Study On Development And Validation Of The In Vitro Propagation System Of Nervilia Fordii (Hance) Schltr

1 ObjectiveTo construct propagation system of Nervilia fordii (Hance) Schltr., based on the maturity of the tissue culture technology, and the cultivated herbs could be expected to supplement or replace the wild resources as one of the stable source of the medicinal materials, finally achieve the sustainable use of the endangered medicinal plant-Nervilia fordii. This issue mainly studied in obtaining effective approaches to control and solve the contamination problems, as well as the comparison and analysis of flavonoids from tissue culture products, cultivated materials and grown wild, which will provide scientific, detailed information and justification to the establishment of the endangered medicinal plant propagation system as well as tissue culture or artificial cultivation of herbs to be alteration of wild resources in future. In short, artificial propagation is an important approach to ensure a sustainable development of Nervilia fordii resource.2 Methods2.1 Decontamination during the tissue culture and construction of propagation system of Nervilia fordii (Hance) Schltr.By referring to existing literatures, the establishment of the propagation system of Nervilia fordii could be repeated in a laboratory, through the review of existing technology of tissue culture and being combined with the actual conditions of the laboratory.To overcome the contamination of the propagation, sterile distilled water (SDW), saline (0.9% NaCl),0.1% mercuric chloride and different concentration of sodium hypochlorite were used as decontamination agents. The growth of the cultured materials after the treatments was recorded. Then the effect of sterile concentration and duration of sodium hypochlorite treatment were studied.2.2 Comparative analysis of flavonoids among various growth patterns and different light quality on cell growth and flavonoids biosynthesis of Nervilia fordii (Hance) Schltr.Taking the total flavones contents of Nervilia fordii (Hance) Schltr. as the criteria, the process was selected by orthogonal experiment, through examination of four main factors-solvent concentration, solvent volume, extraction time, extraction frequency.By analyzing flavonoids of various growth pattern Nervilia fordii using positive ion mode ESI-MS, it changes in the composition of three pattern could be detected.Young plants of Nervilia fordii were planted under various kinds of color films to examine the appropriate light on the growth; as well as phenylalanine ammonia-lyase (PAL) enzyme activity of the cultivated and the wild materials, different stages of the wild products were determined in a period of time.3 Results3.1 Establishment of decontamination and construction of the propagation system of Nervilia fordii (Hance) Schltr.A repeatable in vitro propagation system of Nervilia fordii had been established in laboratory. And the third-generation corms have been successfully harvested; the fourth-generation corms are being multiplied.It showed that 5% sodium hypochlorite with a duration of ten minutes was suitable for decontamination of the plant materials; 0.1% mercuric chloride also showed good effect, but the plants were killed as well. It showed good effect when treated the corms.3.2 Optimum extraction conditions was established; and effects of different growth patterns on the composition of flavonoids, and that of different light quality on culture growth and flavonoids biosynthesis of Nervilia fordii (Hance) Schltr. was conducted.Optimum extraction conditions were established as follows:80% ethanol, ten-fold (v/m) solvent, two times extraction,90 minutes each time. The highest yield of total flavonoids was achieved using the optimum conditions in the study.Influence on the composition of flavonoids of different growth patterns of Nervilia fordii was detected by MS.It showed that green light intended to impact more on the growth of the plants than other colours in the pre-experiment. And it showed that the wild grown plants have 10-20 units higher PAL enzyme activity than the cultivated ones; the wild ones in June have about 100 units higher than the ones in September in the PAL enzyme activity experiments.4 ConclusionsThe propagation system of Nervilia fordii (Hance) Schltr. has been successfully established. The construction of the system may also be developed to a mature technology to provide the medicines, in order to break out the bottleneck of its resource-poor, and lay the foundation for industrial production. The study of decontamination could increase the survival rate of the tissue culture materials of Nervilia fodii, which lays the foundation for setting up an in vitro propagation system. The study of extraction yield provides theoretical basis for the evaluation of Nervilia fodii, which can also act as the indicators for the evaluations and lay the foundation for industrialization large-scale production, and further development of effective component of total flavonoids of Nervilia fodii. Whether cultivation and tissue culture plants can really replace the wild ones or not is still uncertain. Relevant experiments are to be scheduled to determine the pharmacological efficacy.However, the slow growth rate of Nervilia fodii leads to the propagation system lasting more six months for one generation. Whether cultivation and tissue culture plants can really replace the wild ones as a stable source is still under investigation. Similarly, there is not more significant data collating statistical proof to show different light quality had a great influence on total flavonoids yield. All in all, I hope that the above problems and difficulties can be solved in the further research and exploration. Future study will be carried on to solve the problems and explore more interesting results on herbal plant Nervilia fodii.