
One hormone of particular interest in the ornamental plant industry is ethylene gas. Ethylene is involved in many physiological processes in plants including fruit ripening, petal senescence, abscission and seed germination. Floral senescence is of particular interest because quality loss of many important floriculture crops is known to occur due to ethylene gas in the post-harvest environment. Treatment of ethylene-sensitive flowers with chemical ethylene biosynthesis or perception inhibitors delays visual symptoms of corolla senescence (Jones and Woodson, 1997; Whitehead et al., 1984; Serek et al., 1995). Many economically important crops are grown for their floral display; therefore the control of ethylene synthesis and perception is thought to be the key to increasing display life and enhancing visual quality. Since ethylene is such a significant problem in both potted flowering crops and cut flowers there have been several attempts to produce chemical control methods for both ethylene synthesis and sensitivity. Effective control of ethylene synthesis using a chemical approach has been much less effective across the industry than the manipulation of ethylene sensitivity. Ethylene sensitivity has long been managed in floriculture crops through the use of silver thiosulfate (STS), which makes plant tissue insensitive to ethylene, but also has environmental downsides that appear to restrict its commercial use. Another chemical approach gaining popularity within the industry is the use of 1-MCP (1-methylcyclopropene), which is a compound that blocks the ethylene receptor protein and makes plant tissue insensitive to ethylene. Typically applied as a gas, 1-MCP provides a means by which large amounts of tissue can be treated for a short time. Although this compound has proved to be effective in some crops, there is some difficulty with the use of 1-MCP in crops that continue to produce new ethylene receptor proteins through development during post-harvest transit, thus limiting the residual effect of the compound (Cameron and Reid, 2001).

A great deal of research has been conducted to produce floriculture crops that synthesise less ethylene, or are insensitive to ethylene. Transformation with an antisense ACC oxidase has been shown to be effective in producing carnation (Savin et al., 1995) and torenia plants (Aida et al., 1998) that produce significantly less ethylene, and have delayed flower senescence. As has been the case with chemical approaches to control the effects of ethylene in ornamental crops, the method of control receiving the most research attention in terms of genetic engineering has been at the level of ethylene perception. Wilkinson et al. (1997) transformed petunia with a dominant mutant Arabidopsis ethylene receptor, etr1-1, under the control of a constitutive cauliflower mosaic virus 35S (CaMV35S) promoter to produce ethylene insensitivity throughout the whole plant (Figure 45.4). These petunias had an increase in natural and pollination-induced flower longevity compared to wild-type plants, but had physiological side effects that limit their commercial use (Wilkinson et al., 1997; Gubrium et al., 2000). Ethylene-insensitive petunias showed a significant reduction in adventitious root formation, and even exogenous treatments with auxin did not increase adventitious root formation to untreated wild-type controls (Clark et al., 1999). Since many horticultural species are often propagated through vegetative cuttings this characteristic would severely limit the commercial utility of ethylene-insensitive plants propagated by vegetative cuttings. After much experimental effort, it is clear that the key to manipulation of ethylene sensitivity will be the promoters being used to drive transcription of these proven transgenes. Effective temporal and spatial control of transgene expression will ultimately lead to plants that have longer lasting flowers with no negative side effects that would limit commercial production. Bovy et al. (1999) have supported this idea by producing transgenic ethylene-insensitive carnations by driving the etr1-1 transgene with the flower-specific transcriptional promoter fbp1 from petunia. Although they were able to produce plants with a delayed flower senescence phenotype, they did not report heritability of the trait or extensive investigation of other horticultural performance characteristics that would limit commercialisation of these plants. Future experiments directed towards discovering the appropriate promoter to drive transgenes conferring ethylene insensitivity will likely be the key to success for the development of long-lasting flowers in the future. It will be imperative to conduct a significant amount of field and greenhouse trailing of these plants in order to completely eliminate the possibility of negative side effects.

Applications of Plant Biotechnology to Ornamental Crops

2011-07-10T02:09:00.000-07:00

The ornamental plant industry is technically diverse and is characterised by the use of literally hundreds of different plant species. In many cases, numerous different cultivars of the same species may be available, and plants may be reproduced through both sexual and vegetative propagation methods. Due to the large amount of genetic diversity used by this industry, there are often very complex issues that arise during crop production and post-harvest handling throughout the wholesale and retail markets. For example, crop production practices for potted poinsettia and chrysanthemum plants require manipulation of the photoperiod to initiate flowering, and the application of growth regulators to keep the plants compact in habit. This type of production scheme is vastly different from the production methods used for producing bedding plant species such as petunia, geranium and impatiens, but a typical greenhouse grower may be producing all these plants simultaneously. At the post-harvest and shipping end of the industry, cut flower species grown in the tropical regions of Central and South America may have optimal shipping and storage conditions that are vastly different from the optimal conditions needed for cut flower species grown in Northern Europe. However, all the flowers may be exported to a common port such as Miami, then repackaged and shipped together in the same transport vehicle under the same conditions to retailers in virtually any part of the United States. This type of scenario often results in poor product performance when it reaches the florist or is displayed on retail shelves.

In addition to the demands of complex production and marketing systems, the demand from consumers for a constant supply of new and interesting flowering plants with unique characteristics continues to increase. In the past 5–10 years, there has been a dramatic increase in the number of flowering plant species available to consumers worldwide, and this trend is continuing. As a result, there is very little information available on appropriate production and post-harvest schemes for many of these crops. With such a diverse industry it is logical to assume that the application of biotechnology to ornamental crops is going to be very difficult. Applying biotechnology concepts is quite abstract in ornamental plants for several reasons. First, scientists attempting to improve these crops are trying to hit a moving target. Turnover of new cultivars is constant, and there are very few instances of consistent use of inbred lines in commercial breeding programmes. This means that by the time a researcher has transformed a new trait into a particular cultivar and proved that the trait is of commercial interest, the original cultivar may actually have been replaced by a new ‘improved’ cultivar. As a result, it will be of utmost importance to get commercially viable trangenes into breeding stocks, whether the goal is to produce a crop that will be reproduced by seeds or by vegetative propagules. Second, calculating the value added by a trait is very esoteric in ornamental crop species. Calculating increases in corn yield may be relatively straightforward, but determining how much a new colour of pansy is worth, or whether a more fragrant rose is something that someone will buy and actually pay more for, is a bit more cumbersome. Since it is well known that there is significant cost associated with developing an approach for improvement of any plant species using biotechnology, it becomes much harder to determine the potential economic value of a new ornamental cultivar until after it has proved to be successful. Much of the profit to be made in the ornamental plant industry through the use of biotechnology will be made by either the breeding companies who invest in technology or by retailers who have the ability to market the value of a novel trait. Value will also be added for growers if the introduced traits significantly reduce production costs, and this impact should be particularly significant because this segment of the industry currently has lower profit margins than any other. This scenario is very common in many types of plant-based agriculture, but with ornamental plant germplasm being spread amongst so many breeding companies, no one company can justify the investment in biotechnology that has been seen for crops of agronomic scale without making sure that the technology is a financial risk worth taking. It is likely that a vertically aligned strategy capturing as much value as possible from breeder to producer to retailer will be the best approach to making the benefits of biotechnology outweigh the high costs of technology development.

The last major issue that makes the concept of applying biotechnology approaches to ornamental crops more difficult is probably the most important—there is a significant bottleneck in the number of ornamental plant species that have been genetically transformed to date. Although there have been published reports describing the genetic transformation of several of the major floriculture crops such as chrysanthemum, rose, carnation and petunia, other important crops such as poinsettia, hibiscus and impatiens have still not been reliably transformed. Logically, most of the crops that have been the focus of transformation efforts to date have been those with the most value economically, or plants that have been discovered to be easier to transform or culture in vitro. Even in crops that have received a fair amount of research attention for genetic transformation, a significant amount of difficulty has been encountered with developing transformation protocols that can be used successfully on all cultivars of a given plant species. In many cases, transformation of different cultivars or breeding lines has required the development of several different transformation protocols. The more aggressive transformation efforts in ornamental crops are being undertaken by private corporations, with the remainder of the work being taken care of (one species or cultivar at a time) by smaller academic tissue culture laboratories.

In many cases, biotechnology applications are proving to be very difficult, but several advances have been made with engineering a wide variety of genetic traits in floriculture crops and turfgrass. Significant gains have been made in cloning important genes that are proving to be involved with biological processes that scientists hope to manipulate in ornamental crops in the future. The purpose of the rest of this chapter is to provide a status report on the progress that has been made in applying biotechnology to plants grown for their ornamental characteristics, and to project where research efforts will be focused over the next few years.

New Traits—Old Concepts

Flower Colour

Flower colour is a key component that influences consumer choice among crops grown commercially for their ornamental characteristics. Flower colour has been the subject of a large amount of biochemical and applied genetics research for many years (Mol et al., 1998). Flower breeders have been supplying the markets with new colours using traditional breeding techniques and selection, and to a lesser extent mutation breeding. The main biological function of flower colour pigments is to attract pollinators. Various pollinators are attracted to a flower by particular colours, and the patterning of flower coloration makes flowers easy for pollinators to distinguish as they move about.

Although many species have a wide variety of possible flower colours, no single species contains all possible flower colours. Flower colours are produced in plants biochemically as betalains, carotenoids and flavonoids. Betalains are synthesised almost exclusively in the Caryophyllales, and are yellow to red nitrogen-containing compounds derived from tyrosine (Stafford, 1994). Because of their limited presence in the plant kingdom, very little attention has been given to this class of compounds for the purpose of genetically engineering flower colour. Carotenoids are responsible for yellow and orange colours such as those observed in sunflower, marigold and tomato flowers, and over 600 different carotenoid structures have been identified (Straub, 1987). Carotenoids function mainly in photosynthesis by assisting with light harvesting and by quenching singlet oxygen and triplet chlorophyll species that are derived from excessive light energy (Demmig-Adams and Adams, 2000). Carotenoid synthesis and storage occurs in plastids, but all of the biosynthetic genes isolated to date are nuclear-encoded.

Although there are many plant species in which yellow flower colour is not produced and cannot be introgressed through traditional genetics, very little progress has been made in terms of genetically engineering yellow flower colour in plants. The general biochemistry of carotenoid synthesis has been studied for over thirty years, but the carotenoid biosynthetic pathway genes have only been identified and characterised since the 1990s (reviewed by Hirschberg, 2001). There are many different genes involved in carotenoid synthesis, and many of these genes are represented in plants in small multigene families (Zhu et al., 2002; Moehs et al., 2001). Since carotenoids are involved in such a range of diverse biological functions, most of the research on engineering carotenoid synthesis in transgenic plants has focused on the alteration of nutritional characteristics in food plants. Interestingly, rice engineered to over-express the daffodil phytoene synthase gene for increased pro-vitamin A content had a characteristic ‘golden’ colour (Burkhardt et al., 1997). Although these plants may be valuable for helping solve worldwide vitamin A deficiency, they also demonstrate the ability to drive the expression of yellow colour in plants. It is likely that progress in engineering yellow flower colour through the manipulation of carotenoid synthesis will be aided by observations on food crops with altered carotenoid content. It is also possible that attempts to genetically manipulate enzymes of the carotenoid biosynthesis pathway for yellow flower colour may affect unintended physiological processes in the plants leading to undesirable horticultural characteristics. Thus, the choice of appropriate transcriptional promoters to drive the expression of carotenoid biosynthetic genes in specific plant tissues will be imperative in determining the success of engineering yellow flower colour in ornamental plants.

Flavonoids are a class of secondary metabolites that are responsible for pale yellow, red, purple and blue colours in flowers. The flavonoid biosynthetic pathway, in particular, the anthocyanin synthesis pathway, has been extensively reviewed (Heller and Forkman, 1994; Forkman, 1994; Holton and Cornish, 1995). Hundreds of anthocyanins have been purified and their chemical structures have been determined (Strack and Wray, 1994), and virtually all of the genes that encode anthocyanin biosynthetic enzymes have been isolated. Thus, the anthocyanin biosynthetic pathway has served as an excellent target for transgenic manipulation because of the extensive background studies of its chemistry and genetics.

There are six main anthocyanins in plant tissues: pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin. These compounds are usually modified by glycosylation and acylation to produce a broader range of anthocyanins. In addition to the variety of anthocyanin chemical structures, colour variation produced by anthocyanins can be influenced by vacuolar pH, quantities of co-pigments and metal ions, and cell shape (Tanaka et al., 1998; Mol et al., 1998). Increased vacuolar pH is known to correlate with the ‘blueing’ of flowers of many plant species as they age and senesce (Yoshida, et al., 1995), and it is likely that both genetic and environmental factors influence the control of vacuolar pH. Genetic research on petunia has defined seven genetic loci that produce flower blueing when mutated (Chuck et al., 1993; van Houwelingen et al., 1998). Although these mutations are known to lead to measurable increases in the pH of petal extracts, but not an alteration of the actual anthocyanin composition, the actual mechanism of cellular pH control is still unknown. The range of the possible colours produced by anthocyanins can also be affected by the presence or absence of metal ions and co-pigments. In particular, the degree of blueness in anthocyanin pigments can be greatly influenced by metal ions and co-pigments which form stacked complexes with anthocyanins, and change their light absorption spectra (Kondo et al., 1992; Brouillard and Dangles, 1994). Perceived colour is also influenced by the shape of cells that accumulate anthocyanins. Petal epidermal cells more flattened in shape produce fainter colours, while conically shaped epidermal cells produce a sheen that gives the colours a more ‘velvety’ appearance (Noda et al., 1994). Since the underlying molecular mechanisms for changes in cell shape are poorly understood, it may be a few years before a complete understanding can be had of how pH changes and co-pigmentation and cell shape work together to produce an almost infinite array of flower colours through anthocyanins. However, it is well established that many genes involved in anthocyanin synthesis are regulated at the transcriptional level, which suggests that different flower colours and pigmentation patterns in flowers must be largely controlled by the expression patterns of regulatory genes (Holton and Cornish, 1995).

Over the past 10–15 years, many researchers have been able to successfully alter flower colour by manipulating the expression of anthocyanin biosynthetic genes in transgenic plants. Since no species has the ability to produce all possible flower colours, most of the research conducted to date has focused on the modification of already existing anthocyanin production systems in plants, or on the introduction of new biosynthetic enzymes that are not normally found in a particular species to make novel colours. For example, rose and carnation do not produce purple/blue delphinidin derivatives because they lack flavonoid 3′5′-hydroxylase (F3′5′H) activity (Mol et al., 1998). Also, plants such as petunia do not normally produce orange pelargonidin derivatives because the petunia dihydroflavonol reductase enzyme does not use the required dihydrokaempferol precursor as a substrate (Meyer, 1987).

Much of the research, focused on manipulating anthocyanin levels in flowers, has centred around the enzymes chalcone synthase (CHS) and dihydroflavonol-4-reductase (DFR). The CHS enzyme is the first enzyme committed to flavonoid production and catalyses the formation of chalcones, which are the intermediates used in the synthesis of all flavonoids. The DFR enzyme reduces dihydroflavonols to leucoanthocyanidins, another early rate-limiting step in anthocyanin synthesis. Many attempts at producing white flowers by suppression of flavonoid synthesis through the suppression of CHS and DFR activities have been successful. This approach is useful in a practical sense because in many species used as an ornamental crop, it has proved to be rather difficult to produce purely white flowering plants through traditional breeding methods.

Suppression of CHS via antisense or co-suppression has been demonstrated to suppress anthocyanin formation in a wide variety of plant species such as petunia (van der Krol, 1988; Napoli et al., 1990), chrysanthemum (Courtney-Gutterson et al., 1994), gerbera (Elomaa et al., 1993) lisianthus (Deroles et al., 1998) and torenia (Aida et al., 2000a). Similar to CHS, suppression of anthocyanin synthesis has also been achieved through suppression of DFR activity, leading to reduced anthocyanin synthesis in petunia (van der Krol, 1990) and torenia (Aida et al., 2000b). With the suppression of both CHS and DFR, there have also been reports of the generation of novel flower colour patterns in addition to the reduction in anthocyanin in petunia (van der Krol, 1988, 1990), torenia (Aida et al., 2000a, 2000b) and lisianthus (Deroles et al., 1998; Bradley et al., 2000). Interestingly, a consensus developed as a result of suppression of CHS and DFR is that the consistency of new flower phenotypes in these transgenic plants can vary within and between individual transgenic lines (van der Krol et al., 1990; Meyer et al., 1992; van Blokland et al., 1993; Elomaa and Holton, 1994; Deroles et al., 1998). Since colour patterns of some transgenic CHS lines of lisianthus have proved to be more stably inherited than others (Bradley et al., 2000), it is likely that any transgenic approach to the alteration of flower colour or colour patterning will require a significant amount of breeder selection over successive generations to stabilise the phenotype.

Controlled over-expression of anthocyanin biosynthetic genes has resulted in the production of novel flower colours in transgenic plants. One particularly dramatic example has been illustrated with petunia plants engineered for orange coloration. Normally, petunia does not produce orange colour because its intrinsic DFR protein does not accept dihydrokaempferol as a substrate, so pelargonidin-based pigments are usually absent. Maize DFR, which has a different substrate specificity than the petunia DFR, is able to produce pelargonidin derivatives if dihydrokaempferol is present and available. By expressing the maize DFR gene in petunia, Oud et al. (1995) were able to produce flowers with a brick red colour resulting from the accumulation of pelargonidin-derived pigments. Although this colour was not considered commercially acceptable, hybrids based on F4 genetic lines derived from commercial germplasm were obtained that had a unique orange flower colour. By introgressing the maize DFR gene into various breeding lines, DFR expression was stabilised, thus establishing new colour profiles that were not already present in petunia. Once stabilised through successive generations of breeding, the maize DFR gene behaved normally, and was used to successfully develop F1 petunia varieties with orange flower colour with no deleterious side effects (Oud et al., 1995).

Perhaps the best-known example of the production of novel flower colours in plants has been the transgenic ‘blue’ carnations produced by Florigene Ltd. and Suntory Ltd., which have been marketed in the United States, Australia and Japan under the name ‘Moonshadow’. These transgenic carnations contain the petunia flavonoid 3′5′-hydroxylase gene (F3′5′H), which encodes a cytochrome (cyt) P450 enzyme that catalyses the 3′5′ hydroxylation of dihydroflavonols, the precursors of purple anthocyanins. The F3′5′H enzyme activity is normally absent in carnation (and roses), but when the petunia F3′5′H gene is expressed in carnation, the plants produce and accumulate delphinidin, making the flowers a violet-bluish colour. It is likely that further improvements will be possible in producing blue colour using F3′5′H combined with other enhancers of anthocyanin pigmentation. It is known that the addition of cyt b5 activity enhances cyt P450 hydroxylation reactions, and is required for full activity of F3′5′H (de Vetten et al., 1999). Since the substitution pattern of anthocyanin pigments is a main determining factor in flower colour, it is thought that the stimulation of F3′5′H activity by the cyt b5 protein will be useful in enhancing blue flower colour (de Vetten et al., 1999).

Although much research has been conducted to date on anthocyanin biosynthesis and manipulation in plants, many issues related to the mechanisms of flower colouration are still poorly understood. To date, success has been achieved in the manipulation of flower colour through engineering anthocyanin biosynthetic genes and proteins. It is encouraging that most of the transgenic plants resulting from these efforts have been produced through driving transgene expression with constitutive promoters, and few deleterious side effects have been observed (Tanaka et al., 1998). However, the controlled manipulation of co-pigmentation and vacuolar pH have still not been addressed to a great extent. Once these factors are understood, the production of blue colour in flowers may become a reality.

Molecular Farming of Antibodies

2011-06-20T16:44:00.000-07:00

The applications for recombinant antibodies in healthcare are increasing. Being able to produce them on a large scale in plants will make them more affordable, which, in turn, will increase their availability to treat a greater number of diseases than is possible at present.

The contribution of Schillberg and Twyman focuses on the critical molecular factors for antibody expression in plant cells. Expression levels depend on both the structure of the chosen recombinant antibody and where it will be expressed within the organelles of eukaryotic cells. For example, most antibodies are poorly expressed in the cytoplasm of plant cells, but targeting them to the secretory pathway, and especially retaining them in the endoplasmic reticulum, results in higher production levels. The authors review both transgenic plants and cultured suspension cells as production systems for antibodies. The most interesting aspect of plant suspension cells is that they are a biologically contained system, which has advantages for the production of recombinant proteins under controlled conditions.

Antibodies are a diverse family of proteins and it is clear that some forms will have advantages over others in the context of plant-based expression. In addition to their role as pharmaceuticals, one attraction of antibodies is that they can be used to create disease-resistant plant lines. Schillberg and Twyman have pioneered the use of membrane-anchored antibodies to generate plant lines resistant to viral infection and—while this is not molecular farming per se—it demonstrates that antibody expression is, in itself, a useful tool for the improvement of plant characteristics.

Perspectives for Molecular Farming

In bringing these authors together, we have provided a snapshot of molecular farming. It is clear that there is still no consensus on the optimal production system for recombinant proteins in plants. This reflects, in part, the practicalities of the intellectual property situation in molecular farming. However, it is our opinion that consensus will eventually be determined by industry. We believe this to be the case because it will be industry that will commercialise molecular farming, not academic research laboratories. Industry will determine the commercial system that is the most appropriate and financially viable, and this decision will drive the progress of molecular farming.

Public acceptance of molecular farming and plant biotechnology is an issue that we have not discussed here, as our goal is to present an account of the state of the art in this field. We feel that success is the most powerful argument that can be used in favour of the technology. The contributions show that we have made significant progress towards that end. When the first protein from molecular farming is released into the market-place, and patients’ lives are seen to improve as a result, the public will then judge the technology on the basis of its benefits. We estimate that we are 2 to 4 years from that moment.

As with all new technologies, practical problems need to be overcome. Many of these, such as the difference between protein glycosylation patterns in plants and animals, have been discussed in detail in these contributions. We believe that, once defined, these challenges can all be solved. Advances in fundamental research, such as controlling gene silencing or chloroplast-targeted protein expression, will provide benefits for molecular farming. Although fundamental research remains the key tool available for improving the technology, molecular farming is already well advanced and close to product commercialisation.

The last decade has seen dramatic progress in plant biotechnology and this has led to the development of molecular farming. The next decade will see products approved as pharmaceuticals and once this happens molecular farming will finally have come of age.

Plants as a Green Pharmacopoeia

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We all routinely consume plant secondary metabolites—such as caffeine—without necessarily considering the complex biochemistry that is behind their production. Secondary metabolites have been used as medicines or as components of industrial processes for millennia. The methods used to manipulate their production are discussed by Yazaki in Chapter 43. Some secondary metabolites are limited in supply because the natural source is either hard to cultivate or difficult to synthesise chemically. Therefore, an effort has been made to produce these compounds in vitro or increase their production through the metabolic engineering of plants.

Yazaki first discusses how in vitro culture can be exploited to produce secondary metabolites. Thereafter, the strategies for metabolic engineering—that is, the use of genetic engineering methods to manipulate metabolism—are discussed in detail. It is evident from the review that understanding the biochemistry of their biosynthesis is the first step towards engineering the production of metabolites. Metabolic engineering differs from most molecular farming techniques for therapeutic protein production, because the aim is to manipulate complex plant metabolic pathways and to express proteins that are unlikely to have an effect on the plant cell itself. The successes in metabolic engineering reported by Yazaki are impressive.

