Transgenics in crops

© 2001 ElsevierScienceB.V.All rightsreserved. BiotechnologyAnnualReview. Volume7. M.R. EI-Gewely,editor.

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Transgenics in crops Yi Li*, Yan H. Wu, Richard McAvoy and Hui Duan Department of Plant Science, University of Connecticut, Storrs, CT 6269, USA

Abstract. With rapid world population growth and declining availability of fresh water and arable land, a new technology is urgently needed to enhance agricultural productivity. Recent discoveries in the field of crop transgenics clearly demonstrate the great potential of this technology for increasing food production and improving food quality while preserving the environment for future generations. In this review, we briefly discuss some of the recent achievements in crop improvement that have been made using gene transfer technology.

Keywords: genes, plant transformation, herbicide resistance, insect and disease resistance, abiotic stress tolerance, fatty acids, carbohydrates, seedless fruits, extension of shelf life, photosynthetic activities, vitamins and micro-nutrients.

Introduction Throughout human history, attaining an adequate food supply has been one of our biggest challenges. Today there are over 800 million hungry people worldwide, with an additional 185 million malnourished pre-school children owing to disease or lack of food [ 1]. While the world population is expected to reach 7 billion within 25 years and exceed 10 billion by 2050, agricultural production is growing at a much slower rate [2]. The problem of limited agricultural food production has been exacerbated by habitat destruction and continued environmental degradation associated with civilization. Ironically, the greatest threat to the environment comes from the very same agricultural production practices that we rely upon to produce an abundant food supply. The ideal solution to this dilemma would be to increase crop productivity per unit area while reducing the use of agricultural chemicals. For this reason, one could argue that the greatest environmentalists of the past century have been plant breeders since crop improvements resulting from their efforts have spared 800 million hectares of land from agriculture. For example, worldwide cereal production covered about six hundred million hectares (5.5 percent of the land surface) in the 1950's. If crop productivity had remained constant, 1.4 billion hectares, or most of the arable land on earth, would be required to meet the demands of today's population. However, due to improvements brought about by plant breeding technology, the same six hundred million hectares supports cereal production today. During the 1960s the rate of increase in major cereal crop productivity was about 4% per year,

*Addressfor correspondence: Yi Li, Department of Plant Science, University of Connecticut, Storrs, CT 06269, USA. Tel: (860)-486-6780. Fax: (860)-486-6780. E-maih [email protected]

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80 <

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0

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2o. 1998

1997

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1999

Fig. 1. Global Area of Transgenic Crops from 1996 to 1999.

but today the increase in productivity has dropped to about 1% per year in spite of the best efforts of the breeding community. Thus, a new technology is needed to allow crop production to keep pace with population growth without further damaging our environment. The first transgenic plants were produced in the early 1980s [3,4] and thereafter transgenic technology developed rapidly into an important tool for crop improvement. Since the first transgenic food product was marketed in the Untied States in 1994, the production of commercial transgenic plants has increased dramatically worldwide ([5-8] and Fig. 1). For instance, worldwide 3.6 million acres were planted with transgenic crops in 1996 but the acreage increased to 51.3 million in 1998, and 97.4 million in 1999. The major commercialized products are herbicidetolerant or pest-resistant soybean, cotton, corn or canola (Table 1), with many new developments still to be released. Undoubtedly, these achievements have surpassed all previous expectations, and the future is even more promising. A brief review of some of these exciting achievements is presented herein.

Methods of production of transgenic plants Since 1983, Agrobacterium-mediatedand direct DNA delivery methods have been used to transform more than 120 plant species in some 35 families, including major agronomic crops, vegetables, ornamentals, and medicinal, fruit, tree and pasture plants [9]. The first reliable plant transformation system was developed using Agrobacterium tumefaciens [3,4]. Although Agrobacterium-mediatedtransformation is plant genotype-dependent, it is still most efficient and straightforward when compared to other methods. The majority of transgenic plants produced using Agrobacterium-mediatedmethods harbor only one or two copies of a welldefined DNA sequence, which is highly desirable for commercial development of stable transgenic lines. Agrobacteriumis a soil bacterium that transfers it's T-DNA into the host plant genome causing a disease called crown gall in susceptible species. By replacing certain DNA sequences within the T-DNA region, the bacterium has been used to transfer foreign genes into plants. Agrobacterium-mediatedtransformation procedures involve co-cultivation of wounded plant cells with Agrobac-

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Table 1. DominantTransgenicTraits and Crops in 1999 (millionsof acres). Crop

Million Acres

%

Soybean Herbicidetolerant Corn Bt Bt/Herbicide tolerant Herbicide tolerant Cotton Bt Herbicide tolerant Bt/Herbicide tolerant Canola Herbicidetolerant Others (Potato,Squash, Papaya)

53.5 18.57 5.2 3.7 3.21 3.96 1.98 8.67 2.81

54 19 5 4 3 4 2 9
Adapted from James [8].

