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Moving crops biotechnology forward through new germ-line transformation techniques
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[P&F.24] Transgenic rice for food security and sustainable agriculture Q. Zhang Huazhong Agricultural University, China Keywords: Transgenics; Rice Rice is the most important food crop in many parts of the world. Rice improvement has achieved remarkable success in the last halfcentury with the yield doubled in most parts of the world and even tripled in certain regions, which has contributed greatly to food security globally. To secure rice production, large efforts have been made in the last two decades internationally in research and development of rice biotechnology, including genome sequencing, functional genomics and transgenics. From a global viewpoint, a number of challenges need to be met for sustainable rice production: (1) increasingly severe occurrence of insects and diseases and indiscriminate pesticide applications; (2) high pressure for yield increase and overuse of fertilizers; (3) water shortage and increasingly frequent occurrence of drought, and (4) extensive cultivation in marginal lands. A new rice breeding goal, referred to as Green Super Rice, was recently proposed to address these challenges. On the premise of continued yield increase and quality improvement, Green Super Rice would possess resistances to multiple insects and diseases, high nutrient-use efficiency, and drought resistance. Transgenic studies of rice have been conducted in large scale, and most of the target traits are consistent with the goal of Green Super Rice. It is believed that transgenic rice will play a crucial role to ensure sustainable rice production in the future. In the presentation, I will review the main progresses and constraints in the research and development of transgenic rice in a number of fronts. doi:10.1016/j.jbiotec.2010.08.303 [P&F.25] Moving crops biotechnology forward through new germ-line transformation techniques O.I. Kershanskaya ∗ , S.V. Didorenko, A.S. Nurmagambetova, G.L. Esenbaeva, A.T. Tashkenova Institute Plant Biology and Biotechnology, National Center Biotechnology, Kazakhstan Keywords: Germ-line transformation; Techniques; Abiotic stresses resistance; Crop improvement Elaboration of new germ-line biotechnology (genetic transformation by plant germ elements–pollen, ovary, embryo, seed) for crop transgenic plants creation enhance possibility of simple introduction of new valuable genes for plant disease and abiotic stress resistance, genetic modification of photosynthesis, yield crop improvement, benefice of plant biotechnology. We have elaborated methods of genetic germ-line transformation by Agrobacterium Pipetting into wheat ear stigma and soybean pollen tubes. Agrobacterium Pipetting into wheat method is based on unique for wheat mechanism of pollen distance transfer which provided by high level of flavonol glucosides which acts as inducer of the vir-zone of Ti plasmid. Biotechnological protocol includes several stages: Agrobacterium culture grown and optimization; improvement of technique for Agrobacterium pipetting into wheat ears stigma; determination of optimal stage of plant development; screening of putative transgenic seeds on antibiotics resistance using selectable marker genes (nptII encoded kanamycin- and hptII encoded hygromycin resistance); biochemical screening of putative transgenic plants in T1 progeny, in case of
S117
PEPC gene introduction–assay of PEP-carboxylase activity; molecular biological detection of transgenes in wheat genome in T1 and T2 - generations by PCR, Real Time PCR, Northern and Southern Blotting, yield analyses. Totally has been produced about 5000 putative wheat transgenic seeds with efficiency 1.8–2.3% and more than 10000 transgenic plants of first-fifth generations. The method of pollen tube pathway transformation in soybean provides DNA transferring by cutting the stigma following pollination. DNA presumably reaches the ovary by flowing down the pollen tube exactly after anthesis and then integrates into the just fertilized but undivided zygotic cells. Approximately 3000 seeds were produced from the flowers treated with DNA. PPDK, desA12licBM3 and FeSOD valuable genes have been used to provide in soybean plant resistance to different abiotic stresses. Screening on antibiotic resistance by marker (spectinomycin in FeSOD) and reporter (lichenase BM3 in PPDK and des A12lic BM3) genes and molecular biological detection of transgenes by PCR analysis confirm less than 3% of progeny seeds tested expressed a positive reaction in comparison with wild soybean plants. New techniques except waste time and money steps of tissue culture and regeneration are simple, economic and comparatively effective. New methods convert wheat and soybean genetic transformation into routine process, which might be successfully used by many breeders and investigators for increasing of crops yield and moving plant biotechnology forward. doi:10.1016/j.jbiotec.2010.08.304 [P&F.26] Engineering secondary metabolite production in plants using combinatorial genetic transformation B. Miralpeix 1,∗ , R. Hoefer 2 , D. Boewmeester 3 , P. Christou 1,4
Werck 2 , L.
