| Konjaku's soft rot disease is an important disease caused by Erwinia carotovora sub sp. Carotovora. Its occurrence would make a disaster to the product of konjaku, and it's difficult to eradicate. For the shortage of konjaku germplasm resources, the utilizable variety of resistance is relatively few. At present, to control this disease is mostly through the control of agronomic techniques and chemical treatments. But because of the various infection sources and transmission routes, the present techniques always can't catch the ideal control effect.With the improvement in the plant vitro culture techniques and mutants identification methods, the application of those techniques above to select resistance mutants had become a important study area of plant cell engineering. In this paper, the resistance mutant of konjaku's soft rot disease was selected by vitro culture technique, then the manual plant regenerations were gained to establish a base for the application in product.In this research, top buds, bud scales, lateral bud, budlets and scales of artificial corm of Amorphophallus are taken as explants to induce on their respectively suitable culture media. The results demonstrated that the induction rate of scales of artificial corm of Amorphophallus is 90.6% on average, the advantitions bud induction of the formed calluses were taken on the differential media. The induction rate of advantitions buds on the scales of artificial corm is 96.4%, and the reproduction in crenses is 4.9. However, the highest induction rate of other explants is 74.2%, and the reproduction in crenses is just 4.1. Therefore scales of artificial corm of Amorphophallus are the best explants material to induce.At the screening of resistance mutants, the callus were treated with three different method as follows: firstly, Amorphophallus callus of different phases were treated with different concentrations of EMS, the in vitro results showed that: the 4-day preincubation callus were treated with 0.4% EMS, and the average survival frequencies are 47.2%. The survival callus was reproduced, then mutants resistant against Amorphophallus soft rot disease were screened using needle point inoculation method from the callus with EMS treatment and without EMS treatment. In the end, the survival calluses after the inoculation selection were dealt with differentiation, and 19 regeneration plants were obtained. Secondly, bacterium filtrates were used to screen the resistant mutants. The callus were inoculated on the culture mediums which contained 60% the Amorphophallus soft rot disease pathogen filtrates for 1 month, then they were inoculated to the ordinary culture mediums for the 1-month relaxative culture. After 8-time repeats, the survivalcalluses were treated with filed inoculation and identification, 5 regenerated plants were obtained. Thirdly the direct inoculation was used to screen the resistant mutants. The calluses were directly inoculated with the different concentration suspension liquid of Erwinia carotovora sub sp. Carotovora. With time gone, the calluses were covered with a lot of quickly reproduced thalli and its secretion. The inoculation concentration had no obvious effect on the infection of the treated callus. The inoculated callus of this experiment died entirely after 3 weeks.To the common subculture plants and the plants whose phenotype variated during the common subculture, the obtained plants which were selected after EMS induction and inoculation or treated by bacterial filtrates were carried on analysis of peroxidase (POD) zymogram, superoxide dismutase (SOD) zymogram and Polyphenol Oxidase (PPO) zymogram.Results of POD isozyme showed that, the plants treated by bacterial filtrates or selected after EMS induction and inoculation had 3 isozyme bands, with the Rf value of 0.14, 0.18 and 0.23 respectively. But the plants selected after EMS induction and inoculation had 1 special isozyme band with Rf 0.51. The special isozyme bands of the obtained plants with different screening methods maybe have something with the resistance to soft rot disease.Results of SOD isozyme showed that, the plants selected after EMS induction and inoculation had 1 specific isozyme band with Rf value of 0.40. The SOD isozyme bands' Rf value of the 4 sampled plants were at the range from 0.53 to 0.65. So the mobility rates of every isozyme band were very close. It showed that the molecular weights of every isozyme had no obvious difference.Results of PPO isozyme showed that, the new special isozyme bands of the plants treated by bacterial filtrates or selected after EMS induction and inoculation had Rf values between 0.36 and 0.61. The mobility rate of this band area had a big range. It might be the result of the changed molecular weights of the PPO isozyme from the plants, which obtained from the resistance screening. |
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