| Poplar is the general designation for Populus of family Salicaceae. It has a lot of characteristics, as fast-growing in early stage, strong adaptability, wide distribution, many species and varieties, easily hybrid, easily modified genetic characters and easily vegetative propagation. Poplar is widely used for ecological protective forest, three north shelterbelt, agroforestry protective forest and industrial plantation. It is also a excellent species for road greening and garden landscape. Moreover, as one of the fast growing species, poplar is widely cultivated in the world and it has rich genetic resources, strong vegetative regeneration capacity, rapid growth and relatively short rotation period. It is widely considered one of the important species of biological energy in the future. But, if rely on conventional breeding methods, poplar's breeding cycle is long and the process is complex, also effected by climate, geography, and water. It is difficult to breeding new varieties and hard to satisfy the purpose of directive breeding new varieties of poplar. Since the 80's, the development of modern molecular biology techniques, particularly, genetic engineering technology, brings unprecedented reform to the poplar breeding, and it greatly improves the efficiency of breeding and expands the breeding objectives. So far, transgenic breeding of poplar has been carried out 20 years, although the transgenic technology has become more mature, there are still some problems, as the lack of transgenic acceptor systems and the low efficiency of transformation. Therefore, the establishment of a stable and efficient in vitro regeneration system and genetic transformation system of poplar is provided a technical platform for further transgenic breeding and rapid verification system of gene function. It has important scientific significance.In this study, triploid Populus alba×p.berolinensis, P.deltoides Cl.'Zhonglin46'and P.deltoldes Bartr.×P. cathayana Rehd. Cl.'Xifeng-25'were used as explants. On the basis of the establishment of high efficient regeneration system of the three poplars, YUCCA1 gene was transformed to triploid Populus albaxp.berolinensis by using Agrobacterium tumefacien-mediated method. The main progress of this study was as following: 1. Establishment of high efficient regeneration system of three poplars:by using stems with axillary buds of triploid Populus albaxP.berolinensis, P.deltoides Cl.'Zhonglin46'and P.deltoldes Bartr.×P.cathayana Rehd. Cl.'Xifeng-25'as explants, high efficient regeneration system was established in our experiment, the following was the medium and hormones used in the different stage of poplar tissue culture:(1)Triploid Populus albaxP.berolinensis:①The primary culture:1/2MS+TDZ0.02 mg·L-1(same as below)+NAA 0.05, the culture time of axillary bud sprouting is 10 d, the axillary bud sprouting rate is 96%and the mean length of the buds is 6.0 cm after 30 d;②The subculture:MS+BA0.05+ NAA0.02, the differentiation rate is 100%after 30 d, and the plantlet height is 3.5 cm/7.0 cm after 30 d/60 d;③the rooting culture:1/2MS+NAA0.05+IBA0.20, with the rooting rate of 100%after 20 d.(2)P.deltoides Cl.'Zhonglin46':①the primary culture:1/2MS+BA7.5+NAA0.05+TDZ0.02, the culture time of axillary bud sprouting is 10 d, and the axillary bud sprouting rate is 88%and the buds is 4.0 cm after 30 d;②the clump bud culture: MS+BA0.5+NAA0.05+IBA0.1, the differentiation rate is 92%after 30 d, and the average number of adventitious buds is 10.2;③the shooting culture:MS+BA0.1+IBA0.3, the plantlet height is 3.5 cm and the shooting rate is 90% after 30 d;④the rooting culture:1/2MS+NAA0.01+IBA0.3, with the rooting rate of 93%after 20 d.(3)P.deltoldes Bartr.×P. cathayana Rehd. Cl.'Xifeng-25':①the primary culture:1/2MS+TDZ 0.02+NAA0.05, the culture time of axillary bud sprouting is 10 d, and the axillary bud sprouting rate is 94%and the buds is 5.0 cm after 30 d;②the clump bud culture:MS+TDZ0.02+NAA0.05, the differentiation rate is 96%after 30 d, and the average number of adventitious buds is 12.1;③the shooting culture:MS+BA0.02+NAA 0.02, the plantlet height is 7.0 cm and the shooting rate is 92% after 30 d;④the rooting culture:1/2MS+NAA0.02+IBA0.3, with the rooting rate of 90%after 20 d.25 g·L-1 sucrose and 7 g·L-1 agar were added in the medium mentioned above, and pH of the medium is 5.8. Culture condition is illumination time of 12-14 h·d-1, illumination intensity of 2000 lx, and temperature of (25±2)℃.2. Establishment of gene transformation receptor system for triploid Populus alba×P. berolinensis: by using leaf explants of triploid Populus alba×P.berolinensis as explants, adventitious bud regeneration system from leaf explants was established in our experiment, the optimal selection pressures of kanamycin for adventitious bud regeneration and adventitious bud rooting were determined. The concentration of carbenicillin were also determined. The results indicated that the optimal medium for adventitious bud regeneration is 1/2MS+BA0.6+NAA0.1+ 25 g·L-1 sucrose+7 g·L-1agar, with the frequency of adventitious bud regeneration is 99.4% and average adventitious buds per leaf was 10.1; the optimal selection pressures of kanamycin for adventitious bud regeneration and adventitious bud rooting were 50 mg·L-1; 400 mg·L-1 carbenicillin can inhibit the growth of Agrobacterium tumefacien, and will not affect adventitious bud regeneration and adventitious bud rooting.3. Optimization of conditions for genetic transformation of triploid Populus alba×P. berolinensis:using leaf explants of triploid Populus alba×P.berolinensis as the receptor material for transformation, six factors to affect transformation efficiency was studied, such as pre-culture time, Agrobacterium concentration, infection time, co-culture time, adding acetosyringone (AS) concentration as co-culture medium and selection modes. The results indicated that the optimal transform condition for triploid Populus alba×P.berolinensis is:2 days for pre-culture, OD600nm= 0.6,20 minutes for infection,4 days for co-culture, and AS 50μmol·L-1as co-culture medium, and then lasting 14 days before selective culture.62 Km-resistant plantlet were obtained through the adventitious bud regeneration selective culture and the adventitious bud rooting selective culture.4. PCR analysis of Km-resistant plantlet and obtaining transgenic plants:62 Km-resistant plantlet were detected by PCR, and the PCR analysis showed that 16 plantlet took on positive reaction. The result of this research primarily proved the YUCCA1 gene has inserted in the poplar genome.
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