Root Transgenic System Technology Of Tung Tree(Vernicia Fordii)

Tung tree(Vernicia fordii)is an important woody industrial oil tree with a 40%70%oil content in its seeds.The oil extracted from the seed is called tung oil,and as a high-quality dry oil,it can be used to produce the tung oil eco-friendly paint.However,the lack of high-yielding monocultures of tung tree makes it difficult to meet the market demand for tung oil,which seriously restricts the development of the tung oil industry.The cultivation of high-yielding monocultures based on the molecular genetic improvement of tung tree is an effective way to address the low yield of tung oil.In this paper,the first genetic transformation technique for the induction of transgenic hairy roots in tung tree was established using Agrobacterium rhizogenes K599 strain.This technique can provide a convenient platform for the mining and identification of functional genes in tung tree,and is conducive to the rapid development of future molecular breeding work in tung tree.The main findings of the thesis are as follows.1.The establishment of tung tree hair root genetic transformation technology.We took the germination seedlings of the tung tree seeds mixed in the tung tree base as the research object.Agrobacterium rhizogenes K599 containing pSAK277-RFP plasmid(containing a red fluorescent protein reporter gene)was obtained based on the freezethaw method,and the strain was used to prepare the infection bacterial solution,and the young stem segments of tung tree were directly injected in vitro.After two months of culture,the transgenic hairy roots were initially locked with double fluorescent protein observation,and the authenticity of the transgenic hairy roots was further verified after DNA and RNA level identification and laser confocal microscope observation.Based on the treatment results of fifty tung tree seedlings,it was found that:1)the transgenic hairy root transformation efficiency of this technology was 13.96%;2)the continuous moisturizing of the injection site was the key factor for successful adventitious root induction;The injection site may affect the transformation efficiency of positive adventitious roots.2.Optimization of genetic transformation technology of hairy roots of tung tree.The gradient experiment was set up from the OD60 value of bacterial solution concentration(0.05,0.1,0.2,0.3),seedling age(14 d,21 d,28 d,35 d seedling age),stem color(green,red)and injection site(0-2 cm,2-4 cm,4-6 cm,6-8 cm from the root).The results showed that when the OD600 was 0.2,the transgenic hairy root induction rate was the highest(18.15%),the transgenic hairy root induction rate of 21day-old tung tree was the highest(15.94%),and the transgenic hairy root induction rate was the highest(13.96%)when the injection position was 6-8 cm away from the root.To sum up,Agrobacterium rhizogenes K599 with OD600 of 0.2 was injected into the stem segment 6-8 cm away from the root of tung tree seedlings aged 21 days,which could increase the induction rate of transgenic hairy root to 18.61%.3.Study on adventitious root regeneration system of tung tree.In order to obtain transgenic tung trees,we tried to establish a regeneration system by using transgenic hairy roots.At present,the problem of high pollution rate in tissue culture of transgenic hairy roots of seedlings has not been solved,so sterile seedling roots are used to explore the root regeneration system.The current research results are as follows:1)The seed contamination rate is 7.73%,when we disinfect seeds with 0.1%HgCl2 for 8 min.;2)By setting the concentration gradient of plant hormone,it was found that the best combination of callus induction was:1/2 MS+1.0 mg/L 6-BA+1.0 mg/L 2,4-D or 1/2 MS+2.0 mg/L 6-BA+1.0 mg/L 2,4-D;3)Based on the callus obtained from the hairy roots of tung tree,we tried to induce differentiation with different hormone combinations.At present,there is no hormone combination that can promote the callus to differentiate into buds.Finally,this paper established a simple and rapid genetic transformation technology to obtain transgenic hairy roots of tung tree for the first time.Other members of the team will continue to carry out the research on the regeneration of transgenic adventitious roots into complete transgenic plants.