Transformation Of Populus Deltoides With Anti-PLDγ Gene And Chitinase Gene

Poplar is one of the wide-spread cultivated trees in our country. Highgrowth rate, easy propagation and its usefulness in timber industry, pulpwoodmaking, shelter belt and other purpose afforestation make it to be the mostimportant tree for artificial silviculture and for the same reason poplar will bethe main tree species to establish million-hectare forest bases for fast-growingcommercial wood. Via sequential single-gene transformation strategy, antisensephospholipase Dγ(Anti-PLDγ) gene and chitinase (CH5B) gene wereintroduced into Populus deltoides (G2) in proper order by Agrobacteriumtumefacien mediated so as to enhance salt-tolerance and disease resistance. Firstly, the optimal media of Populus deltoides (G2) for buddifferentiation and rooting were established. The high frequency of shootregeneration can be induced on MS medium supplemented with 0.5mg·L-16-BA, 0.1 mg·-1NAA. After 5 days culturing, the explants volum Lbulged. Adventitious buds regenerated in about 15 days. The maximum ofthe buds were observed within 20 to 30 days. When the shoots grew to 2~3 cmhigh, cut them from leaves, and then transferred on to root differentiationmedium (1/2MS+0.02 mg·-1NAA) . The shoots would develop into whole Lplants within 10 days. The sensitivity of explants to cefotaxine, kanamycin and hygromycinwere tested separatly in order to decide a reasonable selective pressure fortransformation. Cefotaxine 400 mg·L-1 could inhibit Agrobacteriumtumefacien growing too rapidly. Kanamycin 20 mg·L-1, hygromycin 10 mg·L-1can be used as the died line concentration of selecting the transgenic bud 3反义磷脂酶 Dγ基因与几丁质酶基因转化杨树的研究during the bud differentiation. In the rooting phase, kanamycin concentrationshoud be 30 mg·L-1, hygromycin concentration shoud be 10 mg·L-1. Populus deltoides (G2) transformation system was reformed. Theprocedure in brief is: ①Selected the healthy and vigorous leaves which hadbeen cultured 2~3 weeks, carved with a piece of scalpel on the contary ofleaflet. Precultured 3 days; ② Diluted the Agrobacterium in log phase(OD600=0.3) with the equal volume of 0.5% sucrose solution (contains a kindof ditergent Silwet-77 0.02%); ③Transferred the leaves into the solution ofabove, shaking the flask gentlely for 5~7 min, put the leaves on filter papersaturated with 0.5% sucrose in a petri dish, placed in dark of 20 ℃ for 12hours; ④Co-cultivated the explants on sucrose liquid medium for 3 days; ⑤Washed the leaves with sterilized distilled water three times, and placed on theregeneration medium with 400 mg·L-1 cefotaxine. Anti-PLDγgene was introduced firstly. 21 kanamycin-resistant rootedplants were obtained through successive selection in shoot and root inductionstage under high level kanamycin pressure. PCR and PCR-Southern analysisshowed that 13 of 21 kanamycin-resistant rooted plants were positive. Thismeant that Anti-PLDγgene was successfully integrated into the genome ofthese 13 plants. Salt-tolerance test demonstrated that 4/13 plants wereenhanced to some extent. The NO.4 plant was selected again to introducechitinase (CH5B) gene. After successive selection in shoot and root inductionstage under high level hygromycin pressure, 6 hygromycin-resistant rootedplants were gained. PCR and PCR-Southern analysis showed that 6 of theserooted plants were positive. This meant that CH5B gene was also successfullyintegrated into the genome of Populus deltoides (G2). The CH5B gene andreporter gene GUS are in the same open reading frame (ORF), so viahistochemical staining of GUS gene test, demonstrated that 6 plants canexpresse CH5B gene normally. Transgenic plants were transplantation into the greenhouse successively.The relative electronic conductivity of the transgenic plants under 1% NaCl 4山东农业大学硕士学...