Plant Regeneration And Agrobacterium Mediated Transformation Of Enhancer Trap On Upland Cotton

Upland cotton (Gossypium hirsutum L.) is one of the most important economic crops.Genetic engineering plays important roles in further improving fiber yield, quality offiber, oil content and increasing the production efficiency and reducing both the monetaryand environmental cost of production, and many excellent cotton cultivars have beendeveloped through since 1987. However, many hinders still exist in cotton biotechnology.The main reason is that it is extremely difficult in somatic embryogenesis andregeneration during genetic transformation. Recalcitrant cotton cultivars, long tissueculture duration, the unpredictability of tissue culture, and a high degree of genotypedependence are troublesome in regeneration of cotton. The long period of cottontransformation results in more somaclonal variation.Enhancer trap system is a powerful approach for identifying novel genes andregulatory elements based on the expression pattern of a reporter gene that randomlyintegrated into the genome of plant.We have optimized some factors that can significantly affect the efficiency ofregeneration and transformation in cotton. Using this high efficiency transgenicprocedure, an enhancer-trapping system has been introduced into cotton genome. And theresult is as follows:1. Various aspects of transformation processes were examined in efforts to improvethe efficiency of production of transgenic cotton. The effects of Agrobacterium strains,acetosyringone (AS), co-cultivation temperature, co-cultivation duration, Agrobacteriumconcentration on transformation efficiency were evaluated. Strain LBA4404 provedsignificantly better than C58C3. Agrobacterium at aconcentration of 0.5×10⁸ cells/ml(OD600=0.5) improved the efficiency of transformation. Relatively low co-cultivationtemperature (19℃) and short co-cultivation duration (48 h) were optimal for developing ahighly efficient method of transforming hypocotyl. Concentration of acetosyringone at 50mg 1^-1 during co-cultivation significantly increased transformation efficiency.2. With decrease of the concentration of hormones (2, 4-D), the calli began to appearthe characteristic of hardness and greenish, which was harmful for somaticembryogenesis3. Putatively transformed calli were analyzed by PCR with specific primers ofselection marker gene npt-â…¡. 29 regenerated plants from independent clones wererevealed by PCR analysis (Figure 3). Evidence collected from PCR analysis suggests the transformation system with hypocotyl via Agrobacterium really effective. Genomic DNAfrom leaves of transformed plants were digested with Hindâ…¢and allowed to hybridizewith npt-â…¡probe. The copy numbers of the transgenic plants ranged from 1 to 4,Southern blotting analysis also indicated T-DNA insertion was randomly integrated intocotton genome.4. The gus gene was expressed in calli, embryogenic callus, somatic embryos and theroot cap of regenerated plant. Especially, the gus gene was significantly expressed inresistant calli with a high expression rate of 49.3%.