terium. The wounded plant materials can be either isolated protoplasts or explants derived from leaves, roots, stems, cotyledons, or floral organs. Transformed cells can develop into shoots and whole plants through tissue culture and regeneration processes. Recently, there have been several significant improvements in Agrobacteriummediated transformation. First, Agrobacterium has been successfully used to transform monocotyledonous species such as rice [10,11], banana [12], corn [13], and wheat [14] with reasonably high transformation efficiencies. This breakthrough resulted from the development of "super binary" vectors, such as pTOK47, which carry extra copies of some virulence genes into the cell along with the binary vector. Agrobacterium carrying super binary vectors may also prove useful in the genetic manipulation of "difficult-to-transform" dicotyledons such as grain legumes. Second, with the use of the bacteriophage P1 Cre/lox sequences, Vergunst et al. [15] developed a system of targeted or location-specific insertion of the Agrobacterium T-DNA into the chromosome of the host plant. Cre/lox, consisting of a recombinase protein (Cre) and a short 34 base pair recombination site (lox), catalyses the precise and stable insertion between the respective DNA target sites. The technology makes it possible to target transgenes to a single pre-selected chromosomal site, thus eliminating variation in transgene expression level. A third significant advance was a new Ti-plasmid vector, the BIBAC (Binary Bacterial Artificial Chromosome) developed by Hamilton et al. [ 16,17], which allows the transfer of large DNA fragments into plant cells. This new Ti-plasmid vector can transfer DNA fragments up to 150 kilobase or about ten times larger than the DNA sequences transferred with conventional Ti-plasmids. With this vector, multi-gene sequences or relatively large-sized DNA fragments can now be engineered into crop plants. The relative difficulty of regenerating whole plants from single cells has been a major obstacle to the production of transgenic lines in many crops. In-planta transformation methods that bypass the tissue culture/regeneration processes have been successfully demonstrated. Feldmann and Marks [ 18] used an Agrobacterium

242 suspension culture to inoculate Arabidopsis seeds and then allowed the seeds to germinate, grow and produce seeds. The resulting F1 seedlings were screened for expression of transgenes or changes in identifiable phenotype. The transformation efficiency of this method was low, presumably because the transformation event occurred late in development, possibly during sexual reproduction. Consequently the Agrobacterium would have had to persist from seed germination until seed formation [19]. Bechtold et al. [20] later developed a more efficient method by vacuum infiltrating developing flowers or inflorescence meristems of Arabidopsis with Agrobacterium. More recently, Trieu et al. [21] successfully extended this methodology to Medicago truncatula by infiltrating flowers, inflorescence meristems, and seedlings with Agrobacterium. If the in-planta transformation methods could be successfully applied to other crops, production of transgenic plants would be greatly simplified and the associated costs greatly reduced. Direct DNA delivery methods using microprojectile bombardment [22,23], polyethylene glycol treatment [24], electroporation [25,26] and silicon carbide fibers [27] have been developed to overcome the limitations associated with Agrobacterium-mediated protocols. Unlike Agrobacterium-mediated techniques, direct DNA delivery methods are plant genotype-independent. The microprojectile bombardment method is the most popular direct DNA delivery method for plant transformation. Inert metal particles, usually gold with diameters of 0.2-4.0 pm, are coated with DNA and accelerated into target cells. The most commonly used instruments for accelerating DNA coated particles are those powered by a burst of helium generated by a rupture-membrane mechanism [28], or by a shock wave generated by a high voltage discharge through a water droplet [29]. Recently, Southgate et al. [30] compared the transformation efficiencies of four direct DNA delivery methods based on microprojectile, tissue electroporation, silicon carbide fibers, and polyethylene glycol treatment. They found high levels of transient GUS expression in maize callus subjected to either microprojectile bombardment or tissue electroporation but only the microprojectile bombardment-mediated method resulted in high GUS activity in immature maize embryos.

Agronomic traits Insect resistance The development of Bt insect-resistant crops stands as one of the most notable successes of transgenic plant technology. These crops were planted on 10 million acres in 1997 and garnered $300 million in benefits from increased productivity and cost-savings resulting from reduced pesticide use [31 ]. Bt is a potent biological insecticide based on the crystal protein endotoxin produced by some strains of soil bacterium Bacillus thurigiensis [32]. When ingested by larvae of sensitive insects, the Bt protein is cleaved and an active part of the molecule is attached to the brush border membrane of the larval intestine. As a result the intestinal wall loses its property of semi-permeability, the insect leaks hemolymph into the intes-

