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GM Soybean

GM Soybean. GM Cotton. The Plant Journal Volume 31 Issue 4 Page 423  - August 2002 Five years of Bt cotton in China: the benefits continue. Adoption of Bt cotton. Yield of Bt and non-Bt cotton in provinces sampled.  Pesticide application (kg ha -1 ) on Bt and non-Bt cotton.

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GM Soybean

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  1. GM Soybean GM Cotton

  2. The Plant JournalVolume 31 Issue 4 Page 423  - August 2002Five years of Bt cotton in China: the benefits continue Adoption of Bt cotton

  3. Yield of Bt and non-Bt cotton in provinces sampled

  4.  Pesticide application (kg ha-1) on Bt and non-Bt cotton

  5. Average costs and returns (US$) per hectare for all farmers surveyed

  6. Impact of Bt on farmer poisoning

  7. Nature Biotechnology  21, 909 - 913 (2003) Genetic manipulation of gibberellin metabolism in transgenic rice The 'green revolution' was fueled by the introduction of the semi-dwarf trait into cereal crop cultivars. The semi-dwarf cultivars--which respond abnormally to the plant growth hormone gibberellin (GA)--are more resistant to wind and rain damage and thus yield more grain when fertilized. To generate dwarf rice plants using a biotechnological approach, we modified the level of GA by overproduction of a GA catabolic enzyme, GA2-oxidase. When the gene encoding GA 2-oxidase, OsGA2ox1, was constitutively expressed by the actin promoter, transgenic rice showed severe dwarfism but failed to set grain because GA is involved in both shoot elongation and reproductive development. In contrast, OsGA2ox1 ectopic expression at the site of bioactive GA synthesis in shoots under the control of the promoter of a GA biosynthesis gene, OsGA3ox2 (D18), resulted in a semi-dwarf phenotype that is normal in flowering and grain development. The stability and inheritance of these traits shows the feasibility of genetic improvement of cereal crops by modulation of GA catabolism and bioactive GA content.

  8. Expression of GA-2 oxidase by actin promoter

  9. Expression of GA-2 oxidase by D18 promoter

  10. Nature Biotechnol March 2001 Volume 19 Number 3 pp 263 - 267 Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time.Citrus trees have a long juvenile phase that delays their reproductive development by between 6 and 20 years, depending on the species. With the aim of accelerating their flowering time, we transformed juvenile citrus seedlings to constitutively express the Arabidopsis LEAFY (LFY) or APETALA1 (AP1) genes, which promote flower initiation in Arabidopsis. Both types of transgenic citrus produced fertile flowers and fruits as early as the first year, notably through a mechanism involving an appreciable shortening of their juvenile phase. Furthermore, expression of AP1 was as efficient as LFY in the initiation of flowers, and did not produce any severe developmental abnormality. Both types of transgenic trees flowered in consecutive years, and their flowering response was under environmental control. In addition, zygotic and nucellar derived transgenic seedlings had a very short juvenile phase and flowered in their first spring, demonstrating the stability and inheritance of this trait. These results open new possibilities for domestication, genetic improvement, and experimental research in citrus and other woody species.

  11. Nature Biotechnol March 2001 Volume 19 Number 3 pp 263 - 267 Overexpression of LEAFY in citrus transgenic plants. (A) Transgenic shoot grafted in vitro on a nontransgenic rootstock showing a precocious terminal flower five weeks after regeneration. (B) Transgenic plant showing a weeping growth habit. (C) Leaves from transgenic plants showing various degrees of curling (top) compared to leaves from nontransformed control plants (bottom). (D) Vegetative shoot from a transgenic plant showing the reduction of thorns and small curled leaves (right) compared to a vegetative shoot from a nontransformed control plant of the same age (left). (E) Transgenic plant flowering 16 months after its transfer to the greenhouse. (F) Ripened fruit from a transgenic plant grown in the greenhouse.