The most telling and inspiring result so far has been the creation of ‘Golden Rice’, a transgenic rice line that has been metabolically engineered to produce high levels of the vitamin-A precursor ββ-carotene. This result exemplifies the potential of metabolic engineering to turn plants into living pharmacies.

Green Vaccines

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Vaccination is one of the most successful developments in preventive human healthcare. In their contribution, Mor and Mason describe how edible vaccines that can be delivered in food plants may make that protection more widely accessible.

Despite their efficacy, most modern vaccines, which are inactivated or attenuated strains of the pathogen delivered by injection, have limitations. This creates constraints on the use of vaccines because many pathogens are difficult, and prohibitively expensive, to culture. Second, the majority of vaccines are delivered to patients by injection. This requires skilled staff and a sophisticated medical infrastructure, which limits their use in the developing world. Therefore, alternative routes for vaccination as well as alternate sources of vaccination antigens have been explored. Orally delivered vaccines that contain a subunit of the pathogen and can elicit a protective immune response are one such alternative. Using transgenic plants for both production and delivery is the major focus of this chapter.

The authors provide a historical overview of the development of strategies for production of recombinant antigens in plants, most of which have been achieved in the laboratories of Arntzen and Mason. They describe their success in producing immune responses in humans with plant-produced hepatitis B surface antigen, the labile toxin B subunit of enterotoxigenic Escherichia coli and the capsid protein of Norwalk virus. Success with these proteins in Phase I/II trials has now prompted larger scale clinical trials. What is compelling is that their approach is successful for proteins from widely different pathogens and indicates that orally delivered vaccines may be successful against a wide range of pathogens. Mor and Mason then discuss the rapid increase of research in the edible-vaccine field and point out that plants can be used to create multicomponent vaccines that can protect against several pathogens at once. This is an aspect of the edible-vaccine approach that further strengthens its impact.

After discussing strategies for vaccine expression, the authors turn their attention to the use of orally delivered antigens both as immunocontraceptive vaccines and in the treatment of autoimmune diseases. They end their chapter with a careful discussion of where the technical challenges in edible-vaccine technology lie, and how they may be solved.

We share their view that orally delivered vaccines are a proven technology, which holds great promise for development within mainstream pharmaceuticals. By their use, entire populations in the developing world will be able to share the same protection from disease that we take for granted in industrialised countries.

Hijacking the Factory Management—Using Viral Vectors for Protein Expression

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There are, of course, alternative ways of exploiting the protein synthesis machinery of plants for the production of foreign proteins. The earliest proponents of this process, however, were not plant molecular biologists, but rather the plant viruses that hijack plant cells and use them for self-reproduction. These viruses provide a direct method for the transient production of recombinant proteins in crop plants, and this is the subject of the chapter written by Grill (see Chapter 40). By inserting target genes into the viral genomes, the hijacker can be turned into a molecular farmer.

In discussing how plant expression can be achieved using stably transformed plants or transient viral expression, Grill uses as examples two plant-produced proteins that have already entered clinical trials in the United States. The protein made in stably transformed plants was a humanised version of the Avicidin antibody for the treatment of prostate cancer, while the transient viral expression approach was used for the production of personalised vaccines produced from the cancerous B cells of non-Hodgkins lymphoma patients. What these examples illustrate is the flexibility of the viral vector system. Essentially, using this approach, personalised plant-based production of treatments for individual patients becomes possible, which is not feasible using stably transformed plants.

Grill then gives a detailed description of how viral vectors work in practice. Most plant viruses have single-stranded positive sense RNA genomes, and those that are assembled into rod-like particles can accommodate large inserted genes because it is possible to increase the length of the virion. Using recombinant viruses to infect plants and then produce the chosen recombinant proteins is rapid, compared to standard transgenic approaches, and has been tested in the field since the early 1990s. Grill argues that the rapid high-level expression of proteins is what sets viral vectors apart from the classical methods. Scaling up the methods for plant inoculation with viral vectors from the laboratory to the field has involved innovative solutions. An example, shown in the review figures, illustrates plants being inoculated using high-pressure sprays containing an abrasive and the recombinant viruses. A further interesting aspect of viral vectors is that there is no requirement for the cultivation of transgenic plant lines in the field. However, proteins transiently produced by viral infection have to be harvested from green leafy tissue and cannot be stored as easily as the seeds produced by transgenic plants.

Grill puts the case, and we agree, that while viral vectors compare well with transgenic plants overall, their real advantage is the ability to produce proteins rapidly. Indeed, the use of viral vectors to produce patient-personalised vaccines against non-Hodgkins lymphoma is such a compelling prospect that we feel viral vectors must remain an important tool for farming molecular medicines in the future.

A Perennial Production System

In their contribution, D'Aoust et al. describe the use of perennial plants for the production of recombinant proteins. Perennial plants are interesting as production systems because, with several harvests possible from the same crop, the plants can be used for repeated extraction of the protein.

The authors argue the case for forage legumes and, in particular, discuss the merits of using alfalfa. Because of the difficulties that had to be overcome to enable protein expression and plant transformation, forage legumes are relatively new to molecular farming. The authors describe the advances in promoter technology that have led to the construction of efficient inducible or strong leaf-specific expression cassettes. The features of the available transformation methods are discussed for alfalfa, which was first transformed more than 15 years ago, with D'Aoust et al. stating that reliable and efficient Agrobacterium-mediated transformation has been made possible by the optimal choice of genetic background and the use of standardised transformation protocols.

Because alfalfa is a fodder crop for ruminants, high-protein-yield varieties have been obtained and large-scale protein extraction techniques have been established by the animal feed industry. The authors describe how they are adapting these techniques for the production of plant-derived vaccines and, through the description of a case study, illustrate successful antibody expression in alfalfa.

Harvesting Recombinant Proteins from Food Crops

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Many recombinant proteins have been produced in those plants that are ideally suited for laboratory experiments, such as tobacco or Arabidopsis thaliana, but these are not suited to long-term or large-scale production. The global agricultural industry has focused on high-yield crops that have been optimised over generations to be cost-effective for the large-scale production of food, animal fodder and other products, such as fibres for clothing and pulp for paper-making. Therefore, research on molecular farming has moved towards evaluating these commercial species for the production of pharmaceuticals and other recombinant proteins.

In their contribution, Stoger et al. first discuss the nature of the recombinant proteins expressed in plants before progressing to a detailed discussion of the issues that are related to choosing the most suitable species for protein expression. These include a meticulous review of the decisions that need to be taken during protein-expression projects and an indication of where the potential advantages lie with each crop species. Their contribution provides a solid framework and entry point for researchers new to molecular farming.

Industrial ‘Plants’ of the Future

In her chapter, Hood describes the exploitation of transgenic plants as a production system for proteins that are of industrial importance. These include recombinant enzymes, the use of which would benefit industrial production processes that are currently based on synthetic chemistry. The high specificity and activity of enzymes is what makes them attractive to industry, and the argument presented in the contribution is that plants, with their high biomass and large-scale production potential, are ideal for mass production of these proteins. The central thesis is that using plants will reduce the cost of these proteins and make them available for a wide number of applications in industries where they were previously unaffordable.

The use of plants to produce industrially relevant enzymes has been shown to be practical in a number of species. For example, phytase and αα-amylase have been produced in tobacco. However, the author argues that the expression of proteins in leaf tissue is not ideally suited to molecular farming. This is primarily because of the expense and the difficulties involved in extracting the proteins from leaves. She discusses, therefore, using other plant species, such as alfalfa or oilseed crops, to produce industrially important proteins. The author makes the case for maize (Zea) as a production system, her well-reasoned argument being based on a comparison of the production costs of various crops. Although alfalfa has the greatest potential for the production of recombinant protein per hectare, this is offset by the need to extract the protein from leaf material. Of the crops surveyed, soybean has the lowest cost for protein production, but the methods required for transformation are labour intensive and expensive. The cereal crops, such as rice and wheat, are shown to have positive advantages for expression but, for pragmatic reasons, maize was their selected production platform. First, maize is the largest crop produced in North America and its two major advantages are the low cost and ease of large-scale production. The rationale for the use of maize rests on the sophisticated existing production and harvesting infrastructure and on the advantages of using a seed-based production system.

One contentious and difficult issue covered by Hood is that of containment, which is of serious concern given the large amounts of maize produced within the United States for both human and animal consumption. The author presents a series of measures that can be used to control inadvertent mixing of corn destined for food or fodder with the transgenic variety. It can be argued that this is where industry has to be at its most vigilant, because the consequences of transgenic crops entering the food chain are potentially very large. Overall, however, with close control over its use, maize could become an acceptable and profitable species for use as an ‘industrial plant’ of the future.

An Introduction to Industrial and Pharmaceutical Protein Production in Plants

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Advances in plant molecular biotechnology do not merely mean that farmers and research scientists alike can now contemplate a more than adequate harvest from newly sown crops. During the last decade, transgenic plants have been shown to provide a practical and feasible method of producing recombinant proteins. This technology has now progressed to the point where plants can be used as a platform for the expression of proteins intended for use in the treatment, or diagnosis, of a number of diseases. Such proteins include recombinant antibodies, cytokines and blood substitutes.

This research area—the combination of molecular biotechnology and agriculture, which is referred to as ‘molecular farming’—focuses on producing valuable proteins in plants, and forms the subject of the following contributions to this handbook. Drawing on expertise from both industry and academia, we present reviews of both the plant species and strategies that are being used to transfer molecular farming from the research laboratory to the field.

It is widely predicted that the world capacity for recombinant protein production will soon be exceeded by the demand and that this demand will continue to increase. Yet, the justification for the use of plants for recombinant protein production may not be immediately obvious. While transgenic cell lines, animals and microbes will continue to have significant roles to play as expression hosts for recombinant pharmaceuticals, the future role of plants should not be underestimated. One reason for this is that plants constitute a mass-production platform that can be used for the economical, large-scale production of proteins for industrial use in processes that were previously unaffordable. This makes them particularly relevant for the production of the recombinant proteins that will be required to treat the diseases we shall be challenged by in the 21st century.

Molecular farming is a fast-developing research area where fundamental research into protein expression and purification is coupled to the practicalities of plant growth and harvesting. This is reflected in the following contributions of how to express proteins, where to express them and how to choose the most appropriate host plant for protein expression. At present, there is no consensus on either the ideal expression method or choice of species, which has, therefore, to be determined empirically in individual cases. It should be emphasised that crops producing recombinant proteins have been in commercial production since 1997 (see Chapter 41) and crops producing recombinant therapeutics have already entered clinical trials (see Chapter 40). The development of edible vaccines using plants, as described by Mor and Mason (see Chapter 39), will have a great impact on world health and protection from disease. It is certain that the impact of molecular farming will increase as the technique develops both scientifically and commercially.

Below, we briefly introduce the contributions to this section and focus on what we regard as the most interesting issues covered in each review.

Cactus, Fleshy Plants Native to America

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Cactus, common name for the family comprising a peculiar group of spiny, fleshy plants native to America. The family contains about 1650 species, most of which are adapted to arid climates. The fruits of cacti are important sources of food and drink in many areas to which they are native. Because cacti require little care and exhibit bizarre forms, they are popular for home cultivation and are coming under increasing pressure as a result. More than 17 kinds of cacti now face extinction (see Endangered Species) because of plundering by avid collectors and professional poachers, especially in the southwestern United States and northern Mexico.

Cactus plants usually consist of spiny stems and roots. Leaves are greatly reduced or entirely absent. Only in two genera are fully formed leaves present. The stems of cacti are usually swollen and fleshy, adapted to water storage, and many are shaped in ways that cause rain to flow directly to the root system for absorption. The roots form extensive systems near the soil surface, assuring that a given plant will absorb the maximum amount of water from a wide area; plants in deserts are usually widely spaced.

The most distinctive vegetative feature of cacti is the areoles, specialized areas on the stems on which stiff, sharp spines usually grow. Some cacti lack spines but have hairs or sharp, barbed structures called glocids on the areoles. Areoles develop from lateral buds on the stems and appear to represent highly specialized branches.

The flowers of cacti are often large and showy and occur singly rather than in clusters of several flowers. The perianth (floral tube) does not consist of sharply differentiated sepals and petals, but rather of a series of bracts (modified leaves), which gradually grade into sepals and finally into showy petals. The flowers have many stamens; the ovary is inferior and fused to the perianth. The fruits are often brightly colored and fleshy.

Most of the 130 or so genera of cacti are found in cultivation, the small, slow-growing species being most popular because of their variety in shapes, colors, and spines. One of the best-known is a group containing beautiful night-blooming flowers and the familiar saguaro plant. In some classifications, this group is split into as many as 10 separate genera (see Cereus). Still more widely grown is the group containing the Christmas cactus. Species of this group, which naturally occur as epiphytes (air plants) in tropical rain forests, do not fit the popular idea of cacti as squat, fleshy plants of desert regions. Examination of their stems, however, reveals the presence of the cactus family’s unique areoles; their flowers have the typical cactus features.

Many groups of plants that are unrelated to cacti have also adapted to survive in arid regions and often resemble cacti in appearance. These offer examples of parallel evolution: Unrelated organisms subjected to similar environmental stresses often evolve similar anatomical and functional characteristics. For example, many spurges that grow in dry parts of Africa, where cacti are not found, exhibit leafless, spiny, fleshy stems (see Spurge).

Scientific classification: Cacti make up the family Cactaceae. Cacti with fully formed leaves are classified in the genera Pereskia and Pereskiopsis. The night-blooming flowers and the saguaro plant are classified in the genus Cereus. The Christmas cactus is classified as Schlumbergera bridgesii.

The Holistic Strory of Coffee as Commercial Crops

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INTRODUCTION

Coffee, common name for any of a genus of trees of the madder family, and also for their seeds (beans) and for the beverage brewed from them. Of the 30 or more species of the genus, only three are important: Arabian, robusta, and Liberian. The shrub or small tree, 4.6 to 6 m (15 to 20 ft) high at maturity, bears shiny green, ovate leaves that persist for three to five years and white, fragrant flowers that bloom for only a few days. During the six or seven months after appearance of the flower, the fruit develops, changing from light green to red and, ultimately, when fully ripe and ready for picking, to deep crimson. The mature fruit, which resembles a cherry, grows in clusters attached to the limb by very short stems, and it usually contains two seeds, or beans, surrounded by a sweet pulp.

Coffee grows well on the islands of Java and Sumatra and in Arabia, India, Africa, the West Indies, and South and Central America. The Americas, where Arabian coffee is grown, produce approximately two-thirds of the world's supply.

PRODUCTION

The soil in which coffee is grown must be rich, moist, and absorbent enough to accept water readily, but sufficiently loose to allow rapid drainage of excess water. The best soil is composed of leaf mold, other organic matter, and disintegrated volcanic rock. Although coffee trees are damaged easily by frost, they are cultivated in cooler regions. The growing temperatures range from 13° to 26° C (55° to 80° F). Altitudes of coffee plantations range from sea level to the tropical frost level, about 1,800 m (about 6,000 ft). Robusta coffee and Liberian coffee grow best at altitudes below 900 m (3,000 ft); Arabian coffee flourishes at the higher altitudes. The seeds are planted directly in the field or in specially prepared nurseries. In the latter case, young selected plants are transplanted later to the fields. Commercial fertilizers are used extensively to promote the growth of stronger, healthier trees with heavier yields. Both the trees and the fruit are subject to insect infestation and microbial diseases, which may be controlled by spraying and proper agricultural management.

Harvesting

The coffee tree produces its first full crop when it is about five years old. Thereafter it produces consistently for 15 or 20 years. Some trees yield 0.9 to 1.3 kg (2 to 3 lb) of marketable beans annually, but 0.45 kg (1 lb) is considered an average annual yield. Two methods of harvesting are used. One is based on selective picking; the other involves shaking the tree and stripping the fruit. Beans picked by the first technique are generally processed, if water is available, by the so-called wet method, in which the beans are softened in water, depulped mechanically, fermented in large tanks, washed again, and finally dried in the open or in heated, rotating cylinders. The so-called dry method, used generally for beans harvested by the second technique, entails only drying the beans and removing the outer coverings. In either case the final product, called green coffee, is sorted by hand or machine to remove defective beans and extraneous material and is then graded according to size.

Commercial Crops

The major types of commercial coffee are the arabicas and the robustas. In the western hemisphere the arabicas are subdivided into Brazils and milds. Robustas are produced in the eastern hemisphere exclusively, together with substantial quantities of arabicas. The Brazils consist principally of Santos, Paraná, and Rio, named for the ports from which they are shipped. Milds are identified by the names of countries or districts in which they are grown, such as Medellín, Armenia, and Manizales coffees from Colombia. Robustas and other arabicas are similarly identified. Green coffee is a major import of the United States; about two-thirds of the 1.2 million metric tons comes from Central and South America, with Brazil and Colombia the two largest suppliers.

Several varieties of green coffee usually are blended and roasted together to produce the tastes, aromas, and flavors popular with consumers. As a rule the beans are heated in rotating, horizontal drums that provide a tumbling action to prevent uneven heating or scorching. Temperatures for roasting range from about 193° C (about 380° F) for a light roast, through about 205° C (about 400° F) for a medium roast, to about 218° C (about 425° F) for a dark roast. The roasted beans are cooled rapidly. Roasted coffee may be packaged and shipped to retail stores, which custom grind it for the customers on purchase, or it may be ground in plate- or roller-type grinding mills before shipment.

Ground coffee loses its unique flavor within about a week unless it is specially packaged. Plastic-and-paper combinations are popular packaging media that afford protection to freshly roasted and ground coffee. Hermetically sealed vacuum, or pressure, cans keep coffee fresh for up to three years.

CHARACTERISTICS

Coffee contains a complex mixture of chemical components of the bean, some of which are not affected by roasting. Other compounds, particularly those related to the aroma, are produced by partial destruction of the green bean during roasting. Chemicals extracted by hot water are classified as nonvolatile taste components and volatile aroma components. Important nonvolatiles are caffeine, trigonelline, chlorogenic acid, phenolic acids, amino acids, carbohydrates, and minerals. Important volatiles are organic acids, aldehydes, ketones, esters, amines, and mercaptans. The principal physiological effects of coffee are due to caffeine, an alkaloid that acts as a mild stimulant. In recent years controversy arose over the possibly harmful effects of coffee beyond those recognized for people who should take few or no stimulants, and the dangers of caffeine for the fetuses of pregnant women. These debated studies were not substantiated, however.

Kind of Coffee:

A Soluble Coffee
Soluble or instant coffee is an important production of the United States coffee industry. In its manufacture an extract is prepared by mixing coarsely ground and roasted coffee with hot water. The water is evaporated from the extract by various methods, including the use of spray driers or high-vacuum equipment. In freeze-dried coffee the coffee extract is frozen, and the water is removed by sublimation. The product is packed in vacuumized, sealed jars or in cans.

B Decaffeinated Coffee
Caffeine can be removed from coffee by treating the green beans with chlorinated hydrocarbon solvents. The beans are roasted by ordinary procedures after removal of the solvents. Decaffeinated coffee is used by people hypersensitive to the caffeine present in regular coffee. In the 1980s nonchemical methods of decaffeination became more common.

C Coffee Substitutes
The use of substitutes for coffee in the United States is limited. The most important substitute is chicory, although chicory is usually used as an extender. Under United States law, the addition of chicory or any other substance must be clearly stated on the brand label.

HISTORY

Exactly where and when coffee was first cultivated is not known, but some authorities believe that it was grown initially in Arabia near the Red Sea about AD675. Coffee cultivation was rare until the 15th and 16th centuries, when extensive planting of the tree occurred in the Yemen region of Arabia. The consumption of coffee increased in Europe during the 17th century, prompting the Dutch to cultivate it in their colonies. In 1714 the French succeeded in bringing a live cutting of a coffee tree to the island of Martinique in the West Indies. This single plant was the genesis of the great coffee plantations of Latin America.

Because of the economic importance of coffee exports, a number of Latin American countries made arrangements before World War II (1939-1945) to allocate export quotas so that each country would be assured a certain share of the United States coffee market. The first coffee quota agreement was arranged in 1940 and was administered by an Inter-American Coffee Board. The idea of establishing coffee export quotas on a worldwide basis was adopted in 1962, when an International Coffee Agreement was negotiated by the United Nations. During the five-year period when this agreement was in effect, 41 exporting countries and 25 importing countries acceded to its terms. The agreement was renegotiated in 1968, 1976, and 1983. Participating nations failed to sign a new pact in 1989, however, and world coffee prices plunged.
Scientific classification: Coffee makes up the genus Coffea of the family Rubiaceae. Arabian coffee is classified as Coffea arabica, robusta coffee as Coffea canephora, and Liberian coffee as Coffea liberica.

Branched-Chain Amino Acids: Valine and Leucine

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Pyruvate (2-oxopropionate) and acetyl CoA provide the carbon backbone for the two ‘branched-chain’ amino acids, valine and leucine. Isoleucine is also a branched chain amino acid, similar in structure to leucine, but the carbon backbone is derived from aspartate. These three amino acids are often grouped together, because they share several common enzymes in their respective biosynthetic pathways (Bryan, 1980; Bryan, 1990). The same enzymes that convert 2-oxobutyrate to isoleucine also convert pyruvate to valine in a parallel but distinct pathway, with no sharing of intermediates. Synthesis of both amino acids, valine and leucine, begins with the formation of acetolactate from two molecules of pyruvate. Acetolactate synthase (ALS) also often denominated acetohydroxyacid synthase (AHAS), catalysing this condensation reaction, contains thiamine pyrophosphate as its prosthetic group. Acetolactate is subsequently reduced, rearranged and the release of water yields 2-oxoketoisovalerate. Finally, a transamination reaction by glutamate produces valine. The branch point in this pathway is 2-oxoketoisovalerate. In a methylation reaction from acetyl coA, isopropylmalate is formed. Isomerisation and decarboxylation produces 2-oxoisocaproate, which is then transaminated to leucine.

These pathways appear to be in the chloroplast since isolated chloroplasts can synthesise valine from 14CO2, and several enzymes of the pathway have been found in isolated chloroplasts. The synthesis of branched chain amino acids is also subject to feedback control by the end products. Isopropylmalate synthase is inhibited by leucine. The first enzyme, ALS, is inhibited by valine and leucine. The sulfonyl urea (e.g. chlorsulphurone) and imidazoline herbicides (e.g. imazethapyr) are very strong inhibitors of ALS, where they bind to the pyruvate-, respectively, 2-oxobutyrate-binding site and antisense mediated repression of ALS also proved ALS to be essential for plant growth and survival (Höfgen et al., 1995). However, upon overexpression of the enzyme there was no increase in the soluble level of valine, leucine and isoleucine indicating that the enzyme is not rate-limiting the biosynthetic pathway (Smith et al., 1988).

