| Conventionally, Agrobacterium-mediated transformation used hypocotyls or cotyledonas explants and Coker 312 as its model cultivar, which was via somatic embryogenesis andwas the preferred method for transgenic cotton. The transformation protocol was severelyrestricted by genotype-dependent response. Although some genotypes have beentransformed successfully, low regeneration efficiency of via somatic embryogenesis, aprolonged culture period, high frequency of abnormal embryo development, lowconversion rate of somatic embryos into plantlets, and a lack of shoot elongation were theproblems associated with cotton regeneration. In this study, standard transformation systemwas optimized and the method of bark grafting was firstly applied to reproduce theregenerated plantlets. The highefficiency transformation system of Simian3, which is amajor cultivar at Yangtze River region, was established. To further improve thetransformation efficiency, a high efficiency transformation system was built and optimizedby transforming the embryogenic calluses of Simian3 and the regeneration ability ofSimian3 and W0 line was studied by means of tissue culture. An anti-herbicide gene (bar)was successfully introduced into the cultivar Coker 312 by the means of optimized protocolof standard transformation system. The results of study are as follows:The conventional transformation system was improved by changing nitrogen (N)sources during the period of callus induction, picking out embryogenic calluses as early aspossible, and controlling the metabolism of embryogenic calluses through metabolic stress.The factors affecting efficiency of transformation were analyzed and it was found that Nsources and metabolic stress were critical in the improvement of transformation system. Itwas the first report to apply the method of bark grafting to reproduce the regeneratedplantlets, and its manipulation processes and skills were also introduced in detail. Theadvantages of bark grafting are characterized its simplicity, easy operation, the highsurvival rate of plantlets and a shorter time for plantlets to build up.Transgenic plants were generated and a highefficiency transformation protocol was built by Agrobacterium-mediated transformation of hypocotyls segments of Simian3,which is one of major cultivars at Yangtze River Valley cotton-growing region.Molecular biology analysis proved the integration of foreign gene into the genome ofSimian 3 and their expression. According to morphology, the calluses which can giverise to plantlets are different from that of Coker 312; the embryogenic calluses ofSimian3 were originated from two kinds of primary calluses. Kanamycine resistance testand PCR of T1 plants were carded out. Abundant mutants of flower and plant type in T0and R0 were obtained. Several genes have been introduced to genome of simian 3 by thehighefficiency transformation protocol.Using the embryogenic calluses of Simian3 as explants, Agrobacterium-mediatedtransformation system has been established and optimized. The transformation efficiencywas enhanced through improving multiplication method of embryogenic calluses andconditions of cotransformation, antibiotic selection and redifferentiation. The status ofembryogenic calluses which served as explants and culture conditions during co-culture andselection culture was highly relative to the transformation efficiency. Yellow, loose andgrain embryogenic calluses had high transformation efficiency. Application of 50rag 1-1acetosyringone (AS) during cocultivation period and coculture under 19℃enhanced thetransformation efficiency. It was found that interaction between AS and cocultivationtemperature can further enhance transformation efficiency. Making embryogenic callusesas dispersive as possible during selection culture with antibiotics was beneficial to inhibitand kill the false positive transformation calluses. Transgenic cottons could regenerate in5-6 months with 55.7％of positive plantlets and 9.7 plantlets per transformation event.Cotton transformation with a high efficiency using embryogenic calluses as explants couldmight be less laborious and expensive, make it possible to produce transgenic cotton in alarge scale, and pave the way for genetic improvement of cotton and functional research toimportant gene in cotton.High-frequency somatic embryogenesis pure lines of W0 were selected by tissue cultureaccording to the ability of regeneration, The efficiency of regeneration in simian 3 wasenhanced from 4.5％to 40％after one cycle of selection. An inherited mutant whichgenerated from tissue culture was selected. High PIT-resistant cottons were successfully regenerated from hypocotyls segments ofCoker 312 by Agrobacterium transformation. PCR, RT-PCR and Southern blottingconfirmed the integration of bar gene into the genome of Coker 312. The expression of bargene was also proved by the application of PPT to the T0 and T1 plants. Selectinghomogenous lines of transgenic cottons and identifying one mutant on phenotype are to befurther studied.
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