Regeneration Of Sea-island Cotton (Gossypium Barbadense L.) And Genetic Transformation

Recently, though cotton tissue culture has been made great progress, these studiesmostly focus on a few varieties, particularly some model varieties. And however, someproblems have been not solved such as low germination rate of somatic embryos, highabnormal embryos' proportion, and long cycle of regeneration and so on. So developingnew regenerative systems of cotton and broadening the number of regenerative varietiesof cotton is presently a new trend to cotton tissue culture. With the development of geneengineering and molecular biology, the method of transformation has been utilized inmodern plant breeding, which led to the realization of so called molecular designedbreeding, which the foreign useful genes could been induced and inherited directionallyand stably in the host plant cells. The reproducible and high efficient plant regenerationtechnique system is a prerequisite for transformation of plants to obtain abundantselective colonies of transgenic plants. So it is very important and significant to establisha high efficient somatic embryogenesis and plant regeneration system for the studies ofthe gene engineering, function genomic and breeding in upland cotton.The research deals with the Gossypium barbadense L. varieties as materials.Through somatic embryogenesis and organogenesis, carry on the research thatregeneration from callus of hypocotyls and shoot apical meristems, inquiry into the cottonsea-island cotton somatic embryogenesis, the shoot apical meristems regeneration and themain influence factor of Agrobacterium-mediated transformation.1. The research deal with the Gossypium barbadense L. varieties with superiormaterials. Hypocotyls, cotyledon, and root act as the explants in vitro. The effect ofdifferent hormones and their combination, carbon source, inorganic salt, amino acid onthe induction of callus, somatic embryogenesis and differentiation were studied. Theresult showed that the combination of NAA (0.1-1.0mg/L) and KT (0.1-1.0mg/L) was themost effective one in the sea-island cotton callus inducting. The length of propagationtime influence somatic embryogenesis and differentiation. Longer callus propagation timepromote crazy growth of callus and inhibit cell differentiation.2. The research deal with the Gossypium barbadense L. varieties with superiormaterials. Shoot apical meristems act as the explants in vitro. Plant cytokinin 6-BA isadded into start-up medium and/or induction medium by two steps for preventing frombrowning effect on explants and we got more than 12 multiple shoots each shoot apicalmeristem. After elongating culture of shoots, rooting was induced in one half of MSmedium added IBA.3. The research deal with the Gossypium barbadense L. varieties with superiormaterials. Shoot apical meristems acted as the explants in vitro. In assistance of ultrasonic,we used Agrobacterium -mediated method to transfer IPT and GFP gene into shoot apicalmeristems and we also got multiple shoots by adding 6-BA into medium. After manytimes experiments, it is most effective that the power of ultrasonic is between 50w-100wand treated time is between 1min-3min. We finally got plantlets with resistance to30mg/L Kan after primary selection.4. In this research, more than 3000 shoot apical meristems were prepared and used asreceptor in the transformation experiment. Through PCR analysis, we detected thatpositive plants reached 2.97ï¼….