  12. Nature423, 823 (19 June 2003) RNA interference: Producing decaffeinated coffee plants The demand for decaffeinated coffee is increasing because the stimulatory effects of caffeine can adversely affect sensitive individuals by triggering palpitations, increased blood pressure and insomnia. Three N-methyltransferase enzymes are involved in caffeine biosynthesis in coffee plants — CaXMT1, CaMXMT1 (theobromine synthase) and CaDXMT1 (caffeine synthase), which successively add methyl groups to xanthosine in converting it into caffeine. Here we describe the construction of transgenic coffee plants in which expression of the gene encoding theobromine synthase (CaMXMT1) is repressed by RNA interference (RNAi). The caffeine content of these plants is reduced by up to 70%, indicating that it should be feasible to produce coffee beans that are intrinsically deficient in caffeine.

  13. Glyphosate (Roundup ready) resistant crops

  14. Global adoption rates of glyphosate-resistant crops since introduction

  15. Manipulation of fruit ripening by genetic modification • Tomato ripening genes • Ethylene biosynthesis genes: ACC synthase and ACC oxidase • Ethylene perception gene: ETR and ERS dominant, mutants insensitive to C2H4 • Cell wall modification C2H4 Ethylene receptor Signal transduction Activation of ripening gene Pectin PG-COOH (Polygalacturonic acid) SAM ACC C2H4 Polygalacturonase (PG) catalyzes the hydrolysis of polygalacturonic acid chains in unmethylated regions of pectin ACC synthase 5’ methyl thioadenosine ACC oxidase color texture flavor

  16. THE FLAVR SAVR ·· Several experiments were performed before the development of the Flavr Savr tomato, which effectively demonstrated that the contiguous expression of antisense PG RNA in transformed tomatoes significantly inhibits the accumulation of PG mRNA and its enzymatic activity during the ripening process. ·· These results confer important agricultural implications due to the role of PG in cell wall degradation and the subsequent softening of tomatoes. Presumably, a tomato with inhibited expression of endogenous PG could remain on the vine longer, thus enhancing flavor, improving processing quality and possibly increasing shelf life. These effects would necessarily impact on commercial sale of such a tomato. ·· Calgene Inc. exploited the antisense RNA technology described above to produce a tomato, the Flavr Savr, with substantially reduced PG expression.

  17. The Flavr Savr tomato (Calgene Inc.) ripens on the vine – resulting in fuller flavour.

  18. Manipulation of seed storage protein 40% of dry wt. of soybean seeds, 8-15% in cereals. Classification Prolamins: cereals, alcohol soluble Albumin: dicots, water soluble Globulin: legumes, saline soluble Processing property: bread making Gliadin and glutenin: gluten Elasticity and extensibility High elasticity suitable for pasta High extensibility suitable for bread All wheat varieties have 6 HMW glutenin but gene silencing renders 1-2 inactive. Positive gene dosage effect. Mixograph Patterns of di-sulfide bonds. Nutritional quality Sulfur content Lysine content Brazil nut 2S albumin

  19. Manipulation of oil seeds Edible oil engineering:Reduce polyunsaturated fatty acids. Linolenic acid (18:3) and linoleic acid (18:2) are somewhat unstable. To reduce the content of these two, precursor 18:1 level will have to be increased, which is the desirable fatty acid. FAD2 FAD3 Acyl-CoA PA DAG PC 18:1 18:2 18:3 pool monounsaturate Antisense FAD2 and FAD3 approach in canola and Soybean has resulted in the increase in 18:1 (oleate) levels. High oleate variety contain 85% of 18:1. Industrial application: high-lauric acid rapeseed Lauric acid used in soap industry is obtained from coconut and palm grown in tropical places. They contain 12:0-ACP thiesterase (california bay) to synthesize 12:0 medium chain fatty acid. Seed specific expression of this gene in rapeseed resulted in the increase in production of 12:0 to 60% level.

