TY - JOUR
AU - Dunwell, Jim M.
AB - Abstract Transgenic crops are now grown commercially on several million hectares, principally in North America. To date, the predominant crops are maize (corn), soybean, cotton, and potatoes. In addition, there have been field trials of transgenics from at least 52 species including all the major field crops, vegetables, and several herbaceous and woody species. This review summarizes recent data relating to such trials, particularly in terms of the trends away from simple, single gene traits such as herbicide and insect resistance towards more complex agronomic traits such as growth rate and increased photosynthetic efficiency. Much of the recent information is derived from inspection of patent databases, a useful source of information on commercial priorities. The review also discusses the time scale for the introduction of these transgenes into breeding populations and their eventual release as new varieties. Genetic modification, field trials, photosynthesis, chlorophyll, stress responses Introduction Over the last few years, transgenic crops have moved from being a laboratory curiosity to providing new varieties grown on large areas throughout the world. Despite opposition to this technology in some countries, the economic benefits to the farming community has ensured a rapid acceptance in North America, and most information on the development of these varieties, and the testing of potential ‘second generation’ products is available from sources in that region. This review is designed to summarize data relating to the commercial growth of transgenic crops, before considering the research trends, particularly those involved in the modification of agronomic performance. Data will be provided from a range of sources including details of applications for the field testing of experimental material, and from recent patent applications which make claims to novel approaches to this problem. Throughout this review emphasis will be given to recent publications, which should be consulted if additional information is required. Present status of transgenic crops The first transgenic plant product marketed commercially was the well‐known ‘Flavr Savr’ tomato which had been modified to contain reduced levels of the cell wall softening enzyme polygalacturonase. Tomato purée with a similar type of modification has been on the market in the UK since February 1996. Since that time, however, there has been a massive expansion in the growth of transgenic field crops, particularly maize, soybean, oilseed rape, and cotton, such that in North America transgenic varieties now represent the majority of the acreage of these crops. For example, it is now estimated that 70% of the Canadian oilseed rape crop in 1999 will be genetically modified. Most of the varieties grown to date have been modified with genes for herbicide or insect resistance, both of which provide a significant economic benefit for the farmer. Material from these modified crops, particularly soybean and maize, is now imported into the European Union in large quantities. However, in several European countries there is now considerable consumer resistance to food products containing such GM constituents, either in the native form or as processed derivatives (e.g. soya lecithin). In the UK this resistance seems to be based primarily on a mistrust of the government regulatory process, allied to a strong desire for choice (to date the commodity GM products have not been segregated from their non‐GM equivalent). These concerns have led to many retailers removing GM soya and maize products from sale, though enzymes and vitamins from GM microbes remain as common components of many foods and drinks. Pending the outcome of ‘farm scale’ trials designed to monitor the environmental impact of GM crops, there has been an associated delay in the granting of permits for the growth of such crops for sale. Summary of applications for field testing of experimental material, 1987–1999 The most complete set of data relating to the growth of transgenic crops is that collated by the US regulatory authority APHIS (Animal and Plant Health Inspection Service) and access to this information is available from http://www.aphis.usda.gov/bbep/bp/database.html., and summarized versions at http://www.nbiap.vt.edu/cfdocs/fieldtests1.cfm. Information on a more general basis is summarized at http://www.isaaa.cornell.edu. Examination of these statistics shows the retrospective increase in trials from 1986 to date (many of which led to commercial products) and it also provides a view of the present range of experimental materials, some of which will reach the market in the next decade. There are several important trends that can be seen from these data. First, the number of US approved trials has increased from 9 in 1987 