Aspartate-Derived Amino Acid Biosynthesis: Lysine, Threonine, Isoleucine, and Methionine

The aspartate pathway is a highly branched pathway, leading to the synthesis of the amino acids lysine, threonine, methionine and isoleucine (Figure 27.5). This pathway is therefore subject to a complex control by enzyme feedback inhibition loops as well as transcriptional and post-transcriptional regulation of expression of genes encoding pathway enzymes (see, Galili, 1995; Saito, 2000; Galili, 2002). Aspartate is formed either through a specific transamination catalysed by glutamate-oxalacetate transaminase or by a deamination of asparagine catalysed by asparaginase. The first two reactions of the aspartate pathway are common to all of its end-product amino acids and include the synthesis of aspartic semialdehyde from aspartate, catalysed by the enzymes aspartate kinase and aspartate semialdehyde dehydrogenase. Aspartate semialdehyde is at an important branch point, since it can either be reduced to homoserine or condensed with pyruvate to give dihydrodipicolinic acid, which subsequently undergoes a series of six enzymatic reactions to yield lysine (see section on ‘Lysine Biosynthesis and Degradation’). The other branch starts with homoserine, an amino acid not found in proteins and usually not present in appreciable concentrations in plants, with the exception of peas, where it can constitute 70&percnt; of the soluble nitrogen in 1-week old seedlings. In most plants homoserine is quickly phosphorylated to O-phosphohomoserine, which represents the next metabolic branch point, since it can be converted in a three step mechanism to methionine (see section on ‘Lysine Biosynthesis and Degradation’) or in a single step to threonine by the enzyme threonine synthase. Threonine can either be used for protein synthesis or is further metabolised to isoleucine, the synthesis of which begins with the deamination and dehydratation of threonine to 2-oxobutyrate catalysed by threonine deaminase (TD). All branched chain amino acids share a number of common enzymes converting either 2-oxobutyrate to isoleucine or pyruvate (2-oxopropionate) to valine and leucine. The steps leading to isoleucine are catalysed by acetohydroxyacid synthase (ALS or AHAS) resulting in 2-acetohydroxybutyrate, acetohydroxyacid isomerase (AHAI) yielding 2,3-dihydroxy-3-methylvalerate, which is oxidised to 2-keto-3-methylvalerate through the activity of dihydroxyacid dehydratase (DHAD). Here the pathway to leucine branches out with four further steps while isoleucine is subsequently formed by transamination through a branched chain amino acid specific aminotransferase (KAAT), specific for 2-keto-3-methylvalerate leading to isoleucine and for 2-ketoisovalerate leading to valine, respectively.

As the biosynthesis of the amino acids lysine and methionine are currently the main focus of plant biotechnology a substantial body of knowledge has accumulated recently. Therefore, we devote a separate section to each of these two pathways.

Aromatic Amino Acids: Phenylalanine, Tyrosine and Tryptophan

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The shikimate pathway provides the basic building blocks for the synthesis of the three aromatic amino acids as well as an array of other aromatic compounds required for functions as different as UV protection, electron transport, signalling, communication, plant defence, structural components and the wound response (Schmid and Amrhein, 1995; Radwanski and Last, 1995; Herrmann and Weaver, 1999). The pathway is firmly rooted in primary metabolism and forms a major link between primary and secondary metabolism in higher plants. From just this brief survey of products derived from the shikimate pathway it is not surprising to find that up to 35&percnt; of the ultimate plant mass in dry weight has its metabolic origins in this pathway as for example lignins are derived from the shikimate pathway. The first seven reactions of the pathway lead from erythrose 4-phosphate and PEP via shikimate to chorismate and are also referred to as the main trunk of the shikimate pathway, or the pre-chorismate pathway.

The shikimate pathway is present only in bacteria, fungi and plants. The absence of the pathway in all other genera has rendered the enzymes catalysing these reactions potentially useful targets for the development of new antibiotics and herbicides (Siehl, 1992).

Some of these reactions are unique in nature: for example, 5-enolpyruvylshikimate 3-phosphate synthase (EPSP-synthase), the sixth enzyme of the pre-chorismate pathway, catalyses the transfer of the intact enolpyruvate to shikimate 3-phosphate.

The first step of the synthesis of these three amino acids is the condensation of erythrose 4′-phosphate (derived from the oxidative pentose phosphate pathway or the Calvin cycle) with phosphoenolpyruvate (from glycolysis) to produce 3′-deoxy D-arabino heptulosonic acid 7′-phosphate (DAHP). This undergoes a series of reactions, including loss of a phosphate, ring closure and a reduction to give shikimic acid, which is then phosphorylated by shikimate kinase. Shikimate phosphate is combined with a further molecule PEP to give 3-enolpyruvylshikimate 5-phosphate (EPSP). The enzyme EPSP synthase, which has received considerable attention because it is inhibited by the herbicide, glyphosate, catalyses this latter reaction. EPSP is converted to chorismic acid, which is at a branch point in this pathway, and can undergo two different reactions, one leading to tryptophan, and the other to phenylalanine and tyrosine.

Anthranilate synthase (AS) catalyses the first reaction in the multi-step tryptophan biosynthesis branch by converting chorismate to anthranilate. AS is feedback inhibited by the end product tryptophan, which binds to an allosteric site on the AS catalytic αα-subunit. The fact that AS is the control point in the tryptophan branch in plant cells is indicated by pathway intermediate feeding and many other studies. But conversion of chorismate to tryptophan has significance beyond amino acid biosynthesis. This is the branch point from which the essential aromatic amino acids as well as many important secondary plant metabolites are derived. Plants use this pathway to produce precursors for numerous secondary metabolites, including the hormone auxine (e.g. indoleacetic acid), indole alkaloids, phytoalexins, cyclic hydroxamic acids, indole glucosinolates, acridone alkaloids, tetrahydrofolate, ubiquinone and vitamine K. These metabolites serve as growth regulators, defence agents and signals for insect pollinators and herbivores. Some of these alkaloids have great pharmacological value, including the anticancer drugs vinblastine and vincristine.

The synthesis of tryptophan from chorismate begins with the reaction of chorismate with the amide group of glutamine to produce anthranilic acid, which subsequently condenses with phosphoribosyl pyrophosphate (derived from ribose 5′-phosphate) to give phosphoribosyl anthranilate. This molecule undergoes a further series of reactions to produce indole glycerol phosphate, which then reacts with serine to produce tryptophan (catalysed by tryptophan synthase).

The synthesis of phenylalanine and tyrosine starts with the rearrangement of chorismate by chorismate mutase to prephenic acid, whose further metabolism has been subject to some debate. For some time the synthesis of phenylalanine and tyrosine from prephenate in plants was assumed to be the same as in bacteria, where the prephenate is either dehydrated to phenylpyruvate (prephenate dehydratase) or oxidatively decarboxylated to hydroxyphenylpyruvate (prephenate dehydratase). Both of these keto acids are subsequently aminated by tranaminases, the former to phenylalanine and the latter to tyrosine. In addition, although phenylalanine, tyrosine and tryptophan are necessary for protein biosynthesis, phenylanine is also a substrate for the phenylpropanoid pathway that produces numerous secondary plant products, such as anthocyanins, lignin, growth promoters, growth inhibitors and phenolics.

Although some of the enzymes involved in this route have been found in plants, there is a growing body of evidence which suggests that another route is either also, or in some plants solely, in operation. Formerly called the ‘pretyrosine’ pathway, it is now generally referred to as the ‘arogenate pathway’, and involves the transamination of prephenate to arogenate, which is then directly converted to either phenylalanine (arogenate dehydratase) or tyrosine (arogenate dehydratase). Arogenate dehydratase has been purified from sorghum and is activity shown to be inhibited by phenylalanine and stimulated by tyrosine, as might be expected from its position in the pathway.

Many of the enzymes of tryptophan synthesis have been found in the chloroplast, and labelling studies with ¹⁴CO₂ have shown that chloroplasts contain the complete pathways for the synthesis of the aromatic amino acids. It is believed that these pathways also exist in the cytosol and perhaps other subcellular compartments. As might be expected, feedback inhibition by tryptophan affects the synthesis of anthranilate from chorismate. Phenylalanine and tyrosine also inhibit their own synthesis, but it is not clear how this occurs. Two isoforms of chorismate mutase exist in a variety of plants, one being sensitive to inhibition by phenylalanine, tyrosine and tryptophan, whereas the other is not. The inhibition, however, is very much dependent on assay conditions and is not well defined.

Glutamine, Glutamate and Asparagine: Ammonia Assimilation

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Plants assimilate inorganic nitrogen into these N-transport amino acids: glutamate, glutamine, aspartate and asparagine. These compounds are used to transfer nitrogen in the phloem from source organs to sink tissues and to build up reserves during periods of nitrogen availability for subsequent use in growth, defence and reproductive processes (Lam et al., 1996; Coruzzi and Zhou, 2001). The concentration of these transported amino acids are not static but are modulated by factors such as light. The most prominent enzymes that synthesise N-transport amino acids are glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH) aspartate aminotransferase (AspAT) and asparagine synthetase (AS). These enzymes are involved in the primary assimilation of inorganic nitrogen from the soil as well as in the re-assimilation (secondary assimilation) of free ammonium within the plant. Nitrogen assimilated into glutamate and glutamine is readily disseminated into plant metabolism, because these amino acids donate nitrogen in the biosynthesis of amino acids, nucleic acids and other N-containing compounds. Because of the high affinity of GS for ammonia, nearly all plant nitrogen is first assimilated into organic form as glutamine. GS acts in concert with glutamate synthase (GOGAT) in what is generally referred to as the glutamate or GS/GOGAT cycle (Figure 27.1). The GS/GOGAT cycle is most likely the principal route of ammonium assimilation in plants. The net effect of this is the amination of 2-oxoglutarate to glutamate. A similar result is achieved by glutamate dehydrogenase (GDH), which also produces glutamate from 2-oxoglutarate and ammonia, but it is generally believed that this enzyme is primarily involved in glutamate oxidation, and contributes little to ammonia assimilation. GS isoforms can be separated into two classes by ion-exchange chromatography—one localised in the cytosol (GS1), the other in the chloroplast (GS2). Plants examined thus far appear to possess a single nuclear gene encoding GS2 and multiple (up to four) nuclear genes encoding GS1 subunits. The plant GS holoenzyme functions as octamer and GS1 polypeptides can assemble into homo- or heterotetramers. Although both GS isoforms do not significantly differ in their biochemical properties, they display distinct in vivo functions. The GS2 holoenzyme is predominant in leaves and very probably involved in primary ammonia assimilation and re-assimilation of respiratory ammonia. GS1 isoenzymes are present at low concentrations in leaves and at higher concentrations in roots suggesting that this enzyme has a role in primary assimilation in roots.

Asparagine synthetase (AS) catalyses the amidation of aspartate to asparagine, using ammonia or glutamine as amino donor. In most cases, it appears that the in vivo substrate for this enzyme is glutamine, not ammonia, and hence AS does not usually constitute a route for ammonium assimilation.

Asparagine synthesis is particularly important in the root nodules of legumes, where much of the nitrogen fixed by the bacteria is rapidly transferred to asparagine through the joint activities of GS and AS. Thus, a lot of nodule-derived nitrogen transported in the xylem is in the form of asparagine. Asparagine levels in plant tissues vary diurnally, and often increase under stress conditions, such as nutrient deficiencies, salt stress or drought. The significance of such increases has not been established but may be a means of storing nitrogen when protein synthesis is limited by the stress that the plant is experiencing.
Proline and Arginine

Glutamate is the precursor of glutamine, arginine and proline (Buchanan et al., 2000). Glutamine synthesis has been handled earlier (in the section on ‘Nitrogen Assimilation and Reduction’ in this chapter and in the chapter on nitrogen fixation of this handbook). The first step of proline synthesis, is the activation of glutamate to an energy rich glutamyl-5′-phosphate consuming ATP and its consecutive reduction to glutamyl-5′-semialdehyde (Aral and Kamoun, 1997). In plants, different from e.g. bacteria, the kinase and dehydrogenase are synthesised as a bifunctional fusion protein, ΔΔ1-pyrroline-5-carboxylate synthetase (P5CS). The semialdehyde spontaneously cyclises to give pyrroline 5′-carboxylic acid, which is then reduced to the imino acid proline. Due to its rigid ring structure proline acts as a chain breaker when inserted into proteins, disrupting regular folding patterns of αα-helices. Physiological features of proline will be discussed in more detail when addressing the biotechnological perspectives.

Arginine biosynthesis resembles very much proline biosynthesis as glutamate is first δδ-phosphorylated by a kinase forming N-acetyl-glutamyl-5′-phosphate and then reduced to N-acetyl- glutamyl-5′-semialdehyde. However, initial N-acetylation of the amino group of glutamate prevents cyclisation. After transamidation of the 5′-semialdehyde with another glutamate as amino group donor the resulting N-acetyl-ornithine is converted in a series of reactions, similar to the animal urea cycle, to the non-protein amino acids ornithine, citrulline and finally arginine. Arginine plays a major role as a basic protein constituent often participating in active centre reactions, e.g. in the substrate-binding site of lactate dehydrogenase where it probably helps to orientate the substrate while a histidine residue acts in the conversion of lactate to pyruvate.

Biosynthesis of Amino Acids in Plants

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CO2 assimilation provides the carbon skeletons required for the synthesis of the various amino acids (Figure 27.3). 3-Phosphoglycerate is the most important carbon precursor for the synthesis of amino acids and from its converted form, phosphoenolpyruvate (PEP), two pathways branch off leading to pyruvate and oxalacetate. Moreover, PEP in combination with erythrose 4-phosphate is the precursor for the synthesis of aromatic amino acids via the shikimate pathway. The pathways proposed for amino acid biosynthesis in plants are inferred in large part from those defined in Escherichia coli and yeast, where the steps and regulatory mechanisms have been identified using a combination of genetics, biochemistry and molecular biology.

Every protein molecule can be viewed as a polymer of the 20 common amino acids. The centre is comprised of a tetrahedral carbon atom forming a chiral centre called the alpha (αα) carbon (Figure 27.4). It is covalently bonded on one side to an amino group and on the other side to a carboxyl group. The third bond is always hydrogen, and the fourth bond is to a variable side chain (R), eventually resulting in the l-configuration typical of most amino acids. d-amino acids also occur in nature but only as specialised forms, e.g. in peptide antibiotics such as actinomycin C1, fungisporin, gramicidin S, polymixin B1 and valinomycin. d-amino acids are formed by racemases as free intermediates and are subsequently incorporated into peptide bonds but these are ordinarily poor substrates for incorporation. Whether the l-amino acid is incorporated first and then the inversion occurs or vice versa has still to be determined in most cases. In neutral solution, the carboxyl group loses a proton and the amino group gains one. Thus, an amino acid in solution, while neutral overall, is a double charged species called a zwitterion (Figure 27.4). Depending on the structure of the side chain the 20 amino acids commonly found in proteins are grouped into the apolar group (alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine), the uncharged polar group (glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine) and the charged group (aspartic acid, glutamic acid, lysine, arginine and histidine). All these amino acids except proline have an ammonium ion attached to the carbon atom. In proline an N-C linkage forming part of a cyclic structure replaces one of the N-H linkages, leading to the formation of an imino acid.
In addition to the 20 commonly occurring αα-amino acids, a variety of other amino acids are found in minor amounts in protein and in non-protein compounds. The unusual amino acids found in proteins result from modification of the common amino acids. In a few cases these amino acids are incorporated directly into the polypeptide chains during synthesis (e.g. selenocysteine). Most frequently the amino acid is modified after incorporation (e.g. modification of proline to hydroxyproline). The types of unusual amino acids found in non-protein compounds are extremely variable and formed by a number of different metabolic pathways.

Amino acids can also be grouped together into ‘families’, each of which is derived from a single ‘head’ amino acid (Figure 27.3). Instead of being grouped according to the functionality of the side group these families are rather based on common precursors of metabolic trees. For example, the ‘aspartate family’ is comprised of asparagine, lysine, threonine, isoleucine and methionine, all synthesised from aspartic acid. Because more than one amino acid may be involved in the synthesis of another, a single amino acid may be assigned to more than one family, and it is not unusual for different authors to differ in their assignment.

The Metabolic Role of Amino Acids in Plants

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Various transaminases transfer the reduced amino group to 2-oxo organic acids to form amino acids. Besides 20 proteinogenic amino acids which are used as building blocks of proteins, a vast number of non-proteinogenic amino acids are formed, either as intermediates of amino acid biosynthetic pathways, e.g. homoserine or homocysteine, or as products of certain pathways where these amino acids serve special functions in metabolism, such as ornithine and citrulline as part of the urea cycle or canavanine A as insect antifeedants.

Furthermore, a huge body of the amino acids are precursors for metabolic pathways (see, Buchanan et al., 2000). Glutamine is the precursor of heme groups and chlorophylls and methionine, respectively its direct activated product, S-adenosylmethionine (SAM), is precursor of the vitamin biotin, or is used directly as a major methyl group donor of numerous reactions of the C1 metabolism in cells, to synthesise the ‘aging’ hormone ethylene responsible for fruit ripening as well as the precursor of polyamines which are involved in stabilising and regulating DNA. The aromatic amino acids are beside other compounds precursors of pigments, phytoalexins, structural compounds as lignins, (here methionine is involved again in methylation reactions) as well as precursor of the plant hormone auxin and an almost innumerable number of secondary metabolites plants being able to synthesise. Furthermore, amino acids are involved in stress responses such as scavenging of active oxygen species, either directly or through regeneration of ascorbic acid by glutathione, a tripeptide (γγ-glutamyl-cysteinyl-glycine) or metal detoxification by phytochelatins (poly-glutathione). Thioredoxins are involved in controlling the redox status of a cell; they control photosynthesis by transmitting the dark-to-light switching signal. These examples are just a glimpse of the role of amino acids and peptides; several more are likely to emerge in the coming years.

Nutrient Uptake and Assimilation

Among the various minerals essential for plant growth the most limiting macronutrients are nitrogen, phosphorus and sulphur. A limitation of any of these minerals limits biomass formation in natural ecosystems and, in particular, plant production in agriculture.

Nitrogen Assimilation and Reduction

Nitrogen represents the mineral nutrient required in the largest quantities by plants and is most limiting where maximal biomass production is desired (Stitt, 1999; Tischner, 2000). The uptake and metabolism of nitrate and ammonia have been extensively investigated and analysed (Figure 27.1). It is now established that all steps of primary nitrogen assimilation are targets of several signal transduction cascades that integrate external stimuli and internal conditions of the plant. Within this regulatory network, nitrate reductase (NR) catalyses one of the most controlled reactions in plants, receiving input from light, photosynthesis, CO2, oxygen availability and nutrient status at the transcriptional and post-translational level. Nitrate uptake systems across membranes exist as high- and low-affinity forms. High-affinity nitrate transporters are either encoded by nitrate inducible or constitutive genes and have Michaelis-Menten constants for nitrate of 6–100 μμM. They are mostly expressed in the outer layers of roots to mediate increased uptake when the external nitrate supply is low. A constitutive low-affinity uptake system operates at nitrate concentrations above ~0.25 mM. Whereas the role of high-affinity transport systems is evident, the function of low-affinity transport systems is less clear. Optimal plant growth is achieved with balanced ratios of nitrate and ammonia but not with nitrogen source alone. The relative contributions of nitrate and ammonia to total nitrogen uptake differ considerably between plant species and ambient availability in soil. Ammonia uptake is mediated by families of active membrane transporters (Km <0.5–40 μμM), which exhibit differential expression in response to light, tissue and nutrient status.
The conversion of nitrate to nitrite is performed by NR, and of nitrite to ammonia by nitrite reductase (NiR). NR is cytosolic, mainly located in root epidermis and cortical cells and leaf mesophyll cells. NiR is chloroplast localised and encoded in the nuclear genome. In photosynthetic tissues reducing equivalents for the reduction of nitrate to ammonia are derived directly from photosynthetic electron transport. Reduced ferredoxin (Fd) is the electron donor that fuels the catalytic activity of NiR. In non-photosynthetic tissue, NADPH derived from the oxidative pentose phosphate pathway can be used to generate ammonia from nitrate. Experimental supplementation with nitrate induces genes of nitrate assimilation and carbon metabolism, mainly to provide carbon skeletons as acceptors for reduced nitrogen. Cross talk between assimilation pathways is further indicated by the finding that low sugar levels are able to repress NR expression, even in the presence of otherwise inductive nitrate concentrations and a down-regulation of NR expression is even observed under conditions of sulphate limitation. However, neither the chemical nature of the signal compounds nor the sensors responsible for these reactions are precisely known. Nitrate and ammonia could be sensed as free ions by extra cellular or intracellular receptors, but downstream products or the carbon could also exert regulation as well as the nitrogen to carbon ratio.

Though the picture is far from being resolved a number of nitrogen sensing and regulatory systems have been described such as a MADS-box like transcription factor responding to local nitrate supply, a transcription factor of the myb structural family and a putative protein kinase gene up regulated by nitrogen deficiency. Furthermore, an extra cellular nitrate reduction system which might act as a nitrate sensor via the release of nitric oxide (NO), GLB1 which is a structural PII homologue of the bacterial glutamine synthetase regulator and up regulated by light and sucrose, respectively repressed by glutamine and glutamate, and 14-3-3 protein family members that reversibly bind to phosphopeptide motifs in diverse target proteins in plant, fungi and animals, resulting in altered activities of enzymes and regulatory proteins.

Sulphur Assimilation and Reduction

Sulphate uptake and assimilation are carried out in plants by a unique pathway that is distinct from that in bacteria and fungi (Anderson, 1990; Leustek and Saito, 1999; Hawkesford, 2000; Hawkesford and Wray, 2000; Leustek et al., 2000; Saito, 2000; Grossman and Takahashi, 2001; Hawkesford, 2002) (Figure 27.2). Beside the most regulatory step, the sulphate uptake by roots which enables the plant to achieve the inner-cellular homeostasis, significant regulatory steps of sulphur incorporation into organic compounds are catalysed by adenosine 5′-phosphosulphate reductase (APR). APR controls the flux of intermediates to yield sufficient reduced sulphur (Suter et al., 2000; Tsakraklides et al., 2002). A further step of control is exerted by the cysteine synthase complex providing the carbon/nitrogen backbone for cysteine formation (Blaszczyk et al., 1999; Harms et al., 2000). Alterations to any of these three processes can have profound effects on cysteine biosynthesis and on the capacity of plants to grow in soils in which nutrient resources are limiting. Essentially most of the reduced sulphur is channelled from cysteine into methionine, Fe/S clusters, vitamin cofactors and proteins required to carry out crucial structural, catalytic and regulatory functions in the cell. Despite this importance for plant biochemistry, plant sulphur metabolism has been much less thoroughly investigated than that of nitrogen. It has, however, gained much more attention in recent years after the unexpected observation of sulphur limitation in agricultural production at least in Europe due to reduced aerial pollution.
Although the uptake and transport of sulphate probably take the same combined apoplastic/symplastic route as, for example, nitrate and phosphate, the sulphate ion seems to be much less mobile after deposition in vacuoles of the source tissue. Sulphate transporters have been cloned and functionally characterised from several species. They can be grouped into high (Km 0.1–1 μμM) and low (Km 1–10 μμM) affinity proton/co-transporters. Expression analysis of sulphate transporters demonstrated that they are present in root hairs and epidermis for sulphate acquisition and in vascular bundles of root and leaf for the allocation of sulphate. Several sulphate transporter genes are induced within hours of sulphate deficiency and are rapidly repressed upon renewed supply of sulphate. Once the sulphate enters the cell it is activated by ATP sulphurylase to form adenosine 5′-phosphosulphate (APS). ATP sulphurylase (ATP-S) isoforms in plants are located either in plastids or in the cytosol. The cDNAs for these isoforms were first isolated from potato. In Arabidopsis there appear to be at least three plastidic and one putatively cytosolic ATP-S. The APS generated by ATP-S can serve as a substrate for sulphate reduction or can be phosphorylated by APS kinase to yield 3′-phosphoadenosine 5′-phosphosulphate (PAPS). PAPS is the substrate of various sulphotransferases to catalyse the sulphatation of a range of metabolites including flavanols, choline, and glucosides.