Dong 3 , H.J.
1
Universitat de Lleida, Spain Université de Strasbourg, France 3 Wageningen University, Netherlands 4 Institució Catalana de Recerca i Estudis avanc¸ats (ICREA), Spain Keywords: Terpenoid indole alkaloid; Catharanthus rouseus; Genetic transfomation; Tobacco 2
Humans have used compounds derived from plants for treating diseases since prehistoric times. Over 25% of new drugs approved for human therapy in the last 30 years are based on molecules of plant origin, and about 50% of the top selling drugs are derived from knowledge of plant secondary metabolism. Catharanthus roseus (Madagascar periwinkle) produces more than 130 different secondary metabolites, including terpenoid indole alkaloids (TIAs). These compounds exhibit diverse biological activities including anti-cancer, analgesic, spasmolytic, antiinflammatory and insecticidal effects. However, such molecules are present in minute amounts in plants such as C. roseus making their isolation from natural sources uneconomical; in addition, their total or partial chemical synthesis is impractical because of their complex structural features. Genetic engineering is thus a viable alternative for the production of such important molecules on a large scale. We had recently reported the development of a combinatorial genetic transformation system which we used to generate a population of plants expressing diverse input transgenes. This methodology was exemplified with the engineering of the carotenoid pathway in corn and resulted in corn germplasm accumulating very high levels of nutritionally important carotenoids. The approach also permited the dissection of the pathway, thus revealing as yet unkown rate limiting steps.
[P&F.24] Transgenic rice for food security and sustainable agriculture Q. Zhang Huazhong Agricultural University, China Keywords: Transgenics; Rice Rice is the most important food crop in many parts of the world. Rice improvement has achieved remarkable success in the last halfcentury with the yield doubled in most parts of the world and even tripled in certain regions, which has contributed greatly to food security globally. To secure rice production, large efforts have been made in the last two decades internationally in research and development of rice biotechnology, including genome sequencing, functional genomics and transgenics. From a global viewpoint, a number of challenges need to be met for sustainable rice production: (1) increasingly severe occurrence of insects and diseases and indiscriminate pesticide applications; (2) high pressure for yield increase and overuse of fertilizers; (3) water shortage and increasingly frequent occurrence of drought, and (4) extensive cultivation in marginal lands. A new rice breeding goal, referred to as Green Super Rice, was recently proposed to address these challenges. On the premise of continued yield increase and quality improvement, Green Super Rice would possess resistances to multiple insects and diseases, high nutrient-use efficiency, and drought resistance. Transgenic studies of rice have been conducted in large scale, and most of the target traits are consistent with the goal of Green Super Rice. It is believed that transgenic rice will play a crucial role to ensure sustainable rice production in the future. In the presentation, I will review the main progresses and constraints in the research and development of transgenic rice in a number of fronts. doi:10.1016/j.jbiotec.2010.08.303 [P&F.25] Moving crops biotechnology forward through new germ-line transformation techniques O.I. Kershanskaya ∗ , S.V. Didorenko, A.S. Nurmagambetova, G.L. Esenbaeva, A.T. Tashkenova Institute Plant Biology and Biotechnology, National Center Biotechnology, Kazakhstan Keywords: Germ-line transformation; Techniques; Abiotic stresses resistance; Crop improvement Elaboration of new germ-line biotechnology (genetic transformation by plant germ elements–pollen, ovary, embryo, seed) for crop transgenic plants creation enhance possibility of simple introduction of new valuable genes for plant disease and abiotic stress resistance, genetic modification of photosynthesis, yield crop improvement, benefice of plant biotechnology. We have elaborated methods of genetic germ-line