Studies On Isolation And Culture Of Protoplast Of Gentiana Straminea Maxim

Gentiana straminea Maxim, commonly known as “Mahua Qinjiao”, is a perennialherb in the family Gentianaceae. It distributed mainly in the Qinghai-Tibet Plateau,and is one of the key-protected wild medicinal materials in China. Demand for G.straminea in the pharmaceutical industry is met from the wild. As a result of overharvesting and lack of organized cultivation, the wild resources of G. straminea havereduced rapidly. Therefore, it is imperative to develop appropriate tissue culturetechniques for this species. In this study, G. straminea growing naturally in theQinghai-Tibet Plateau was chosen as experimental material. Based on the study ofsomatic embryogenesis and plantlet regeneration, the isolation and culture ofprotoplast of G. straminea were studied. These would provide scientific references forthe further researches on the artificial reproduction, selective breeding of goodvarieties, genetic transformation, and exploiting of medicine resources in G.straminea.The results obtained were as follows:1. An in vitro protocol for efficient plant regeneration through somaticembryogenesis was developed from leaf explants of G. straminea. MS mediumsupplemented with2.0mg·L^-12,4-D and0.5mg·L^-16-BA was the best medium forembryogenic callus induction. After subculture of embryogenic calli on MS mediumcontaining0.5mg·L^-12,4-D, the calli were transferred to the regeneration medium.The highest regeneration frequency （93.8%） and average number of plantlets （31.7）per1g of calli were obtained on the MS medium containing3.0mg·L^-16-BA. Whengreen plantlets with or without primary roots were transferred to the half-strength MSmedium without growth regulators, thick white roots were developed. After theplantlets with well-developed roots were transferred to pots containing soil, about90%of the regenerated plants survived and exhibited normal growth.2. The gentiopicroside contents at different stages of somatic embryogenesis in G.straminea were determined by HPLC. The contents of gentiopicroside increased withthe development stages of somatic embryos. In extracts of globular-shaped embryoids,the concentration of gentiopicroside was13.8mg·g^-1dry weight, while theconcentration was22.2mg·g^-1in cotyledon-shaped embryoids. The gentiopicrosidecontent was30.7mg·g^-1in regenerated plantlets, which was higher than the mother-plants.3. The protoplasts were isolated through enzyme digestion. The effects ofdifferent explants, concentration of mannitols, enzymatic digestion time,pre-treatment, and culturing methods on protoplast isolation, divisions andregeneration were studied. The regeneration protocol was developed from protoplastsisolated from embryogenic calli of G. straminea. The optimal concentration ofmannitols was0.5⁰.6mol·L^-1for isolating leaf protoplasts. The optimal enzymedigestion time was5hours for isolating leaf protoplasts. The yield and viability ofprotoplasts were increased after the leaves were pre-treated by0.6mol·L^-1mannitols.The yield of protoplasts isolated from the leaves was higher than the embryogeniccalli, but their viability was lower than the latter. Among the three culturing methodstested, the agrose-liquid double layer culture and the thin layer liquid culture werebetter than the solid culture. On culture of leaf protoplasts, the first division wasobserved after6days, the division frequency after14days of culture was17.6%, thecolony formation frequency after28days of culture was2.4%, and no calli wereformed. On culture of protoplasts isolated from the embryogenic calli, the firstdivision was observed after3days, the division frequency after14days of culture was31.7%, the colony formation frequency after28days of culture was7.9%, and thecalli were formed.4. The genetic fidelity of the regenerated calli from protoplasts of G. stramineawere analyzed by chromosome examination, esterase isozyme and molecular marker.The number of chromosomes in the regenerated calli was26, which was same as themother-plants. The esterase isozyme were5bands in the regenerated calli, whereasthat was6bands in the mother-plants. Except a few of minor bands, the major bandsof DNA have not obvious differences between the regenerated calli and themother-plants. The results indicated that the calli regenerated from protoplasts of G.straminea maintained high genetic fidelity, but there were still some somaclonalvariation.