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Larch (Larix Mill) is a fast-growing northern China an important timber tree species, larch molecular breeding to carry out research, accelerate the process of genetic improvement is a modern and efficient plantation cultivation is an important task. Somatic embryogenesis similar to zygotic embryos, is a plant embryo research and application of artificial important way is tree species improvement, asexual reproduction and ideal experimental system for basic research, but also the genetic engineering of the bridge. Create and optimize larch stem cell-based model system of transgenic technology, efficient foreign gene into the genome larch, make important target traits and gene expression regulation of functional verification, speed larch biological breeding process has important scientific significance and application value. In this paper, larch as experimental material, according to the 39 cell lines of the original embryo development and maturation of somatic embryos screened cell lines 638,119,113 other three cell lines as transformed cell lines; and make their antibiotic susceptibility testing, the use of Agrobacterium-mediated genetic transformation system research, the main findings are as follows: 1. Through the 39 cell lines of the original larch embryo tissue and somatic embryo maturation and developmental status screening studies indicate that Japanese larch (Larix leptolepis) 638,113 two cell lines and larch (L. olgensis) 109 cell lines regeneration ability, three cell lines can be used for genetic transformation, Agrobacterium transformation can be used as receptor material. (2) By screening antibiotics - kanamycin (Kan), hygromycin (Hyg) and bacteriostatic antibiotics - cefotaxime (Cef) and carbenicillin (Cb) studies show that: (1) Kan on cell The sensitivity lines 638,109,113 critical concentration of 25mg / L, 20mg / L, 15mg / L; Hyg were 5mg / L, 3mg / L, 3mg / L; the critical concentration, Hyg faster action than the Kan ; (2) Cef appropriate concentration inhibiting the growth of Agrobacterium 300mg / L; at 300-500mg / L concentration range, Cef contribute to the growth of cell lines, and Cb is inhibited. 3 by Agrobacterium-mediated genetic transformation larch cell lines receptor material genotypes, training methods, bacteria disseminated concentration and time of incubation time and other factors, studies show that: (1) In carrying plasmid pCHF3-gp and pSuper1300-HC Agrobacterium transformed cell lines 638,109,113, 54 resistant cell lines obtained by molecular detection gain further 10 transgenic lines; (2) to 638 on solid medium for the receptor, the concentration OD600 of 0.4-0.6 carrying plasmid of Agrobacterium dip pSuper1300-HC 10-20min, co-cultured 2d, conversion efficiency of up to (0.94 / g), showed that cell line 638 is an ideal conversion receptor; (3) Kan screening less resistant cell lines obtained, and the high false positive rate; while Hyg screening is able to receive more resistant cell lines, and the false-positive rate. Show Kan and Hyg screening efficiency varied. In summary, we established and optimized Agrobacterium-mediated genetic transformation system larch cell lines, summing up the first three parts and optimize research, elaborated established stem cell-based model of Agrobacterium-mediated genetic larch transformation system for the development and further molecular genetic larch breeding provides a scientific basis.
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