The sulphate of APS is reduced to sulphite by the plastid-localised APS sulphotransferase, also termed APS reductase (APR). The reductant used by the enzyme is probably reduced glutathione; a domain of the enzyme resembles a glutathione-dependent reductase. APR transcript accumulates during S-starvation, suggesting that a key juncture for controlling assimilatory processes occurs at the point at which APS interacts with either APS kinase or APR. The sulphite generated in the APR catalysed reaction is reduced to sulphide by plastidial sulphite reductase (SiR), the gene of which has been recently identified. Electrons used for sulphite reduction are donated by reduced ferredoxin. Sulphide is finally transferred to activated serine, O-acetyl serine, yielding cysteine through the activity of the enzyme O-acetyl serine-(thiol) lyase (OASTL). Cysteine is the first organic compound carrying reduced sulphur and the precursor of all following metabolic steps carrying a sulphur or thiol moiety.

The allocation of reduced sulphur proceeds via the phloem. Glutathione (GSH) and S-methylmethionine (SMM) appear to have a role in transport as well as being an interorgan signal for the sulphur status from the shoot to the root. In plants such as wheat, substantial amounts of reduced sulphur are transported as SMM from source leaves to sink tissues. Similar to nitrate assimilation, either sulphate or its downstream metabolites are suspected to trigger changes in the mRNA levels of the sulphate transporter and APR genes. An activating effect on uptake and APR activity has been demonstrated for O-acetylserine (OAS), an intermediate in cysteine biosynthesis (for review see Buchanan et al., 2000). An investigation of changes in OAS levels may suggest a link between sulphur, nitrogen and carbon metabolism, because external supplies of these macronutrients mutually affect at least single steps within the assimilatory activities of each pathway and photosynthesis.

As a number of amino acid related topics will be covered through other sections of the handbook, this chapter will highlight examples in which combined molecular, biochemical and genetic approaches have helped to define the pathways and uncover regulatory mechanisms of amino acid biosynthesis in plants. Especially, we will focus on biotechnology driven research and its implications for both basic and applied research. Rational engineering of amino acid biosynthesis is exploited for as diverse aspects as herbicide design and quality improvement of crop plants. A comprehensive review of the general biochemistry of amino acid synthesis can be found elsewhere (Miflin and Lea, 1990; Singh et al., 1992; Singh, 1999; Buchanan et al., 2000).

Amino Acids in Plants

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In plants, amino acids fulfil a wide variety of functions. Their common role is to serve as building blocks of proteins, which exert manifold functions in plant metabolism, and as metabolites and precursors they are involved in plant defence, vitamin, nucleotide and hormone biosynthesis, and as precursors of a huge variety of secondary compounds. One way or the other, as active catalysts or as precursors, amino acids are essentially involved in all metabolic, regulatory, and physiological aspects of plant metabolism (for comprehensive reviews see, Miflin and Lea, 1990; Singh et al., 1992; Singh, 1999; Buchanan et al., 2000).

Uncovering plant specific aspects of amino acid biosynthesis contributed both to fundamental and applied research. The goals are to understand the biosynthetic pathways and their regulation and to understand the regulation of genes controlling growth-limiting processes (e.g., the assimilation of inorganic nitrogen into amino acids). A second huge field of investigations are the pathways and factors regulating the synthesis of secondary plant compounds derived from amino acid precursors. However, these areas are mainly covered in other chapters of this handbook. In addition to their obvious role in protein synthesis, amino acids perform essential functions in both primary and secondary plant metabolism. Some amino acids as glutamine and asparagine serve to assimilate and transport fixed nitrogen from sources to sinks. The aromatic amino acids serve as precursors to secondary products such as hormones and compounds involved in plant defence. Thus, the synthesis of amino acids controls directly or indirectly various aspects of plant growth and development. Such studies should also provide a framework for manipulating amino acid biosynthesis pathways in transgenic plants. For example, enzymes in several pathways have been identified as targets for herbicides. In some cases, the genes encoding these enzymes have been used for engineering herbicide resistance in plant of the first generation of transgenic plants. Eventually, future progress in amino acid biosynthesis research may provide enhanced crop resistance to osmotic stress and improved food protein composition. Therefore, the structural and regulatory genes controlling amino acid biosynthesis in plants are of interest not only to biochemists but also to agricultural biotechnologists, breeders and agrochemical industry to develop new transgenic plants with benefit to the consumer, sometimes befitted as the ‘second generation’ of genetically modified organisms (GMOs).

Beside the proteinogenic amino acids, many plants channel large amounts of nitrogen into amino acids that are not usually constituents of proteins (Herrmann, 1995; DellaPenna, 1999; Grusak and DellaPenna, 1999). These non-protein amino acids comprise a diverse and often complex group of compounds: several hundreds of them have been found in plants, most often in seeds (especially those of legumes), where they can accumulate to high levels. Non-protein amino acids are found in all plant tissues as intermediates in the synthesis of protein amino acids (e.g., homoserine, diaminopimelic acid, ornithine, citrulline), and in a more restricted range of plants as metabolic ‘end products’. The function of the latter one is often unclear, but they are frequently toxic to animals and found in seeds where they appear to serve both as a storage reserve of reduced nitrogen and as a feeding-deterrent to herbivores. The mode of toxicity varies, but is usually based on interference with regulation, transport or protein synthesis. One amino acid fairly common in legumes is canavanine A, which can account for up to 6&percnt; of the fresh weight of seeds of the Jack bean. It is very similar in structure to arginine and is thus able to interfere with arginine metabolism in animals that ingest those seeds. When metabolised, canavanine A causes a variety of toxic effects, including pupal malformation in insects and immune dysfunction in vertebrates. Other toxic arginine analogues include homoarginine and indospicine, also found in legumes.

Carbon and nitrogen are the principal constituents of amino acids. The carbon backbones are derived at different branching points from primary carbon metabolism while reduced nitrogen is transferred to these 2-oxo acids to form the common amino acid head group. The unique feature of this amino acid head group is the ability to form peptide bonds and thus polymerise to proteins. The side groups determine the chemical properties of the respective amino acids and later the derived protein structures (Table 27.1). These side groups are either directly derived from the carbon moiety or modified, e.g. through binding of reduced sulphur (cysteine and methionine), or through cyclisation (proline and aromatic amino acids). Actually amino acids are the entry port of the macronutrients N and S into plant metabolism. Nitrogen and sulphur assimilation first results in amino acids, i.e. glutamine and cysteine, respectively. These two amino acids serve as precursors of all further organic molecules containing any of these macronutrients.

Potentials and Prospects of Cryopreservation of Plant Cell, Tissue and Organ Culture and Establishment of 'Germplasm Bank'

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Due to gradual disappearance of economic and rare plant species the necessity for storage of genetic resources of plant realm in general and agricultural plants in particular was realized by the biologists (Bajaj and Reinert, 1977; Wilkins and Dodds, 1983). The conventional methods of storage fail to prevent from losses caused by (i) attack of pathogens and pests, (ii) climatic disorders, (iii) natural disorders, and (iv) political and economic causes. However, the conventional methods could not save the viability of short lived seeds of economic plants, for example, oil palm (Elaeis guineensis)i rubber (Hevea brasiliensis), Citrus sp. and Coffes sp. (Dodds, and Roberts, 1985).

These materials are stored at low temperature, due to which growth-rate of cells retards; consequently biological activities are conserved for long time.

Cryobiology deals with the study of metabolic activities and their responses in plant materials (and animal ceils) stored at low temperature (- 196°C) by using liquid nitrogen in the presence of cryoprotectants. Dodds and Roberts (1985) have discussed 3 principal methods used for growth suppression in plant tissue culture (i) the alteration of physiological conditions of culture i.e. temperature or gas composition within the vessel; (ii) changing the composition of basal medium e.g. using sub or supra-optimal concentrations of nutrients (some factors essential for normal growth may be either omitted or employed at a reduced level) and (iii) supplementing the medium with growth retardants (e.g. abscisic acid) or osmocegulatory compounds (e.g. mannital, sorbital, etc.).

Storage at reduced temperature has been very affective for tissue culture of most of the plant species such as potato, cassava (Manihot esculentum), pea (Pisum sativum), chickpea (Cicer arietinum), rice (Oryza sativa), wheat (Triticum vulgare), coconut (Cocos nucifera), oil palm (E. guineensis) strawberry (Fragaria vesca) and sugarcane (Saccharum officinarum) (Bajaj, 1987).

Content

⇒ Difficulties in cryopreservation

⇒ Methods for cryopreservation

⇒ Plant cell bank

⇒ Pollen bank

⇒ Achievements through cryopreservation

Difficulties in Cryopreservation
A number of reviews available during the last two decades illustrate the significant progress made in this field and also the outline of the existing problem (Witherr, 1980; Henshaw, 1982). The difficulties are (i) high specific feature of plant cells, such as their large size, strong vacuolization and abundance of water, (ii) cell damage during freezing and subsequent thawing caused by ice crystals formed inside the cells and by cell dehydration, and (iii) gradual formation of large crystals of more than 0.1mm whose facets rupture many cell membranes (Shimada and Ashahina, 1975). However, in the presence of cryoprotectants (the chemicals decreasing cryodestructJ0h) and reduced temperature, free water has enough time to leave the cells. Therefore, it can freeze on the crystal surface in the solution (Samygin, 1974). This results in marked dehydration and protoplast shrinkage (Muzur, 1977). Excessive time and degree of plasmolysis are the reasons of cell destruction during slow freezing, since they cause irreversible contraction ofthe plasmalemma (Wiest and Steponkus, 1978).

Methods of Cryopreservation
The freezing-storage-thawing cycle is an external procedure consisting of the following basic stages:

(i) Selection of Materials. For selecting the plant materials a number of factors are taken into account ; the important ones are, nature and density of cells in the vials/ampules to be cryopreserved ; because the cryoability of the cell cultures depends on these. Young meristematic, highly cytoplasmic and small cells which are non-vacuolated and thin walled and in small aggregates, are good materials to be selected for this purpose. Cell density in vials or amples should be high, as it shows prolonged survival at high cell density.

(ii) Addition of Cryoprotectors. Cryoprotectors are the chemicals which decrease cryodestruction. These are sugars, glycols, sugar alcohols, alcohols, polyvinylpyrrollidone, polyethylene glycol (PEG), polyethylene oxide (PEO), dextrans, hydroxystarch, glycerine, sucrose, and some amino acids (e.g. proline). Bajaj (1987) has advised to use a mixture of two or three cryoprotectants at low concentrations rather than a single cryoprotectant at a high concentration as it could be toxic. During treatment, the cultures should be maintained in ice to avoid deleterious effects.

(iii) Freezing. Freezing should be done in such a way that it does not cause intracellular freezing and crystal formation, as it is possible in sudden freezing. To avoid this problem, regulated rate of cooling or pre-freezing is done. Moreover, freezers have also been developed which allow the uniform temperature decrease at a desired rate, commonly not less than 1°C per minute (Popov, 1985), In 1987, the Institute of Cryobiology and Cryomedicine of the Ukrainian Academy of Sciences (erstwhile U.S.S.R.) devised the programme freezer which envisaged lower rate (0.5°C per minute) of temperature decrease.

(iv) Storage in Liquid Nitrogen. If the cells are not stored at sufficiently low temperature, an additional injury to the cultures may be caused. The storage temperature should be such that it stops all metabolic activity and prevents biochemical injury (Bajaj 1987). Prolonged storage of frozen materials is possible only when the temperature is lower than -130°C. This can be simply achieved with the help of liquid nitrogen, which keeps the temperature -196°C. Popove (1988) stored the cultures of carrot cells for about 5 years by doing so.

(v) Thawing. Thawing is the process of releasing the vials containing cultures from the frozen state to elevate the temperature between 35 and 40°C. It should be done quickly but without overheating. As soon as the last ice crystals disappear, the vials are tranferred into a water bath at 0°C (Popov, 1985).

(vi) Washing and Reculturing. Washing of plant materials is done to remove the toxic cryoprotectants. When low toxic or non-toxic cryoprotectants are used, the cultures should not be washed, but simply recultured. Washing becomes necessary only when cryoprotectants have toxic effects on cells. Washing follows the following procedure : dilution, resuspension, centrifugation and removal of cells. It is, however, possible that some cells die due to storage stress and the most stable ones survive. Therefore, determination of cell viability by culturing them on growth medium is essential.

vii) Regeneration of Plantlets. The viable cells are cultured on non-specific growth media to regenerate into plantlets. Bajaj (1987) has given an extensive list of works on cryopreservation of cells, tissue, and organ culture of various plants e.g. potato, cassava, sugarcane, soybean, groundnut, carrot, cotton, citrus, coconut, etc.

Plant Cell Bank / Germplasm Bank / Cell Crygbank

Cryopreservation of genetic stock i.e. germplasm (or vegetatively propagated crops, recalcitrant producing plants, rare plant species, medicinal, horticultural and forest plants, and VAM fungi) is a novel approach for their conservation in liquid nitrogen on a long term basis. To achieve this goal, a plant cell bank ( = germplasm bank and cell cryobank) has been suggested by Bajaj (1977 a), Bajaj and Reinert (1977) and Popov (1985). Suggestions have also been made that germplasm bank should be attached to some of the International Research Institutes (e.g. IRRI) that would hold responsibility for the storage, maintenance, distribution (at national and international level), and exchange of these disease free germplasm of the important plants. Fig. 10.1. shows the potential and prospects of cryopreservation of plant cell, tissue and organ and establishment of germplasm bank.

Facilities for storage of genetic stock of plants can be developed in large sized cylinders (30-50 liters capacity) where liquid nitrogen does not require refilling for 6-8 months (Bajaj, 1987). Potentials and prospects of cryopreservation of plant cell, tissue and organ culture and establishment of .'Germplasm Bank' (after Bajaj, 1977a).

Fig. 10.1. Potentials and prospects of cryopreservation of plant cell, tissue and organ culture and establishment of .'Germplasm Bank' (after Bajaj, 1977a). Thus, germplasm bank is such a device where facilities of cryopreservation of genetic resources of a variety of plants are available and on demand, the germplasm can be supplied nationally and internationally.

Pollen Bank
Besides germplasm bank, the storage of pollen grains in liquid nitrogen and establishment of pollen bank have also been suggested to retain their viability for various lengths of time. The freeze storage of pollen would enable (i) hybridization between plants with flowers at different times, (ii) growth at different places, (iii) reducing the dissemination of diseases by pollination vectors, and (iv) maintenance of germplasm and enhancement of longevity (Bajaj, 1987).

Achievement made through Cryopreservation
Various forms of plant materials viz. cell suspensions clones, callii, tissues, somatic embryos, root/shoot tips propagules (tubers) pollen grains, etc. have been preserved in liquid nitrogen for prolonged time and tested for their survival and regeneration potential.

No doubt, in most of the cases, the cells/tissues, organs regenerated into plants. Bajaj (1987) has described a number of plant species that have been successfully cryopreserved. Some of the observations made are as below :

(i) Cryopreservation of cell lines : For example, cell suspensions (soybean, tobacco, dhatura, carrot) and somatic hybrid protoplasts (rice x pea, wheat x pea).

(ii) Cryopreservation of pollen and pollen embryos : For example, fruit crops, trees, mustard, carrot, peanut, etc.

(iii) Cryopreservation of excised meristems : For example, potato, sugarcane, chickpea, peanut, etc.

(iv) Cryopreservation of germplasm of vegetatively propagated crops: potato, sugarcane, etc.

(v) Cryopreservation of recalcitrant seeds and embryos: Large sized seeds that are shortlived and abortive, such as oil palm, coconut, walnut, mango and cocoa.

The Prospects of Developing Medicinal Plants (Present & Future)

2011-04-27T17:06:00.000-07:00

Medicinal plants are the local heritage with global importance, World is endowed with a rich wealth of medicinal plants. Herbs have always been the principal form of medicine in India and presently they are becoming popular throughout the developed world, as people strive to stay healthy in the face of chronic stress and pollution, and to treat illness with medicines that work in concert with the body's own defense. People in Europe, North America and Australia are consulting trained herbal professionals and are using the plant medicines. Medicinal plants also play an important role in the lives of rural people, particularly in remote parts of developing countries with few health facilities.

The variety and sheer number of plants with therapeutic properties is quite astonishing. It is estimated that around 70,000 plant species, from lichens to towering trees, have been used at one time or another for medicinal purposes. The herbs provide the starting material for the isolation or synthesis of conventional drugs.

In Ayurveda about 2,000 plant species are considered to have medicinal value, while the Chinese Pharmacopoeia lists over 5,700 traditional medicines, most of which are of plant origin. About 500 herbs are still employed within conventional medicine, although whole plants are rarely used.

In India, medicinal plants have made a good contribution to the development of ancient Indian Material Medica. One of the earliest treatises on Indian medicine, the Charak Samhita (1000 B.C.), records the use of over 340 drugs of vegetable origin. Most of these continue to be gathered from wild plants to meet the demand of the medical profession. Thus, despite the rich heritage of knowledge on the use of plant drugs, little attention had been paid to grow them as field crops in the country till the latter part of the nineteenth century.

Medicinal plants help in alleviating human suffering. These plants "are being integrated to the field of foods as additives, beverages and cosmetics. They are widely used as sweeteners, as biters, as spices, as natural colouring agent and as insecticides. Mass selection recurrent selection, hybridization, clonal selection mutation and biotechnology are some of major techniques at their use for many proven medicinal plants. There are still several constraints.

During the past one century there has been a rapid extension of the allopathic system of medical treatment in India. It generated commercial demand for pharmacopoeial drugs and their products in India. Efforts have been made to introduce many of these drug plants to farmers. Several research institutes have undertaken studies on the cultivation practices of medicinal plants, which were found suitable and remunerative for commercial cultivation. The agronomic practices for growing poppy, isabgol, senna, cinchona, ipecac, belladonna, ergot and few others have been developed and there is now localized cultivation of these medicinal plants commercially.

Medicinal plants have curative properties due to the presence of various complex chemical substances of different composition, which are found as secondary plant metabolites in one or more parts of these plants. These plant metabolites, according to their composition, are grouped as alkaloids, glycosides, corticosteroids, essential oils, etc. The alkaloids form the largest group, which includes morphine and codeine (Poppy), strychnine and brucine (Nux vomica), quinine (Cinchona), ergotamine (Ergot), hyocyamine (Belladonna) scolapomine (Datura), emetine (Ipecac), cocaine (Coco), ephedrine (Ephedra), reserpine (Rauwolfia), caffeine (Tea dust), aconitine (Aconite), vascine (Vasaca) santonin (Artemisia), lobelin (Lobelia) and a large number of others. Glycosides form another important group represented by digoxin (Foxglove), stropanthin (Strophanthus), glycyrrhizin (Liquorice), barbolin (Aloe), sannocides (Senna), etc. Corticosteroids have come into sannocides (Senna), etc. Corticosteroids have come into prominence recently and diosgenin (Dioscorea), solasodin (Solanum sp.), etc. now command a large world demand. Some essential oils such as those of valerian kutch and peppermint also possess medicating properties and are used in the pharmaceutical industry. However, it should be stated in all fairness that our knowledge of the genetic and physiological make-up of most of the medicinal plants is poor and we know still less about the biosynthetic pathways leading to the formation of active constituents for which these crops are valued.

Medicinal and aromatic plants are found in forest areas throughout South Asia, from the plains to the high Himalayas, with the greatest concentration in the tropical and subtropical belts and arid region of Thar desert. India recognizes more than 2,500 plant species as having medicinal value, Sri Lanka about 1,400, and Nepal around 700. Some of these, found at high altitudes in particularly stressful environments, grow very slowly and cannot live elsewhere. Others are more broadly distributed and adapt more easily to different ecological conditions.

During the past decade, a dramatic increase in exports of medicinal plants attests to worldwide interest in these products as well as in traditional health systems. In the last 10 years, for example, India's exports of medicinal plants have trebled. But with most of these plants being taken from the wild, hundreds of species are now threatened with extinction because of over-harvesting, destructive collection techniques, and conversion of habitats to crop-based agriculture. For instance, the small coniferous Himalayan yew (Taxus baccata) has recently become a heavily traded species. Similarly, senna is being grown extensively in arid region of India.

The pharmaceutical industries have made massive investment on pharmacological, clinical and chemical researches all over the world in past five decades. Efforts have been made to discover still more potent plant drugs. In fact, a few new drug plants have successfully been passed the tests of commercial screening. The benefits of these efforts would reach to the masses in future if farmers initiate commercial cultivation of medicinal plants. In fact, agricultural studies on medicinal plants, by its very nature, demand an equally large investment and higher priority. India, in particular, has a big scope for the development of pharmaceutical and phytochemical industry.

The subcontinent, India is blessed with varieties of aromatic and medicinal plants. The agroclimatic conditions and rainfall favouring this bio-availability. More than 7,500 species of medicinal plants are grown in India. Owing to this India is considered as the botanical garden of the world and treasure house of the biodiversity. Ayurveda, our indigenous system of health care is accepted everywhere especially abroad. Vedas and other ancient scriptures give cleanout evidences of using herbs and medicinal plants. Ayurveda alone describes about 2000 species of plants, which constitutes more than 10,000 formulations.

Over the past 10 years there has been a considerable interest in the use of herbal medicines in the world. Regarding the export of medicinal plants India's contribution to the international market is comparatively very low. Utilizing our biodiversity and proper planning, Indian products can very well enter the overseas markets. This can be achieved only through proper development of medicinal plants, standardization of the extracts and keeping the quality. WHO has recognized the effectiveness of traditional system of medicine and its safety.

The Indian Pharmacopoeia (1966) recognized eighty five drug plants whose ingredients are used in various pharmaceutical preparations. The text is however; confine to a few important commercially grown medicinal plants whose cultivation deserves priority in out national economy.

According to R. B. S. Rawat and R. C. Uniyal, National Medicinal Plants Board Department of ISM&H (Agrobios News Letter, Vol. 1, No. 8, January 2003) the use of medicinal plants is as old as human civilization. India has a glorious tradition of health care system based on plants, which dates back to Vedic era. In Rig Veda which is the oldest known repository of human knowledge and wisdom (4500-2500 B.C.) mentions about hundred medicinal plants used by the Aryans while in Atharva Veda (2500-2000 B.C.) elaborate description of medicinal plants are given. Later in Samhita period the science of medicine systematically organized with clear concept and theories based on the treatises the Charak Samhita - 2000 B. C, Sushruta Samhita - 1000-800 B.C. Besides this there are other works on Ayurveda and medicinal plants by Nagarjun, Chakradatta, Sharangadhar and Bangasen - 1000-500 B.C. Vaghabhatta Junior - 800 A. D. complied most of the books on Ayurveda and wrote Ashtanga Hariday Samhita.