243 tinal cavity, and eventually leading to dehydration and death. Although Bt proteins are active against several types of insects, no toxic effects on humans and animals have been observed. Tobacco was the first plant to express the Bt protein [33], but tomato, potato, cotton, and corn expressing Bt endotoxin genes were subsequently produced [31]. Meanwhile, improvements to optimize activity, stability and expression level of Bt proteins have led to an even broader activity spectrum. For example, a number of strains have been identified which produce insecticide crystal proteins that are effective against the larvae of lepidoptera, coleoptera and diptera [34] and even against nematodes [35]. In an effort to combat insect resistance to Bt proteins, Kota et al. [36] discovered that a combination of very high transgene expression, via insertion in the chloroplast genome, coupled with improved protein stability resulted in mortality of even Bt-resistant insects. A single laboratory study on non-target effects of Bt corn pollen on monarch butterflies [37] triggered a great deal of public debate over transgenic plants, but subsequent studies demonstrated that Bt corn poses little threat to butterflies [38-40]. On the other hand, non-target beneficial effects of Bt crops have been well documented. For example, Bt corn hybrids are significantly less likely to contain harmful mycotoxins than their non-Bt counterparts [41]. Insect feeding increases the incidence of diseases such as corn ear and stalk rots, resulting in reduced yield and quality. Some fungal diseases produce mycotoxins, which poses a significant problem worldwide, affecting an estimated 25% of grain crops. Ariatoxins are the major mycotoxins in corn. Both aflatoxins and fumonisins can be fatal to livestock and are probable human carcinogens. Symptoms of Fusarium and Aspergillus ear rots are often highly correlated with insect damage [42]. Munkvold et al. [41] demonstrated that Bt transgenic hybrids experience significantly less Fusarium ear rot and yield corn with lower fumonisin concentrations than their non-Bt counterparts. Lower mycotoxin concentrations represent a benefit to consumers of Bt grain, whether the intended use is for livestock or human food products. During the past several years, other naturally insecticidal proteins have been discovered, including lectins, protease inhibitors, antibodies, wasp and spider toxins, microbial insecticides, and insect peptide hormones [43-45]. For instance, toxin produced by the gram-negative bacterium Photorhabdus luminescens, which lives in the gut of entomophagous Nematodes, represents potential alternatives to Bt for transgenic deployment. In insects infected by the nematode, the bacteria are released into the insect hemocoel; the insect dies and the nematodes and bacteria replicate in the cadaver. The toxin consists of a series of four native complexes encoded by toxin complex loci tca, tcb, tcc, and tcd. Both tca and tcd encode complexes with high oral toxicity to Manduca sexta [46]. Photorhabdus toxin from a single bacterial cell can kill an insect. Even picomolar quantities of Photorhabdus toxin can be lethal to caterpillars, mealworms, cockroaches, and ants. Combined production of Photorhabdus toxins and Bt toxins in transgenic crops can be used to combat insect resistance.

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Disease resistance Viral, fungal and bacterial disorders are responsible for major crop losses worldwide. Transgenic approaches, which were described in detail in several recent review articles, provide powerful tools for the development of disease resistance in crops [44-50]. One approach involves expression of viral genes that interfere with the successful completion of the life cycle of viruses. In 1986, Powell-Abel et al. [51] discovered that when the coat protein gene for tobacco mosaic virus (TMV) was expressed in a host plant, it interfered with the replication and systemic spread of the virus. Plants expressing the TMV coat protein gene were resistant to TMV infection. Coat protein-mediated resistance is now widely used to protect many crop plants from a large number of viruses [52]. China was the first country to commercialize virus-resistant transgenic crops with the introduction of virusresistant tobacco in 1992 [6]. Then the same approach was used to create virusresistant tomato, squash, and watermelon plants [53]. More recently, the coat protein strategy was successfully used to protect Hawaiian papaya production from a devastating virus called papaya ring spot virus (PRSV). PRSV is a significant disease of papaya. The spread of PRSV is extremely difficult to control and once a plant is infected there is no effective treatment. In Hawaii, PRSV caused a 38% drop in fresh papaya production from 1993 to 1997. Transgenic papaya resist PRSV infection in the field and yield at levels equal to or higher than the industry standard. The fruits are viewed favorably by consumers in both appearance and taste [54,55]. Other strategies have also been used to create disease resistant plants. For example in oilseed rape, over-expression of a tomato chitinase gene with a strong gene promoter resulted in plants with increased resistance to fungal attack [56]. The plants exhibited increased resistance to the pathogens Cylindrosporium concentricum and Phoma lingam, and chitinases catalyze the hydrolysis of chitin, a polymeric component of fungal cell walls. Cao et al. [57] used a "master-switch" gene, NPR1 that regulates expression of a set of pathogenesis-related (PR) genes, to activate a number of PR genes simultaneously. PR genes, which individually may not provide adequate protection, can work collectively to provide a modest but long term resistance against certain pathogens. The NPR1 transgenic plants showed increased expression of many PR genes, and more importantly a dramatic increase in resistance to the bacterial pathogen Pseudomonas syringae and the fungal pathogen Pernospora parasitica. For example, 3-days after infection bacterial growth was inhibited more than a thousand-fold in transgenic plants [57]. The availability of NPR1 may make it possible to create crops that possess broadspectrum resistant to many destructive diseases using just one gene.

Herbicide tolerance The developed world relies almost entirely on herbicides to control weeds in agricultural crops. Many of the early herbicides produced undesirable environmental

