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Exploitable results
Long term objective of this joint research project and the resulting network activities is to develop insect and pathogen resistant commercial FSU cotton varieties through genetic transformation. Specific objectives are:
development of an efficient in vitro regeneration system for local FSU cotton varieties,
cloning and construction of vectors conferring insect and pathogen resistance in cotton,
development of an efficient transformation system for local FSU cotton varieties.
The protocol for plant regeneration through somatic embryogenesis developed for Gossypium hirsutum L. cv. Coker (Trolinder and Goodin, Plant Cell Tissue and Organ Culture, 1988) was applied to commercial FSU cotton cultivars. Factorial experiments have been done to analyze the influence of explant (cotyledon, hypocotyl, immature zygotic embryo, anther, buds), media composition (plant growth regulators and oligosaccharides) and environmental conditions (temperature and light) on callus induction and further regeneration and resulted in a general protocol . Callus development was obtained for all tested cvs., plant regeneration was obtained for one local FSU cv. (Ok Oltyn) and is at a final stage for a second one (C 6524). A protocol was optimized for the long term conservation of embryogenic cotton cell lines.
Various vectors harbouring Bacillus thurigensis genes or protease inhibitor genes (OCI and CII) have been constructed. A native cryIA(b) gene encoding a toxin active against Lepidoptera and particularly Helicoverpa armigera which is one of the most important pests in cotton in numerous countries, OCI active against Anthonomus grandis (a coleoptera) and CII active against H. armigera were introduced in the vector pKYLX71 35S2 and a potyviral 5' untranslated sequence was added to enhance the translation efficiency. Analysis of the expression of OCI gene in cotton has shown that the level of expression is quite high. These vectors have been used in Agrobacterium tumefaciens-mediated transformations. Bioassays with the T1 Coker transformed plants will be performed during summer 97. To obtain a good expression level for Bt genes, modified or synthetic genes were constructed. Two synthetic genes, cryIA(c) and cryIA(b), (obtained from Prof Altosaar, Ottawa University, Canada) and a partially modified cryIC gene have been introduced in the same pKYMX71 35S2 vector and transferred to cotton. A totally synthetic cryIC gene has been constructed and will be introduced in the cotton genome during summer 97.
Chitinase genes are recognized as potential pathogen resistance genes for plants. The coding chitinase region from potato was used as probe for in situ hybridisation with genomic cotton DNA. The most intensive signal was obtained with Gossypium herbaceum cv. Accala maxxa. Restriction analysis was done to determine the physical map of the insert. The promotor and coding regions with sequence homologous to the probe were identified. Six Sau3AI fragments were cloned. The clone containing the 100 bp insertion was sequenced and vectors will be used for transformation experiments.
In addition, polyglucanhydrolase (PGH) genes of thermophillic bacteria have been chosen as potential pathogen resistance genes. Genes from thermophilic bacteria Clostridium thermocellum and Thermotoga neopolitana coding for laminarinase (beta-1,3 -glucanase), lichenase (beta-1,3-1,4-glucanase), cellulase (beta-1,4-glucanase) and xylanase (beta-1,4-xylanhydrolyse) have been cloned in vectors pTT26, pCU402, pCU110, pTT32 respectively. These gene products possess high endoglucanase activity and thermostability. Vectors with lic A (lichenase gene) and cel 7 (cellulase gene) for Agrobacterium mediated plant transformation were constructed. Transgenic tobacco plants expressing the PGH under control of different regulatory elements were obtained. These vectors are ready to be used in transformation of cotton and other plants.
The Agrobacterium-mediated transformation of the model variety G.hirsutum L. cv. Coker was further optimized and is now routinely applied even though it takes much time. The genes of interest can be introduced into commercial cotton lines by back crossing. A genotype independent transformation system using Agrobacterium-mediated transformation of apical meristems was studied for the local FSU cv. C 6524. Shooting and rooting was obtained for non transformed plants; after transformation, only shooting but no rooting was observed on kanamycin enriched medium.
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