They further indicated that India is bestowed with unique diversity in culture and natural vegetation exhibiting rich plant diversity. It has all known types of agro-climatic, ecologic and edaphic conditions. It also have unique biogeographical positions having all known types of ecosystems ranging from coldest place, the dry cold desert of Ladakh (Nubra Valley with - 57°C), to temperate, alpine and sub-tropical regions of north-west and trans-Himalayas; rain forests with high rainfall; wet evergreen humid tropics of western ghats and arid and semi-arid regions of peninsular India; dry desert conditions of Rajasthan and Gujarat to the tidal mangroves of Sunderban. It harbors 17500 flowering plants out of which 2000 plants are used in various classical system of medicine like Ayurveda, Siddha and Unani. The tribal and other communities use about 8000 species of wild plants as traditional medicine. The drugs used in ISM are 90% based on plant material and are considered to be safe, cost effective and with minimal or no side effects when genuine ingredients are used.

Medicinal plants are living and irreparable resource, which is exhaustible if over used and sustainable if used with care and wisdom. The importance of medicinal plants has been overlooked in the past. However, at present medicinal plants are looked upon not only as a source of affordable health care but also as a source of income. According to WHO report, over 80% of the world population relies on traditional medicine largely plant based for their primary healthcare needs.

The forest areas have been the traditional source of medicinal plants and herbs. The position cannot be sustained much further because on the one hand the areas under forests have been steadily shrinking and on the other the requirement of medicinal plants and herbs has increased steeply. This has resulted in unscientific and over exploitation of medicinal plants in the forests. One indication of the scarcity of some medicinal plants is their steep prices. The Ministry of Environment and Forests have already banned 29 endangered species of medicinal plans from their natural habitat. While medicinal plants are being utilized in the preparation of a number of modern drugs, there is a new trend worldwide of using natural products. Besides medicinal values, Pharmaceuticals, herbal food supplements, toiletries and cosmetics are growing in consumption in the international market.

Gene Mapping, DNA Marker-Aided Breeding and Genetic Transformation in Africa

2011-04-22T02:52:00.000-07:00

It appears very likely that DNA marker-assisted breeding for a range of traits—particularly to control diseases and pests, and overcome abiotic stresses—is the second most important application of agrobiotechnology in the mid-term in Africa. Once biosafety laws and appropriate regulatory frameworks and systems are enacted in order to ensure food safety and minimise human health risks and environmental hazards, transgenic crops can be added to the tool-kit of plant breeders working in that region.

The International Center for Agricultural Research in Dry Areas (ICARDA) began operations in Aleppo, Syria, in 1977. The ICARDA mandate covered dry areas in West Asia and North Africa (WANA). The WANA region includes the primary centres of diversity of the ICARDA-mandated crop species: barley, lentils and broad beans (global mandate), and wheat, chickpea and a number of forage species [regional mandate, in collaboration with the International Maize and Wheat Improvement Center (CIMMYT) for wheat and the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) for chickpea].

In the ICARDA Medium-Term Plan for 1990–94, it was stated that, although food self-sufficiency would prove impossible during the 20th century in the WANA region, self-reliance for food should be enhanced through a combination of new technology, better farm practices, more favourable government policies and a more rational land-use pattern. While acknowledging that major increase in food production would come from lowlands with over 350 mm of rainfall annually, ICARDA focused its work on the highlands and driest areas.

A strategy has been developed for integrating biotechnologies into the ICARDA crop-enhancement activities, with a view of providing the National Agricultural Research Systems with well-targeted biotic and abiotic stress-tolerant cultivars and genetic stocks, through the evaluation, adaptation and application of novel genome analysis techniques (DNA marker technology). This approach is applied to crops as well as to the corresponding pathogens, viruses and pests, and should ultimately lead to a more efficient and effective use of existing genetic variability in the ICARDA-mandated crops. Genome analysis also allows for a better estimation of the diversity in these crops, and helps to improve management of the germplasm collections. In cases where insufficient genetic variability exists in the cultivated gene pool, wide crossing with the help of tissue-culture techniques is being explored to bridge species barriers. Double haploid techniques are used to achieve, in a short time, the homozygous state of segregants for fast trait evaluation and selection. Double haploid lines are also considered a useful material for DNA-marker linkage analysis. This strategy was incorporated within ICARDA's Medium-Term Plan for 1994–98 (Sasson, 2000).

While genetic transformation of broad bean (Vicia faba) is difficult to achieve, producing herbicide-resistant broad bean would allow the farmers to better control the invasion of their fields by the Orobanchae weeds (Baum et al., 2002).

With respect to chickpea (Cicer arietinum), genetic transformation aims at producing lines resistant to the blight caused by Ascochyta. Chickpea is cultivated on 11 247 723 ha (FAO Statistics, 1998) worldwide and its production reaches 8 829 095 tons, the average yield being 785 kg/ha. The yield range is 500 kg/ha (Algeria) to 1 800 kg/ha (Egypt). The Ascochyta blight is the most devastating disease of chickpea; the fungal pathogen is highly variable, at least three to six races have been identified; there are limited genetic resources for resistance in the chickpea gene-pool. Fertile transgenic Kabuli-/desi-type chickpea lines have been obtained by the ICARDA scientists, using Agrobacterium-mediated transformation of decapitated zygotic embryos and npt-II/pat as selectable markers. Other genetic constructs will be introduced, followed by the assessment of the resistance to the blight by the GM lines (Baum et al., 2002).

Fertile transgenic lentil (Lens culinaris) lines have also been obtained at ICARDA, using a transformation system developed at the Cooperative Research Centre (CRC) for Mediterranean Agriculture (CLIMA, based in Western Australia) and transferred to the WANA region (Baum et al., 2002).

Wide crossing in wheat and barley has been carried out in collaboration with the University of Cordoba, Spain. The transfer of desirable genes from wild species of Aegilops was carried out at ICARDA as well as in collaboration with the University of Tuscia, Viterbo. Interspecific and intergeneric hybridisation in winter cereals aims to transfer genes of abiotic stress tolerance such as drought, cold, heat and salinity from wild types to cultivated forms by expanding the genetic base against diseases, improving the quality and total biomass of Triticum and Hordeum in moisture-stressed areas and providing specific genetic stocks to national programmes for use in their breeding programmes (Sasson, 2000).

In the case of barley and wheat, following anther culture, inter-specific crosses and embryo rescue, the first double haploid lines were tested under field conditions by the early 1990s. The bulbosum technique was used for this purpose. Hordeum bulbosum is a wild barley species found throughout WANA; it can be crossed with wheat and barley (for barley only in the diploid form); however, after crossing, the bulbosum chromosomes are eliminated and the young embryo is cultured to produce haploids. After selection against biotic and abiotic stresses, double haploids are produced. These techniques could skip a number of intermediary breeding generations (Sasson, 2000).

An ovule-embryo rescue technique has been developed in order to cross the cultivated lentil species, Lens culinaris, with Lens nigricans, a wild species adapted to dry environments (Sasson, 2000).

With cooperation of the institutions involved in the North American Barley Genome Mapping Network Project, ICARDA is developing RFLP markers for barley breeding in low-rainfall environments. This would allow a more efficient and accurate selection of drought-tolerant barley germplasm. Drought tolerance is not a single trait, but the collective result of many traits of a plant which interact with each other positively or negatively. RFLP markers could be used for the identification and selection of single-gene traits associated with drought tolerance (such as osmotic adjustment, photoperiodic response in wheat, water-use efficiency). These were the main findings of a technical study carried out at the request of the Dutch Government's Directorate General for International Cooperation. Another project supported by the German Agency for Technical Cooperation (GTZ) aims to develop molecular markers (RFLP and RAPD/PCR) for barley breeding, in order to effectively select disease-resistant barley germplasm (Sasson, 2000).

The Centre d'étude regional pour l'amélioration de l'adaptation à la sécheresse (CERAAS, Regional Centre for Studies on the Improvement of Plant Adaptation to Drought) was set up in 1982 as a partnership between the Institut sénégalais de recherches agricoles (ISRA, Senegalese Institute for Agricultural Research, Dakar, Senegal), the French CIRAD and Universities of Paris VII and XII, with a view of improving and/or stabilising groundnut production in Senegal. In 1987, the Conference of African Agricultural Research Executives for West and Central Africa (CORAF/WECARD) made CERAAS a regional centre under its umbrella. Nowadays, CERAAS receives funds from the European Commission, other development investors and staff secondment from CIRAD.

CERAAS' general objective is to develop crop cultivars adapted to drought and provide methods of analysis and decision-making tools which will improve agricultural production in arid and semi-arid zones. CERAAS researchers are investigating the mechanisms which allow cowpea (Vigna unguiculata) to adapt to drought and they are trying to map the genes associated with this trait. They are also in the process of mapping cowpea population segregating for drought tolerance with the aim of identifying genetic markers associated with this trait. Micro-satellite markers are being used for this research (Ortiz, 2002).

Among CERAAS' development products, it is worth citing the following:

*
Creation, in collaboration with the Senegalese Institute for Agricultural Research (ISRA), of a new groundnut variety with a very short life cycle, GC 8-35; this variety will eventually replace the oil-producing variety 55-437, and cultivated in Senegal on about 130 000 ha; the increase in yield estimated for one growing season will reimburse the investments made in research work conducted over 15 years for creating the new variety.
*
Selection, in collaboration with ISRA, of about 30 groundnut varieties potentially more interesting than varieties GC 8-35 and 55-437 in terms of their production and their drought-resistance capacity; from this improved germplasm, several countries (Burkina Faso, Botswana and Brazil) have selected lines whose agronomic and physiological response to drought are superior to those of local varieties.
*
Creation and registration of eight sorghum varieties of agricultural importance in Mali, which often cover up to 95&percnt; of the area cultivated with sorghum; one of them, Migsor 86-30-03, is particularly resistant to drought and beating down by the wind; it is also used as a genitor in Africa and the USA.
*
Development of a plant model (AraBHy), coupled with a geographic information system (GIS), that allows the estimation of groundnut production 1 month before harvest; initially developed for groundnut, this model can be adapted to pearl millet, cowpea and soybean, and to other environments, as has been done in Argentina. At the country level, this tool can considerably reduce the costs of identifying agricultural calamity zones and, therefore, contribute to a more effective management of food security.

The IITA (Ibadan, Nigeria), a CGIAR Future Harvest Center, through its Strategic Plan (2001–10), aims at targeting donors' investments to stimulate innovations (e.g., agrobiotechnology) needed to alleviate rural poverty, protect the environment and other natural resources, empower rural peoples and promote economic growth. More specifically, IITA conducts biotechnological research to address the food and income needs of sub-Saharan African countries. Priority is given to genetic transformation of cowpea and plantains/bananas; cassava and maize are a second priority. Molecular mapping of important genes associated with conventional breeding aims at enhancing tolerance or resistance to stresses, e.g. cassava mosaic disease, plant parasitic nematodes or the witchweed Striga. Priority is also given to DNA marker-assisted selection of plantain/banana, cassava and cowpea, whereas cocoa, maize and yams, in which DNA maps are also available, are second tier crops. IITA may also benefit from research advances in the genomics of soybeans, a major legume, also a model crop system. Gene discovery and cloning of functional DNA elements such as promoters will provide non-proprietary tools needed for genetic transformation.

IITA transfers, where appropriate and in collaboration with overseas partners and within the continent, biotechnological products from the laboratory to the market. One well-known example is micropropagation and clonal multiplication of vegetatively propagated crops. Another example is the assistance provided to the emerging private sector to use DNA fingerprinting of cultivars to protect proprietary rights, or to use molecular mapping for identifying new genes relevant to end-user needs.

IITA serves as a platform for technology transfer between overseas advanced research institutes and sub-Saharan African countries. By the end of 2002, 10 internationally-recruited staff were working on biotechnology at IITA laboratories in Cotonou (Benin), Ibadan (Nigeria), Namulonge (Uganda) and Yaounde (Cameroon), as well as at the high throughput genomics laboratory of the International Livestock Research Institute (ILRI) in Nairobi.

Finally, IITA enhances the capacity of national selected partners in order to apply and monitor biotechnology, e.g. IITA, together with research-for-development partners and development investors, is working towards the approval of biosafety guidelines concerning GMOs, as has been achieved in Nigeria (Ortiz, 2001).

Partnerships with African researchers are reinforced through group and individual training. For instance, with funding from the USDA and USAID, IITA initiated a project for developing and updating skills of biotechnologists from Nigeria and Ghana to address farmers' needs. This project deals with biotechnological capacity building and research, adapts available approaches for developing or strengthening bioinformatics databases; and conducts research on potential risks associated with the introduction of transgenic crops into Africa (Ortiz, 2002b).

In 2002, a visiting scientist assessed the status of, and needs for agrobiotechnology in West and Central Africa (thanks to a USAID grant given to IITA). This assessment will lead to the design of a regional agrobiotechnology programme for West and Central Africa. In the last quarter of 2002, IITA initiated, as implementing agency, the Nigerian Biotechnology Programme with an agenda driven by the Nigerian stakeholders and funding from the USAID and the Nigerian Government. This programme includes capacity building on genetic transformation—including testing biosafety guidelines, crop genomics and livestock biotechnology, as well as creating unbiased public awareness of biotechnology in Nigeria (Ortiz, 2002b).

Diagnostic Tools for Plant Disease in Africa

2011-04-22T02:47:00.000-07:00

Diagnostic tools, particularly for viruses in crops such as yams, cassava and plantain/banana, and for pathogen strain-fingerprinting were developed by the IITA (Ibadan, Nigeria) researchers through investments of unrestricted funds or small grants from a pool of development investors, e.g. the Bundesministerium fur Wirtschaftliche und Entwicklung Zusammenarbeit (BMZ, Germany), Danish International Development Agency (Danida), Gatsby Charitable Foundation, Rockefeller Foundation and USAID, among others. These tools include both protein-based diagnostics (including polyclonal and monoclonal antibody technology) as well as nucleic acid based diagnostics, either alone using polymerase chain reaction (PCR) tests or combined with protein-based tests in combined immunocapture PCRs (IC-RT-PCR). They are routinely used for indexing plant material (germplasm exchanges and production of cleansed planting material), monitoring distribution of biocontrol agents, developing control strategies for disease epidemics, and detecting pathogens and other pests studied by the institute. The links with laboratories in West Africa on the application of diagnostic methods are particularly effective (Ortiz, 2002b).

In Tunisia, researchers of the Faculty of Science Laboratory of Genetics and Molecular Biology, the National Institute for Applied Science (National Institute for Applied Sciences and Technology; INSAT) Laboratory of Biological Engineering and the National Institute for Agricultural Research (National Agricultural Research Institute of Tunisia; INRAT) Laboratory of Virology are working on grapevine viral diseases. One of these diseases, the rugose wood complex, is widely distributed and causes significant reduction in yield and quality of the crop. Phloem-limited filamentous virus particles are closely associated with these diseases. Two trichoviruses, grapevine virus A (GVA), and grapevine B (GVB) are thought to be involved in the kober stem grooving and corky bark diseases of the rugose wood. Instead of relying on the ELISA method, the Tunisian researchers could successfully detect GVA and GVB from infected grapevine tissue by standard reverse transcription-PCR (RT-PCR) or RT-PCR coupled with immunocapture. The efficiency of the latter method resides on a few rapid steps used to obtain the purified viral RNA preventing its degradation before the reverse transcription reaction. Immunocapture can, therefore, be commonly used in the molecular detection of the two trichoviruses in Tunisian vineyards.

Grapevine infectious degeneration disease affects both productivity and longevity of the plant. One causal agent is a nepovirus with bipartiteRNA-genome: the GFLV (grapevine fanleaf virus). All virus serotypes (fanleaf, yellow mosaic virus or vein banding) are transmitted by the nematode vector Xiphinema index. So far, the only control strategy against this disease has been to select and produce virus-free stocks and avoid the use of contaminated soil to eliminate virus reservoirs and to decrease nematode population. The Tunisian researchers were able to identify the virus in its nematode vector underground, in infected grapevines in the northern Tunisian vineyards, using molecular biology techniques, RT-PCR and IC-RT-PCR. The IC-RT-PCR is more efficient in detecting the GFLV in its nematode vector than other serological and molecular biology techniques.

Grapevine leaf roll is one of the widespread and economically important viral diseases of grapevine in the world. Seven serologically distinct types of grapevine leaf roll associated closteroviruses have been described, but the GLRaV3 is the most important and abundant closterovirus in Tunisian grapevine cultures. It is transmitted by mealy bug species, Pseudococcus and Planococcus. The Tunisian researchers described the implication of Pseudococcus citri in the transmission of the virus in Tunisian vineyards. GLRaV3 in mealy bugs was detected by the IC-RT-PCR technique most efficiently, and the latter is to be used on a large scale to detect the virus in grapevine cultures (Marzouki and Marrakchi, 1998).

In Vitro Micropropagation and Clonal Multiplication of Crops in Africa

2011-04-22T02:44:00.000-07:00

Among the agrobiotechnology tools, in vitro micropropagation of plant tissues or organs, followed by clonal multiplication of the in vitro plants, ranks first in the propagation of a wide range of herbaceous and tree crop species.

In the case of the banana tree, the example of Kenya is very illustrative of the benefits provided by agrobiotechnology. Unlike large parts of Latin America and other banana exporting countries, small farmers, mostly women, are the main producers in Kenya. They grow bananas for home consumption and the national market. It is the most popular fruit in Kenya, and cooking varieties are also an important staple food. Yet, the average banana yield in Kenya—14 tons per ha—is less than one-third of the crop potential under favourable conditions of the humid tropics. The main problem is the infestation of banana stock with weevils, nematodes and fungi, which cause severe diseases, such as Panama disease and black sigatoka. The resulting yield losses make banana a relatively expensive item for consumers. Producers also suffer reduced cash earnings, and the crop potential to contribute to the food security of rural households is undercut. A biotechnology project for the benefit of small-scale banana producers was facilitated by the International Service for Acquisition of Agri-Biotech Applications (ISAAA) and hosted by the Kenya Agricultural Research Institute (KARI) with funding from the Rockefeller Foundation and the International Development Research Centre, Ottawa (Wambugu, 2001). This model project included tissue culture technology for banana propagation and was awarded the 2000 Medal Prize Award by the Global Development Network—an initiative of the World Bank and the Japanese Government. The project benefited from a private–public partnership that demonstrated the feasibility of North–South technology transfer and the ability of resource-poor farmers to have access to research and technology innovations ensuing from appropriate linkages among partners. The project also benefited from a micro-credit programme that allowed small-scale farmers to buy superior pest- and pathogen-free planting materials.

The potential impact of bananas derived from tissue culture was analysed on three types of farms: small, medium and large (although even large-scale farmers have a mean banana area of only about 2 ha). Large farms increased average yields by 93&percnt; and medium-scale farmers gained 132&percnt;. For small holders, however, the increase was 150&percnt;. One farmer (Esther Gachugu) made up to $300 in one-day sale—more than she could earn in a year from a traditional banana orchard (Wambugu and Kiome, 2001). Other farmers built new houses, installed water tanks or sent their children to school. This success story shows the benefits African farmers can draw from horticultural crop biotechnology.

In addition to the direct impacts of the project, biotechnology distribution channels were established in order to facilitate the development of future innovations. For instance, as the international availability of transgenic banana varieties with resistance to major biotic stresses is expected by 2009, the project opens up avenues for the quick introduction of these and other promising biotechnologies for resource-poor farmers (Qaim, 1999).

In Uganda, substantial investments in research on banana and plantain have been made in recent years. This has culminated in the development of a biotechnology project in which the Government of Uganda provides the largest funding. The US Agency for International Development (USAID), the Rockefeller Foundation and Directorate General for International Cooperation (DGIC, Belgium) also allocate resources. The hub of the project is the National Agricultural Research Organization (NARO) together with Makerere University. Important partners include the Katholieke Universiteit Leuven (KULeuven), International Institute of Tropical Agriculture (IITA, Ibadan), French Agricultural Research Institute for Overseas Development (CIRAD), University of Pretoria and International Plant Genetic Resources Institute (IPGRI), which through its International Network for the Improvement of Bananas and Plantains (INIBAP) coordinates the project (Ortiz et al., 2002). The project aims at creating a biotechnological centre in Uganda and using genetic transformation for enhancing the resistance of the local East African highland bananas to the wide range of pests and diseases currently affecting the crop. The IITA provides technical backstopping for gene mapping of banana weevil resistance through an associated project, funded by a grant given to IPGRI/INIBAP by the Rockefeller Foundation.

In Morocco, with a production capacity of more than 1 million banana in vitro plants per annum, the company Domaines Agricoles, based near the city of Meknès, can meet the national needs for banana plants. These in vitro plants are raised in the nurseries located near Rabat and in the Massa area (Agadir region), which are the main banana-producing regions (Sasson, 2000).

Shoot-tip micrografting, together with thermotherapy, is the technology selected to clean citrus varieties from their viral, mycoplasmic and bacterial pathogens. Cleansed plant material can be obtained in 3 to 6 months instead of 10 to 15 years using conventional technologies such as nucellar selection or mass selection through indexing. Thus, since 1994, Morocco's Domaines Agricoles Unit of Plant Control has been able to clean about 20 commercially important citrus varieties, and produce certified and well-performing plant material (Sasson, 2000).

In air-conditioned greenhouses, one can experimentally control almost all viral diseases which affect citrus plants, i.e. about 30. These facilities enable the company to play an important role at both national and regional level for controlling the tristeza viral disease, a major threat to citrus cultivation. They also serve as quarantine facilities for introduced citrus species or varieties, in full cooperation with the Ministry of Agriculture Services of Plant Protection. The company took the initiative of undertaking a programme for the biological control of a citrus borer, Phylacnistis citrella, which originated from South-East Asia and invaded all citrus-growing Mediterranean countries in less than 3 years, causing heavy losses. Two natural enemies of the insect pest were introduced from Florida and Australia, Ageniaspis citricola and Semielacker petiolatus; hundreds of thousands of Ageniaspis citricola were produced and disseminated throughout the citrus-growing regions of Morocco. Algeria, Egypt and Spain also benefited from the Moroccan experience concerning the breeding of the useful insects. Another example of biological control is that against Aonidiella auranti, one of the oldest and major pests of citrus plants; chemical control is expensive, rather inefficient and can harm the exports of fruits because of the pesticide residues remaining on the fruit surface. A pilot unit was set up at the Domaines Agricoles to produce Aphitis milinus insects and to disseminate them in order to control the Californian lice (Aonidiella auranti) (Sasson, 2000).

The date palm is part of the landscape and a key element of land-use planning in large areas of African countries. It is also found beyond the eastern boundaries of the North African region, in the Near and Middle East, and has been introduced in several sub-Saharan countries, such as Namibia. It is the typical multipurpose tree crop of the oases from Morocco to Egypt, not only to supply dates (for local consumption and export), leaves and trunks as building materials, but more so to provide shade for barley and alfalfa cultivation under irrigation, and animal husbandry (sheep, goats and camels). Except for the case of plantations managed for exporting dates, e.g. in Algeria, Tunisia and Egypt, most date palm groves belong to families of resource-poor farmers and are the pivot of horticulture-type farming. Maintaining this tree crop is, therefore, a crucial socioeconomic issue for the development of marginal areas where date palm is growing; it is a way of controlling rural exodus and of mitigating rural poverty.

Although the date palm varieties grown throughout North Africa are quite sturdy, a fungal disease caused by soil-inhabiting Fusarium oxysporum subsp. albedinis, locally named as bayoud (meaning whitening, because white streaks appear on the leaves of the diseased tree), is causing havoc among the palm groves, particularly in Morocco. In this country, tens of millions of trees have been killed since the beginning of the 20th century. There is no effective chemical remedy for eradicating the fungus whose filaments penetrate through the roots and multiply in the vascular bundles, finally choking the tree (tracheomycosis). In addition to Morocco, which is severely affected, Algeria is also affected by this disease, although slightly less. The fungus may spread to Tunisia and even farther (in the Middle East, especially in the Gulf area, where the main pest of the date palm is the red weevil and the bayoud disease is unknown).

