Establishment Of A Rice Enhancer Trap Mutant Library By T-DNA Insertion

The tissue culture technique of Cremastra appendiculata, Pleione bulbocodioides, Pleione yunnanensis has been sophisticated, but their industrialization is hampered by low survival rate and slow growing of transplanted seedlings. According to the fact that Orchidaceae symbiosed with mycorrhizal fungi, the study on dynamic changes, physiological and biochemical characteristics, application, classification and identification of mycorrhizal fungal from these Orchidaceae were carried out. The growth and development relationship between mycorrhizal fungi and these orchids should be revealed to establish scientific and reasonable application of mycorrhizal fungi and improve the survival rate of transplanted seedlings.The aim of this study is to realize the industrialization of this type of Chinese herbal medicines. Major findings and conclusions were as follows:1,An accurate and efficient technique to isolate orchid mycorrhizal fungi from a single peloton was set up.Some key problems of isolating orchid mycorrhizal fungi were resolved by this technique. This made it possible to study changes of orchid mycorrhizal fungi species and quantities quantitatively.2,The relationship between dynamic changes in the characteristics of mycorrhizal fungi and the growth and development of orchid was revealed.The mycorrhizal fungi changes in different growth phase and the vertical and horizontal changes were studied in the research. The fungi in early new root grew quickly and developed more aerial hyphae. But those in old roots grew slowly and little aerial mycelium developed. From root tip to the base and from the root epidermis to inward, the fast growing mycorrhizal fungi with more aerial hyphae declined and those growing slowly and developing little aerial hyphae increased.3,Physiological and biochemical characteristics of mycorrhizal fungi and their influence on orchid growth and development were studiedPhysiological and biochemical characteristics of mycorrhizal fungi from three Orchidaceae, such as utilization capacity of carbon and nitrogen sources, producing capacity of B vitamins and GA, cellulase and polyphenol oxidase activity were studied. Those fungi in vegetative growth stage had weak cellulase activity, positive or weakly positive polyphenol oxidase activity; the formation of B vitamins and the ability to use starch, sucrose, maltose and inorganic nitrogen were high. But in Reproductive growth stage, Cellulase activity of them was high and polyphenol oxidase activity was weak; the formation of B vitamins was low and the ability to use cellulose, starch and organic nitrogen was high.4,How to improve the survival rate of transplanted tissue cultures was revealed from the perspective of mycorrhizal fungi Nutrition. Hypothesis of "Three nutrition library" and "three strategies" to increase survival rate of transplanted tissue cultures was brought forward.Mycorrhizal fungi of three Orchidaceae were co-cultured with tissue cultures in different stages. From the results, we found it was the predominant fungi in the special stage to promote the growth the seedlings. The survival rate of rhizomes and seedlings with rhizomes or bulbs was higher than seedlings without rhizomes or bulbs. The mycorrhizal fungi played a key role in protecting and keeping the activity of the root.According to these results, "three nutrition library" hypothesis was given. They were rhizome, leaves and bulbs. The success of transplanting tissue cultures relied on changing from "rhizome nutrient library" to " leave photosynthetic nutrition library ", and then to " bulbs nutrition library " successfully. " three strategies " to increase survival rate of tissue cultures were summed up. That is chooseing rhizome, seedlings with rhizome or seedlings with bulbs and then symbiosed with mycorrhizal fungi. The seedling changing into " leave photosynthetic nutrition library " and through dormancy to "bulbs nutrition library" were promoted by the protection of fungi and the nutrition library role of rhizomes or bulbs.5,The model of mycorrhizal fungi being used in transplanting tissue culture seedlingsAccording to the dynamic characteristics of mycorrhizal fungi in orchid plant, physiological and biochemical characteristics of mycorrhizal fungi and characteristics of, a model of transplanting orchid seedlings were summed up: Seedlings with a storage organ were selected out and were symbiotic cultured with the predominant fungi in early new roots to meet the nutrition requirement and formation of new roots. When the symbiosed relationship formed and a stable photosynthesis established, followed by application of dominant fungi in the vegetative growth stage to transfore Orchidaceae photosynthate into storage and to maintain high root vigor, so as to improve survival rate.6,Classification and identification of mycorrhizal fungi Mycorrhizal fungi of three Orchidaceae had been classificated and identificated by using morphology and enzyme activity characteristics, phylogenetic analysis of ITS sequence. The method to classificate and identificate Orchidaceae Rhizoctonia was given and the keys of some genera and species were brought forward. A third Epulorhiza population, New genus, was found, which was not named. The most important characteristics of species in this population were growing very slowly, without monilioid cells or with irregular monilioid cells and with high ability to use starch. They are important in promoting storeage, conversion and utilization of photosynthate in Orchidaceae.The "three nutrition library" hypothesis in the study will enrich the symbiotic theory of orchid with their mycorrhizal fungi. The classification and identification method proposed and keys for relevant genera and species will enrich the classification and identification system of orchid Rhizoctonias. In this study, the establishment of isolating orchid mycorrhizal fungi from a single peloton, "three strategies" to increase survival rate of transplanted tissue cultures and model of transplanting tissue cultures with fungi, will achieve industry of Cremastra appendiculata, Pleione bulbocodioides, Pleione yunnanensis to provide theoretical and technical support.