| Combining excellent traits of the parents,hybrid larch performs thoroughly better than it’s parents in growth,resistance and breeding.Therefore,building a suitable system of Tissue Culture and Rapid Propagation will provide technical support for large-scale production of tissue-cultured seedlings of hybrid Larch.In the other hand,proven tissue culture technology will provide a stable and controllable platform for further basic research on larch growth and development.Take Japanese larch×Larix olgensis hybrid clones-day wing 8x long mix 18-10as an object,which was breded by Forestry Research Institution,Chinese Academy of Forestry,good in both growth traits and rooting capacity,direct regeneration of tissue culture in vitro of an organ.The research focused on shoots Study on Tissue Culture Technique,also try to find a suitable culture conditions of proliferation.Using experimental Regeneration System of hybrid Larch built in the early organization,hybrid larch group cultivated strains of screening and Cloning and Expression Analysis of AP2 gene were conducted.The main results are as follows:1.Study on Tissue Culture of hybrid LarchWith shoots as explants,sodium hypochlorite 15%for 10 minutes treatment showed the best sterilization effect,with a result of 91.7%bacteria-free survival rate of materials.Total Nitrogen concentration of culture medium had a big impact on the rate of twigs into the branches of the Axillary buds.In a BEMB medium with 29.4 mmol·L-1total nitrogen concentration,the highest axillary buds rate was up to 38.4%.With dormant bud as material,Adventitious Bud induction was carried out in MCM medium.ZT and KT working in conjunction can significantly affect the rate of Adventitious Bud induction of dormant bud.Adventitious Bud induction reached a best result in 1.5mg·L-1ZT+0.15mg·L-1KT treatment,where Adventitious Bud induction rate and the average number of individual germination of dormant bud formation of adventitious buds were respectively 39.1%and 2.6.In bottle rooting experiment on the material,compared with one-step method,two-step method was proven more suitable..When performing Rooting experiment using two-step method in medium with1/3 macromineral and 2.0 mg·L-1 NAA,it was found that sucrose concentration had a significant impact on the rate of rooting materials,and similarly,sucrose concentration and glutamate concentrati on material survival in the process of rooting,and Medium pH value on Average number of Roots of materials.Rooting induction conducted in L9 medium with 1/3macromineral and a pH value of 6.5,containing 2 mg·L-11 NAA、0.25 mg·L-11 IBA、5 g sucrose、5 mg·L-1glutamate performed best,with a result of 92.9%rooting rate,100%material survival rate,average days of rooting 27.9 and average number of roots 11.8.2.Study on Tissue Culture of hybrid LarchUsing qPR-PCR technology,screening of reference gene was carried out in the process of tissue-cultured root,between the different organs of the plant tissue culture and different organs under Acid alkaline stress.The results indicated that among 11 genes such as ACT7,EF-1a,EF-1b,CYP19-2,TUBA5,TUBB6,UBQ10,TIP41,PXM16,UKN2,GAPC-1,TUA5,TUB6and EF-1a were the best fit reference genes for Tissue Culture process of hybrid Larch,while UBQ10.EF-1b and TUA5 were the best fit reference genes for different organizations such as root,stem,leaf and stem apex in tissue culture plant.And in the process of acid-stress on tissue culture plants,EF-1b,PXM16 and UKN2 were the best fit reference genes for roots,ACT7 and GAPC-1 for stems,and ACT7 and GAPC-1 for leaves.Meanwhile,in the process of Alkaline stress on tissue culture plants,EF-1a,PXM16 and TUA5 were the best fit reference genes for roots,EF-1a,PXM16 and EF-1b for stems,and EF-1a,EF-1b and ACT7 for leaves.With data of all samples being analyzed,EF-1a,EF-1b and UBQ10 turned out to be the best fit reference genes.3.Cloning and Expression Analysis of 7 Hybrid Larch genesThrough the race technology,the four genes cloned from euANT genome,AP2 subfamily,AP2/ERF family in Hybrid Larch were LkBBM1,LkBBM2,LkAIL1 and LkAIL2,the three genes from basalANT genome were LkWRI2,LkWRI3 and LkWRI4.By gene sequence conjecture of proteins and comparative analysis of amino acid sequences,it was found that LkBBM1 and LkBBM2 contained BBM-Motif which was BBM protein-specific.Likewise,in the comparative analysis,conservative BBM-PLT Motif1 and BBM-PLT Motif2 were found between homologous proteins BBM and PLT,as well as conservative AIL6/7 Motif between homologous proteins PLT3 and AIL7,WRI2 Motif1 and WRI2 Motif2 among homologous proteins WRI.Predictions on the 3D structure of the seven speculated protein indicated that AP2domains of all seven proteins contained three antiparallelβ-pleated sheet and a a-spiral.And based on sequence analysis of AP2 domain in the seven proteins,it was speculated that R1domain evolved more conservatively than R2 domain during the evolution of AP2 subfamily genes in larch,with the changing of R2 domain during evolution as the significant cause of functional differences among AP2 family genes.LkBBM1 express exclusively in roots among the 4 tissues as roots,stems,leaves and stem apexes,on the eighth day of rooting process.Analysis on a combination of promoter elements,genes expression pattern and Homologous genes function showed that it is likely that LkBBM1 gene is the character gene of root meristem in Hybrid Larch,and thereby the period of its initial expression in the rooting process is the initial formative period of root primordium.On the other hand,LkBBM2 might play an important role in regulating the growth and development of Hybrid Larch. |