to 1045 in 1998 (Table 1). Secondly, there is an unsurprising dominance of a small number of biotechnology companies (Table 2); indeed the number of different companies has now been reduced still further by the reported acquisition in March 1999 of Pioneer Hi‐Bred by Du Pont. Thirdly, the range of transgenic species being tested has increased from the initial small number, principally tomato, tobacco, corn, and soybean to a list of more than 50, including row crops, fruit, vegetables, trees, and ornamental species (Table 3). Similarly, there has been a great expansion in the number of different genes introduced and the traits encoded by them. In the first few years there was a great emphasis on single gene traits, especially herbicide and insect resistance. For example, data for trials in Canada show that in 1994, 72% of trials concerned herbicide tolerance, and that this figure had declined to 24% by 1998. This has now been superseded by experimental material expressing quality traits such as alterations in seed protein and carbohydrate composition, and also by more complex physiological changes which will form the basis of the analysis below. Table 1. Number of approved field trials of transgenic crops in the US for the period 1987–1998, taken from the Environmental Releases Database For further details see http://www.nbiap.vt.edu/cfdocs/biomon1.cfm. Year   Number of trials   1987  9  1988  18  1989  38  1990  58  1991  107  1992  150  1993  306  1994  593  1995  681  1996  626  1997  743  1998  1045  Year   Number of trials   1987  9  1988  18  1989  38  1990  58  1991  107  1992  150  1993  306  1994  593  1995  681  1996  626  1997  743  1998  1045  View Large Table 2. Summary of US field trial applications by institution (first 25) Institution   Total   Issued   Pending   Denied   Monsanto  1097  146  7  46  Pioneer  564  43  6  30  AgrEvo  310  14  4  14  Du Pont  309  13  DeKalb  190  9  1  7  Calgene  170  73    2  ARS  112  35  1  3  N. King  88  11    3  Upjohn  85  63  Asgrow  81  26    1  DNAP  68  50  15  1  Cargill  60  11  1  1  Agracetus  60  2    1  Frito Lay  58  18    2  Seminis  54  18  PetoSeed  51  10  Novartis  51      2  PGS  46      1  Ciba‐Geigy  45  10  Univ. Idaho  43  14  1  Zeneca  41  2    3  Delta Pine  41  Agritope  40  6  2  2  NC State U.  38  6  Holdens  38  9  Institution   Total   Issued   Pending   Denied   Monsanto  1097  146  7  46  Pioneer  564  43  6  30  AgrEvo  310  14  4  14  Du Pont  309  13  DeKalb  190  9  1  7  Calgene  170  73    2  ARS  112  35  1  3  N. King  88  11    3  Upjohn  85  63  Asgrow  81  26    1  DNAP  68  50  15  1  Cargill  60  11  1  1  Agracetus  60  2    1  Frito Lay  58  18    2  Seminis  54  18  PetoSeed  51  10  Novartis  51      2  PGS  46      1  Ciba‐Geigy  45  10  Univ. Idaho  43  14  1  Zeneca  41  2    3  Delta Pine  41  Agritope  40  6  2  2  NC State U.  38  6  Holdens  38  9  View Large Table 3. List of plant species for which field trials have been conducted in the US Species   Number of releases   Corn  2046  Potato  502  Soybean  440  Tomato  439  Cotton  297  Oilseed rape  144  Tobacco  134  Melon  101  Sugar beet  79  Squash  64  Rice  57  Wheat  55  Creeping bentgrass  31  Poplar  27  Alfalfa  26  Cucumber  20  Grape  20  Strawberry  16  Lettuce  15  Sugarcane  14  Walnut  13  Sunflower  12  Apple  11  Barley  10  Brassica oleracea  10  Peanut  10  Carrot  9  Papaya  9  Pepper  9  Arabidopsis  7  Petunia  7  Sweet potato  7  Atropa belladonna  6  Eggplant  6  Rubus idaeus  6  Watermelon  6  Pea  4  Cucurbita texana  3  Pelargonium  3  Sweetgum  3  Chrysanthemum  2  Chicory  2  Kentucky bluegrass  2  Plum  2  Amelanchier  1  Cranberry  1  Gladiolus  1  Oat  1  Onion  1  Pine  1  Pineapple  1  Populus deltoides  1  Total no. species  52  Species   Number of releases   Corn  2046  Potato  502  Soybean  440  Tomato  439  Cotton  297  Oilseed rape  144  Tobacco  134  Melon  101  Sugar beet  79  Squash  64  Rice  57  Wheat  55  Creeping bentgrass  31  Poplar  27  Alfalfa  26  Cucumber  20  Grape  20  Strawberry  16  Lettuce  15  Sugarcane  14  Walnut  13  Sunflower  12  Apple  11  Barley  10  Brassica oleracea  10  Peanut  10  Carrot  9  Papaya  9  Pepper  9  Arabidopsis  7  Petunia  7  Sweet potato  7  Atropa belladonna  6  Eggplant  6  Rubus idaeus  6  Watermelon  6  Pea  4  Cucurbita texana  3  Pelargonium  3  Sweetgum  3  Chrysanthemum  2  Chicory  2  Kentucky bluegrass  2  Plum  2  Amelanchier  1  Cranberry  1  Gladiolus  1  Oat  1  Onion  1  Pine  1  Pineapple  1  Populus deltoides  1  Total no. species  52  View Large Summary of recent field trial applications and patent applications Much valuable research data, from both academic and commercial laboratories, are published in patent applications or patents (in the case of the USA) before being published in conventional scientific