Study On The Tissue Culture And Protoplast Isolation From Nervilia Fordii

Nervilia fordii is an endangered plant in China. With corms as the reproductive organ, the proliferation rate of N. fordii is very low. Because of extinct excavation, the wild resource of N. fordii is beginning to tail off. In recent years, the wild resource of N. fordii cannot meet the requirement of increasing domestic and international market demand. Tissue cultures and domestic cultivation are considered effective ways to protect wild resources, and alleviate the contradiction between supply and demand. Build up multiplication system of N. fordii can provide technology basis for domesticate cultivation. The current study aims to advance and optimize modification for setting up a more stable and integrated multiplication system based on previous study.Plant cell suspension culture can be used to conduct protoplast isolation and culture, somatic cell hybridization, gene transfer, produce plant secondary metabolites, etc. Preliminary study was carried out which is based on the stable callus culture system of N. fordii and some progresses were made. To evaluate whether the regenerated plant of N. fordii can substitute wild resources, the differences in morphology and chemical composition between tissue cultured corms and cultivated plant were also compared.Fresh leaf of N. fordii was used to study the protoplast isolation for the purpose of providing new experimental system of gene transfer, protoplast fusion and fundamental theory research in biology.The main results in this thesis are as follows:1. Using the fresh corm of N. fordii as explants, better culture can be achieved by decontamination of the plant materials with1.0g·L-1mercuric chloride for10minutes, and induction of callus in glass petri dishes; lots of clustering buds and callus can be obtained by culturing N. fordii amphitropous stem and callus on MS basal medium supplemented with1.0mg·L-16-BA [M(3)] for a few months. After several times of successive subcultures, regenerated plantlet was produced; the regeneration mode of N. fordii tissue culture seedling is: bulb explant-white amphitropous stem-green amphitropous stem-regenerated corm-regenerated seedling. But this process lasts long period, and the regenerated plantlet was too weak to carry on seedling adaption and mass-production.2. After several times of successive transfers of culture, granular and loose yellow embryonic callus with strong vitality was obtained. Callus grew on1/4MS and1/2MS medium were considered superior to other groups; the cultures grew on1/2MS medium got the highest dry weight increasing rate, lowest MDA content and highest POD content, which indicated1/2MS was the most suitable medium for callus growth and accumulation of organic matter among the tested. The callus floated under the light got more flavonoids contents; however, the dry weight increasing rate was higher when cultured under the dark. For the purpose of obtaining large amounts of culture, the sucrose concentration should be adjusted to1%, and to3%with the goal of getting more flavonoids content. Adding1.0g·L-1MES in medium with a pH6.0could increase the growth of suspended tissue culture and yields of flavonoids.3. The differences between tissue culture regenerated corms and cultivated plant are in the following respects:3.1Microscopic identification:ten vascular strands is observed in cross section of tissue culturing bud with no obvious trachea, arranged in a irregular circle. The number of vascular strands in cross section of N. fordii was six; the trachea inside vascular strands is clearly visible. There are lots of needle-like calcium oxalate crystals in cultivated plant, however, it was not seen in tissue cultured corms.3.2Chemical components identification:Comparing the spectroscopic characteristics of N. fordii, tissue culture may contain much more chemicals. Quantitative analysis remains further investigation.4. The highest yield of protoplast was obtained using the mixed enzyme solution containing1.0%cellulase R-10+0.6%macerozyme R-10, and incubated for12hours. The optimum process for protoplast isolation was pretreating leaves for1hour with CPW solution containing13%mannitol, then incubating the leaves at (25±1)℃in enzyme solution under dark. Purification of protoplasts was conducted as follows:after the enzymolysis, the enzyme liquid was filtrated through200mesh screen and centrifuged at800rpm for8minutes. The filtration and centrifugation were repeated twice before microscopic examination. Under the above conditions, large amount of intact and clean protoplast with very few cell debris was obtained.