Establishment Of Regeneration System Of Zizania Latifolia In Vitro And Study On Inoculation Of Ustilago Esculenta

Zizania latifolia is a perennial herbaceous plant of Zizania,which is the second largest aquatic vegetable in China.It is not only nutritious,but also has health and medicinal value.There are three phenotypes in the field,named male Zizania latifolia,grey Zizania latifolia and white Zizania latifolia,which are closely related to the growth of Ustilago esculent in Zizania latifolia.At present,the method of separating and screening is used to cultivate Zizania latifolia.The propagation coefficient is low and the propagation speed is slow,meanwhile,the germplasm resources are few and the propagation process is limited by seasons.If the production is not selected for two consecutive years,the occurrence rate of male stem and grey Zizania will reach 30%,which will seriously affect the economic benefits.This subject choose three kinds of Zizania latifolia cultivated varieties as the research object which called "E jiao No.1","E jiao No.2" and "Da bie shan No.1".Through the study of different genotypes,different explants and different hormone ratios,the callus induction and differentiation were studied to explore the regeneration in vitro.At the same time,the male seedlings of "E jiao No.1" were used as the experimental materials,and the Ustilago esculenta were inoculated by injection method.The Ustilago esculenta in the plant were detected by molecular detection,and the pregnant conditions of the inoculated strains were investigated and counted during the pregnant period.The main results are as follows:1)Different genotypes of Zizania latifolia had no significant effect on callus induction rate.In a single repeated experiment,the average induction rate of callus induction was slightly higher by using "E jiao No.1" as test material.2)The callus induction rate of "E jiao No.1" from different explants was significantly different.The average callus induction rate of lateral buds was significantly higher than leaves and roots.3)The optimal combination of hormone ratio of callus induced by lateral bud of Zizania latifolia was as follows: NAA 1 mg/L + 6-BA1mg/L + 2,4-D 1mg/L;the optimal hormone combination for callus differentiation induction of Zizania latifolia was as follows: 6-BA 2 mg/L + NAA 1mg/L + KT 0.1mg/L.4)7.8 % of the plants inoculated by the injection method could be pregnant normally,among which 105 spores /ml suspension of Ustilago esculenta had the best effect. The Ustilago esculenta was also detected in the 3.3% of plants that failed to pregnant normally.5)The results from the paraffin section method showed that in the same field of view of microscopic observation,the cell size of the growing point after inoculation and the growing point after expansion of Zizania latifolia were significantly larger than that of the male,and the gap between the cells was also significantly larger than that of the male.In conclusion,the effects of different genotypes,different explants and different hormone ratios on callus induction and differentiation of Zizania latifolia were studied in this paper.Meanwhile,the effects of Ustilago esculenta inoculation on the infection and pregnancy of Zizania latifolia were discussed.The results provided a theoretical basis for in vitro plantlet regeneration of Zizania latifolia,and were of great significance to further study on the interaction between Ustilago esculenta and Zizania latifolia and the mechanism of the pregnancy of Zizania latifolia.