Cloning Of Lignin Biosynthesis-Related Genes And Regeneration Of Wild Forage Legume Viciaunijuga Al.Br.

Vicia unijuga A. is a good,nutritious and adaptable forage legumes and has great promotional value.As an important forage legumes in Gannan alpine meadow grassland, Vicia unijuga A. is able to adapt to local special natural environment and also the main protein source for ruminants.Thus,their seeds are hard to germinate and the production of Vicia unijuga A. is not high under natural condition.In the last growth stage, Vicia unijuga A. lignificates seriously and is not suitable to feed ruminants,otherwise,it will lead to indigestion.So, Appropriate improvements are necessary.This study is aimed to construct plant regeneration system of Vicia unijuga A. and get enough tissue culture seedling by micropropagation for further study.Meanwhile,we extracted total RNA from tissue culture seedling and reverse transcripted,amplificated lignin biosynthesis-related genes fragment of caffeoyl coenzyme AO-methyltransferase (CCoAOMT), ferulic acid-5-hydroxylase (F5H), cinnamyl alcohol dehydrogenase (CAD),laid a solid foundation for research of Vicia unijuga A. The main results are as follows:1) Seeds were treated by 98%sulfuric acid for 25min,soaked in 28℃steriled water,transferred to MS medium,dark cultured 5d,the germination rate can be up to maximum 98%.2) Bud induction medium is 1/2MS+IBA 0.5mg/L+6-BA 0.5mg/L; the best stem-cutting medium is MS;the best callus induction medium is MS+2,4-D 4.0mg/L+6-BA 0.5mg/L; the best callus subculture medium is MS+2,4-D 2.0mg/L+6-BA 0.5mg/L+hydrolyzed casein (300mg/L).In MS+2,4-D 1.0mg/L+ 6-BA0.5mg/L,stem seeding rate could be up to 66.7%;the best root induction medium is 1/2MS and the average number of root is 1.33.3) Explants were soaked in 2.0g/L soluble polyvinylpyrrolidone (PVP) for 15min,treated in 4℃for 12h,transferred to 1/2MS medium after 5d dark culture.This 1/2MS medium contains 4.0mg/L 2,4-D,0.5mg/L 6-BA,0.2g/L Vc,1.0g/L PVP 0.04%DTT and 0.3 g/L hydrolyzed casein.Browning would delay and reduce if did like this in callus induction and subculture.Shorten the culture cycle time is also an effective way to inhabit browning.4) Tender stems and leaves from wild grow plant and tissue culture seedling (including frozen) could be used to extract the total RNA.Integrity and purity of RNA extracted from the 4th generation of tissue culture seedling is the best,and the multi-generation (including 15th) seedling and variation seedling is not suitable for RNA extraction.5) Gene fragment length of CAD,F5H and CCoAOMT is 325bp,361bp and 339bp and has the highest homology with alfalfa up to 88%,84%and 92% respectively.According to these results,we can make sure these gene sequences are the just fragment of corresponding gene of Vicia unijuga A.