The Study Of Somatic Hybridization Between Brassica Napus And Sinapis Arvensis By Protoplast Fusion

Xinjiang wild rape 18 (Sinapis arvensis L.)is a wild colony distributing in Xinjiang of china , shattering resistance and sclerotinia rot resistance capacity is strong , erucic acid and glucosinolate content is lower , possessing cytoplasmic male sterility , it is excellent germ plasm for breeding of Cruciferae .Somatic hybridization not only can overcome sexual hybridization incompatibility barriers , but also quickly lead some excellent agronomic traits and some useful traits of wild wariety into cultivated species , have unique function in germplasm resources innovation and variety improvement . Protoplast fusion has been involved with both parents cytoplasm , not only the cytoplasmon transferring to absolute novel nuclear background , but also the genome of chloroplast or mitochondria can combinate over again . Through adopting protoplast fusion can achieve the transfer of useful traits under the control of cytoplast ,such as cytoplasmic male sterility , herbicide resitance ,cold resistant , etc .Through this study ,we obtain the results as fellows.1.Established the cotyledon protoplasts regeneration system of cultivar Zhongshuang 6 of Brassica nupus L.Protoplasts were prepared from the cotyledon of Brassica napus cultivar Zhongshuang 6, cultured with solid-liquid combined method. The optimized conditions about the isolation, culture and regeneration of protoplast were tested and ultimately the regeneration plantlets were obtained. The results indicated that high recovery of protoplast could be achieved with enzyme mixture (0.5% cellulase, 0.1% pectolyase + 0.2 M mannitol + 80mM CaCl2) diluted with SCM solution as 1.5:3.5 ratio. It is fine to culture protoplast on solid-liquid combined media B and C. The optimum differentiation medium was E+1.0 mg/ml 6-BA+0.1 mg/ml NAA+0.02 mg/ml GA3+30μM AgNO3. The best rooting media was MS without hormones.2. We explored the conditions in which isolating the mesophyll protoplasts of Xinjiang wild rape 18(Sinapis arvensis L.), and performed further culture.This study isolated the protoplasts of 18 hao mesophyll of Xinjiang wild rape 18 (Sinapis arvensis L.) , adopted solid conbine liquid culture method , investigated the condition about isolating, culturing and regenerating. The results indicated that high recovery of mesophyll protoplasts of xinjiang wild rape could be achieved with enzyme mixture (2% cellulase, 1% pectolyase + 0.2 M mannitol + 80mM CaCl2) diluted with SCM solution as 1.5:3.5 ratio for 14 hours, while when cultured in the B culture medium designed by the anterior ,we discovered that only a minority of protoplasts can go through the first division , but they ultimately dead. 3.Optimized the condition for protoplasts fusion between Brassica napus L. and Xinjiang wild rape 18(Sinapis arvensis L.).We indicated that when fused the density of the protoplasts among the two cultivars at 1×105mL-1-5×105mL-1and PEG at 30% , can obtain the relatively better fusion frequency .when cultured in the B solid combine liquid culture medium , the fused protoplasts scarcely go through division , only expanded in the size, and eventually brown.4. The clonal propagation of Xinjiang wild rape 18 (Sinapis arvensis L.) could be performed with in vitro culture combined with conventional cultivation method under subtropical monsoon humid climate condition in Wuhan.