| Cotton is an important economic crop. In the past century, significant improvements in yield and quality of cotton have been made through traditional breeding methods. However, traditional breeding is based on the random gene combination, most of time, desirable and undesirable genes combine together, so it needs a long time for selection. Also, only genes in the same species can be used by cross since there is a sexual incompatibility in different species. This greatly restricts the use of the gene resource. Genetic engineering provides novel approaches to facilitate cotton breeding by introducing genes from various plants and other organisms into cotton to generate new germplasms with higher yield, better fibre quality and resistance against insects and diseases.There are several methods, such as Agrobacterium-mediated system, particle bombardment and pollen-tube pathway system, for stable introduction of novel genes into cotton. Agrobacterium-mediated system remains the most efficient method for cotton transformation in the world. Hypocotyl and cotyledon are commonly used as target material in the previous Agrobacterium-mediated system. The time required to regenerate a transgenic plant from explants, such as hypocotyls and cotyledons which have been cocultivated with Agrobacterium, is extremely long (9-12 months). In the process of regenerating transgenic plants, only few of transgenic cells have the ability to produce embryogenic callus and further develop into normal plants, which result in low transomation efficiency. An availability of highly regenerable embryogenic cell cultures for transformation would reduce the time involed in the regeneration oftransgenic cotton plants. In the present method, cotton embryogenic cultures are used as an ideal target material for Agrobacterium-mediated transformation. Most of the Km resistant and GUS positive embryogenic cultures regenerated on the selection medium have the ability to develop into transgenic plants, which increasing the transformation efficiency. The step of inducing transgenic embryogenic callus, which is a pivotal step in the regeneration of transgenic plants, is elimited and thereby the period of regenerating transgenic plants reduces at least 3~4 months. We introduced afp-spCEMA gene into the cotton genome with the improved transformation system. The main results are as follows:1. Development of efficient Agrobacterium-mediated transformation system of cotton embryogenic cultures.The gus-intron gene was used in experiments to study the factors influencing transformation efficiency by transient expression. An efficient Agrobacterium-mediated transformation system of cotton embryogenic cultures was developed. The research indicated that 100[imol/L acetosyringone (AS) supplied in the inoculation and co-cultivation medium increased the transient expression efficiency greatly for all the 3 Agrobacterium tumefaciens strains investigated (LBA4404, EHA101 and EHA105). The supervirulent strain EHA101 gave the highest transient efficiency in the presence of 100 mol/L AS or not. The maximum transient expression frequency was obtained when embryogenic cultures cocultived with Agrobacterium tumefaciens strain EHA101 for 4 days. The cotyledon embryo was easily infected by Agrobacterium tumefaciens.2. Stable transformation of interest gene and regeneration of transgenic plants.After optimizing the factors influenced transformation frequency, interest gene was introduced into the cotton genome with Agrobacterium tumefaciens strain EHA101 in the presence of 100 mol/L AS. Following co-cultivation, the embryogenic callus was transferred onto selection medium for selecting transformed cells. After 7 to 8 weeks, kanamycin resistant cultures formed on the selection medium and were verified to transgenic cells by GUS histochemical assay. GUS positive frequency were varied greatly from 20%~90% with different repetition. A great deal of embryos produced after the Km resistant and GUS positive embryogenic callus cultured in liquid MSB for two we...
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