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Genetic Ingeeniring of Crop Chloroplast Genome

#2161


Improvement of Economically Important Traits of Agricultural Crops by Foreign Gene Expression in Chloroplasts

Tech Area / Field

  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • AGR-PPR/Plant Protection/Agriculture

Status
3 Approved without Funding

Registration date
02.04.2001

Leading Institute
Institute of Bioorganic Chemistry (Branch), Russia, Moscow reg., Puschino

Supporting institutes

  • Phytopathology Research Institute, Russia, Moscow reg., Bolshie Vyazemy

Collaborators

  • University of Central Florida / Department of Molecular Biology and Microbiology, USA, FL, Orlando\nDow AgroSciences LLC, USA, IN, Indianapolis

Project summary

Current interest in crop biotechnology
The plastid genome of higher plants is an attractive target for crop engineering, owing to the feasibility to express transgenes at high levels. Efficient containment of the transgenes can be an additional advantage because, in most crops, foreign genes are not transmitted by pollen.

A remarkable feature of chloroplast genetic engineering is the observation of exceptionally large accumulations of foreign proteins in transgenic plants. Recently, foreign gene expression as high as 46% of total soluble protein has been accomplished in transgenic chloroplasts (DeCosa et al. 2001, Nature Biotechnology 19:71-74). Stable expression in chloroplasts of GVGVP, a protein-based polymer with varied medical applications (e.g., the prevention of postsurgical adhesions and scars, wound coverings, artificial pericardia, tissue reconstruction, and programmed drug delivery), 100 times higher than nuclear expression has been shown (Guda et al. 2000). Also, human somatotropin was expressed 300 times more in transgenic chloroplasts than via nuclear genome (Staub et al. 2000, Nature Biotechnology, 18: 333-338). These observations make chloroplast genetic engineering a valuable tool for the production of pharmaceutical proteins in plants.

The presence of antibiotic resistant genes in genetically modified (GM) crops is another serious area of public concern. Engineering GM crops without the use of antibiotic resistance genes should eliminate the potential risk of their transfer to the environment or gut microbes. Therefore, the betaine aldehyde dehydrogenase (BADH) gene from spinach was used by Daniell et al (2001, Current Genetics, in press; see attached article) as a selectable marker. The selection process involves conversion of toxic betaine aldehyde (BA) by the chloroplast BADH enzyme to nontoxic glycine betaine, which also serves as an osmoprotectant (thereby conferring drought or salt tolerance). Chloroplast transformation efficiency was 25 times higher in BA selection than spectinomycin. In addition, rapid regeneration was obtained. Transgenic shoots appeared within 12 days in 80% of leaf discs (up to 23 shoots per disc) under BA selection compared to 45 days in 15% of discs (1 or 2 shoots per disc) under spectinomycin selection. Southern blots confirmed the stable integration of foreign genes into all of the chloroplast genomes (~10,000 copies per cell), resulting in homoplasmy. This is the first report of genetic engineering of the higher plant chloroplast genome without the use of antibiotic selection. Use of genes that are naturally present in spinach for selection, in addition to gene containment, should ease public concerns regarding genetically modified crops.

High levels of expression of several foreign proteins in transgenic tobacco chloroplasts have not significantly affected growth rates, photosynthesis, chlorophyll content, flowering, or seed set (Daniell, H. 1999). However, long-term tests using agronomically important crops, grown under field conditions, are needed to confirm this observation. Recent success in potato plastid transformation should pave the way for studies on such agronomic traits (Sidorov, V., 1999). All of these findings augur well for chloroplast genetic engineering of economically useful crops. Thus, several environmentally friendly approaches have been opened for new advances in plant biotechnology.

The purpose of the project is to develop an efficient transformation system for expression of foreign proteins in plant chloroplasts in local crops. Particularly, we propose to transfer and express native bt-toxin genes from Bacillus thuringiensis (Bt) into chloroplast genomes.