journals. This timing, which is designed to ensure the maximum level of confidentiality required for the patenting process, means that much important information is overlooked by the academic scientist who is often only concerned with material available in their institution's library or in the usual databases. Until comparatively recently it was difficult and expensive to consult the patent databases. Now, however, much of the relevant information is available on‐line at no cost. Useful sites with patent information include the US Patent Office (http://patents.uspto.gov/access/search‐adv.html), an extensive site supported by IBM (http://www.patents.ibm.com/), a World Intellectual Property Organization database (http://pctgazette. wipo.int/), and a site provided by Derwent, one of the major commercial providers of patent information (http://www.derwent.co.uk/plweb‐cgi/fastweb?searchform+view1). All these sites have searchable databases which can provide either summary details, or images of complete applications in some cases. In addition to providing an up‐to‐date review of progress, they are a very useful means of avoiding repetition of research already completed elsewhere. They should therefore be consulted on a regular basis. The summary below has been assembled from a recent survey of these databases, together with the field trials database referred to above. The subdivision of information is necessarily somewhat arbitrary in nature, but it does provide a framework in which to demonstrate the range of material and methods being produced and tested at present. Details of the many enabling technologies such as methods for transgenic plant production (Adams et al., 1999), and promoters providing leaf‐specific (Sonnewald et al., 1998) or general (Baszczynski et al., 1998) expression will not be considered in detail, though of course they do have significant commercial importance in determining the cost of applying proprietary techniques in the development of a particular transgenic product. Photosynthetic enhancement and yield increase There is a persistent hope amongst plant breeders, whether they be concerned with conventional or transgenic varieties, that the photosynthetic efficiency of crops can be improved. After all, the first three priorities for a breeder are yield, yield, and yield, and it is assumed that improved energy capture can be translated into greater harvestable yield. Although there is still little reliable information that relates transgenic modification of specific photosynthetic genes to performance under agricultural conditions, results from many preliminary tests have been published. The scale of tests underway is demonstrated in Table 4, which summarizes those US field tests of crops claimed to have an enhanced level of photosynthesis, and Table 5 which included the more numerous trials of plants with ‘enhanced yield’. Despite the incomplete details of some applications due to the presence of CBI ( confidential business information) aspects, it is clear that many different types of transgenics have now reached the field stage of testing, and that more information of agronomic performance should be available soon. Many of the specific transgenes represented in these trials are discussed below. Amongst the most radical experimental approach is the concept of introducing gene(s) involved in the C4 type of photosynthesis into a C3 plant such as Arabidopsis (Ishimaru et al., 1997) and potato (Ishimaru et al., 1998). The justification for this approach is based on the fact that C3 photosynthesis suffers from O2 inhibition due to the oxygenase reaction of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco), and the subsequent loss of CO2 from photorespiration. In contrast, C4 plants such as maize and many weedy species, have evolved a biochemical mechanism to overcome this inhibition. A key feature of this mechanism is the activity of phosphoenolpyruvate carboxylase (PEPC), an enzyme that fixes atmospheric CO2 in the cytosol of mesophyll cells. Using an Agrobacterium‐mediated transformation system, the intact maize PEPC has recently been transferred into the C3 plant rice (Matsuoka et al., 1998a, b; Ku et al., 1999). Analysis of the transgenics produced showed that PEPC activity in some plants was two‐ to three‐ times higher than in maize, with the enzyme comprising up to 12% of soluble protein. Physiologically, these plants exhibited reduced O2 inhibition of photosynthesis and had photosynthetic rates comparable to those of control, untransformed plants. Similar claims have been made previously (Arai et al., 1998), and a granted patent on the maize PEPC gene and its promoter has been published recently (Grula and Hudspeth, 1999). Investigations into the manipulation of the key photosynthetic enzymes, Rubisco, pyruvate phosphate kinase (PPDK) and NADP malate dehydrogenase (NADP‐MDH) in the C4 dicotyledonous species Flaveria bidentis have been reported (Furbank et al., 1997), whilst an alternative strategy to reduce photorespiration by manipulating catalase amounts in tobacco has also been described (Brisson et al., 1998). Several other approaches to yield improvement have been suggested. For example, it is claimed (Barry et al., 1998) that improved yield can be achieved by manipulation of fructose‐1,6‐bisphosphate aldolase (FDA), an enzyme that reversibly catalyses the reaction converting triosephosphate to fructose‐1,6‐bisphosphate. Leaves of transgenic plants which express the FDA from E. coli in the chloroplast show significantly enhanced starch accumulation and lower sucrose concentration; they also had a significantly higher root mass. In addition to improvements in the activity of specific photosynthetic genes, a more generic method of changing plant performance may be to modify plastid number, a feasible approach since genes controlling this character are now available (Osteryoung, 1998). Another proposed method (Grimm, 1998) for increasing chlorophyll content, without increasing plastid number, is the expression of a hybrid protein comprising a yeast gene encoding 5‐amino levulinic acid synthase and an N‐terminal transit sequence for the small subunit of carboxydismutase. Similarly, manipulation of chlorophyll a/b binding genes has been used to modify chlorophyll amounts, as well as other characteristics. It is claimed that overexpression of this protein leads to increases in plastid proteins and also plant biomass. Conversely, the down‐regulation of chlorophyll synthesis has also been claimed to have practical value, for example, in the degreening of oilseed rape seeds (Johnson‐Flanagan et al., 1998), a problem caused by sublethal freezing during seed maturation. This degreening was accomplished by anti‐sense reduction of the type I chlorophyll a/b binding protein of light‐harvesting complex II, whereby transgenic oilseed rape showed reductions of chlorophyll content from 260 μg g−1 fresh weight in controls to 152 μg g−1 in the modified plants. Manipulation of the promoter from this gene has also been used as a means to modify plant development (Kirchanski, 1998). Specifically, the gene encoding a phytochrome‐regulated transcription factor CCA1 that binds to the promoter of the Arabidopsis gene has been isolated and overexpressed. This leads to disruption of the normal circadian rhythms and extended vegetative growth, a beneficial effect for some crops. Other, non‐photosynthetic, approaches to increasing yield of both shoot and root include overexpression of a cyclin gene, preferably the cyc1a gene from Arabidopsis (Doerner and Lamb, 1998). Table 4 Summary of records for all US field trial applications subdivided for category ‘Photosynthesis enhanced’ All applications are from Monsanto. Further details including location and area of test site are available (see site in Table 1 caption). APHIS No.   Species   Gene(s)a   Phenotypeb   98‐128‐22N  Corn  CBI, NptII  PQ  98‐078‐01N  Corn  CBI, NptII  PQ  97‐322‐05Nc  Corn    AP  97‐302‐11N  Corn  CBI, NptII  AP  97‐289‐08N  Wheat  CBI, NptII  AP  97‐051‐05N  Corn  CBI, GUS, NptII  AP  96‐306‐02N  Corn  CBI, NptII  AP  96‐094‐04N  Corn  CBI, NptII  AP  APHIS No.   Species   Gene(s)a   Phenotypeb   98‐128‐22N  Corn  CBI, NptII  PQ  98‐078‐01N  Corn  CBI, NptII  PQ  97‐322‐05Nc  Corn    AP  97‐302‐11N  Corn  CBI, NptII  AP  97‐289‐08N  Wheat  CBI, NptII  AP  97‐051‐05N  Corn  CBI, GUS, NptII  AP  96‐306‐02N  Corn  CBI, NptII  AP  96‐094‐04N  Corn  CBI, NptII  AP  aCBI (Confidential business information), NptII (neomycin phosphotransferase II) provides resistance to kanamycin. bPhenotype is described as PQ (Product quality) or AP (Agronomic performance). cApplication ‘denied’. View Large Table 5 Summary of records for all field trial applications subdivided for category ‘Yield increased’; reverse chronological order; other details as above APHIS No.   Species   Applicant   Gene(s)a   Phenotypeb   99‐020‐06N  Rice  Monsanto  CBI, CBI  AP—Yield increased  99‐006‐14N  Tomato  Zeneca  Trehalase, NptII  PQ—Dry matter increased          PQ—Yield increased  98‐279‐06N  Corn  Pioneer  CBI, CBI  AP—Yield increased  98‐275‐04N  Corn  Pioneer  CBI, PAT  AP—Yield increased  98‐273‐13N  Corn  Pioneer  CBI, PAT  AP—Yield increased  98‐264‐04N  Cotton  Monsanto  CBI  AP—Yield increased  98‐254‐11N  Rapeseed  Calgene  CBI, SPS, NptII  AP—Yield increased  98‐252‐07N  Cotton  Monsanto  CBI  AP—Yield increased  98‐107‐01N  Corn  Pioneer  CBI, PAT  AP—Yield increased  98‐078‐03N  Tomato  Zeneca  Trehalase antisense,  PQ—Dry matter increased        NptII  PQ—Yield increased  98‐075‐29N  Tomato  Calgene  CBI, NptII  PQ—Yield increased  98‐064‐25N  Rapeseed  Calgene  SPS, NptII  AP—Yield increased  98‐064‐20N  Rapeseed  Calgene  SPS, NptII  AP—Yield increased  98‐037‐03N  Rice  Monsanto  CBI, CBI  AP—Yield increased  98‐035‐04N  Wheat  Monsanto  CBI, NptII  AP—Yield increased  97‐275‐02Nc  Wheat  Monsanto    AP—Yield increased  97‐275‐01Nc  Wheat  Monsanto    AP—Yield increased  97‐261‐03N  Corn  Pioneer  CBI, PAT  AP—Yield increased  97‐261‐02N  Corn  Pioneer  CBI, PAT  AP—Yield increased  97‐241‐04N  Rapeseed  Calgene  CBI, SPS, NptII  AP—Yield increased  95‐093‐14N  Cotton  Calgene  CBI, NptII  AP—Yield increased  APHIS No.   