Establishment Of Plant Regeneration System And Genetic Transformation Of Tartary Buckwheat

Tartary buckwheat (Fagopyrum tataricum Gaertn.) is an autogamous and dicotyledonous plant belonging to the Polygonaceae family. It is also a cultivated crop contains many nutritional substances, and the requirement to it has been increasing year after year. However tartafy buckwheat could be consumed not only as food but also for medical purposes. Traditional breeding of tartary buckwheat has the problems such as long cycles and degeneration etc, and genetic transformation could be adopted as an alternative pathway in the improvement of yield and quality.The establishment of high-frequency regeneration system is the key factor forplant transgenic manipulation. In this study, an efficient and rapid plant regeneration system was established for tartary buckwheat using hypocotyl and cotyledon segments of seedlings as explants via somatic embryogenesis and organic pathway, The experiment results showed that high shoot differentiation ratio was obtained from hypocotyl-derived calli on 1/2 MS medium supplement with 2mg·L^-1 BA + 0.5 mg·L^-1 KT + 0.1mg·L^-1 NAA and from cotyledon-derived calli on 1/2 MS medium with 1mg·L^-1 BA + 0.1mg·L^-1 KT + 0.1mg·L^-1 NAA. Roots were induced on MS medium containing 1mg·L^-1 NAA.High soil salinity is one of the major abiotic stresses which constraints crop growth, development and output. The improvment of salt tolerance of crops has become an important project. The tonoplast Na⁺/H⁺antiporter is responsible for the compartmentalization of Na⁺ from cytoplasm into vacuole and plays an important role in plant salt tolerance. In the present work, hypocotyl and cotyledon segments of tartary buckwheat were transformed by Agrobacterium tumefaciens LBA4404 containing AtNHX1 gene, kanamycin-resistant calli were obtained and transgenic plants were regenerated. Integration and transcription of AtNHX1 gene were comfirmed by PCR,RT-PCR and Southern blotting in transgenic plants. The examination of resistance to NaCl of transgenic calli and plants demonstrated that under the different NaCl concentration, the growth states and relative growth rate of the transgenic calli and plants were better than that of untransformed materials.Rutin content was determined by HPLC in tartary buckwheat, the results showed that rutin content declined from 16.8 mg/g to 6.3 mg/g in the calli induced from cotyledon, and from 7.4 mg/g to 3.9 mg/g in the calli induced from hypocotyl. The decrease rate were 62.5% and 47.3% respectively. Rutin content had no significant differences in root, leaf, stalk and seed between regenerated plants and plants from field. Rutin content in root and stalk of transformed plants had no significant differences in comparison with untransformed tissues under 1% Nad stress, but in leaf the rutin content increased significantly up to 9.2%, and in seed rutin content increased slightly up to 2.5%.