Tasks

The project will address the following objectives:

– creation of plasmid vectors for foreign gene introduction into plastomes;


– construction of recombinant plasmids to confer important traits via plastomes;
– development of chloroplast transformation protocol using a Gene Gun;
– development of efficient selection and regeneration systems for selection of cells containing transformed plastomes and for transgenic plant production;
– evaluation of foreign protein expression levels in transgenic plants by immunoassays;
– testing suitability of transgenic plants in laboratory and greenhouse conditions;
– field experiments for evaluation of transgenic plants for insect resistance in native conditions in accordance with biosafety rules.
Methodology

During last six years at artificial climate station "Biotron", the authors have developed efficient transformation protocols for several horticultural crops, including fruit trees (apple, pear, sour cherry), small berries (strawberry, actinidia), ornamentals (chrysanthemum, carnation) and carrot. Transgenic plants expressing foreign genes conferred economically important traits. These are apple, pear, strawberry and carrot plants with gene of super-sweet protein thaumatin II, plant defensin gene, antifreeze protein from winter fish, and apple and pear clonal rootstocks, which are resistant to the "Basta" herbicide. Also, successful modification of chrysanthemum plant architecture and flower color have been achieved by introduction of rolC and chalcone synthase genes.

Based on cloning of the bt toxin gene sequence at the State Research Institute of Applied Microbiology, the plant transformation vector with its truncated form was created. Transgenic chrysanthemum plants expressing this gene demonstrated improved resistance to spider mites and Spodoptera exigua. It is the first representative evidence of Bt protein toxicity for a new kind of vermin - arachnids. Unfortunately, owing to its native origin, the toxin level in plants and their field resistance that was tested in CPRO-DLO is not adequate for commercial development. Development of a plastid transformation system and expression of the Bt-toxin gene via the plastome should allow very high levels of expression, adequate for commercial development of agricultural crops resistant to this injurious vermin. This is especially important not only for ornamentals, but also for other greenhouse crops, such as tomato and pepper.

Furthermore, the authors have prior experience in biolistic method application for plant biotechnology. An efficient transformation protocol for sunflower transformation has been recently developed at "Biotron" by particle gun usage.

Expected results

Based on the aforementioned facts, the authors propose to develop an efficient system for introduction and expression of foreign genes via plastome. This method, for the first time, should open the possibility to achieve a high expression level of prokaryotic genes without very expensive modification of DNA sequences. The authors propose, particularly, to obtain transgenic potato, tomato and chrysanthemum plants with CryIAb1 and CryIII bt-toxin genes, to confer resistance against important pests (Colorado potato beetle and spider mite). Toxicity of these genes has been confirmed by previous research, but nuclear expression levels are not adequate for commercial development.

Technical –scientific and commercial value

The application of genetic engineering techniques to commercial agriculture will benefit not only crop breeders, but also the agricultural industry as a whole. Moreover, transgenic plants that exhibit interesting novel traits, but which are unmarketable due to additional unwanted changes, can be used in narrowly defined classical breeding programs. In addition to improving the competitiveness of the Russian agriculture industry in the world marketplace, another important outcome of this proposal will be an improvement in the basic knowledge of processes involved in the control of plastome gene expression and the mechanism of plant insect resistance. Particularly, plastid transformation is used to understand plastid molecular biology, to characterize promotor strength, trans-splicing and mRNA stability, to manipulate biosynthetic pathways for amino acids, fatty acids or photosynthesis, to achieve targeting disruption of plastid genes, and to optimize the requirements for expression of foreign genes.


Work on the project will contribute to the increased efficiency of agricultural production and improved quality of food-stuffs in Russia and other CIS countries, and to a reduction in environmental pollution from chemical pesticides or nuclear transgenic plants.

Foreign collaboration
Together with foreign collaborators (University of Central Florida and DowAgroSciences LLC), joint research, testing and evaluation of equipment/technologies, and training of Russian scientists will be performed.

Meeting ISTC goals and objectives
This work is in line with the goals of the International Science and Technology Center, as it enables skilled specialists, earlier engaged in defense R&D, to direct their activity towards the solution of fundamental and applied problems of plant protection and to promote their integration into the international scientific community.


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