Regeneration And Agrobacterium-mediation Gennetic Transformation In Oilflax (Linum Usitatissimum L.)

The study on hypocotyls of oilflax as expant, Establish high frequency and stable regeneration system by adventitious shoots induced,callus regeneration,anther callus regeneration. The study of affect genetic transformation on oilflax,order to establish gennetic transformation system.The main results are as follows:1.The Study of regeneration system on oilflax(1) adventitious shoots inducedAdventitious shoots induced on add 6-BA and NAA to MS basic medium, inproved shoots induced and shoot numbers.The best shoots induced medium was YP4:MS basic medium add 0,2 mg/L 6-BA and 0.5 mg/L NAA.An oilflax regeneration system was established through optimization of basic medium, genotype, seedling age of oilflax. Data showed that the best medium for adventitious shoots induced was MS basic medium,in YP 4 medium Shoot induction rate of "longya10", "longya9", "longya8", "81A350", "873", "S1", "hongmu65"were 93.73%,38.13%,90.49%,76.26%,23.90%,73.26%,29.17%;While the average shoot numbers per expants were:16.89,7.66,16.44,13.67,4.44,11.78,3.67;Hypocotyls taken from 7 to 8 days old seedlings showed the highest regeneration ability.(2) callus regenerationAdd 6-BA and NAA or 2,4-D to MS basic medium induced callus, the growth of callus was indeterminate on add 6-BA and 2,4-D to callus regeneration medium; the power of callus differentiation was greater on add 6-BA and NAA to callus regeneration medium.The best callus regeneration medium was:MS basic medium add 2 mg/L 6-BA,0.5 mg/L IAA and 200 mg/L LH,the rate of callus regeneration was 27.04%.(3) anther callus inducedThe effects of hormone combination and low temperature pretreatments on anther callus induction. lower temperature pretreatment with 4℃for buds.Before inoculating were beneficial to callus induction.Synthetical study callus induction rate and quality, result as follow:the anther of F1 about 1S×95005, buds lower temperature pretreatment with 4℃for 5 days, the highest callus induction was 37.29%. (4) regenerated shoot rootAdd NAA and IB A to 1/2MS basic medium induced root formation, root induction rate and roots per shoot significant difference.The best root formation medium was N2:1/2MS basic medium add 1 mg/L IBA,sucrose 20 g/L.It was optimum for root formation of regenerated shoot with 78.82% root induction rate and 14.44 roots per shoot.2.The Study of genetic transformation system(1)The optimum procedure in oilflax transformation as follows:longyal0 hypocotyls, the hypocotyls were precultured for 2 days on YP4, infected 10-15 min with OD600=0.6, co-cultivated for 4 days in shoots induced medium with 20-24℃.(2) Detection of transforming regenerated oilflax:We used PCR to detect the transforming shoots.2/3 were positive with bar gene; The results of PCR demonstrated the integration of target gene into oilflax genome primarily. Establishment of oilflax (huma) gennetic transformation system by Agrobacterium mediation was primarily accomplish.