Species   Applicant   Gene(s)a   Phenotypeb   99‐020‐06N  Rice  Monsanto  CBI, CBI  AP—Yield increased  99‐006‐14N  Tomato  Zeneca  Trehalase, NptII  PQ—Dry matter increased          PQ—Yield increased  98‐279‐06N  Corn  Pioneer  CBI, CBI  AP—Yield increased  98‐275‐04N  Corn  Pioneer  CBI, PAT  AP—Yield increased  98‐273‐13N  Corn  Pioneer  CBI, PAT  AP—Yield increased  98‐264‐04N  Cotton  Monsanto  CBI  AP—Yield increased  98‐254‐11N  Rapeseed  Calgene  CBI, SPS, NptII  AP—Yield increased  98‐252‐07N  Cotton  Monsanto  CBI  AP—Yield increased  98‐107‐01N  Corn  Pioneer  CBI, PAT  AP—Yield increased  98‐078‐03N  Tomato  Zeneca  Trehalase antisense,  PQ—Dry matter increased        NptII  PQ—Yield increased  98‐075‐29N  Tomato  Calgene  CBI, NptII  PQ—Yield increased  98‐064‐25N  Rapeseed  Calgene  SPS, NptII  AP—Yield increased  98‐064‐20N  Rapeseed  Calgene  SPS, NptII  AP—Yield increased  98‐037‐03N  Rice  Monsanto  CBI, CBI  AP—Yield increased  98‐035‐04N  Wheat  Monsanto  CBI, NptII  AP—Yield increased  97‐275‐02Nc  Wheat  Monsanto    AP—Yield increased  97‐275‐01Nc  Wheat  Monsanto    AP—Yield increased  97‐261‐03N  Corn  Pioneer  CBI, PAT  AP—Yield increased  97‐261‐02N  Corn  Pioneer  CBI, PAT  AP—Yield increased  97‐241‐04N  Rapeseed  Calgene  CBI, SPS, NptII  AP—Yield increased  95‐093‐14N  Cotton  Calgene  CBI, NptII  AP—Yield increased  Abbreviations and notation as Table 4. In addition, SPS (sucrose phosphate synthase), PAT (phosphinothricin acetyltransferase) provides resistance to glufosinate and similar compounds). View Large Chloroplast transformation Obviously, a large proportion of the photosynthetic efficiency of a plant is under the control of genes located in the chloroplast genome, making them a target for manipulation (Shikanai and Hashimoto, 1997) ever since methods became available for the introduction of genes into this organelle. Such approaches, which depend on homologous recombination (Bae et al., 1998), are being aided by the determination of the complete sequences of plastid genomes from more than a dozen species (for a summary of these issues the reader is referred to Rochaix, 1997). The two principal methods for plastid transformation are particle bombardment, as used in the majority of studies (Lee et al., 1998; Sikdar et al., 1998; Tomizawa et al., 1998) or PEG‐mediated uptake (Kofer et al., 1998a, b). In the early studies, success was limited to a small number of amenable species such as tobacco, but this range has now been extended to Arabidopsis (Maliga et al., 1997b; Sikdar et al., 1998) and claims have been made that the method can now be applied more universally to crops including monocots (Blowers and Sanford, 1999; Daniell, 1999). Specific elements of the technique including polymerase promoters (Maliga et al, 1997a, 1998) and specific targeting (Gray and Knight, 1997; Ko and Pang, 1998), as well as the general method are the subject of many patent applications or granted patents (Maliga and Maliga, 1995; McBride and Maliga, 1996; McBride and Stalker, 1996a, b; Maliga et al., 1999). Amongst the specific photosynthetic targets for this method have been RuBisco (Tomizawa et al., 1998) and reaction centre proteins (Lee et al., 1998), and of course the plastid is the favoured site for the expression of insecticidal proteins (McBride and Maliga, 1996; Kota et al., 1999) and proteins that confer herbicide tolerance (Daniell et al., 1998). Recently, the use of an inducible, transactivator‐mediated system has been described as a method suitable for the chemical regulation of cellulase genes in chloroplasts (Lebel et al., 1998). Sugar and starch metabolism This area of metabolism has also been a focus for much fundamental and applied research recently, and it will not be considered in detail here. Of all the specific enzymes whose activity has been modified, possibly the most well studied is sucrose phosphate synthase (SPS), a key enzyme in the regulation of sucrose metabolism, being responsible for the synthesis of sucrose 6‐phosphate from fructose 6‐phosphate and uridine 5′‐diphosphate‐glucose. Evidence of the effects of modifying the amount of this enzyme (Sonnewald, 1998) come from many experiments including one on Arabidopsis (Signora et al., 1998). In this study, transgenics