245 impacts. These have been replaced with new chemicals such as glyphosate that is highly effective at low concentrations and more environmentally friendly because it is degraded rapidly by soil microorganisms [32]. However, like many other herbicides, glyphosate is a non-specific herbicide and kills any green plant, therefore it can only be used prior to seed emergence. By introducing glyphosate tolerance genes into crops, the herbicide can now be applied over the top of crops during the growing season to control weed populations more effectively. In 1998 and 1999, plants expressing transgenic herbicide tolerance accounted for 71% of all transgenic crops grown worldwide [8]. Herbicide tolerant soybean, corn, cotton and canola represent the major transgenic products [8,31]. Recent surveys indicate that 9-23% less herbicide was used on herbicide tolerant soybeans in the USA [31,58,59], resulting in a US$15.40 per acre increase in revenue [60]. Other advantages of herbicide tolerant crops include no carryover of herbicide residues, more flexibility in agronomic management, more selective use of herbicides, better control of weeds, and promotion of reduced tillage soil conservation practices [31 ]. Two approaches have been used to create herbicide-tolerant crops: one is to decrease the sensitivity of the plant to the herbicide by modifying the level or the sensitivity of the target enzyme, and the second is to engineer a herbicide-detoxifying pathway in to the plant [ 19]. Examples of the first approach include glyphosate and acifluorfen tolerance. Glyphosate is a board-spectrum, rapidly biodegradable herbicide with low toxicity to animals and humans. Glyphosate binds specifically to the enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS), which is responsible for synthesis of aromatic amino acids in plants. When a bacterial gene encoding glyphosate-insensitive EPSPS is used to create transgenic plants, tolerance to glyphosate is achieved [61]. Transgenic plants tolerant to the herbicide acifluorfen, which inhibits chlorophyll biosynthesis, have been produced through over-expression of the target enzyme involved in chlorophyll biosynthesis [62]. In comparison, resistance to glufosinate and bromoxynil is based on the second approach. By introducing genes that enhance metabolism of these herbicides the active compounds are converted to products that are non-toxic to the crop [5]. Similarly, in the case of the herbicide Ignite/Basta, the bar resistance gene from Streptomyces hygroscopicus was used to detoxify the herbicide. The bar gene was introduced into tobacco, tomato, potato, oilseed rape, alfalfa, sugarbeet, aspen and poplar plants [63-67]. Critics of herbicide resistant crops fear the overuse of herbicides and the development of herbicide resistant weeds. However, herbicide resistant weeds can be controlled by rotating crops with different transgenic modes-ofaction. Availability of various environmentally friendly herbicides and their corresponding resistance genes now make effective crop rotation practices possible. Abiotic stress tolerance Abiotic stresses such as salt, drought, flooding, extreme temperatures, and oxidative stresses often diminish plant growth and reduce yield. Agricultural productivity could be increased dramatically if crops were redesigned to better cope

246 with environmental stresses. Transgenic regulations of solutes such as mannitol and proline have been used to promote stress tolerance in plants [68]. Recently, Hayashi et al. [69] used the choline oxidase (codA) gene cloned from a soil bacterium to produce transgenic Arabidopsis plants. Expression of the Choline oxidase gene increases glycinebetaine production, which preserves the osmotic balance in cells and is known to help plants acclimate to various stresses. The codA transgenic plants were tolerant to high salt and low and high temperatures, and were able to maintain photosynthetic activity under stress. Studies with rice confirmed that chloroplast targeting of the codA gene is a more effective way to enhance tolerance to these abiotic stresses [70]. Van Camp et al. [71] demonstrated that overproduction of a superoxide dismutase (SOD) gene resulted in increased chilling tolerance in plants. This is presumably due to the fact that many stress conditions (including cold, high light intensity and pathogens) exert damaging effects through the production of reactive oxygen species. Enzymes, such as SOD, catalases and peroxidases, are involved in neutralizing the effects of reactive oxygen species [191. Plants can tolerate salt by compartmentalizing sodium into the vacuole. Apse et al. [72] demonstrated that over-expression of a vacuole Na+/H÷ antiporter could enhance salt tolerance in Arabidopsis. While wild-type plants were stunted, chlorotic, and had smaller leaves in response to high salt concentrations, the transgenic plants grew well and set seed normally in soil irrigated with up to 200 millimolar saline water. Salt tolerance was correlated with increased expression levels of the Na+/H÷ antiporter gene and increased Na÷/H÷ antiporter activity in the vacuole. These results show that it is possible to redesign plants to remain productive under saline conditions, thus, opening the possibility of modifying economically important crops to grow in saline conditions. The fatty acids composition of cellular membranes, particularly chloroplast membranes, is a critical factor in plant tolerance to temperature stresses. Plants grown in colder temperatures have higher concentrations of trienoic fatty acids in the chloroplast membranes while many desert and evergreen plants have lower levels of trienoic fatty acids. On this basis, Murakami et al. [73] used gene transfer techniques on tobacco to silence the gene encoding for chloroplast omega-3 fatty acid desaturase, the enzyme that produces trienoic fatty acids. These transgenic tobacco plants contained lower trienoic fatty acid concentrations and were able to withstand higher temperatures compared to the wild-type plants. In contrast, expression of a broad-specificity delta 9-desaturase gene in tobacco led to a reduced saturated fatty acid content in most membrane lipids and an increased tolerance to chilling stress [74]. The delta 9-desaturase gene was cloned from the cyanobacterium Anacystis nidulans and targeted into plastids by the transit peptide of the pea RuBisCO small subunit. The delta 9-desaturase introduces a cis-double bond at the delta-9 position of both 16 and 18 carbon saturated fatty acids, both fatty acid species are associated with many types of cellular membrane lipids. "Master switch" genes, transcription-regulating-factor genes that normally control expression of a number of genes, have been used to improve plant tolerance

247 to various stresses. Using a cis-acting element called the Dehydration Response Element (DRE), a regulatory element found in the promoter sequences of many drought- and cold-induced stress genes, Kasuga et al. [75] cloned a DRE-binding protein (DREB 1A) gene from Arabidopsis. When the coding sequence of DREB 1A gene was placed under the control of a stress-inducible promoter (rd29A) and the fusion gene was delivered into Arabidopsis, the transgenic plants showed a slight growth retardation in the absence of stress but were otherwise similar to the wildtype. In the transgenic plants, a number of stress genes were rapidly and simultaneously expressed in response to stress, and consequently the transgenic plants showed a greater tolerance to drought, salt, and freezing temperatures. Thomashow and his colleagues cloned a "master switch" gene from Arabidopsis called CBF1 that regulates expression of a set of low temperature inducible genes [76]. Transgenic Arabidopsis plants over-expressing CBF1 displayed an enhanced tolerance to low temperature [76]. The transgenic plants survived exposure to lethal low temperatures while the nontransgenic plants turned yellow and quickly died under the same experimental conditions (-5°C for two days).

