Biotechnology applications of Populus micropropagation

摘要

Six aspects involved in biotechnology applications of Populus alba x P. grandidentata micropropagation were studied: (1) the influences of medium consistency and shoot density on in vitro shoot proliferation, (2) protoplast isolation and culture of in vitro cultured leaf explants, (3) morphogenetic potential of in vitro cultured leaf, internode, and root explants, (4) influences of subculturing period and different culture media on cold storage of the plantlets, (5) in vivo and in vitro polyploidy induction, and (6) transformation by an Agrobacterium binary vector;For achieving optimum in vitro shoot proliferation from axillary bud culture, liquid medium and shoot density were defined. The results of protoplast culture indicated that the yield of viable protoplasts from in vitro cultured leaf explants was consistently greater than that from leaves of greenhouse grown stock plants. In vitro cultured leaf, internode, and root explants exhibited remarkable morphogenetic potential that can be readily manipulated to form shoots, roots, or calli. Abaxial side culture of entire leaf explants was best suited for inducing adventitious shoots;Subculturing period preceding cold storage, plantlet condition, and culturing medium all had important influences on survival at 4°C in darkness. Plantlets possessing 4-6 axillary shoots that were subcultured on shoot proliferation medium for one month preceding cold storage could be stored at 4°C in darkness for 24 months;Colchiploid plant material could be obtained by in vivo colchicine treatment of axillary buds and by in vitro adventitious shoot induction from chimeric leaf discs, but not by in vitro colchicine treatment of axillary buds of the Crandon clone. The established micropropagation of the chimeric hybrid poplar can probably be used to isolate tissue and produce plants with the desired ploidy level via leaf explant culture and protoplast culture;Transformed kanamycin resistant calli were obtained by co-culturing an Agrobacterium binary vector pGA472 containing a neomycin phosphotransferase gene with leaf discs on a selective regeneration medium. Of the two wild type Agrobacterium strains tested, strain A281, containing plasmid pTiBo542, appears to be better suited to serve as a helper plasmid for binary vector systems.