  20. Tree biotechnology Pulping process: conversion of ligno-cellulosic material to pulp. Generates large quantities of by-products which have adverse effect on the environment. Consumes a lot of energy. Two approaches to address these problems: Improving wood quality:wood contains 40-50% cellulose and 15-30% hemi- cellulose and 20-35% lignin. Lignin is the undesirable content for the pulping process. The main approach is to reduce lignin with minimum damage to the fibre. Lignin is a complex aromatic biopolymer composed of various combination of p-hydroxyphenyl, guaicyl or syringyl monomers. Many genes involved in lignin biosynthesis have been cloned. Antisense expression of the gene for cinnamyl alcohol dehydrogenase (CAD) in tobacco decreased CAD activity but had no effect on total lignin content. However, chemical structure of lignin in CAD-reduced plants was altered due to an increase in cinnamyl aldehydes in the polymer. The lignin has improved pulping characteristics. These results were confirmed in transgenic poplar.

  21. Production of biodegradable thermoplastics and elastomers Polyhydroxyalkanoates (PHA) polyesters produced by bacteria is biodegradable. Eg. PHB (BiopolTM Monsanto) produced by bacterial fermentation is a copolymer of 3-hydroxy butyrate and 3-hydroxy valerate (3HB-3HV). This copolymer is produced by bacteria Alcaligenes eutrophus. Bacterial fermentation process is very expensive. PHB Synthase (phaC) Acetoacetyl Reductase (phaB) 3-ketothiolase Acetyl CoA acetoacetyl CoA R-(-)-3-hydroxybutyryl-CoA PHB Cost of production Biopol: $15/ kg Synthetic plastic: <$1/ kg Acetyl-CoA Aceto malonyl acetylCoA CoA flavonoids Sterols plastid Acetyl-CoA lipids PHB production in Arabidopsis 35S-phaB and 35S-phaC expressed. Plants produced 0.1% (dry wt.) of PHB. Stunted plants. Therefore, phaA, phaB and phaC were fused with ssRubisco to be targeted into plastid. PHB was 14% of dry wt. and plants were normal. mitoch peroxisome Acetyl CoA TCA cycle Fattyacids acetylcoA ß-oxidation

  22. Production of antibodies in plants Antibody protein consists of two heavy chain and two light chain polypeptides. Production of a mouse IgG in tobacco was first reported in 1989. Transgenic plants was made with a gene for heavy chain and a gene for light chain each. F1 progenies were found to contain functional IgG (1% of total soluble protein). Natural IgG contain ER signal peptide. Omission of this sequence from the transgene Results in instability of each polypeptide formation. The heavy and Light chains are bound with disulfide bonds. ER contains protein disulfide isomerase (PDI), which is necessary for these proteins. scFv fragments vs whole antibody • Application: • Antibody farming • In situ application

  23. Golden rice technology Engineering the Provitamin A (  -Carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm Science, Vol 287, 303-305 , 2000 Turning carotene into gold. The carotenoid biosynthetic pathway of plants. Carotenoids are synthesized in the central isoprenoid pathway within plastids. All isoprenoids (more than 20,000 different compounds exist in plants) are built from the common precursor isopentenyl diphosphate (IPP). IPP is thought to be synthesized in plastids from pyruvate and glyceraldehyde-3-phosphate. The first committed step in the carotenoid pathway is the head-to-head condensation of two molecules of geranyl geranyl diphosphate (GGPP) to produce phytoene. Phytoene synthase is encoded by psy, phytoene desaturase by crtI, and lycopene beta-cyclase by lcy. Golden rice engineered to produce beta-carotene accumulates lutein and zeaxanthin as well.

  24. Immature rice endosperm is capable of synthesizing the early intermediate geranylgeranyl diphosphate, which can be used toproduce the uncolored carotene phytoene by expressing the enzymephytoene synthase in rice endosperm. The synthesisof -carotene requires the complementation with three additionalplant enzymes: phytoene desaturase and  -carotene desaturase,each catalyzing the introduction of two double bonds, and lycopene -cyclase, encoded by the lcy gene. To reduce the transformationeffort, a bacterial carotene desaturase (crtl), capable of introducingall four double bonds required, can be used.

  25. Structures of the T-DNA region of pB19hpc used in single transformations, and of pZPsC and pZLcyH used in co-transformations. Representative Southern blots of independent transgenic T0-plants are given below the respective Agrobacterium vectors. LB, left border; RB, right border; "!", polyadenylation signals; p, promoters; psy, phytoene synthase; crtI, bacterial phytoene desaturase; lcy, lycopene -cyclase; tp, transit peptide.