The threat is, therefore, very serious, and the whole ecosystem and way of life is being threatened, at least in the Moroccan oases. Fortunately, the Moroccan scientists of the National Institute for Agricultural Research (INRA) have been identifying, selecting and gathering many bayoud-tolerant date palm varieties, the clonal multiplication of which could be a viable solution. These scientists, with bilateral and multilateral assistance, have succeeded, many years ago, in micropropagating the date palm, starting from the caulinary meristems of offshoots (the tree produces a few offshoots over its life-span), and leading to uniform in vitro plants (through organogenesis). A private corporation, working in collaboration with INRA, is producing around 250 000 date palm in vitro plants per annum. This figure is far from meeting national needs, which are estimated as several millions per year if the medium-term objective is to replace the dead trees by tolerant varieties, and rehabilitate the oases and their specific agriculture (a National Plan for the Rehabilitation of Palm Groves was initiated in 1978).

The only agrobiotechnological tool available for such a purpose is that of somatic embryogenesis, which has been successfully used for several crop species, including the oil-palm, coconut and other tree species. Starting from leaf or inflorescence explants, using liquid instead of semi-solid medium, the production figures are of an additional order of magnitude compared to the organogenesis process. A difficulty relates to the inadvertent production of somaclonal variants, which may have abnormal inflorescences. Recent research overcomes this problem, and in fact several research teams in Europe have been able to produce large populations of normal date palm plants in vitro. Time has, therefore, come for the Moroccan relevant institutions to make the appropriate decisions in order to meet the huge needs of oasis farmers and to contribute to solving a very important socioeconomic problem.

Morocco has cooperated with Mali to introduce tissue culture-derived bayoud-free date palms into the north-eastern region of this country (Menaka), within the framework of an FAO project. Similar cooperation has been established with Libia (Sasson, 2000).

In Egypt, at El-Menoufia University, Sadat City, research is being carried out on the production of plants in vitro. A number of scientists belonging to this university have been trained in Germany and in the USA. The Genetic Engineering and Biotechnology Research Institute of this University succeeded in cloning date palm (Phoenix dactylifera cv. Zaghloul) through somatic embryogenesis and organogenesis. Successful regeneration of plantlets from the shoot-tip and leaf primordia derived from adult plants were reported. The Egyptian researchers are of the opinion that this method holds good chances for achieving mass production of true-to-type plants from adult date palm since the callus stage is avoided (Sasson, 2000).

Moving to somatic embryogenesis for multiplying bayoud-tolerant varieties does not preclude the pursuance of basic research on the molecular basis of the host–parasite relationship as well as on the genome of these varieties in order to identify a resistance gene(s) and stimulate plant-defence mechanisms. Any breakthrough achieved in date palm propagation, physiology and genetics will have a great impact on the socioeconomic development of the whole North Africa region and beyond.

In the Republic of South Africa, the Vegetable and Ornamental Plant Institute of the Agricultural Research Council (ARC-Roodeplaat) has developed tissue-culture protocols during the last 25 years for many vegetable crops—including root and tuber crops such as cassava and the research-neglected Livingstone potato (Plectranthus esculentus). Meristem culture and thermotherapy are used routinely for eliminating viruses in potato, sweet potato, cassava, garlic and indigenous ornamentals. The ARC-Roodeplaat provides all the virus-free material of sweet potato in South Africa. Its in vitro gene bank contains cultivars and breeding materials of potato, sweet potato, cassava and the ornamental Lachenalia spp. The Institute also carries out genetic transformation and molecular marker-aided selection (Ortiz, 2002b).

Agricultural Biotechnology in Africa

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The North Africa region stretches from Morocco to Egypt and includes the Maghreb countries (Morocco, Algeria and Tunisia), Libya and Egypt. It extends eastwards to West Asia which includes Turkey, Pakistan and other countries and share several weather, soil and agricultural features in common. Thus, the percentage of arable land is low (around 13&percnt;), while steppes and deserts are quite extensive.

The main crops in the North Africa region are cereals (wheat, barley and some maize generally under irrigation), grain legumes (lentils, chickpeas and broad beans), forage species (alfalfa, berseem—Trifolium alexandrinum—mainly in Egypt), irrigated crops such as vegetables (tomato, green peas, artichoke, etc.), citrus,sugar-beet, olive tree, almond and date palm. Sunflower and groundnut are also cultivated, while cotton is the main non-food crop species.

Despite some good harvests during years with adequate rainfall, the region is a net importer of food, especially cereals. In the West Asia and North Africa (WANA) region, with a population growth rate of 3&percnt; and per capita food production at 1.1&percnt;, the shortfall in cereal production has been projected to be more than 50 million tons by 2005 (Weigand and Baum, 1997). This situation may worsen due to an aridification trend under global climatic change.

However, self-reliance for food would be enhanced through a combination of new technology, better farm practices, more favourable government policies and a more rational land-use pattern. While acknowledging that major increases in food production would come from the high rainfall lowlands (over 350 mm of rain annually), highlands and drier areas should not be neglected.

In sub-Saharan Africa, afflicted by hunger, periodically or endemically, food availability, access and absorption are the key issues relating to the food deficit existing in this region. Increasing the availability of food demands improvements in agricultural productivity such as those achieved during the ‘green revolution’ of the 1960s in Asia. Regarding access to food, this can be restricted by economic or environmental criteria; food is available, but people cannot purchase it, or it is unequally distributed within families. The major problem here is poverty and more so the ‘feminisation of poverty’. Finally, even with high and sustainable agricultural productivity and equitable access to food, people may still go hungry if the food is not safe to consume or if its nutritional quality is low. This is the issue of food absorption (M.S. Swaminathan, personal communication, 2002).

Some argue that the problem is not one of quantity of food but of its unequal distribution. However, even if we resolve the issue of distribution in the short run, the future growth in food demand will require increase in productivity from a decreasing stock of arable land. The challenge, therefore, is not only to feed more people (population growth is still rather high throughout Africa), but to do so with less available land, fewer non-renewable resources and less water.

Such facts, combined with the commitment to fighting poverty, indicate that the main thrust of national (and international) policies aimed at solving issues of rural poverty and food insecurity must include significant increases in local food production. Because the rural poor represent a significant percentage of the total population in Africa, an innovation that increases productivity will have a major impact on food security efforts and a nation's poverty.

Any strategy designed to eliminate food deficit and poor nutrition, and also to accelerate the evolution from household production to more commercial farming entrepreneurs, should comprise two interdependent approaches: developing commercial opportunities for the less vulnerable farmers by developing or enhancing markets for agricultural and horticultural products with high added value; and increasing food security by reducing the reliance on monocultures through encouraging diversified crop–livestock–forestry systems, which may be more environmentally resilient, nutritionally superior and commercially attractive (Ortiz, 2002b).

For the African countries, it may be more appropriate to specifically target biotechnology innovations that will increase productivity in marginal areas, where an increase in food production is needed and crop yields are significantly low. Raising productivity requires action in several areas: adoption of technologies that combat low productivity levels, decrease in post-harvest losses, control of pre-harvest pests and increase in yields on soils.

Some argue that the poor farmers and consumers stand to benefit very little from biotechnology. Indeed, this is part of the criticism directed at biotechnology, in particular, genetically modified (GM) crops. It is true that many modern biotechnological applications are geared towards market-based economies or used for commodities in highly productive environments. It is also true that in the current environment of declining public investments in agricultural research, one may wonder whether there is a positive impact of biotechnology on the livelihood of the rural poor. The following examples or case studies show that modern biotechnologies can benefit small holders and consumers in a positive way: increase in farm output; higher nutritional value of specific crops and livestock; provision of employment opportunities and higher incomes to small farmers and landless rural labourers; improvement of the rural environment through the decreasing use of chemicals; and lower food prices in urban and rural areas.

Genetically Modified Crops in Developing Countries

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By early 2003, genetically modified (genetically enhanced, as qualified by many scientists in developing countries) crops were already established in the third world: two-thirds of the 5.5 million farmers growing these crops are in developing countries, which demonstrates that small and poor farmers are also involved. In addition to maize, soybeans, rapeseed (canola) and a few horticultural crop varieties, genetically modified cotton is the fastest spreading non-food GM crop. It is currently cultivated in India, China, Indonesia, Thailand, Argentina and South Africa, and the prospects are very promising.

Illustrative regional examples of GM crops are given in later chapters. From the strategic viewpoint, the adoption of GM crops by an increasing number of developing countries, and particularly by the larger ones (e.g. China, India, Argentina), reflects the need to acquire the relevant technologies before they are completely in the hands of the industrialised countries. It also reflects the will to participate in the so-called biotechnology revolution and even to become formidable competitors in some areas, instead of just purchasing and adapting biotechnologies. China's huge commitment to plant biotechnology, through increasing five-fold the funds devoted to this area of endeavour (US $500 million annually by 2005) is illustrative of this trend.

Another key element in the strategy of developing countries is to improve their competitiveness in international commodity and agricultural product markets. For those who are big exporters and whose agriculture is not subsidised, GM crops and agricultural biotechnology can contribute to decreasing production costs (e.g. through the reduction of use of biocides) and to increasing farmers' incomes. This aspect has been clearly demonstrated for GM soybeans in Argentina and GM cotton in South Africa and China.

This strategy also requires the design and update of biosafety regulations, the establishment or revision of intellectual property legislation and active participation in the negotiations on trade-related issues at the World Trade Organization.

Adopting GM crops is not synonymous with exclusion of other forms of agriculture, particularly the so-called biological or organic agriculture. A number of developing countries, e.g. Argentina and Chile, have an important and prosperous organic agriculture sector, which they wish to preserve and even extend because of its commercial benefits (e.g. Chile exports high volumes of ‘organic’ products to Japan and the European Union). Nevertheless, the advantages offered by GM crops enable developing countries to meet more rapidly the need to establish higher yielding, stress- and pest-resistant crop varieties, particularly when one has to deal with pathogens and pests against which there is no known natural resistance or tolerance.

Naturally, the developing countries are carefully following the controversy on GM crops in the European Union member countries as well as the disaccord between those countries and the USA in this respect. They are vigilant at the World Trade Organization, the Codex Alimentarius Commission on GM organisms and their impact on health and nutrition, in order to safeguard their interests. They generally consider that agricultural biotechnology and GM crops can help them to face the challenges of sustainable agricultural development. In this respect, their position is not far from that of the representatives of farmers in industrialised countries who welcome these technologies and maintain the highest standards of biosafety and biovigilance. They also consider, to a large extent, that the precautionary principle (now called the precautionary approach, since the 2002 Earth Summit in Johannesburg) should not become a dogma that hampers research, trials and large-scale cultivation. They agree on the need for biovigilance as in the case of medicines.

With regard to labelling and traceability of GM or biotechnology-derived products, developing countries tend to refer to substantial equivalence of these products compared to conventional ones, and to adopt labelling when there are substantial differences in composition. Thus, sugar, starch or vegetable oils derived from GM crops should not be labelled as GM. They are pragmatic in discussing the minimum percentage of GMOs in foodstuffs and agricultural products, the threshold of 0.9&percnt; (proposed by the European Union's Council of Ministers) being considered as unrealistic.

Finally, developing countries support the strengthening of regulatory institutions and biosafety measures, but they wish to avoid over-regulation, which will hinder their competitiveness. There is also a growing trend of improving public perception and social acceptance of agricultural biotechnology in developing countries, involving the participation of all sectors of society.
Confronted by the urgent need to feed their people and make their agriculture more competitive on international commodity markets, the developing countries, be they food exporters or not, have resisted the adoption of a moratorium on the cultivation of GM crops like that in Europe. In contrast, they wish to draw benefits from modern agricultural biotechnology and seize the opportunities offered to them.

In addition to the competitive edge provided to the commodity-exporting developing countries, agricultural biotechnology must reach resource-poor farmers—a large majority in developing countries. For such a purpose, it is necessary to carry out the social analysis of these technologies, when they are transferred to the farming communities. It is also necessary to pay great attention to the so-called orphan crops such as sorghum, millet, cassava, yams, sweet potato, etc., which do not attract the big seed corporations, but which play a vital role in local and national economies.

While favouring a sustainable diversified agriculture, including agricultural biotechnology, and making special efforts to help the resource-poor farmers, developing countries can protect their biological diversity (e.g. through the conservation of potentially useful varieties), clone crops on a large scale and participate in the selection of new varieties with the appropriate traits. Many projects being carried out in developing countries reflect these goals, while at the same time key issues, such as biosafety regulation, risk assessment and management, intellectual property rights and training of human resources are dealt with.

There are undoubtedly, in this vast area of research and development, opportunities for collaboration among the developing countries but also between them and industrialised countries' public research centres, enterprises and professional associations. In this regard, we are dealing not only with solidarity, but also with mutually beneficial cooperation in important international markets.

Agricultural Biotechnology for Developing Countries

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Since the early 1970s, when the exploitation of biotechnology started to soar in the industrialised countries, developing countries—representing about 80&percnt; of the world's population—have progressively adopted and adapted biotechnology as a contribution to solving their social and economic development problems. At the beginning of the 21st century, most developing countries use biotechnology in one form or another, at scales and complexities that depend on their economic, scientific and technological status. In particular, they often rely on agricultural biotechnology, such as in vitro micropropagation of plant tissues or organs, followed by clonal multiplication of herbaceous or tree crops to produce virus and pathogen-free plants. They also use a wide range of food fermentations.

Many developing countries, for example India, China, Thailand, Brazil, Mexico, Egypt and South Africa, utilise the so-called modern biotechnology, based on genetic engineering and genomics. Agricultural biotechnology is the most widespread biotechnology in developing countries, but only a few of them are able to carry out all of the research and development activities leading to the commercialisation of genetically modified seeds. These include basic research in molecular and cell biology and genetics; greenhouse and field trials according to internationally agreed biosafety standards; risk assessment and management; respect for intellectual property rights relating to the transferred genes and to the creation of new crop varieties; production of genetically modified (GM) seeds by private corporations or working in cooperation with the public agricultural research sector; extension activities aiming at delivering the new seeds to the farmers and biovigilance in the fields of GM crops so as to detect any abnormalities or any hazards caused to the environment and to conventional crops. It is therefore important to follow the strategy of the countries capable of going through all these steps in order to understand how agricultural biotechnology supply meets economic and social demand (Sasson, 2000). A number of these countries are considered in more detail in later chapters in this section. Unfortunately, due to time constraints, it was not possible to include all of the key countries, so that the geographical coverage of this section is not complete. In particular China, where agricultural biotechnology has a rapidly growing role, is not covered in a separate chapter. The editors believe that the current coverage presents the reader with a detailed discussion of the major issues and opportunities of agricultural biotechnology in developing countries but they plan to extend the coverage in future editions of the Handbook.

It should be emphasised that the developing countries whose economies still largely depend on their food supply, exports and employment on agriculture that is not (or very little) subsidised by the government, must face the following challenges:

*
increase in production and productivity, and in competitiveness at national, regional and international levels (within the framework of the rules being established or revised by the World Trade Organization);
* protection of the environment and biological diversity, while reducing agricultural inputs (water, fertilizers and biocides), improving soil fertility and conservation (e.g. biological nitrogen fixation), increasing nitrogen and phosphorus absorption by crops, without significantly decreasing yields;
* diversification of agro-food production so as to meet the evolving needs of consumers and the food industry.

These challenges are similar to those faced by industrialised countries whose intensive agriculture employs, nevertheless, a very small proportion of the active population and is generally heavily subsidised (which leads to unfair competition with food-exporting developing countries).

Although food self-sufficiency is not an intangible rule anymore, and countries can devote land to high value-added export products and buy cereals or legumes on international markets at rather low prices, it is important to keep in mind the strategic role of efficient agriculture.

Population Growth and the Food-Production Challenge

Norman Borlaug (2002, 2004) has analysed the ways in which the birth of agriculture some 10 000–12 000 years ago, led to a stable food supply and enabled humankind to increase its population from some 15 million at that time to about 250 million by the start of the Christian era. Borlaug (2004) noted that that population doubled by 1650, then doubled again (to one billion) by 1850, redoubled by 1930 and doubled again by 1975, when the global population reached four billion. The next doubling is projected by 2020 and this will represent a 530-fold increase since the origin of crop improvement by selection of seeds from the best plants for sowing to deliver the next generation. Although the rate of increase of the world's population is now decreasing, the current rate in much of the developing world is still so high that the world's population is likely to increase to at least 10 billion people over the next 50 years, with 90–95&percnt; of them living in low-income developing countries and under conditions of poverty. Although it is hoped that the world's population will stabilise at 11–12 billion by the end of the 21st century, we have to confront a situation today where more than two billion people have no food security and 840 million of them are chronically malnourished. Six million children under the age of five die each year as a result of hunger and malnutrition. Of these millions, relatively few are the victims of famines. Most die unnoticed, killed by the effects of chronic hunger and malnutrition that leaves them weak, underweight and vulnerable. Health and mortality indicators are closely correlated with the prevalence of hunger. Common childhood diseases are far more likely to be fatal in children who are even mildly undernourished, and the risk increases sharply with the severity of malnutrition. Eliminating hunger and malnutrition could save millions of lives each year (FAO, 2002).

There are two major challenges that mankind must confront. The first is to produce enough food to satisfy the needs of the huge population. The second, even more complex problem is to ensure that the food is equitably distributed. The chief impediment to equitable food distribution is poverty (lack of purchasing power). Some 42&percnt; (2.6 billion people) of the world's population live on the land and rely on their own efforts to feed themselves. Only increases in agricultural productivity in food-deficient areas can enable the millions of rural poor to become food-secure.

The possibility of expansion of arable land area is limited for most regions of the world and the International Food Policy Research Institute (IFPRI) has estimated that more than 85&percnt; of the essential increase in cereal production (which represents two-thirds of human calorific intake) must come from increasing yields on land that is already in production. These productivity increases must come from varieties with higher genetic yield potential and greater tolerance of drought, insects and diseases. Crop management must emphasise soil and water conservation, reduced tillage, fertilization, weed and pest control and post-harvest handling.

Irrigated crops, which account for 70&percnt; of global water withdrawals, cover some 17&percnt; of cultivated land and yet provide nearly 40&percnt; of the world's food production. The rapid increase in land irrigation and in urban and industrial water usage has resulted in growing water shortages. It seems likely that two-thirds of the world's population will be suffering from water stress by 2025 (Borlaug, 2004).

The efficiency of water use in agriculture can be improved by several technologies. Wastewater treatment enables use for irrigation, especially for peri-urban agriculture. New improved varieties which require less water can achieve significant savings, especially if they are used in systems with more efficient crop rotation and more timely planting. Technologies are now available for saving water by increasing water productivity (yield per unit of water used). Reduction of soil salinity is now a matter of the highest priority. Borlaug (2004) has emphasised the need to bring about a ‘blue revolution’ by marrying water-use productivity to land-use productivity.

The conclusion that cereal yields must be increased on lands currently farmed, using less water and biocides means that in addition to conventional agricultural techniques, other techniques relating to protection of the environment and preservation of natural resources, drastic reduction of postharvest losses and control of biotic and abiotic stresses should be utilised (Borlaug, 2002). This is where agricultural biotechnology will help; it is not a panacea, nor a substitute for established agronomic techniques, but it represents another tool for increasing productivity and improving food quality.

During the World Food Summit, organised in 2002 in Rome by the Food and Agriculture Organization of the United Nations (FAO), it was again emphasised that developing countries should rely on agricultural biotechnology along with other agricultural technologies, while respecting internationally agreed biosafety standards. One year earlier, in its Report on Human Development, the United Nations Development Programme (UNDP) recommended the widest application of biotechnology (and other advanced technologies) in developing countries.

There is indeed an overall consensus on the utility of in vitro production of plantlets, derived from plant tissue or organ micropropagation, that are free of viruses and other pathogens and can contribute to increasing agricultural production, provided that small and poor farmers can purchase them at a low cost. In vitro production, which also concerns ornamental and forest species, is widespread in developing countries. It has become an important element of agro-food production, as it is applied to potato and several other tuber and root crops, high value-added horticultural varieties, oil palms and date palms, banana and plantain, which are the staple food of several hundred million people worldwide (Sasson, 2000).

IRRI Create Golden Rice to Overcome Vitamin A Deficiency

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To help address the devastating impacts of vitamin A deficiency, particularly on the poor in Asia, the International Rice Research Institute (IRRI) and its national and international partners are now developing Golden Rice – a new type of rice that contains a source of vitamin A.

Vitamin A and human health

Vitamin A is an essential micronutrient that helps the body to fight diseases and maintain healthy eyesight. Vitamin A deficiency lowers immune system function, causing people to get sick more often and have a higher risk of dying from infections. Vitamin A deficiency can also cause night blindness and is a leading cause of preventable blindness in children.

Vitamin A deficiency particularly affects infants, young children, and women who are pregnant or nursing. According to the World Health Organization (WHO), an estimated 250,000 to 500,000 vitamin A-deficient children become blind every year, half of them dying within 12 months of losing their sight. With adequate vitamin A, young children are up to 30 percent less likely to die from infections and the death rate for women during or shortly after pregnancy can be reduced by approximately 40 percent.

Vitamin A deficiency can be reduced by eating more foods that are naturally high in vitamin A or beta-carotene (a form of vitamin A), by eating foods that have had these micronutrients added to them, or by taking supplements.
Vitamin A deficiency in rice-consuming populations

Vitamin A deficiency can be particularly severe in countries where the staple food contains no forms of vitamin A and other nutritious food is scarce, unavailable, or too expensive.

Rice is the staple food crop for more than half of the world’s population, and is especially important in Asia, where more than 60% of the world’s 1 billion poorest live. Rice is an affordable and filling food, yet it contains no source of vitamin A. More than 90 million children in Southeast Asia suffer from vitamin A deficiency, more than in any other region.
Golden Rice

Golden Rice is a type of rice that contains beneficial amounts of beta-carotene, which is used by the human body to make vitamin A. Beta-carotene gives Golden Rice its yellow color. Many fruits and vegetables, such as papaya and carrots, also get their color from beta-carotene. Golden Rice was bred using a combination of genetic modification and other breeding methods. It contains genes from maize and other sources that together produce beta-carotene. Golden Rice is expected to taste the same as other rice, be cooked in the same way, and have the same eating quality of other popular rice varieties.

According to research published in the American Journal of Clinical Nutrition, one cup of Golden Rice could supply half of the vitamin A needed every day. Golden Rice could be used in combination with existing ways of overcoming vitamin A deficiency through diet, fortification, and supplements.

Researchers have already found that the body turns more than 25% of the beta-carotene in Golden Rice into vitamin A, a better conversion rate than for many green, leafy vegetables.
Developing Golden Rice

Work to develop Golden Rice currently includes laboratory, greenhouse, and field studies at IRRI, national agricultural research institutions, and other institutions around the world to

* breed Golden Rice varieties that are well suited for different rice-growing environments and consumer preferences in Asia,
* confirm the nutritional benefits of Golden Rice in combating Vitamin A deficiency, and
* evaluate the safety of Golden Rice.