Photosynthetic activities Increasing C O 2 fixation or reducing photorespiration has been a dream for breeders because of the potential to enhance crop productivity. Many important crops, including rice and wheat, assimilate atmospheric CO 2 by the C3 pathway of photosynthesis. The C3 plants assimilate CO 2 into sugars but part of the potential for sugar production is lost to respiration, termed photorespiration. Photorespiration can reduce net carbon gain and productivity of C3 plants by as much as 40%. In contrast, C4 plants, with some modifications in leaf anatomy and a mechanism to concentrate atmospheric CO 2 in the leaf, exhibit high photosynthetic rates, fast growth and highly efficient use of water and minerals. Ku et al. [177] transformed rice plants with maize genes encoding the C4 photosynthetic pathway enzymes phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK), or NADP-malic enzyme (ME). PEPC and PPDK transgenic rice plants exhibit higher photosynthetic capacity than untransformed plants, mainly due to an increased stomatal conductance. Preliminary field trials conducted in China and Korea produced 10-30% and 30-35% increases in grain yield for PEPC and PPDK transgenic rice plants, respectively [78]. These results were surprising since only one of the maize C4 pathway enzymes was present in each transgenic rice line and one would not expect that the activity of a single C4 enzyme would be sufficient to concentrate CO 2 as in a typical C4 plant. It is believed that increased synthesis of organic solutes by the enzymes in the guard cells may be responsible for enhanced stomatal conductance of CO 2 [78]. Further enhancement of the photosynthetic capacity may be possible if all three maize C4 genes can be properly engineered into a single transgenic line. If successful, this technology would hold great potential for improving production in a wide array of agronomic, horticultural, and forestry crops.

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Dwarfism In the 1960s, substantial increases in world wheat and maize production resulted from the development of dwarf varieties that partitioned a greater percentage of their biomass into grain at the expense of straw. These varieties were also more resistant to wind and rain damage. Recently, the molecular basis of the dwarf phenotype was discovered as Peng et al. [79] demonstrated that mutations within the gibberellin insensitive (GAI) gene caused dwarfism in wheat and maize. The plant hormone gibberellin is an important regulator of stem elongation and dwarf plants have a reduced response to gibberellin [80]. Six different orthologous dwarfing mutant alleles encode proteins that are altered in a conserved amino-terminal gibberellin signaling domain [79]. Transgenic rice plants expressing a mutant GAI gene show reduced response to gibberellin and are dwarf, suggesting that a mutant GAI gene may be used commercially to reduce height growth and increase crop productivity.

Quality traits Amino acid composition A plant-based diet is healthy, but most plants are deficient in one or more of the amino acids essential to humans and animals. For instance, cereal seed proteins are deficient in lysine and tryptophan, while legume seed proteins are deficient in cysteine and methionine. Thus, grain legume fed to pigs and poultry is supplemented with synthetic methionine, which these non-ruminant animals can then convert to cysteine. Adding a synthetic amino acid to animal feed is expensive but classical plant breeding has had little success in changing the amino acid composition of plants [81]. Previously it was thought that storage proteins had no catalytic activity and that this problem could be solved by introducing DNA sequences encoding for nutritionally valuable amino acids within the coding sequence of a seed storage protein gene. Unfortunately, this solution was not as straightforward as originally thought. For instance, when six methionines were inserted into phaseolin, a seed protein of the common bean Phaseolus vulgaris, the altered protein was synthesized in transgenic seeds but the protein was unstable and degraded rapidly [82]. Falco et al. [83] took a different strategy to increase the amount of lysine in seed crops. Two key enzymes, aspartokinase (AK) and dihydrodipicolinic acid synthase (DHDPS) in higher plants are subject to natural feedback mechanisms that control lysine synthesis. However, the bacterial DHDPS and AK genes are insensitive to lysine feedback. When canola expressed the bacterial DHDPS and AK genes under the control of a seed- specific phaseolin promoter, free lysine concentrations in the transgenic seeds increased by more than 100-fold. The proportion of lysine in total seed amino acids (12%) was nearly twice that of non-transgenic canola. Another approach is to transfer a gene encoding a protein rich in an essential amino acid

249 into a target species from another plant species. Altenbach et al. [84] used a Brazil nut gene, encoding a 2S storage protein, which contains 18% methionine, to transform canola. Expression of the nut gene resulted in canola seeds with a 30% increase in methionine. Unfortunately, because the 2S storage protein is a major Brazil nut allergen, some individuals are likely to be allergic to the transgenic seeds and the research was discontinued [85]. Molvig et al. [86] avoided this problem by using the seed 2S albumin (2SA) gene from sunflower (a seed that is not known to cause allergic reactions) to engineer lupin. Five percent of the total protein in the transgenic lupin seeds was the 2SA protein and the transgenic seed had 95% more methionine than the untransformed control line. Rats fed on transgenic lupin meal gained 17% in body weight within eight days while the reference animals receiving standard lupin food increased their weight by only 3%. The study also demonstrated that the transgenic lupin seeds contained 23% higher utilizable protein than the control, thus the transgenic protein was digested and the nitrogen was well absorbed in the animal body. Similarly, soybeans transformed to express a methionine-rich zein protein from maize had an 80-100% increase in methionine content [5].