  26. We used Agrobacterium-mediated transformation to introduce the entire β-carotene biosynthetic pathway into rice endospermin a single transformation effort with three vectors. The vector pB19hpc combines the sequences for aplant phytoene synthase (psy) originating from daffodil (Narcissus pseudonarcissus; GenBank accession number X78814)with the sequence coding for a bacterial phytoene desaturase (crtI)originating from Erwinia uredovora (GenBank accession number D90087)placed under control of the endosperm-specific glutelin (Gt1)and the constitutive CaMV (cauliflower mosaic virus) 35S promoter,respectively. The phytoene synthase cDNA contained a 5'-sequencecoding for a functional transit peptide, and thecrtI gene contained the transit peptide (tp) sequence of the peaRubisco small subunit. This plasmid should directthe formation of lycopene in the endosperm plastids, the siteof geranylgeranyl-diphosphate formation.

  27. Phenotypes of transgenic rice seeds. Bar, 1 cm. (A) Panel 1, untransformed control; panels 2 through 4, pB19hpc single transformants lines h11a (panel 2), h15b (panel 3), h6 (panel 4). (B) pZPsC/pZLcyH co-transformants lines z5 (panel 1), z11b (panel 2), z4a (panel 3), z18 (panel 4).

  28. Nature Biotechnology23, 482 - 487 (2005) Published online: 27 March 2005; | doi:10.1038/nbt1082 Improving the nutritional value of Golden Rice through increased pro-vitamin A content 'Golden Rice' is a variety of rice engineered to produce β-carotene (pro-vitamin A) to help combat vitamin A deficiency, and it has been predicted that its contribution to alleviating vitamin A deficiency would be substantially improved through even higher β- carotene content. We hypothesized that the daffodil gene encoding phytoene synthase (psy), one of the two genes used to develop Golden Rice, was the limiting step in β-carotene accumulation. Through systematic testing of other plant psys, we identified a psy from maize that substantially increased carotenoid accumulation in a model plant system. We went on to develop 'Golden Rice 2' introducing this psy in combination with the Erwinia uredovora carotene desaturase (crtI) used to generate the original Golden Rice1. We observed an increase in total carotenoids of up to 23-fold (maximum 37 microgg/g) compared to the original Golden Rice and a preferential accumulation of β-carotene.

  29. Expression of a psy transgene increases the carotenoid content of maize callus.

  30. Carotenoid enhancement of the rice endosperm by transformation with psy orthologues and crtI.(a) Schematic diagram of the T-DNAs used to generate transgenic rice plants. The T-DNA comprised the rice glutelin promoter (Glu) and the first intron of the catalase gene from castor bean (I), E. uredovora crtI functionally fused to the pea RUBISCO chloroplast transit peptide (SSUcrtI) and a phytoene synthase from each of five plant species (psy), with a nos terminator, as well as a selectable marker cassette comprising the maize polyubiquitin (Ubi1) promoter with intron, hygromycin resistance (hpt) and nos terminator. (b) Photograph of polished wild-type and transgenic rice grains containing the T-DNA (as above) with the daffodil psy (Np) or maize psy (Zm) showing altered color due to carotenoid accumulation. (c) Histogram showing the total carotenoid content of T1 rice seed containing a T-DNA (as above) with the psy gene from either rice, maize, pepper, tomato or daffodil from the five events with the highest carotenoid content for each T-DNA. Measurement error tended to be proportional to absolute carotenoid content and pooling across all 25 transformants resulted in a measurement standard error of 6.3% approximately. dwt, dry weight. (d) Schematic diagram of the T-DNA in pSYN12424 used to create Golden Rice 2. The T-DNA components were as described above with a selectable marker cassette comprising the maize polyubiquitin (Ubi1) promoter with intron, phosphomannose isomerase gene (pmi) and nos terminator. The use of an intron was abandoned because it was shown to have no effect on carotenoid accumulation (data not shown).

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