This research on Golden Rice will ensure that any approved Golden Rice varieties will grow just like other rice crops, with comparable yields and pest resistance, and with the same environmental impacts. It is expected that Golden Rice will be planted, harvested, threshed, and milled like current rice varieties.
All Golden Rice research is conducted according to national biosafety regulations and additional biosafety conditions established by the institutes carrying out the research.
IRRI’s role

IRRI coordinates the Golden Rice Network and works with national agricultural research institutes and other partners with expertise in agriculture and nutrition to research and develop Golden Rice. IRRI’s support for partners includes initial breeding of the Golden Rice trait into selected varieties, which involves laboratory work, greenhouse tests, and some preliminary field evaluation. These advanced breeding lines are being transferred to national partners for further development and assessment.

IRRI also works with national partners to

* provide technical support and training to help with breeding and development and build scientific capacity at the national level,
* help develop locally adapted plans to deliver Golden Rice to farmers and consumers, and
* research and collate biosafety data.

National partners

National agricultural research institutes in Bangladesh, China, India, Indonesia, the Philippines, and Vietnam are leading their in-country development of Golden Rice. They manage varietal development and selection, do field evaluations, and undertake biosafety research for science-based regulatory review of Golden Rice in the country. National partners will also interact with other public- and private-sector institutions and government to advance the release and adoption of Golden Rice by farmers and consumers.
Availability of Golden Rice

Golden Rice will be available to farmers and consumers only after it has been authorized by the agricultural, environmental, health, and food safety agencies of their countries. Public health officials, nongovernment organizations, grain traders, and private industry will be consulted in each country before Golden Rice is introduced.

Golden Rice may be approved in the Philippines and Bangladesh as early as 2013 and 2015, respectively, and introduced to the public in those countries soon after. Other countries developing Golden Rice in local varieties are India, Indonesia, and Vietnam.

Golden Rice will be made available to people with vitamin A deficiency in different ways depending on community needs and preferences.

Golden Rice will cost no more than other rice for farmers and consumers.
Funding for Golden Rice

Because of its enormous potential to benefit public health, the technology behind Golden Rice has been donated by its inventors, Professor Ingo Potrykus and Dr. Peter Beyer, for use by public institutions. Different governments and private charities are supporting the development and testing costs.

A one-time investment to develop a biofortified crop such as Golden Rice can generate new varieties for farmers to grow for years to come, in many different countries. There will be some recurrent expenditure for monitoring and maintaining the high beta-carotene trait in Golden Rice, but these costs will be relatively low compared with the ongoing costs of traditional supplementation and fortification programs.

Soource: http://irri.org

Adoption of GM Crops in The Future

2011-04-04T15:28:00.000-07:00

The experience of the past is often the best guide for the future. The experience of the first seven years, 1996–2002, during which a cumulative total of over 235 million hectares (over 580 million acres) of transgenic crops were planted globally in 19 countries, has confirmed that the early promise of biotechnology has been fulfilled. GM crops can deliver substantial agronomic, environmental, economic and social benefits to farmers and, increasingly, to society at large. GM crops have met the expectations of large and small farmers planting transgenic crops in both industrial and developing countries.

The most compelling case for biotechnology, and more specifically GM crops is their capability to contribute to increasing crop productivity, particularly in the developing countries (James, 2002a, 2002b; www.isaaa.org; Pinstrup-Andersen and Schioler, 2001) where they can make a crucial contribution to food, feed and fibre security; conserving biodiversity, as a land-saving technology capable of higher productivity; more efficient use of external inputs and thus a more sustainable agriculture and environment; increasing stability of production to lessen the suffering during famines due to abiotic and biotic stresses; and improve economic and social benefits and the alleviation of abject poverty in developing countries (James, 2002b; UNDP, 2001). It is critical that a combination of conventional and biotechnology applications be adopted as the technology component of a global food, feed and fibre security strategy that also addresses other critical issues including population control and improved food, feed and fibre distribution. Adoption of such a strategy will allow society to continue to benefit from the vital contribution that plant breeding offers the global population.

With significant progress in the first seven years of the first decade, 1996–2005, when GM crops are being commercialised, what can we expect during the remaining three years, 2003–2005, at the dawn of a new era in crop biotechnology? The latest GM crop indicators for 2003 and beyond augur well for the future of crop biotechnology. In 2002, coincidental with increased political, policy and institutional support for GM crops, due to their acknowledged essential contribution to global food security, the global area of transgenic crops in 2002 benefited from continued growth of 12&percnt;. The number of farmers who benefited from GM crops in 2002 was approximately six million of which five million were resource-poor farmers planting Bt cotton, mainly in eight provinces in China and also in the Makathini Flats in the KwaZulu Natal province in South Africa (Ismael et al., 2002). The well-documented experience of China with Bt cotton (Huang et al., 2002, Pray et al., 2001, 2002) presents a remarkable case study where five million small resource-poor farmers in 2002 already benefited from significant agronomic, environmental, health and economic advantages—this is a unique example of how biotechnology can impact on poverty alleviation as advocated in the 2001 UNDP Human Development Report (UNDP, 2001). The China experience with Bt cotton lends itself for introduction and replication to carefully selected developing countries in Asia, Latin America and Africa where resource-poor farmers can learn, share and benefit from the rich experience of China—the majority of the hectarage of global cotton is in the developing countries of the world. Following a successful launch of Bt cotton by Indonesia in 2001, India, the largest cotton-growing country in the world, grew Bt cotton for the first time in 2002.

The opportunities and constraints associated with public acceptance of transgenic crops continue to be important challenges facing the global community. Because of our thrice-daily dependency on food, agriculture touches the life of every individual in the global community of over six billion. Unlike industrial countries, such as the United States and countries of the European Union, with few exceptions, all developing countries are net importers rather than exporters of food, and where a high percentage of the population employed in agriculture are either small resource-poor farmers practising subsistence farming or the rural landless who are dependent on agriculture for survival; 70&percnt; of the world's 1.3 billion poorest people are rural people, the majority of them are resource-poor farmers and their families. Agricultural employment, as a percentage of total employment, was 80&percnt; in the developing countries in 1950, and is still projected to be 50&percnt; in 2010 when the population of the developing countries will be approximately six billion, equivalent to the global population of today. Improved food, feed and fibre crops derived from appropriate conventional and biotechnology applications for small resource-poor farmers are vital for increasing productivity and income to provide access to food in the rural areas where the majority of the poverty, hunger and malnutrition exists. Crops are not only the principal source of food but are also the livelihood of farmers and agricultural workers. Increased crop productivity provides more employment and acts as the engine of economic growth in the rural communities. Producing more food, feed and fibre on small resource-poor subsistence farms, where most of it is consumed, has the significant advantage that the inevitable infrastructure constraints associated with transport can, to a large extent, be circumvented in that the produce is largely consumed at the same locations where it is produced.

Global society must seek equitable solutions that meet the different needs of people and nations and respect differing opinions regarding GM crops. Implementing an equitable policy is a challenge in a world where globalisation, a web of international protocols and international trade are all impacting on the ability of sovereign nations in the developing world to access and utilise biotechnology and GM crops in their national food, feed and fibre security strategies, to meet domestic and export needs. This does not imply that biotechnology and GM crops are panaceas. Biotechnology, like any other technology, has strengths and weaknesses and needs to be managed responsibly and effectively. Biotechnology represents one essential link in a long and complex chain that must be in place to develop and deliver more productive crops, which are urgently required by small resource-poor farmers in developing countries. This will require the political will, goodwill and unfailing support of both the public and private sectors in the industrial and developing countries to work together in harmony, as pledged during the 2002 World Summit on Sustainable Development in Johannesburg.

The Challenge of Global, Food, Feed and Fibre Security

2011-04-04T15:23:00.000-07:00

The global population reached six billion on 12 October 1999, and is expected to reach nine billion by 2050, when approximately 90&percnt; of the world's population will live, or survive, in the three continents of the South: Asia, Africa and Latin America where today malnutrition results in 24 000 deaths per day. Thus, in the next 50 years, the population will increase by 50&percnt;, or three billion, and food production will need to be doubled on the same area of arable land (1.5 billion hectares), by 2050. The magnitude of the challenge of feeding tomorrow's world is difficult to conceive and the enormity of the task is probably best captured by the statement that: ‘In the next fifty years mankind will consume twice as much food as mankind has consumed since the beginning of agriculture, 10 000 years ago’ (James, 2002a, 2002b).

Crops are the major source of food globally. There is a widely held view in the international scientific and development community that conventional crop improvement alone will not allow us to meet the global food demands of 2050. What is being advocated is a global strategy that integrates both conventional crop improvement and biotechnology, including transgenic crops, which are often referred to as genetically modified (GM) crops; adoption of such a strategy would allow society to harness and optimise the contribution of biotechnology and GM crops to global food security. There is cautious optimism that such a strategy would contribute significantly to the alleviation of poverty and malnutrition which afflict 1.3 billion people and 815 million people, respectively, today, and that the global food demands of 2050 and beyond can be met.

China was the first country to commercialise transgenic crops in the early 1990s. The first approval for commercial sale of a genetically modified product for food use in an industrialised country was in the United States in 1994, but significant commercialisation did not actually begin until 1996. The unprecedented rapid adoption of transgenic crops during the initial seven-year period, 1996–2002 (Figure 63.1), when GM crops were first adopted, reflects the significant multiple benefits realised by large and small farmers in the industrial and developing countries that have grown transgenic crops commercially. Between 1996 and 2002, a total of 19 countries, 10 industrial and 9 developing, contributed to a more than 35-fold increase in the global area of transgenic crops from 1.7 million hectares in 1996 to 58.7 million hectares in 2002 (James, 2002a). The accumulated area of transgenic crops planted globally in the seven-year period, 1996–2002, totals more than 235 million hectares, equivalent to more than 575 million acres, an area equivalent to 25&percnt; of the land area of China or the United States, and 10 times greater than the land area of the UK.

In 2002, the global area of transgenic crops continued to grow for the sixth consecutive year at a sustained rate of growth of more than 10&percnt; between 2001 and 2002. The estimated global area of transgenic or GM crops for 2002 was 58.7 million hectares or 145 million acres, grown by approximately 6.0 million farmers in 16 countries, up from 13 countries in 2001. The increase in area between 2001 and 2002 was 12&percnt;, equivalent to 6.1 million hectares or 15 million acres, and 2002 was the first year when more developing countries (9) grew GM crops than industrial countries (7), Table 63.1. More than one quarter (27&percnt;) of the global transgenic crop area of 58.7 million hectares in 2002, equivalent to 16.0 million hectares, was grown in developing countries where growth continued to be strong. Whereas the absolute growth in GM crop area between 2001 and 2002 was higher in industrial countries (3.6 million hectares) compared with developing countries (2.5 million hectares), the percentage growth was more than twice as high in the developing countries of the south (19&percnt;) than in the industrial countries of the north (9&percnt;).

In 2002, four principal countries grew 99&percnt; of the global transgenic crop area (Table 63.1). The United States grew 39.0 million hectares (66&percnt; of the global total), followed by Argentina with 13.5 million hectares (23&percnt;), Canada 3.5 million hectares (6&percnt;) and China 2.1 million hectares (4&percnt;). Of the four leading GM crop countries, China had the highest year-on-year growth with a 40&percnt; increase in its Bt cotton area from 1.5 million hectares in 2001 to 2.1 million hectares in 2002, equivalent to 51&percnt; of the total cotton area of 4.1 million hectares; this is the first time for the Bt cotton area in China to exceed more than half of the national cotton area. Despite the economic crisis in Argentina, its GM crop area grew at 14&percnt; from 11.8 million hectares in 2001 to 13.5 million hectares in 2002. A growth rate of 9&percnt; was achieved in both the United States (equivalent to 3.3 million hectares) and Canada (0.3 million hectares). GM crop hectarage increased in South Africa by over 20&percnt; to 0.3 million hectares. Three developing countries, India, Colombia and Honduras grew transgenic crops for the first time in 2002. Notably, India, the largest cotton growing country in the world, with 8.7 million hectares equivalent to 25&percnt; of the world cotton hectarage, planted 45 000 hectares of commercial Bt cotton for the first time in 2002. Colombia also planted an introductory pre-commercial area of up to 2000 hectares of Bt cotton for the first time in 2002. Honduras became the first country in Central America to grow an introductory pre-commercial area of approximately 350 hectares of Bt corn in 2002. Thus, the number of countries that grew GM crops increased from 13 in 2001 to 16 in 2002—these include nine developing countries, five industrial countries and two Eastern European countries.

Globally, in 2002, the principal GM crops were: GM soybean occupying 36.5 million hectares (62&percnt; of global area), up from 33.3 million hectares in 2001; GM corn at 12.4 million hectares (21&percnt;), up from 9.8 million hectares in 2001; transgenic cotton at the same level of 6.8 million hectares (12&percnt;); and GM canola at 3.0 million hectares (5&percnt;), up from 2.7 million hectares in 2001, (James, 2002a). During the seven-year period 1996–2002, herbicide tolerance has consistently been the dominant trait with insect resistance being second. In 2002, herbicide tolerance, deployed in soybean, corn and cotton, occupied 75&percnt; or 44.2 million hectares of the global GM 58.7 million hectares, with 10.1 million hectares (17&percnt;) planted to Bt crops. Stacked genes for both herbicide tolerance and insect resistance deployed in both cotton and corn occupied 8&percnt; or 4.4 million hectares of the global transgenic area in 2002. The two dominant GM crop trait combinations in 2002 were: herbicide-tolerant soybean occupying 36.5 million hectares or 62&percnt; of the global total and grown in seven countries, and Bt maize, occupying 7.7 million hectares, equivalent to 13&percnt; of global transgenic area and also planted in seven countries. Notably, South Africa grew 58 000 hectares of Bt white maize for food, up 10-fold from 2001; herbicide-tolerant canola was planted in Canada and the United States occuping 3.0 million hectares equivalent to 5&percnt; of global transgenic area; the other five GM crops, herbicide-tolerant maize and cotton, Bt cotton and Bt/herbicide-tolerant cotton and maize, each occupied 4&percnt; of global transgenic crop area.

Another useful way to portray the adoption of GM crops is to express the global adoption rates for the four principal GM crops in 2001, soybean, cotton, canola and corn (James, 2002b). The data indicate that for the first time the GM soybean area exceeded 50&percnt; of the global hectarage of soybean. In 2002, 51&percnt; of the 72 million hectares of soybean planted globally were transgenic—up from 46&percnt; in 2001. Twenty per cent of the 34 million hectares of cotton were GM, the same as last year; decreases in total plantings of cotton in the United States (down by approximately 10&percnt;) and Australia (down by approximately. 50&percnt; due to a severe drought) were offset by a significant increase in GM cotton in China and the first planting of Bt cotton in India. The areas planted to transgenic canola and maize, both increased in 2002. Of the global 25 million hectares of canola, the percentage of GM increased from 11&percnt; in 2001 to 12&percnt; in 2002. Similarly, of the 140 million hectares of maize grown globally, 9&percnt; were GM in 2002—up significantly from 7&percnt; in 2001. If the global areas (conventional and transgenic) of these four principal GM crops are aggregated, the total area is 271 million hectares of which 21&percnt;, up from 19&percnt; in 2001, was transgenic in 2002. The biggest increase in 2002 is a 3.2 million hectares increase in GM soybean equivalent to a 10&percnt; year-on-year increase, followed by a 2.6 million hectares increase in GM maize equivalent to a significant 27&percnt; year-on-year growth.

Crops are the major source of food globally. There is a widely held view in the international scientific and development community that conventional crop improvement alone will not allow us to meet the global food demands of 2050. What is being advocated is a global strategy that integrates both conventional crop improvement and biotechnology, including transgenic crops, which are often referred to as genetically modified (GM) crops; adoption of such a strategy would allow society to harness and optimise the contribution of biotechnology and GM crops to global food security. There is cautious optimism that such a strategy would contribute significantly to the alleviation of poverty and malnutrition which afflict 1.3 billion people and 815 million people, respectively, today, and that the global food demands of 2050 and beyond can be met.

China was the first country to commercialise transgenic crops in the early 1990s. The first approval for commercial sale of a genetically modified product for food use in an industrialised country was in the United States in 1994, but significant commercialisation did not actually begin until 1996. The unprecedented rapid adoption of transgenic crops during the initial seven-year period, 1996–2002 (Figure 63.1), when GM crops were first adopted, reflects the significant multiple benefits realised by large and small farmers in the industrial and developing countries that have grown transgenic crops commercially. Between 1996 and 2002, a total of 19 countries, 10 industrial and 9 developing, contributed to a more than 35-fold increase in the global area of transgenic crops from 1.7 million hectares in 1996 to 58.7 million hectares in 2002 (James, 2002a). The accumulated area of transgenic crops planted globally in the seven-year period, 1996–2002, totals more than 235 million hectares, equivalent to more than 575 million acres, an area equivalent to 25&percnt; of the land area of China or the United States, and 10 times greater than the land area of the UK.

In 2002, the global area of transgenic crops continued to grow for the sixth consecutive year at a sustained rate of growth of more than 10&percnt; between 2001 and 2002. The estimated global area of transgenic or GM crops for 2002 was 58.7 million hectares or 145 million acres, grown by approximately 6.0 million farmers in 16 countries, up from 13 countries in 2001. The increase in area between 2001 and 2002 was 12&percnt;, equivalent to 6.1 million hectares or 15 million acres, and 2002 was the first year when more developing countries (9) grew GM crops than industrial countries (7), Table 63.1. More than one quarter (27&percnt;) of the global transgenic crop area of 58.7 million hectares in 2002, equivalent to 16.0 million hectares, was grown in developing countries where growth continued to be strong. Whereas the absolute growth in GM crop area between 2001 and 2002 was higher in industrial countries (3.6 million hectares) compared with developing countries (2.5 million hectares), the percentage growth was more than twice as high in the developing countries of the south (19&percnt;) than in the industrial countries of the north (9&percnt;).

In 2002, four principal countries grew 99&percnt; of the global transgenic crop area (Table 63.1). The United States grew 39.0 million hectares (66&percnt; of the global total), followed by Argentina with 13.5 million hectares (23&percnt;), Canada 3.5 million hectares (6&percnt;) and China 2.1 million hectares (4&percnt;). Of the four leading GM crop countries, China had the highest year-on-year growth with a 40&percnt; increase in its Bt cotton area from 1.5 million hectares in 2001 to 2.1 million hectares in 2002, equivalent to 51&percnt; of the total cotton area of 4.1 million hectares; this is the first time for the Bt cotton area in China to exceed more than half of the national cotton area. Despite the economic crisis in Argentina, its GM crop area grew at 14&percnt; from 11.8 million hectares in 2001 to 13.5 million hectares in 2002. A growth rate of 9&percnt; was achieved in both the United States (equivalent to 3.3 million hectares) and Canada (0.3 million hectares). GM crop hectarage increased in South Africa by over 20&percnt; to 0.3 million hectares. Three developing countries, India, Colombia and Honduras grew transgenic crops for the first time in 2002. Notably, India, the largest cotton growing country in the world, with 8.7 million hectares equivalent to 25&percnt; of the world cotton hectarage, planted 45 000 hectares of commercial Bt cotton for the first time in 2002. Colombia also planted an introductory pre-commercial area of up to 2000 hectares of Bt cotton for the first time in 2002. Honduras became the first country in Central America to grow an introductory pre-commercial area of approximately 350 hectares of Bt corn in 2002. Thus, the number of countries that grew GM crops increased from 13 in 2001 to 16 in 2002—these include nine developing countries, five industrial countries and two Eastern European countries.

Globally, in 2002, the principal GM crops were: GM soybean occupying 36.5 million hectares (62&percnt; of global area), up from 33.3 million hectares in 2001; GM corn at 12.4 million hectares (21&percnt;), up from 9.8 million hectares in 2001; transgenic cotton at the same level of 6.8 million hectares (12&percnt;); and GM canola at 3.0 million hectares (5&percnt;), up from 2.7 million hectares in 2001, (James, 2002a). During the seven-year period 1996–2002, herbicide tolerance has consistently been the dominant trait with insect resistance being second. In 2002, herbicide tolerance, deployed in soybean, corn and cotton, occupied 75&percnt; or 44.2 million hectares of the global GM 58.7 million hectares, with 10.1 million hectares (17&percnt;) planted to Bt crops. Stacked genes for both herbicide tolerance and insect resistance deployed in both cotton and corn occupied 8&percnt; or 4.4 million hectares of the global transgenic area in 2002. The two dominant GM crop trait combinations in 2002 were: herbicide-tolerant soybean occupying 36.5 million hectares or 62&percnt; of the global total and grown in seven countries, and Bt maize, occupying 7.7 million hectares, equivalent to 13&percnt; of global transgenic area and also planted in seven countries. Notably, South Africa grew 58 000 hectares of Bt white maize for food, up 10-fold from 2001; herbicide-tolerant canola was planted in Canada and the United States occuping 3.0 million hectares equivalent to 5&percnt; of global transgenic area; the other five GM crops, herbicide-tolerant maize and cotton, Bt cotton and Bt/herbicide-tolerant cotton and maize, each occupied 4&percnt; of global transgenic crop area.

Another useful way to portray the adoption of GM crops is to express the global adoption rates for the four principal GM crops in 2001, soybean, cotton, canola and corn (James, 2002b). The data indicate that for the first time the GM soybean area exceeded 50&percnt; of the global hectarage of soybean. In 2002, 51&percnt; of the 72 million hectares of soybean planted globally were transgenic—up from 46&percnt; in 2001. Twenty per cent of the 34 million hectares of cotton were GM, the same as last year; decreases in total plantings of cotton in the United States (down by approximately 10&percnt;) and Australia (down by approximately. 50&percnt; due to a severe drought) were offset by a significant increase in GM cotton in China and the first planting of Bt cotton in India. The areas planted to transgenic canola and maize, both increased in 2002. Of the global 25 million hectares of canola, the percentage of GM increased from 11&percnt; in 2001 to 12&percnt; in 2002. Similarly, of the 140 million hectares of maize grown globally, 9&percnt; were GM in 2002—up significantly from 7&percnt; in 2001. If the global areas (conventional and transgenic) of these four principal GM crops are aggregated, the total area is 271 million hectares of which 21&percnt;, up from 19&percnt; in 2001, was transgenic in 2002. The biggest increase in 2002 is a 3.2 million hectares increase in GM soybean equivalent to a 10&percnt; year-on-year increase, followed by a 2.6 million hectares increase in GM maize equivalent to a significant 27&percnt; year-on-year growth.

Bioethics in Plant Genetic Engineering

2011-03-27T07:53:00.000-07:00

The GM crops are fast becoming a part of agriculture throughout the world because of their capacity for increased crop productivity and their use in health-care and industry. However, there are conflicting schools of thought regarding the safety issues related to the use of GM crops and foods. The major concerns about GM crops and GM foods are:

- Are GM foods fit for human and animal consumption?

- What will be the effect of GM crops on biodiversity and environment?

- the risk of transgenes escaping through pollen to related plant species (gene pollution) which may lead to the development of highly resistant super weeds.

-The GM crops may change the fundamental vegetable nature of plants as the genes from animals (e.g. fish or mouse) are being introduced into crop plants.

- The transfer of antibiotic resistance marker genes present in transgenic crops into microbes which can induce the problem of antibiotic resistance in human and animal pathogens.

- The GM crops may cause changes in the evolutionary pattern.