expressing the maize SPS under the control of the promoter from the small subunit of tobacco Rubisco showed increased foliar sucrose/starch ratios in leaves, and decreased foliar carbohydrate when plants were grown with CO2 enrichment. The value of this type of modification is presently being tested in several field trials including those with oilseed rape, tomato, potato, cotton, and corn (Tables 5, 6). In addition to the plant version of SPS, various bacterial genes are also available including one from the filamentous cyanobacterium Anabaena (Haselkorn et al., 1998). An alternative approach to increasing the concentrations of starch in a plant is by modifying the activity of the metabolites of the TCA cycle, specifically by reducing the amount of the NAD‐malic enzyme (Leaver et al., 1998). Other related aspects of sugar metabolism that may have potential as a target for manipulation include introduction of the E. coli inorganic pyrophosphatase in order to alter the amount of sugar (Sonnewald and Willmitzer, 1996), and modification of hexokinases (Sheen and Jang, 1997), enzymes which affect the sugar‐sensing capacities of a plant, and sucrose‐binding proteins (Grimes and Chao, 1998), a class of cupin protein (Dunwell, 1998a) implicated in sugar unloading in developing legume seeds. Since few crops are grown under optimum conditions, there is a close relationship between improving the yield of a plant and its response to stress conditions. For example, much attention has been focused recently on maize shrunken2 (Sh2) mutants with altered ADP glucose pyrophosphorylases. Various claims, including increased starch production, increased yield, increased plant size, increased growth rate, increased seed number, and improved response to heat stress have been made for the heat stable (Hannah and Greene, 1998), and/or up‐regulated allosteric mutants (Giroux and Hannah, 1998a, b; Okita et al., 1999). Other transgenic approaches to stress resistance will be considered below. Table 6 Summary of other agronomic traits included in US field trial applications Phenotype   APHIS No.   Species   Institution   Gene(s)   (a) Altered  98‐082‐17N  Tobacco  Univ. Kentucky  Isopentenyl transferase  senescence        NptII    97‐176‐04N  Tobacco  Univ. Wisconsin  Isopentenyl transferase        /Madison  NptII  (b) Altered  98‐273‐14N  Corn  Pioneer  CBI, PAT  maturation  98‐114‐03N  Corn  Pioneer  CBI, PAT    97‐275‐04N  Cotton  Monsanto  CBI, NptII    97‐265‐02Nc  Cotton  Monsanto    97‐260‐03N  Cotton  Monsanto  CBI  (c) Altered  Various  Soybean  Du Pont  Galactanase/UDP glucose  carbohydrate        glucosyltransferase  metabolism    Corn  Du Pont  Starch branching enzyme      Corn  Du Pont  Starch branching enzyme II antisense      Corn  Du Pont  Levan sucrase      Corn  Du Pont  Fructosyl transferase      Cotton  Texas Tech.  SPS      Tomato  Univ. Wisconsin  SPS      Potato  Univ. Wisconsin  SPS      Tomato  Calgene  SPS      Corn  Univ. Florida  SPS  (d) Growth  Various  Corn  Stine/Pioneer  CBI  rate/form      /Holdens      Corn  Garst  CBI  (e) Salt  98‐103‐24N  Creeping  Rutgers Univ.  Betaine aldehyde  tolerant    bentgrass    dehydrogenase, HPT  Phenotype   APHIS No.   Species   Institution   Gene(s)   (a) Altered  98‐082‐17N  Tobacco  Univ. Kentucky  Isopentenyl transferase  senescence        NptII    97‐176‐04N  Tobacco  Univ. Wisconsin  Isopentenyl transferase        /Madison  NptII  (b) Altered  98‐273‐14N  Corn  Pioneer  CBI, PAT  maturation  98‐114‐03N  Corn  Pioneer  CBI, PAT    97‐275‐04N  Cotton  Monsanto  CBI, NptII    97‐265‐02Nc  Cotton  Monsanto    97‐260‐03N  Cotton  Monsanto  CBI  (c) Altered  Various  Soybean  Du Pont  Galactanase/UDP glucose  carbohydrate        glucosyltransferase  metabolism    Corn  Du Pont  Starch branching enzyme      Corn  Du Pont  Starch branching enzyme II antisense      Corn  Du Pont  Levan sucrase      Corn  Du Pont  Fructosyl transferase      Cotton  Texas Tech.  SPS      Tomato  Univ. Wisconsin  SPS      Potato  Univ. Wisconsin  SPS      Tomato  Calgene  SPS      Corn  Univ. Florida  SPS  (d) Growth  Various  Corn  Stine/Pioneer  CBI  rate/form      /Holdens      Corn  Garst  CBI  (e) Salt  98‐103‐24N  Creeping  Rutgers Univ.  