Regulation Of Somatic Embryogenesis Of Cotton (gossypium Hirsutum L.) And Regeneration Of Transgenic Salt-resistant Plants

Soil salinity is a major limiting factor to productivity of crops. High salinity causes ion imbalance and hyperosmotic stress in plants. The United Nations Environment Program estimates that approximately 20% of agricultural land and 50% of cropland in the world is salt-stressed. Many researches show that overexpression of the Na+/H+ antiporter gene and H+-PPase gene confers salt tolerance to transgenic plants.Cotton is an important cash crop for its natural fiber. Therefore, enhancing the salt tolerance of cotton is of great importance to improve cotton production and strengthen the development and utilization of salt-stressed land. However, many hinders still exist in cotton transformation. The main reason is that cultivars of transgenic cotton, which requires transformation of appropriate tissue followed by regeneration, remains extremely difficult in somatic embryogenesis and regeneration. Recalcitrant cotton cultivars, long tissue culture duration, the unpredictability of tissue culture, and a high degree of genotype dependence are more troublesome in regeneration of cotton. The length of time in cotton transformation can result in a higher rate of somaclonal variation. We have examined some factors that can significantly affect the efficiency of regeneration and transformation in cotton. The regeneration of salt-resistant and drought-resistant plants is developed through cotton hypocotyl imediated by Agrobacterium tumefaciens, and transgenic plants are examined and analyzed on the molecular level in this dissertation. The main results obtained are listed below.1 KT is one of the main factors determining the tissue culture response in cotton. Calli and embryogenic calli are induced in media containing 0.1 mg/L 2,4-D and 0.1 mg/L KT. Somatic embryogenesis from calli derived from hypocotyls segments are observed only on inducing original calli medium containing NH4NO3. The large number of somatic embryogenic calli are recorded on the medium containing double KNO3 but free of NH4NO3. The efficiency of induction of embryogenic calli from the tissue culture can reached 80% in the medium supplemented with 2.0 g/L Gln and 1.0 g/L Asn of Xinluzao 17. Somatic embryos in the medium with 2.0 g/L Gln and 1.0 g/L Asn facilitated maturation and regeneration.2 plant expression vectors pCABMIAL2300/HcNHX1 and pCAMBIAL2300/HcVP1 were constructed and transformed to the agrobacterium strain, EHAl05, respectively. This provided vectors for further genetic transformation and optimizing the transformation condition.3 Hypocotyl was proved to be a valuable explant to cotton transformation. Various aspects of transformation proeesses were examined in efforts to improve the efficiency of production of transgenic cotton. OD (A600) value 0.3~0.5, 75 mg/L Kanamycin and 200 mg/L Cefotaxine were proved to be significantly better than other coneentrations.4 Two salt-resistance genes, HcNHX and HcVP1 were introduced into cotton genome via Agrobacterium tumefaciens. PCR results showed that there were transgenes in the cotton genome.