| Flax?Linumusitatissumum L.? is an important economic crop,Linseed is a kind of important oil crop,and Fine linen is a significant source of natural fibre in China.Compared with those developed nations;there is still great discrepancy in flax output and quality level in China.The reasons are various.However,one of the most important reasons is the relative drawback in flax varieties.Therefore breeding improvement is an urgent task of scientific research in China.In this paper,resistant abiotic stress gene At NDPK2 is introduced to linen genome mediated by Agrobacterium and the induction of linen tetraploid by chromesome doubling technology is applied to foster new flax germplasm so as to lay a solid foundation for breeding new flax variety with high-level output and quanlity.The primary research results are listed as follows:1. The establishment of Flax sterile rapid propagation system:Optimun disinfection method of Selecting two varieties of flax cultivars Shuang Ya-7 and Jin Ya-7 was being treated by chlorine which comes from 100m L Na Cl O and 4m L concentrated HCl for 24h in closed container;as for Shuang Ya-7,Medium for bud was MSB;optimun subculture propagation medium was MSB with 6-BA 1.486 mg·L-1,IAA 0.293 mg·L-1 and KT0.223mg·L-1,the proliferation coefficient got to 13.15;Optimun medium of tissue culture seeding elongation and growth was MSB with 6-BA 0.1 mg·L-1 and IBA 0.2 mg·L-1;optimum medium of rooting was MSB with IAA0.01 mg·L-1,the average number of root was 20.6,the ratio of rooting was 100%.As for Jin Ya-7,the medium for bud was MS;optimum medium of stem tip subculture propagation was MS with 6-BA 1.17 mg·L-1and NAA 0.14 mg·L-1,the proliferation coefficient was 6.80;optimum medium of hypocotyl subculture propagation was MS with 6-BA 1.17 mg·L-1 and NAA 0.2 mg·L-1,the proliferation coefficient was 5.29;Optimun medium for tissue seeding elongation and growth was MSB with 6-BA 0.1 mg·L-1 and IBA 0.1mg·L-1;Optimun medium of rooting was 1/2 MS with IAA 0.01 mg·L-1,the average number of rooting was15.6,the ratio of rooting was 100%.2.The transformation of gene At NDPK2 into flax and the effect of transgenic callus resistane stress:Stable genetic transformation system was established by selecting genetic conformation infactors:the cultured flax seeding of Shuang Ya-7 and Jin Ya-7 as material,hypocotyls was cut to 0.5 cm and then precultured for 2d in preculture medium,transformeded into Agrobacterium-mediated bacterium fluid with 0.6-0.8 OD and shaked for 10-15min.The Agrobacterium on the hypocotyl surface were blotted up with steriled filter paper.The hypocotyls were cocultured for 3d in coculture medium without antibiotic.And then the hypocotyls were transformed into the mdium with 50 mg·L-1 and 300 mg·L-1.at about 30d,most of the callus formed from hypocotyls and differentiated buds died,a minority of little buds can survive,the remained little buds transformed into the medium for elongagtion of buds so as to make the buds with high resisitance.The buds were divided into individualplant and incubated into medium of rooting with 50 mg·L-1,after 10d,about 15root present and the plant with resistance form.The primary analysis of the resistance to stress of transgenic callus showed the stress of Na Cl and mannitol did certain harm to transgenic and nontransgenic callus,but the degree of harm of transgenic callus was lower than that of nontransgenic callus.3.Flax polyploidy were induced by chromosome doubling technology:The sterile seed and stem tip of Shuang Ya-7 variety were immersed in colchicin solution so as to induce flax polyploidy.To begin with,the range of every factor was selected by singe factor assay,then the optimum condition of every factor was selected by orthogonal test.The results showed that the optimum condition induced polyploidy by sterile flaxseeds were the ones which were immersed in water for 36h,0.075%of colchicin was applied to be treated for 24h,the ratio of heteromorphosis got to 40.7%.the optimum condition induced flax ployploid by steril stem tip was that the stem tip cultivated for 8d was treated with 0.10%colchincine solution for 18h,the ratio of heteromorphosis got to 48.7%;the stem tip of the variant plant with perusal after induction was examined for chromosome.The results showed that the number of chromosome was 2n=4x=64;the stem of tetraploid was stronger than that of diploid,the leaf of tetraploid was thicker than that of diploid,the color of tetraploid was deeper than that of diploid,the surface of tetraploid was a little rougher than that of diploid.The root of tetraploid was stronger than that of diploid and the root number of tetraploid was less than that of diploid by way of observing the external features. |
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