There is a need for public debate on these aspects of using GM foods and crops. The researchers and scientists are accumulating a large number of authentic and reproducible evidence about the safety of these products by doing field trials. The transgenic crops e.g. cotton, tomato, corn and soybean are already being used commercially after the risk assessment for environmental safety. However one cannot deny the importance of the assessment of the risks associated with the use of transgenic plants for animals and humans before they are released in to the environment. According to some people the use of GM crops and plant genetic engineering will be a very effective tool to sole the problems of poverty and hunger.

Introduction to tissue culture callus

2011-03-26T15:43:00.000-07:00

In 1902, G. Haberlandt noticed that the plant cells can be grown in synthetic media. The discovery by Haberlandt that the plant cells have the capacity to grow in a nutrient medium in presence of sufficient light made an impact in plant propagation and crop improvement. This has become possible with the development of techniques to regenerate whole plants from the tissue cultured cells.

The ability of a plant cell to give rise to a whole plant is called totipotency. In this method, instead of taking cuttings from a plant, a cell (or few cells) or a tissue is taken from a plant and cultured in suitable containers in which nutrient medium is present. Through these methods new plants can be obtained from single cells or a clump of cells or a bud or other organ of a plant. The portion of the plant that is taken from the desired plant is referred as Explant. By this method, thousands of plants can be produced from a single desirable parent inone generation. These techniques require maintenance of sterile conditions in the medium. The explant need to be sterilised before it is introduced into the medium. All the elements that are required for the growth of the plant are provided in the medium. In addition, the media will contain one or more growth regulators as supplements, depending on the purpose for which the explant is introduced in to the medium. The medium is kept free from bacteria and fungi. Cells or tissues are grown n culture under aseptic conditions.

Tissue Culture Callus Description

Culture techniques are now available for cells, tissues or organs from the plant. In the tissue culture, the explant often divides to give rise to an unorganised mass of tissue called Callus. Te callus or the explant as such, will undergo differentiation into shoots; roots or embryo like structures (Embryoids). This differentiation is dependent on the concentration and combination of the plant growth substances like auxins, kinetin, gibberellin etc., in the medium. Some times numerous independent shoots (multiple shoots) differentiate from the explant or callus.

Plants raised through tissue culture are routinely used in agriculture, horticulture and forestry. When we use a diploid explant, we get a diploid plant like in any of the vegetative propagation methods. Similarly, if we want to develop a haploid plant, it is possible to obtain it through tissue culture. For this, we need to select a haploid cell in the plant. The best choice of a haploid cell is pollen grain . Development of haploid plants through tissue culture was discovered by Indian Scientists Shipra Guha and Satish Maheswari.

How to Produce Haploid Plant ?

2011-03-26T15:37:00.000-07:00

Haploids are defined as saprophytes with gametophytic chromosome number and have been produced in a variety of plant species using a variety of methods.

Although, the significance of haploids in genetics and plant breeding has been recognized for long time, with the advent of new biotechnology it has received renewed emphasis, so that the production of haploids has become an important component of biotechnology programmes in different countries.
Although, haploids could be produced following delayed pollination, irradiation of pollen, temperature shocks, colchicine treatment and distant hybridization, the most important methods currently being utilized under biotechnology programmes include

(i) anther or pollen culture and ovule culture and
(ii) chromosome elimination following interspecific hybridization (bulbosum technique).
Factors Affecting Haploid Production

- A number of factors influence androgenesis in vitro. The genotype of the donor plant plays a significant role in determining the frequency of pollen plant production. Anther wall factors also support pollen embryo development.

Histological studies support this view. As induction of the pollen into embryoids occurs most easily within the confines of an anther, the anther wall seems to provide a nursing effect. There are two schools of thought regarding the role of the anther wall. One is that it may have a stimulatory effect on the growth of pollen embryos (probably due to the presence of enhanced levels of some amino acids such as glutamine and serine); the other view holds that it may emanate some inhibitory substances into the culture medium thereby blocking the growth of more pollen into embryos.
The culture medium also plays a vital role since the requirements vary with the genotype and probably the age of the anther as well as conditions under which donor plants are grown. The medium should contain the correct amount and proportion of inorganic nutrients to satisfy the nutritional as well as physiological needs of the many plant cells in culture.

In addition to basal salts and vitamins, hormones in the medium are critical factors for embryo or callus formation. Cytokinins (e.g. kinetin) are necessary for induction of pollen embryos in many species of Solanaceae except tobacco. Auxins, in particular 2,4-0, greatly promote the formation of pollen callus in cereals. For regeneration of plants from pollen calli, a cytokinin and lower concentration of auxin are often necessary.

Sucrose has been considered the most effective carbohydrate source which cannot be substituted by other disaccharides. Glucose can be used in anther culture in some cases but fructose is far less effective. The concentration of sucrose also plays an important role in induction of pollen plants. Activated charcoal is also added to the culture medium.
It helps in the removal of inhibitors from the agar used for gelling the medium. Another role assigned to activated charcoal is the adsorption of 5¬hydroxymethylfurfural, a product of sucrose dehydration during autoclaving, assumed to bean inhibitor of growth in anther cultures.

Certain organic supplements added to the culture medium often enhance the growth of anther cultures. Some of these include the hydrolyzed products of proteins such as casein (found in milk), nucleic acids, and others. Coconut milk obtained from tender coconuts is often added to tissue culture media. It contains a complex mixture of nucleic acids, sugars, growth hormones and some vitamins.

The physiological state of the parent plant plays a role in haploid production. Success in haploid induction is in part dependent on knowledge of the physiology of the pollen yielding plant. In various plant species it has been shown that the frequency of androgenesis is higher in anthers harvested at the beginning of the flowering period and declines with plant age.
This may be due to deterioration in the general condition of the plants, especially during seed set. The lower frequency of induction of haploids in anthers taken from older plants may also be associated with a decline in pollen viability. Seasonal variations, physical treatment, and application of hormones and salts to the plant also alter its physiological status, which is reflected in a change in anther response.

Temperature and light are two physical factors which play an important role in culture of anthers. Higher temperatures (30°C) yield better results. Temperature shocks also enhance the induction frequency of microspore androgenesis. Frequency of haploid formation and growth of plantlets are generally better in light.
Certain physical and chemical treatments given to flower, buds or anthers prior to culture, can be highly conducive to the development of pollen into plants. The most significant is cold treatment.
The developmental stage of pollen greatly influences the fate of the microspore, Androgenesis occurs when a microspore or pollen is induced to shift from a gametophytic pathway to a sporophytic pathway of embryo formation.

Anthers of some species (Datura, tobacco) give the best response if pollen is cultured at first. mitosis or later stages (postmitotic), whereas in most others (barley, wheat, rice) anthers are most productive when cultured at the uninucleate microspore stage (premitotic). Anthers at a very young stage (containing microspore mother cells m tetrads) or a late stage (containing binucleate, starch filled pollen) of development are generally ineffective, albeit some exceptions are known.

Source

Rice Research

2011-03-17T16:32:00.000-07:00

From the 1950s to 1970s, in an effort to combat world hunger, plant breeders at the International Rice Research Institute (IRRI) in the Philippines developed new rice varieties that were, when fertilized, higher yielding than traditional varieties. The new varieties were shorter and less likely to fall over, which made them easier to harvest mechanically. They also ripened sooner, reducing the risk of poor weather affecting yield, and enabling farmers to harvest and replant several times during the growing season. While successful in many areas, the new varieties required more money for fertilizer and chemical pesticides, and in some cases, machines for sowing and harvesting—tools often too costly for peasant farmers. In some areas a single new rice variety replaced diverse, centuries-old varieties adapted to thrive in a particular climate and soil type and with some resistance to local insects and diseases. The new variety was not able to thrive in these areas, and the crop yields were not always greater.

Rice breeders at IRRI and other research facilities are now trying to increase yields through genetic engineering. They hope to create rice varieties that are genetically designed to require less fertilizer, resist insects and diseases, tolerate poor soil, require less irrigation, and photosynthesize more efficiently.

Scientific classification: Rice is an annual grass in the grass family, Poaceae (formerly Gramineae). Asian rice is classified as Oryza sativa and African rice as Oryza glaberrima.

History of Rice

2011-03-17T16:29:00.000-07:00

According to the most widely accepted theory, rice cultivation originated as early as 10,000 BC in Asia. Archaeological evidence shows that rice was grown in Thailand as early as 4000 BC, and over the centuries spread to China, Japan, and Indonesia. By 400 BC rice was cultivated in the Middle East and Africa. The invading armies of Alexander the Great probably introduced rice to Greece and nearby Mediterranean countries around 330 BC. Rice was brought to the American colonies in the early 1600s, and commercial production began in 1685.

Rice cultivation, a very demanding process, has shaped values and changed history. For example, rice encouraged populations to crowd together to take advantage of a reliable food supply. The labor-intensive process of growing paddy rice requires large numbers of people to work together to level fields, build and maintain bunds, and care for the crop. Where paddy-rice cultivation has been introduced, hard work, organization, persistence, and above all, cooperation, have been encouraged.

In the United States, rice played an important role in establishing slavery in the coastal Southeast—the Carolinas, Georgia, and north Florida. For instance, rice exportation was deemed necessary for economic survival in Georgia, and as a result, slavery was legalized in that state to create a work force to clear swamps, install dikes, and plant, grow, harvest, and thresh the rice.

Production And Using of Rice

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Asian countries produced about 90 percent of the 576 million tons of rice grown worldwide in 2002. Typically, China and India together produce about 50 percent of the world’s rice, and it is a significant agricultural crop in more than 50 other countries. About 96 percent of the rice grown worldwide is consumed in the countries where it is produced, with some exceptions. The United States, for example, exported about 37 percent of the 8.7 million tons it produced in 2000, and Pakistan exported about 28 percent of its 7.2 million tons, according to the FAO. In the same year, Thailand exported significantly more rice than any other country—6.6 million tons, or about 26 percent of its total, while India exported 1.5 million tons, or about 1.1 percent of total production. Major rice-importing countries include Côte d'Ivoire, Nigeria, Philippines, Iran, Saudi Arabia, Brazil, Senegal, Japan, and Indonesia. Some rice-importing countries buy rice on a regular basis, others buy when drought, floods, or other conditions reduce the yield of their own rice crop.

Rice is used for a variety of food and nonfood products. Foods include cooked rice, breakfast cereals, desserts, and rice flour. Rice is also used in beer and in sake, a Japanese fermented brew. The inedible rice hull is used as fuel, fertilizer, and insulation, while the bran is a source of cooking oil. Straw from the leaves and stems is used as bedding for animals and for weaving roofs, hats, baskets, and sandals.

Growing Rice

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Although rice originally flourished in the dry climate of Central Asia, it spread to the flood plains of tropical regions, resulting in evolution of varieties with the capacity to grow with roots submerged in water. The African and Asian varieties that are flooded during the growing season are more productive than the varieties that are not flooded, partly because the submerged roots easily extract needed nutrients from the water. Flooded rice, also known as lowland rice, is grown in paddies, which are fields that contain water enclosed by low walls of earth called bunds. Paddy rice fed by rainfall alone accounts for about 50 percent of all rice grown worldwide, while paddies flooded by a combination of rainfall and irrigation provide about 35 percent of rice produced. The major rice-producing countries, including China, India, and Vietnam, primarily cultivate paddy rice.

Other African and Asian rice varieties, known as upland rice, are grown in regions of low rainfall that do not provide enough moisture for paddies, or in tropical rain forests where high humidity prevents the ripening of other edible grains. Upland rice is less productive than paddy rice, partly because it depends on nutrients that are dissolved in the soil moisture. When soil moisture is low, few nutrients are available, compared to the storehouse of nutrients typically found in paddy waters. Upland rice accounts for about 15 percent of world rice production, and is particularly important in Laos, where it accounts for between 20 and 22 percent of the rice harvest.

Rice, grown in more than 100 countries, is particularly productive in tropical regions with abundant moisture, but it also grows successfully under widely different climate conditions. Rice farmers choose varieties adapted to the region’s length of growing season, soil, altitude, and, for paddy farmers, the depth of water in the fields. Paddy rice farmers in developing countries usually sow seeds in small seedbeds, then hand-transplant the seedlings into flooded fields that have been leveled by water buffalo or oxen-drawn plows. One advantage of transplanting seedlings instead of planting from seed is that the young plants help limit weeds by shading them from needed sun. In industrialized countries, seed is sown with a planting drill or cast from an airplane into machine-leveled fields that are then flooded. Herbicides are the primary method of weed control.

Depending on the rice variety and the climate, rice grains are ready for harvest in three to six months. In developing countries, farmers harvest rice with sickles or knives, tie it in bundles, and let it dry in the field. They then remove the grain from the plant, a process called threshing, by hitting the plant against a slatted screen or walking animals over it. Farmers in industrialized countries use combines, which are machines that move through fields and harvest, thresh, and clean the grains. The grain is then dried in sheds with heated air.
Rice is susceptible to a range of diseases and pests, which annually destroy about 55 percent of rice crops. The most common diseases are caused by the fungi sheath blight and rice blast, and the stalk borer is a common insect pest. Weeds compete with rice for nutrients and water and are a serious problem, especially in upland rice farming. Rodents and birds also feed on rice grains before they are harvested. Disease-causing fungi, insects, and a variety of other pests infest rice during storage and transport.

When rice is processed, the hull is removed, exposing the bran. Rice at this stage is brown rice. The fibrous bran of brown rice is rich in oil; protein; the B vitamins thiamin, riboflavin, and niacin; and the minerals iron, phosphorus, and potassium. To make white rice, the bran is removed. White rice is less nutritious than brown rice and, when feasible, is enriched with the addition of vitamins and minerals to increase its nutritive value. Without the tough bran layer, white rice cooks faster and stores longer than brown rice, so it is often preferred in regions where fuel is limited and refrigeration is not readily available. Polished rice is made by passing white rice kernels through a machine with a brush that smoothes and shines them.

Rice as an edible grain

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Rice, plant that produces an edible grain; the name is also used for the grain itself. Rice is the primary food for half the people in the world. In many regions it is eaten with every meal and provides more calories than any other single food. According to the United Nations Food and Agricultural Organization (FAO), rice supplies an average of 889 calories per day per person in China. In contrast, rice provides an average of only 82 calories per day per person in the United States. Rice is a nutritious food, providing about 90 percent of calories from carbohydrates and as much as 13 percent of calories from protein.

Of the 20 known species of rice, only two are cultivated—the widely grown Asian rice and the hardier African rice. Asian rice, if managed with modern techniques such as fertilizers, irrigation, and chemical pesticides, produces significantly more grain per plant than African rice, and for this reason is the preferred type in the majority of rice-growing countries. African rice, however, is more productive than Asian rice in traditional farming systems where modern techniques are not used or poor growing conditions are present. About 50,000 varieties exist within these two species, only a few hundred of which are cultivated.

covering, surrounds the bran, which consists of layers of fibrous tissue that contain protein, vitamins, minerals, and oil. Beneath the bran is the endosperm, which makes up most of the rice grain. The endosperm contains starch, the energy source used by the germinating seed. The bran and endosperm are the edible portions of the grain.

A rice plant, a type of grass, has narrow, tapered leaves and grows from about 60 to 180 cm (about 2 to 6 ft) tall. Several flower stalks emerge from the plant, and in most varieties, a loose cluster of branching stems, called a panicle, radiates from the top of each stalk with small green flowers hanging from each stem. When the grain has developed, the panicle droops under the weight of the ripened kernels. Depending on the variety, one panicle provides about two handfuls of rice.

The Cartagena Protocol on Biosafety

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At the international level, the Cartagena Protocol for Biosafety (Bail et al., 2002, hereafter referred to as the Protocol) came into force on September 11, 2003, 90 days after the fiftieth instrument of ratification had been deposited by signatory countries to the Convention on Biological Diversity (CBD). The Protocol is the most important single international treaty, and will play a dominant role in shaping the future of transgenic technology in the world. It is the first legally binding international regulatory framework for the transboundary movement of living modified organisms (LMOs). The need to have an international regulatory framework for GM organisms was discussed by countries during the drafting of the CBD, which was adopted at the 1992 Earth Summit in Rio de Janeiro. At that time, the first GM crops were coming to the market in the USA, and their use would increase exponentially during the following years. In the final document, the CBD did not specifically address biosafety, but Article 19 called for the signatories to consider the ‘need for and modalities of a protocol setting out appropriate procedures, including, in particular, advance informed agreement, in the areas of the safe transfer, handling and use of any living modified organism resulting from biotechnology that may have adverse effect on the conservation and sustainable use of biological diversity’.

The international community will need time to gain experience as to how the various instruments and mechanisms of the Protocol can operate effectively to ensure the safe transfer of LMOs from country to country, taking into account biodiversity considerations. In carefully worded language, the Protocol obviates antagonism with national regulatory frameworks, by explicitly giving precedence to national standards regarding the safety assessment of LMOs, and in this sense does not lay any claims to standard setting. Parties are sovereign under the Protocol to have their own national standards. What the Protocol brings forth is a set of ideals or socio-ethical endpoints that parties should consider in deciding about LMOs and their applications, particularly concerning a precautionary approach, sustainable development and the conservation of biological diversity.

One of the Protocol's goals is to serve as a minimal baseline regulatory framework for countries that do not yet have domestic frameworks. The Protocol distinguishes three types of LMO applications: for food, feed or processing (LMO-FFP); for either contained use or for intentional introduction into the environment. For LMO-FFP and contained use with LMOs (Article 18), parties are obliged, under the Protocol, to inform the other parties of their decision regarding transboundary movement, but domestic standards and regulations prevail for the approval process. On the other hand, LMOs intended for introduction into the environment, for example, as seeds, are subject to the advance informed agreement (AIA) procedure of the Protocol, applicable to the first transboundary movement. As such, the Protocol is both product and process based. According to the AIA, the party of export must notify the competent authority of the country of import, prior to the first transboundary movement of the LMO. The exporter is required to supply the party of import with all necessary information regarding the LMO, including a risk-assessment report and the regulatory status of the particular organism in the exporting country. Within 270 days after the receipt of notification, the party of import communicates, in writing, to the exporter and to the Biosafety Clearing House (BCH) on the decision it has taken. The AIA procedure is meant to prevent an uncontrolled dissemination of LMOs, for example, in developing countries that do not yet have the appropriate biosafety framework for regulating these products.

An ambiguous case for the regulation of LMO-FFPs would be the import of grains as food by farmers in developing countries that do not have domestic regulatory frameworks, particularly when the grains could also be used as seed material. The solution put forth in the Montreal negotiations of the Cartagena Protocol was to make reference to the precautionary approach and to require precise wording ‘not intended to be used in the environment’ on the accompanying documentation (Article 18.2a). If a domestic framework does not exist, developing countries or countries with economies in transition can indicate that the final decision will be taken according to a risk assessment undertaken in accordance with the provisions of the Protocol and within a predictable time frame.

There is also an information-sharing aspect included in the AIA procedure, since the party of import is required to communicate with other parties through the BCH. In addition, the party of import may also request the opinions of independent biosafety experts or seek out further sources of information. The BCH is, therefore, the main information-sharing mechanism of the Protocol and is meant to assist parties in its implementation. It is an Internet-based platform for the exchange of scientific, technical, environmental and legal information about LMOs at national, regional and international levels. Information available in the BCH includes contact information for national competent authorities, rosters of biosafety experts, and risk-assessment reports, as well as national decisions regarding the import of LMOs (www.biodiv.org).
The Cartagena Protocol is a de facto trade agreement, since its scope includes export and import activities. The Protocol could be positive for trade: trade rules would be clearer with AIA; trade could be fairer; scientific risk assessment would be used systematically for decision making and a basic, operative regulatory framework for LMOs would be available for countries without domestic regulations. Seed companies would benefit from a system with mutual acceptance of safety evaluations and science-based risk assessments (de Greef, 2000). The Protocol is a decentralised approach that recognises national standards and allows them to be more restrictive. At the same time, and as the Preamble states, the Protocol is not subordinate to other international agreements and its implementation should work in accordance with them.

National and International Frameworks for the Safety Assessment of Transgenic Crops

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The controversy regarding GM crops has less to do with their scientific safety assessment, which demonstrates that these crops are at least as safe as their conventional counterparts, than with the wider social, political, ethical and economic endpoints related to their commercialisation. As a result, national regulatory frameworks for the authorisation of GM crops often combine science-based risk assessments with public policy. The word ‘biosafety’ has come to denote the entire process of coming to a decision about the safe use of biotechnology products. Alternatively, Wolt and Peterson (2000) call the process of considering both scientific and social issues ‘risk analysis’ to distinguish it from risk assessment, which considers only technical risks. Applied to GM crops, this means that the consideration of potential export markets for these products would belong to the wider risk analysis, while the consideration of outcrossing with native species would belong to the technical risk assessment. Widening the risk discussion to include alternative framing of risk, such as the availability of export markets, invites public debate regarding the wider impacts of applied technology, such as political accountability, equity, ethics and economics. ‘Public debate is a way of isolating what is strictly scientific from what is socially or politically determined in the development of scientific activity’ (Touraine, 1997).

National regulatory frameworks for GM crops are all alike, in that they seek to ensure an adequate level of protection for human and environmental health, based on the best available science. There is general agreement, for example, on the characterisation of donor and recipient organisms, environmental impact assessments and toxicity issues. On the other hand, national regulatory frameworks can also be diverse, depending on the basic assumptions about the novelty of using recombinant DNA techniques to modify plants. The margin separating natural from unnatural gene modification certainly exists but, because traditional plant breeding also involves the transfer of genes, on a scale that is magnitudes greater than during the genetic engineering of plants; it is less tangible than we would wish it to be. An important distinction must be made, in that there is both a need and a desire to know the kind of regulatory data requirements for risk assessment. Requesting unnecessary (nice to know) data will only add to the cost of technological development of transgenics, which in most cases is already becoming prohibitive.

Where gene modification is considered inherently safe, regulations tend to be product based, and only the final product is assessed, not the process that produced it, as in the United States. In almost all countries, the regulations governing transgenics are process based due to the fact that the organisms have been developed using modern genetic engineering techniques. Whenever gene modification is considered inherently dangerous, regulations are likely to be process based, and the trigger for regulation is the process itself. Adopted in February 2001, European Directive2001/18/EC, on the deliberate release of GM organisms, repealed Directive 90/220/EC as a comprehensive regulatory framework for environmental applications of GM organisms and their commercialisation. In both directives, regulations are process driven, since all ‘plants obtained through the techniques of genetic modification’ are subject to regulatory approval. Genetic modification in the Directive is understood as ‘the introduction of new combinations of genetic material by the insertion of nucleic acid molecules, the direct introduction of heritable material prepared outside the organism, cell fusion or hybridisation techniques’. In the US regulatory system, risk assessment for the environmental release of GM plants examines the likelihood of a GM plant to becoming a pest (7CRF340), based on the assumption that it has the potential to become one due to the presence of genetic elements from defined plant pests (CRF340.2). For example, the 35S promoter from cauliflower mosaic virus or regulatory elements from Agrobacterium used to transform plants. The Canadian approach, which is both product and process based, regulates both GM and conventional plants provided they ‘demonstrate neither familiarity nor substantial equivalence to those present in a distinct, stable population of a cultivated species of seed in Canada PNTs [plants with novel traits] include those derived from both recombinant DNA technology and plants derived through traditional plant breeding’ (Regulatory Directive Dir 94-08).