Fatty acids Vegetable oils are important for both human nutrition and industrial use, with a current worldwide annual production of approximately 65 million metric tons. Although the quality of vegetable oils has been dramatically improved by traditional breeding, nutritional value, oxidative stability, and functionality of most vegetable oils are still not ideal [31 ]. Two major transgenic approaches have been used to change oil composition: alter the concentration of a major fatty acid, or add a unique fatty acid component. The major fatty acids of vegetable oils include palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3). By manipulating enzymatic activities in the rate-liming steps of lipid biosynthesis, the relative content of the three maj or fatty acid constituents ( 16: 3, 18:2 and 10: 3) can be altered to create new oils. One example of oil modification through transgenic technology is the development of high-oleic-acid soybean oil through antisense suppressing and/or co-suppression of oleate desaturase [5]. The new oil has an oleic acid content of 80% or higher, compared with 24% in normal soybean oil. The oil has a good oxidative stability and is targeted for frying or cooking applications [5,87]. Also, an effort has been made to develop high-stearate canola, soybean and corn oils using gene transfer technology [5,59,88-91]. These high stearate oils can be used to replace hydrogenated oils in margarine, fluid shortening, and confectionery items to produce zero trans-products. Industrial oils represent another important target for transgenic improvement because of the limitations of conventional breeding. Oils with short-, medium-, or very-long-chain fatty acids and those having double bonds at an unusual position or carrying a hydroxy or epoxy group are targeted mainly for industrial uses. These specialized oils often sell at prices considerably higher than common oils

250 since their production entails great cost. Therefore, introduction of unconventional fatty acids into common oilseed crops can reduce the cost of these materials and increase the value of oilseeds. Since a number of unusual fatty acids accumulate in the seed oil of various plants, one strategy is to transfer the genes encoding the key biosynthetic enzymes into oilseed crops [92,93]. A well-known example of this strategy is the laurate (12:0) thioesterase gene from the California bay laurel tree that was introduced into canola to generate laurate-rich oil [94]. Transgenic canola seeds contained 38% or more laurate while traditional canola oil was devoid of laurate. Acetylenic and epoxy fatty acids are also critical raw materials used in the production of polymers such as plastics and certain chemicals. These unusual fatty acids are modified forms of fatty acids commonly present in edible oils, however, they are derived from either non-renewable petroleum or chemically processed vegetable oils. Lee et al. [95] used genes involved in the synthesis of acetylenic and epoxy fatty acids cloned from Arabidopsis to produce transgenic Arabidopsis seeds that contained up to 15% epoxidated vernolic acid (by weight), or up to 25% acetylenic (crepenynic) acid. Control plants contained not even a trace of these compounds in seeds. Polyhydroxyalkanoates produced by bacterial fermentation have been used to produce biodegradable plastics. However, production of bacterial PHAs is substantially more expensive than synthetic plastics made from fossil fuels. Transgenic Arabidopsis plants produced polyhydroxybutarate (PHB) when the biosynthetic genes for PHB from A. eutrophus was inserting into the chloroplast genome [96]. Although the concentration of PHB reached 10 mg/g fresh weight, no major deleterious effects on growth or seed yield were observed. The plant-based production of PHBs could dramatically reduce production costs for biodegradable plastics. These studies clearly demonstrate that plants could provide a low-cost, renewable, biodegradable source of these high-value specialty products.

Carbohydrates Considerable success has been reported in modifying the starch content and/or structure of starch in several food crops [97]. Three enzymes are important in starch biosynthesis, ADP glucose pyrophosphorylase (ADPGPP), starch synthase, and branching enzyme. One strategy used to increase starch concentration in potato was to circumvent the normal feedback regulation of ADPGPP by inserting a feedback-insensitive bacterial enzyme [31,98]. An average increase in starch concentration of 20-30% was achieved in transgenic potato tubers. These transgenic tubers produced a fried product with more potato flavor, improved texture, reduced calories, and a less greasy taste [98]. The same strategy has been applied to tomato since a high-solids tomato is desirable for production of tomato sauces. The ability to alter starch composition or to design new types of starch will enhance the use of starches as thickeners, bulking agents, caloric sources, and stabilizers in food products [32].

