| Cotton is one of important economical crops in China, and verticillium wilt is akind of most serious diseases in cotton production. In this research, a lysozyme generesistant to fungal diseases had been introduced into the cotton cultivar "Xiangmian10" and "Xiangmian 15" through the pollen tube pathway transformation method tobreed transgenic cotton lines resistant to verticillium wilt. In addition, A regenerationin vitro system of "Xiangmian 10" for Agrobacterium-mediated transformation hadbeen preliminarily established. The main results were as follows:1 The establishment of cotton regeneration in vitro systemThe explant types were an important factor to cotton calli induction. For"Xiangmian 10", the calli induction rate of explants was hypocotyl＞Seed leaf＞Root.The optimal culture medium was MSB+ZT0.1mg/ml for calli induction, subcultureand differentiation from the hypocotyl explants with the optimal carbon source beingglucose. The shoot regeneration rate of cotyledon-shape embryos reached 13.6% inmedium SH+IAA0.2mg/l+KT0.2mg/l. The optimal rooting medium wasMS+IAA0.1 mg/l with a rooting rate of 100%. The speed of calli induction dependedon the explant types, culture temperature and hormone combinations. Increasing the 2,4-D concentration was advantageous to calli induction, however, it could not be ableto directly induce embryo calli, and seperately supplement of ZT could effectivelyinduce embryo calli. The optimal culture temperature was 28℃or so. Cotton callibrowning could be reduced by selecting the suitable basic mediums and hormonecombinations, as well as by adding active carbon and PVP. The hormone combinationof KT+6-BA could obviously reduce calli browning, but solely using IAA, KT or6-BA hormone could not reduce browning.2 Obtaining of transgenic cotton resistant to verticillium wilt via pollen tube pathwaytransformation method A foreign lysozyme gene had been introduced into the cotton cultivar "Xiangmian10" and "Xiangmian 15" via the pollen tube pathway transformation method Thesuitable position for DNA injection was the 2nd fruit spur to the 6th fruit spur, and theoptimal time for injection was at the approximately 24 hours after self-crossing Thetransformation method was to vertically inject DNA into ovary along the center oftheaxile placenta, the transformation rate being up to 9%. In this research, 23anti-antibiotic T0 plants from transformed "Xiangmian 10" and 9 T0 plants fromtransformed "Xiangmian 15" were selected, respectively. The results of PCR andPCR-southern blotting analyses indicated that there were 25 plants integrated withboth of lysozyme gene and NPTⅡgene, 6 plants only with NPTⅡgene and 1 plantonly with lysozyme gene.3 preliminary disease resistance evaluation of the transgenic plantsA preliminary resistance testing of the transgenic cotton plants to verticillium wilthad been conducted by root sprinkling and leaf pricking and spreading withverticillium wilt strain suspention in the greenhouse, respectively, and then the wiltingsymptom of the leave was observed. The results suggested that the transgenicindividuals grew normally, but the leaf blade of the control individuals produced thebrown spots, then turn yellow and wilt, furthermore the plant growth was stoped. Asinitially indicated that the cotton plants transformed with lysozyme gene hadproduced resistance to verticillium wilt.
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