transformation by Agrobacterium Pipetting into wheat ear stigma and soybean pollen tubes. Agrobacterium Pipetting into wheat method is based on unique for wheat mechanism of pollen distance transfer which provided by high level of flavonol glucosides which acts as inducer of the vir-zone of Ti plasmid. Biotechnological protocol includes several stages: Agrobacterium culture grown and optimization; improvement of technique for Agrobacterium pipetting into wheat ears stigma; determination of optimal stage of plant development; screening of putative transgenic seeds on antibiotics resistance using selectable marker genes (nptII encoded kanamycin- and hptII encoded hygromycin resistance); biochemical screening of putative transgenic plants in T1 progeny, in case of
S117
PEPC gene introduction–assay of PEP-carboxylase activity; molecular biological detection of transgenes in wheat genome in T1 and T2 - generations by PCR, Real Time PCR, Northern and Southern Blotting, yield analyses. Totally has been produced about 5000 putative wheat transgenic seeds with efficiency 1.8–2.3% and more than 10000 transgenic plants of first-fifth generations. The method of pollen tube pathway transformation in soybean provides DNA transferring by cutting the stigma following pollination. DNA presumably reaches the ovary by flowing down the pollen tube exactly after anthesis and then integrates into the just fertilized but undivided zygotic cells. Approximately 3000 seeds were produced from the flowers treated with DNA. PPDK, desA12licBM3 and FeSOD valuable genes have been used to provide in soybean plant resistance to different abiotic stresses. Screening on antibiotic resistance by marker (spectinomycin in FeSOD) and reporter (lichenase BM3 in PPDK and des A12lic BM3) genes and molecular biological detection of transgenes by PCR analysis confirm less than 3% of progeny seeds tested expressed a positive reaction in comparison with wild soybean plants. New techniques except waste time and money steps of tissue culture and regeneration are simple, economic and comparatively effective. New methods convert wheat and soybean genetic transformation into routine process, which might be successfully used by many breeders and investigators for increasing of crops yield and moving plant biotechnology forward. doi:10.1016/j.jbiotec.2010.08.304 [P&F.26] Engineering secondary metabolite production in plants using combinatorial genetic transformation B. Miralpeix 1,∗ , R. Hoefer 2 , D. Boewmeester 3 , P. Christou 1,4
Werck 2 , L.
Dong 3 , H.J.
1
Universitat de Lleida, Spain Université de Strasbourg, France 3 Wageningen University, Netherlands 4 Institució Catalana de Recerca i Estudis avanc¸ats (ICREA), Spain Keywords: Terpenoid indole alkaloid; Catharanthus rouseus; Genetic transfomation; Tobacco 2
Humans have used compounds derived from plants for treating diseases since prehistoric times. Over 25% of new drugs approved for human therapy in the last 30 years are based on molecules of plant origin, and about 50% of the top selling drugs are derived from knowledge of plant secondary metabolism. Catharanthus roseus (Madagascar periwinkle) produces more than 130 different secondary metabolites, including terpenoid indole alkaloids (TIAs). These compounds exhibit diverse biological activities including anti-cancer, analgesic, spasmolytic, antiinflammatory and insecticidal effects. However, such molecules are present in minute amounts in plants such as C. roseus making their isolation from natural sources uneconomical; in addition, their total or partial chemical synthesis is impractical because of their complex structural features. Genetic engineering is thus a viable alternative for the production of such important molecules on a large scale. We had recently reported the development of a combinatorial genetic transformation system which we used to generate a population of plants expressing diverse input transgenes. This methodology was exemplified with the engineering of the carotenoid pathway in corn and resulted in corn germplasm accumulating very high levels of nutritionally important carotenoids. The approach also permited the dissection of the pathway, thus revealing as yet unkown rate limiting steps.