251 Impressive progress has also been made in altering the sugar composition in crops. For instance, fructans are of commercial interest as replacements for high caloric sweeteners and fats. Short chain fructans have the same sweet taste as sucrose but without the caloric load since humans lack the fructan-degrading enzymes necessary for digestion. The same is true for the longer chain fructans, which form emulsions with fat-like textures. Commercial production of small fructans via enzymatic conversion of sucrose is extremely expensive, resulting in a final cost that prohibits their use in general foodstuffs. In transgenic sugar beet plants that expressed the 1-sucrose : sucrose fructosyl transferase gene from Jerusalem artichoke, short chain fructans accounted for about 40% of taproot dry weight [99]. In this study, over 90% of the endogenous sucrose had been converted to small fructan molecules when compared with wild-type lines. Thus, sugar beets were converted into "fructan beets" without a discernible impact on plant growth or phenotype. Similarly, expression of a bacterial fructosyl transferase gene in sugar beets resulted in increased production of long chain fructans [100]. As an added benefit, the plants expressing the bacterial gene performed significantly better under drought stress when compared to wild-type plants. Increased drought tolerance was not surprising since it has long been suggested that fructans (of all lengths) have a role in drought stress tolerance in species that naturally accumulate these compounds.

Seedless fruits The seedless trait is highly desirable in many fruit crops but traditional methods of producing seedless fruits are expensive and laborious. It has been believed that using transgenic technology to produce seedless fruits from woody plants and vegetables will be one of the five very important areas for crop improvement within next 10-20 years [ 101 ]. Recently, transgenic techniques have been used to produce seedless eggplant, tomato, and watermelon ([102-105] and Y. Li, unpublished data). The seedless trait was achieved by manipulating either auxin concentration or auxin sensitivity of specific cells. It was previously demonstrated that seedless fruits could be induced in many species by applying auxin to developing flower [106]. In the case of tomato, Li et al. ([105] and Y. Li, unpublished data) observed that the size, weight, and concentrations of acids, sugars, and solids of the transgenic seedless fruits were significantly increased compared to those of the wildtype seeded fruits. Production of seedless fruits offers many advantages. First, poor pollination is a major cause of poor fruit set and undersized fruits in many crops including greenhouse and field-grown tomatoes. Diseases and environmental stresses such as low and high temperatures, low light intensity, and drought cause poor pollination and a reduction in fruit yield. Production of high-yielding fruits independent of pollination can therefore reduce or eliminate the yield reduction associated with poor pollination. Second, the seedless fruit technology can be used to produce consumer-desirable seedless fruits, such as watermelons, cucumbers, green peppers and grapes. Third, for tomato, a significant increase in solids

252 contents is beneficial to the processing industry. The tomato is 95% water with the remaining 5% consisting of pulp, seeds, and soluble solids (mostly sugars, organic acids, and flavor compounds). During processing of tomato products such as ketchup, paste and sauce, the seeds and most of the water are removed. To a large degree the price of these tomato products is determined by the cost of removing water. Thus, increasing the solid content of tomato from 5% to 6% would save the US tomato industry about US$75 million a year [107]. Postharvest qualities

Senescence is a natural developmental process in plants. In the case of fruits, senescence is called ripening and it is associated with desirable changes in the fruit such as softening, conversion of starch to sugar, reduction in chlorophylls and increases in red or yellow pigments and aromatic compounds. In tomato, genes for enzymes involved in cell wall degradation (polygalacturonase and pectinesterase) and ethylene synthesis (ACC synthase) have been identified and used to alter fruit characteristics [108]. The best examples include expression of anti-sense RNAs to the rate-limiting enzyme in the biosynthetic pathway of ethylene, 1-aminocyclopropane-l-carboxylate synthase [109], and cell wall degradation enzymes polygalacturonase and pectinesterase [110]. For example, by suppressing the cell wall-degrading enzyme, polygalacturonase, ripening of the tomato is significantly delayed. This allows the fruit to stay on the vine longer for greater flavor development, and also to extend the shelf-life [93]. Klee et al. [111] inhibited fruit ripening by removing ACC, an immediate precursor to ethylene, from plant cells. A gene encoding ACC deaminase cloned from a soil bacterium was introduced into tomato plants and the expression of the ACC deaminase gene resulted in a dramatic reduction in ethylene synthesis in the transgenic plants and significant delays in fruit ripening. Mature fruits that express the ACC deaminase gene remained firm for at least 6 weeks longer than the nontransgenic control fruit. Delaying senescence or extending shelf life of flowers, leaves and whole plants is of great commercial importance. Initially, delayed leaf senescence was observed in transgenic plants expressing the IPT gene under control of either a heat inducible gene promoter or an auxin inducible gene promoter [112,113]. The IPT gene encodes for isopentenyltransferase, the enzyme that catalyzes the rate-limiting step in cytokinin biosynthesis. In tobacco plants transformed with IPT under the control of an auxin inducible promoter (the SAUR promoter), Li et al. [ 113] observed that the IPT transgenic leaves remained green and healthy even when detached and held in the dark for 6 months. Later, Gan and Amasino [114] were able to autoregulate cytokinin production in transgenic plants by using a senescence-specific promoter to control expression of the IPT gene. Transgenic tobacco plants expressing IPT under the control of the senescence-specific promoter did not exhibit the developmental abnormalities usually associated with constitutive IPT expression. The leaves of the transgenic tobacco plants exhibited a prolonged, photosynthetically active life-span. More recently, Klee's group used the etrl gene of Arabi-