Betaine aldehyde  tolerant    bentgrass    dehydrogenase, HPT  Abbreviations as Tables 4 and 5. In addition, HPT (hygromycin phosphotransferase) provides resistance to hygromycin. View Large Alteration in senescence It has long been argued that a reduction in senescence (Smart et al., 1996; De Nijs et al., 1997) would improve the performance of a plant and thereby increase its yield. This suggestion is also linked to the interest in ‘stay green’ cultivars of maize. In addition to the claimed benefit of introducing the farnesyl transferase (McCourt et al., 1999) and the isopentenyl transferase (Table 6) genes as possible means of delaying senescence, two senescence associated promoters (SAG1 and SAG2) may also have value in the production of transgenic plants with improved performance (Amasino and Gan, 1997). Improvement in responses to stress There are many proposed transgenic routes for the improvement of stress related responses in plants. In the summary here, emphasis will be given to those strategies concerned with improving photosynthetic efficiency (Sato et al., 1998) and crop yield. Of the stress‐related compounds, two of the most extensively studied are the sugar alcohol mannitol (Adams et al., 1998) and trehalose, a sugar known to play a role in drought resistance of many organisms including the resurrection plant. In bacteria this sugar is produced by action of the two enzymes trehalose phosphate synthase which produces trehalose phosphate, and trehalose phosphate phosphatase, which degrades T‐6‐P into trehalose. When these two enzymes are expressed in plants (Goddijn et al., 1997; Pilon‐Smits et al., 1998) the transgenics have larger leaves, altered stem growth, and improved response to stress. For example, when grown under drought stress, two selected transgenic tobacco plants had total dry weights that were 28% and 39% higher than the controls. Chlorophyll fluorescence measurements showed a more efficient photosynthesis in the transgenics under these stress conditions. More recently (Goddijn et al., 1998), it has been claimed that similarly beneficial results can be achieved by modifying T‐6‐P via the inhibition of endogenous trehalase, an enzyme that hydrolyses trehalose into two glucose moieties. Transgenic tomato lines with various levels of trehalase are now being field tested (Table 5). Overexpression of various glutamate dehydrogenases (GDH) is also claimed to improve growth and stress tolerance. Specifically, plants have been transformed with genes encoding the α‐ and β‐subunits of the chloroplast‐located GDH from the alga Chlorella sorokiniana (Schmidt and Miller, 1997). An alternative approach (Ellis et al., 1998) concerns the introduction of the uridine diphosphate glucose pyrophosphorylase gene from the bacterium Acetobacter xylinum. These transgenics, which have modified concentrations of cellulose precursors, are claimed to have increased growth rates and yield, and improved response to stress conditions. Similar improvements in performance are reported for rice plants transformed with the barley late embryogenesis (LEA) gene (Wu and Ho, 1997). Modification of calcium‐related proteins is another method claimed to improve crop performance. For example, introduction of a protein kinase domain‐containing gene, a calcium‐dependent protein kinase gene, or a calcium/calmodulin‐dependent gene, are all reported to be beneficial (Sheen, 1998). The most recent approach of this type is that involving the introduction of functional calcineurin activity as a means of providing a salinity tolerant plant (Pardo et al., 1999). Another recently published claim (McCourt et al., 1999) is that involving the introduction of a gene encoding a plant farnesyltransferase (Pei et al., 1998), and also inhibitors of this enzyme, which when expressed in plants will enhance drought tolerance, improve resistance to senescence and modify growth habit. Two other related strategies are based on the premise that many of the deleterious effects of stress are mediated through the accumulation of reactive oxygen species. First, it has been suggested that the accumulation of these radicals is dependent on the presence of free iron in the cell; consequently, it has been proposed that controlling the amount of free iron could reduce oxidative damage. Convincing evidence of the value of this strategy has been shown recently in a study of transgenic tobacco expressing the alfalfa ferritin, an iron‐binding protein (Deák et al., 1999). The second approach of this type is the recent claim (Altier et al., 1999) that plants may be protected from the effects of stress‐induced reactive oxygen species by the introduction of a transgene encoding an enzyme such as oxalate oxidase (Dunwell, 1998a) that generates hydrogen peroxide. This molecule is known to stimulate the endogenous defences of the plant, for example, by the induction of PR proteins. An additional recent method for providing general non‐specific protection by up‐regulating or pre‐activating an existing defence pathway is exemplified by the introduction of a gene encoding the DREB1A ( dehydration response element B 1A) transcription factor from Arabidopsis (Kasuge et al., 1999) This factor is induced by a range of stresses and its introduction into transgenic Arabidopsis under the control of various promoters improved the tolerance to stress. The best results were achieved with transgenics expressing the gene under the