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Head tilt dish is an excellent forage legumes, nutrient-rich, adaptable, great promotional value. Gansu alpine meadow grassland, head tilt vegetables have a natural ability to adapt to the local natural environment, is also an important source of protein in animal feed. Natural growth of head tilt vegetable production is not high, growing season late lignified serious, animal palatability, easy to cause livestock indigestion, its seed germination under natural conditions is not easy. Therefore, it is necessary to make improvements Waitoushan dish. This study was constructed the head tilt dishes plant regeneration system, for a large number of tissue culture and micropropagation, laid the foundation for the the head tilt dishes molecular genetics research. Total RNA was extracted for the material to tissue culture and reverse transcription and amplification of the lignin synthesis related genes coffee coenzyme AO-methyl aminotransferase (CCoAOMT), ferulic acid -5 - hydroxylase (F5H), cinnamon alcohol dehydrogenase (CAD) fragment, to lay the foundation for the the head tilt dishes studying gene function. The main results are as follows: 1) head tilt vegetable seeds in 98% concentrated sulfuric acid processing 25min, 28 ° C sterile water soaked to fully imbibed transferred to the MS medium dark culture 5d, the germination rate can reach 98%; 2 ) multiple shoots induction medium 1/2MS IBA 0.5mg / L 6-BA 0.5mg / L; stem segments cuttings best medium was MS medium; callus induction best medium for the MS 2,4-D 4.0mg / L 6-BA 0.5mg / L; callus Following the generation of the best medium for MS 2,4-D 2.0mg / L 6-BA 0.5mg / L casein hydrolyzate (300 mg / L); in MS 2 ,4-D 1.0mg / L 6-BA0.5mg / L medium, the stem segments directly into a the seedling rate up to 66.7%; different sources of explants root induction the best medium were 1/2MS medium average the rooting number up to 1.33. 3) explants with 2.0g / L of soluble polyvinyl pyrrolidone (PVP) to soak for 15min, 4 ° C low temperature for 12h, and then dark culture 5d after transfer to containing 4.0mg / L 2,4-D, 0.5mg / L, 6 - BA, 0.2g / L Vc, 1.0g/LPVP 0.04% DTT and 0.3g / L hydrolysis the casein 1/2MS medium, callus induction and subculture process of browning time will be postponed, the degree will reduced. Shorten the seedlings cycle turn against browning also have a certain role. 4) natural growth of head tilt dish young shoots and plantlets (including frozen) stems and leaves can be used for total RNA extraction, but the 4th generation tissue culture extracted RNA integrity and purity, multi-cultured ( After 15 generations) tissue culture and phenotypic variation seedlings unsuitable material as RNA extraction. 5) amplification of the CAD gene sequencing fragments long 325bp the F5H gene fragment length of 361bp CCoAOMT gene fragment length 339bp with alfalfa CAD, F5H, and CCoAOMT genes highest sequence homology, respectively, up to 88% 84%, 92%; to judge, these three gene fragments are Waitoushan dishes corresponding part of the gene sequence.
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