In Vitro Selection Of Anti-black Shank Cell Mutants In Tobacco Callus

Tobacco black shank,a ruinous disease caused by Phytophthora parasitica var nicotianae, has influenced the tobacco production seriously.Therefore, incubating cell mutants of tobacco against black shank by biotechnology of plant tissue, then exploring and establishing a selecting system is of significance to tobacco quality and output.In this thesis, leaf explants from Hongda and Yunyan85 were inoculated on Mass Scan(MS) culture medium with different doses of crude toxin from tobacco black shank to screen cell mutants, with a combination of tissue culturing technology and in vitro screening technology. Several cell mutants and regenerating plants of tobacco against black shank of Hongda and Yunyan85 are obtained. Finally,The selecting system was primarily established based on the result of experiment.1. Determination of the proper toxin concetration of screening Hongda cell mutants resisting to black shank. With the increasing in the concentration of crude toxin, the growing pace of explant and the pace of callus formation were decreasing, compared with the CK. But while the concentration is up to 6%, the ratio of callus differentiation was less than 20%, and the sprouts were seldom generated, which indicated that this toxin concentration is the limitation of the great majority of callus. 9% concentration of crude toxin extremely affected the growth of leaf explants, making all of them died. So,6% concentration of crude toxin is considered as the proper concentration of screening Hongda anti-black shank cell mutants.2. Determination of the proper toxin concentration of screening Yunyan85 cell mutants resisting to black shank. The same as Hongda, The effect of small toxin concentration wasn't significant. While the concentration is up to 7%, the ratio of callus differentiation was less than 20%, and sprouts weren't appeared, which showing that this concentration this toxin concentration is the limitation of vast majority of callus. The toxin concentration of 9% made all leaf explants dead. So,7% concentration of crude toxin is determined as the proper concentration of screening Yunyan85 anti-black shank cell mutants. 3. Differences in vitro screening between the two varieties. On the same black shank toxin concentration, the Yunyan 85's pace of callus is higher than Hongda. The principal reason of different toxin concentration in screening two varieties'cell mutants resistant to black shank is the difference in their tolerance to black shank.The study shows that the tolerance of Yunyan85 is stronger than Hongda.4. Leaf explants of Hongda were inoculated on MS culture medium with crude toxin concentration of 6%, and Yunyan85 were that of 7%. A total of 253 regenerating plants were obtained. The test of resistance showed that 16 Hongda and 23 Yunyan85 regenerating plants were moderately,with only 2 Yunyan85 regenerating plants were strongly resisting to black shank.