control of the stress inducible promoter rd29A. Such transgenics showed better survival than the controls, when exposed to salt, freezing and drought. The only field trial of material claiming to have salt tolerance is that conducted on Agrostis containing a betaine aldehyde dehydrogenase gene (Table 6). Future commercial trends Quality traits Inspection of the field trials data from the last decade demonstrates clearly a change of emphasis from the single gene agronomic traits of herbicide and insect tolerance, towards traits such as modified seed quality where specific carbohydrates, proteins, and oils have been changed. These targets are driven by end users, processors, and retailers, as much as by seed or agrochemical companies. As such they exemplify an upheaval in the traditional relationship between the suppliers of agricultural products, and the consumers of those products. Pyramiding of genes In the same way that plant breeders are continually developing new varieties that contain the most effective combination of existing characters, there is now a similar trend with transgenic crops. Perhaps the most obvious example of this is the potato line being tested by Monsanto (APHIS Application 98–069–23N). This line contains seven transgenes, namely three selectable markers (gus, npt II, CBI), a cry IIIA Bt gene to provide resistance to Colorado potato beetle, virus coat protein and replicase genes to give resistance to two viral diseases, and another CBI gene that presumably is linked to one or more of the following transgenic traits, resistance to Verticillium, improvement in bruising resistance and altered carbohydrate metabolism. This accumulation of transgenes will inevitably become an increasing feature of new varieties. Integration with functional genomics Now that transgenic crops have been accepted by the growers (at least in North America) the plant breeding community is preparing to integrate the output from extensive (and expensive) genome programmes into its future objectives. Increasingly, knowledge gained from genomic and post‐genomic projects is providing information from which to design the next targets for transformation. Time‐scale for transgenic breeding programmes It is important in any review of progress in the area of transgenic crop development to consider the process by which experimental material of the type described above reaches the commercial market (Dunwell, 1996). It often assumed that this procedure is automatically rapid and predictable, but in fact, in common with other breeding programmes, it can be a lengthy, frustrating and often unsuccessful process. Success depends upon the identification of a transgenic plant whose progeny, whether produced from seed or by vegetative propagation, expresses the desired trait in a stable manner, both over generations and in a range of environments. The phenomenon of genotype×environment interaction is as true with transgenics as with any conventional breeding line. This time‐scale, that takes 10–12 years from initial planning to final release of a new variety is demonstrated in Fig. 1. Fig. 1. View largeDownload slide Time‐scale for the production of transgenic winter wheat, showing period required for gene identification and cloning, gene introduction, and multi‐site field testing. Fig. 1. View largeDownload slide Time‐scale for the production of transgenic winter wheat, showing period required for gene identification and cloning, gene introduction, and multi‐site field testing. Public perception and acceptance Recent attention in the media and elsewhere has shown that many sections of the general public in the UK are uneasy about the safety of genetically modified crops and food derived from such crops (see above) (Dunwell, 1998b). This issue is forcing a political reappraisal of the rate of introduction of these crops, and of course, eventual commercial success of this technology depends completely on public acceptance. Conclusion This relatively brief review has summarized the present status of transgenic crops with especial emphasis on data available from US and patent databases, which both provide a good perspective on the potential next generation of products. The range of material undergoing testing is now extensive and there seems little doubt that some of these experimental plants, including those with altered photosynthetic traits, will prove to be the progenitors of many successful breeding lines in the next century. 1 Fax: +44 118 931 6577. 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TI - Transgenic approaches to crop improvement
JF - Journal of Experimental Botany
DO - 10.1093/jexbot/51.suppl_1.487
DA - 2000-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/transgenic-approaches-to-crop-improvement-03osAtWusX
SP - 487
EP - 496
VL - 51
IS - suppl_1
DP - DeepDyve
ER -