253 dopsis that encodes for a mutated receptor and confers dominant ethylene insensitivity, to extend shelf life of several crops [115]. Expression of the etrl-1 gene delayed fruit ripening, flower senescence, and flower abscission in tomato and petunia. Harvested fruits from transgenic tomato plants retained their original golden yellow color even when stored for 100 days while tomato fruits from nontransformed plants turned red, softened, and started to rot soon after harvest. Similarly, when petunia plants expressing the etr-1 gene were exposed to ethylene, the flowers stayed fresh for nine days while flowers on non-transformed petunias wilted within just three days. Vitamins and other micro-nutrients The health-protective properties of vitamins and micronutrients have received much attention. In recent years, progress has been made to improve the nutritional value of food crops. Vitamin E is a powerful antioxidant that detoxifies unstable oxygen molecules, the culprits linked to many ailments including aging. Vitamin E also reduces the destruction of LDL cholesterol and is essential for numerous bodily functions. Among the four types of vitamin E found in food, alpha-tocopherol has the highest vitamin E activity but is less abundant than it's precursor gamma-tocopherol [116]. In soybean oil, which provides 80% of the edible oil consumed in the U.S., the alpha-version accounts for only 7% of the total tocopherol pool. When a gamma-tocopherol methyltransferase gene cloned from Arabidopsis was over-expressed in seeds of Arabidopsis, an 80-fold increase in the level of alpha -tocopherol was observed - accounting for more than 95% of the total tocopherol pool in the seeds [117]. The methyltransferase enzyme converts gamma-tocopherol to alpha-tocopherol. Vitamin A is also essential to human development. Widespread dietary deficiency of vitamin A in rice-eating Asian countries causes an eye disease called xerophthalmiar. This disorder affects five million children in South East Asia every year, and 250,000 of them eventually become blind. Improved vitamin A nutrition would alleviate this serious health problem and could also prevent up to two million infant deaths. Beta-carotene, the carotenoid that gives tomato, carrots and sweet potatoes their orange color, is a precursor to vitamin A. Both vitamin A and beta-carotene (provitamin A) are antioxidants which neutralize cancer-causing compounds known as free radicals, thus destroying their ability to damage cells [118]. Rice is a staple that feeds nearly half the world's population, but milled rice is devoid of beta-carotene and its' precursors. Transgenic rice plants over-expressing phytoene desaturase and lycopene cyclase genes (cloned from a flower plant called daffodil) accumulate a large amount of beta-carotene in the seed endosperm [119,120]. This development has important implications for the long-term prospects of overcoming worldwide vitamin A deficiency. Transgenic technology has also been used to increase the iron content of rice seeds. Iron is the most commonly deficient micronutrient in the human diet and iron deficiency affects an estimated 1-2 billion people worldwide. Anemia char-

254 acterized by low hemoglobin is the most widely recognized symptom of iron deficiency, but there are other serious problems such as impaired learning ability in children, increased susceptibility to infection and reduced work capacity. Rice feeds half of the world, and is eaten every day in those parts of the world where iron deficiency is most prevalent [121]. Iron enhanced rice plants have been developed by expressing the gene for ferritin, an iron-rich soybean storage protein, under the control of an endosperm-specific promoter in rice [ 122]. Seed grain from transgenic rice plants contained three times more iron than normal rice. Iron-fortified rice could be selectively targeted to segments of the population at greatest risk of iron deficiency such as infants, school children and pregnant mothers. In addition to improving the nutritional value of rice, the ferritin gene also appears to help the plant overcome its' own iron deficiency problem. Either too much or too little iron in the soil can cause decreased plant productivity. Tobacco plants expressing the ferritin gene grew well under conditions of wide ranging iron concentrations in the soil.

Concluding remarks In this limited review, many exciting discoveries of the last 17 years were not discussed, and several active areas such the use of transgenic plants for phytoremediation and as bioreactors were omitted. Nevertheless, from this review it is evident that transgenic plant technology has great potential and that it is just beginning to deliver on that potential to increase food production, to improve food quality, and to address specific industrial and medicinal needs while preserving the environment for future generations. With rapid world population growth, declining availability of fresh water and arable land, there is a pressing need to dramatically improve crop productivity and nutritional quality without dramatically increasing the use of agricultural chemicals and lands. Transgenic plant technology combined with traditional breeding methods can offer a solution. Similar to plant breeding, transgenic technology introduces new genes to crops but in a more selective, precise, and controlled manner. Transgenic plant technology holds great promise for humankind because it has the potential to deliver another "green revolution" with dramatic increases in crop productivity and quality. Furthermore, transgenic plant technology is also a source of innovation for the food processing, chemical, and pharmaceutical industries. Like many other new technologies, transgenic plant technology also faces some challenges and problems as briefly mentioned in this review. However, these potential problems should be minimized as the technology evolves. To take full advantage of this exciting new technology, we need to overcome many technical, economic and social constraints, such as the limited investment capital, the relatively long time required for commercialization of new transgenic products, the risk associated with uncertain profitability of the products, the complex issue of intellectual property rights, safety issues, legislation and regulation, international trade, and consumer acceptance [123]. Based on the worldwide need for food, the

255 huge potential of the technology, and the initial commercial success of some first generation transgenic products, it is certain that transgenic plant technology will play a major role in improving quality-of-life in the new century.

Acknowledgement The authors thank financial support from US-NASA, USDA, Connecticut Innovation Program, US-DOE/CPBR and the Storrs Agricultural Experiment Station. This is scientific contribution number 1986 of the Storrs Agricultural Experiment Station.

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