| Objectives: I engaged in the investigation of resources distribution of Gentianastraminea Maxim, improved the seeds germinating ability of G. straminea, and establishedthe vitrification cryopreservation protocols on the basis of the dormant buds of G.straminea.Methods: According to the collected materials, specimens and samples,6surveyroutes which contained30sites were designed. G. stramine protection strategies were putforward by studying the plant development history, status and prospect. On the purpose ofimproving the ability of seeds germination, chemical substance KMnO4and ultrasonictechnology were adopted. Adopting single factor test design, the vitrificationcryopreservation protocols based on G. straminea dormant buds were built.Results:1. G. straminea species mainly distributes in Lanzhou, Dingxi, GannangTibetan autonomous prefecture, Jiuquan, Wuwei, Zhangye, Linxia of Gansu province, andHuangyuan, Gonghe, Guide, Huzhu of Qinghai province. The wild resources of G.straminea mainly distribute in the natural protected regions and the afforestation area ofanimals and plants. At part of the traditional Tibetan areas, as southwestern of Gansu andQinghai province, the wild resources of G. straminea are protected well. In central region ofGansu province, as the roots of the plants are applied in medication, and influenced byharvesting, herding and reclamation, the wild resources only can be found in the protectedregions.2. The seeds of G. straminea were put into Distilled water,24h,20℃, then treatedby by ultrasonic instrument at40kHz and60W for20min, last, were soaked in1.5%KMnO4for10min; this method can significantly improve the germination ability.3. Established the vitrification cryopreservation system based on G. straminea dormantbuds that should be undergone the winter exercise and had not be stirring. The buds used inthis study were picked in March. First, the dormant buds which were10~11mm long werecultured on MS medium for1day without light and supplemented with0.3mol·L-1,0.5mol·L-1and0.7mol·L-1sucrose, respectively. Second, the buds were immersed in loading solution (2mol·L-1glycerin+0.4mol·L-1sucrose) for20min, with a temperature of20℃,and then treated with PVS2solution (30%glycerin+15%glycol+15%DMSO+0.4mol·L-1sucrose) for40min,0℃. Subsequently, they were plunged into liquid nitrogen (LN)which is-196℃. After24hours of freezing，the buds were rapidly thawed in water bath at40℃for2~3min and then washed twice with MS medium supplemented with1/2MSliquid medium which contain1.2mol·L-1sucrose for10min, each time. Finally, the budswhich were dried by the sterile filter papers were transferred to the regeneration medium(MS+0.5mg·L-16-BA+0.1mg·L-1NAA+3%sucrose+0.7%agar)，cultivated withoutlight for7days at22℃. After cultivation in the regeneration medium, the buds wereexposed to normal lighting cultivation and the buds could successfully acclimatize normalconditions with up to83.3%survival rate.Conclusions:1. Based on the resources distribution and the present reserves situationof G. straminea, suggestions are to carry out the protection research of G. straminea asfollowing, building wild reserves and constructing seedling nursery fields to provideexcellent seedlings for production. In addition, we can popularize artificial cultivationtechnique in appropriate areas to accelerate strain breeding and standardized cultivationresearch.2. Combined application of KMnO4solution and ultrasonic could acquire synergisticeffect in improving the seeds germination ability.3. Vitrification cryoprsecation method used in this study has significant guidance in theplant germplasm resources preservation of G. straminea group and provides an advancedmethod in the preserve of G. straminea germplasm resources. |
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