| Poplar, with the largest area of artificial cultivation, is one of the species of tree which are most widely distributed.It is not only regarded as an important economic woody specie, but also plays a significant role in soil and water conservation and maintaining ecological balance. Like other tree species, poplar has traits like long breeding cycle, high degree of heterozygosity and complicated genetic characters, which make the conventional breeding technology is far from enough to meet the needs of modern genetic improvement.Therefore, plant tissue culture and genetic engineering to solve many problems which can not be settled by classical breeding methods, which possesses a very significant value in science. In this research, three variety, Populus.cathayana Rehd.var.Qinghai, Triploid P. xliaohenica and "Langfang No.3"(P.Langfangensis) were used to establish regeneration system; on the base of establishment of the regeneration system, we took Populus.Cathayana Rehd.var.Qinghai as receptor material and transform the gene YUCCA1 by Agrobacterium-mediated transformation to establish genetic transformation system.. The main results were as follows:(?) The experiment established regeneration system of three poplars:①Take the shoot of Populus.cathayana Rehd.var.Qinghai as explants and establish its stable and efficient regeneration system through selecting the optimal culture medium in different period of growth. The results indicated that:The highest differentiation rate of adventitious buds was on the culture medium with 1/2MS+0.5 mg·L-16-BA+0.05mg·L-1NAA+25g·L-1sucrose+7g·L-1agar, under the condition of (25±1)℃,light intensity 1500-2000 lx,and light period 12-14 h/d,under this condition,the ratio of adventitious buds differentiation reached 82%; the optimal medium for adventitious buds elongation was with MS+0.005mg·L-1NAA+25g·L-1sucrose+7g·L-1agar, which has a better capability to improve plant growth; the best subculture medium was added with MS+0.1 mg·L-16-BA+0.3 mg-L-1 NAA+25g·L-1sucrose+7g·L-1agar, and coefficient of proliferation reached to 5.1 in 30d; the optimized medium for rooting was 1/2MS+0.3mg·L-1IBA+25g·L-1 sucrose+7g·L-1agar, the rooting rate can reach 100%.;②Take the shoot of Triploid P.×liaohenica as explants; design 4 kinds of different phytohormone combination to select the optimal culture medium for inducement and differentiation of adventitious buds. The results indicated that:to combine 6-BA with TDZ has the best inducement effect, on the culture medium with 1/2MS+0.6mg·L-16-BA+O.O1mg·L-1TDZ+0.02 mg.L-1NAA+25 g·L-1sucrose+7g·L-1agar adventitious buds grew healthy with the differentiation rate of 82%; on the same culture medium, we could subculture the plantlet which would be moved on to another medium with MS+0.005mg·L-1NAA+25g·L-1sucrose+7g·L-1agar after adventitious buds differentia-ted in large quantities, then inoculated on culture medium with 1/2MS+0.2mg·L-1IBA+ 25g·L-1sucrose +7g·L-1agar, then grew into complete plantlet;③Take the shoot of "Langfang No.3"(P.Langfangensis) as explants; based on the above establishment of tissue culture of Populus.cathayana Rehd.var.Qinghai, Triploid P. xLiaohenica and design different kinds of culture mediums to induce adventitious buds differentiation, multiplication and rooting. The results indicated that:In the medium of 1/2MS+1.Omg·L-16-BA +0.1mg·L-1TDZ+O.1mg·L-1NAA+25g·L-1 sucrose+7g·L-1agar the effects on differentiation were obvious, the differentiation rate reached to 80.0%; in the culture medium of MS+0.1mg·L-16-BA+0.1mg·L-1 TDZ+0.3mg·L-1NAA+25g·L-1 scurose+7g·L-1agar, the callus is induced to differen -tiate adventitious buds to multiply, and coefficient of proliferation reached to 8.2; in the medium of 1/2MS+0.05 mg·L-1NAA+O.1mg·L-1 IBA+25g·L-1sucrose+7g·L-1agar,which is favorable to transplant successfully.On the base of establishment of regeneration system of Populus.cathayana Rehd.var.Qinghai, we studied and selected that the optimal culture medium for differentiation rate of adventitious buds of explants was 1/2MS+1.0mg·L-16-BA+0.05mg·L-1NAA+0.05mg·L-1IBA+25g·L-1sucrose+7g·L-1 agar, the differentiation rate reached to 90%, which was qualified to genetic transformation; we studied the sensitivities of Populus. cathayana Rehd.var.Qinghai to antibiotic in different culture stages, and the sensitivities of leaves to Kanamycin were studied, it was found that Kanamycin concentiation of 20mg·L-1 was the tolerant concentration for leaves,15mg·L-1 for plantlet rooting; we studied the bacteriostatic effects of car after infected with Agrobacterium, and discussed the favorable using methods of antibiotic, namely,4 days after the leaves were infected with Agrobacterium, added 400mg·L-1 car to the culture medium, which can effectively inhabit the excessive multiplication of Agrobacterium and has slight effect on the normal growth of explants. After manipulated as above, a stable acceptor system for gene transformation had been established, and a sound foundation for further gene transformation had been constructed.Several factors affecting YUCCA1 gene transformation of Populus.cathayana Rehd.var. Qinghai by A.tumefaciens was studied, and a simple and effective with optimized condition for transformation of Populus.cathayana Rehd.var.Qinghai was establised. The resule was when pre-cultivated for 1 day, infected in OD600=0.4 for 1Omin, and co-cultured for 3 days,no AS in the co-culture medium, then selected 14days after co-culture, the genetic transformation efficiency was the highest.By using Agrobacterium-mediated transformation, this research obtained 159 Kanamycin-resitant adventious buds. After selecting at different levels, we got 49 Kanamycin-resitant rooted plants. In conclusion, we selected and obtained 8 PCR positive plants of transgenic YUCCA1 by PCR molecular detection.Summary, in this research, three variety, Populus.cathayana Rehd.var.Qinghai, Triploid P.×liaohenica and "Langfang No.3"(P.Langfangensis) were used to investigate the optimal culture mediums in different stages, and establish efficient and stable regeneration system to basis of large-scale production and genetic engineering; on the base of establishment of the regeneration system, we took Populus.cathayana Rehd.var.Qinghai as receptor material and transform the gene YUCCA1 by Agrobacterium-mediated transformation. We discussed and researched into the main factors which produce effects on the efficiency of genetic transformation of Populus cathayana Rehd.var.Qinghai, which promote the efficiency of genetic transformation, and establish genetic transformation system to provide an experimental basis for the molecule aggregation breeding.
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