The Molecular Breeding Of Ramie(Boehmeria Nivea L.)

In order to improve the efficiency of ramie breeding and to create new germplasm, it was studied to improve ramie cultivars with the plant gene engineering or the total exogenous DNA introducing. The results are as follows. 1.Establishing the receptor system for ramie genetic transformation. The redifferentiation abilities of different explants of ramie were compared, and the hypocotyl was considered as a favorable receptor for genetic transformation because of its high redifferentiation potential and easy preparation. Under different tissue culture conditions, the regeneration frequency of adventitious shoots from hypocotyls varied as: that of light culture in alternating temperatures of day 26-28X2 and night 18-20C > that of dark culture in constant temperature of 26-28XD > that of light culture in constant temperature of 26-28 0C, which revealed that the differentiation of shoots was more sensitive to temperature than to illumination. The only addition of BA in medium was not benefit for dedifferentiation of hypocotyls. However, the combination of BA with IAA promoted the dedifferentiation and redifferentiation, though the regeneration frequency differed in kinds of combinations, among which the combinations of 2.0-3.0 mg/i BA + 0.5 mg/I IAA produced a highest differentiation frequency of 70 % or so. The addition of 1 mg/I AgNO3 in medium also improved the regeneration of shoots. 2. Producing herbicide-tolerant transgenic ramie via A. tumefaciens mediated transformation. A system for genetic transformation of ramie was developed by systematically optimizing the factors affecting A. tumefaciens mediated transformation efficiency. It was showed that ramie was a kind of plant susceptiable to herbicide, whose critical tolerance concentration to herbicide asta was 0.3 mg/I or so, and that the herbicide inhibited the regeneration of shoots especially. N4eanwhile, the carbecillin facilitated the emergency of clustered shoots but had no obvious repression for rooting of adventitious shoots. Hypocotyls had a high sensitivity to the strains of A. tumefaciens, with a GUS instantaneous expression frequency close to 100 % when co-cultivated 1 or 2 days with the bacteria. The strain of E1-1A105 was suitable for transformation of hypocotyl by inoculating 5 minutes in a bacterial concentration of 0D600=0.1-0.2 (the bacteria were resuspended and diluted with 1/2MS liquid medium). The preculture of hypocotyis and their co-cultivation with the bacteria improved greatly the transformation efficiency, and a preculture duration of 6-9 days and a co-cultivation duration of 4 days were the best choices for hypocotyl of ramie. In addition, the GUS instantaneous expression frequency was not closely positively correlated to the GUS stable expression frequency and to the ratio of positive transformants to herbicide-tolerant calli, which indicated it necessary to combine these three indexes for the optimization of transformation system of ramie. The oxidization of ramie materials in histochemical GUS assay could be effectively reduced by adding 6 % PVP in the X-GIuc solution. Through histochemical GUS assay and DNA dot blotting, 8 phophinothricin-tolerant transgenic plants were obtained. 3. Obtaining variants by introducing exogenous cotton DNA into ramie. The exogenous cotton DNA was successfully introduced into ramie by over-dried seed soaking. In the D1 generation, two plants 97-24 and 97-25 whose leave had extraordinary morphology, were selected out. The D2 generation of 97-24 segregated, with most of p...