Establishment Of Safflower Tissue Culture System And Cloning Of BAK1 Gene

Carthamus tinctorius L.(safflower)is an economic plant of the Compositae family.It is widely distributed and planted in Xin Jiang,Yun Nan,An Hui and other provinces in China.The filaments of safflower contain flavonoids,safflower polysaccharides,fatty acids,lignans,polyacetylenes,alkaloids,sterols and other compounds,which have medicinal value.Moreover,safflower seeds have high content of unsaturated fatty acid and are good edible oil resources.Safflower has high economic value as cooking oil and medicine plant,but its growth cycle is long,and the yield is affected by factors such as diseases and waterlogging in the field.The establishment of safflower tissue culture system can lay a foundation for breeding excellent safflower varieties with disease and stress resistances,and for building a technical platform for transgenic research.The SERK gene family can produce somatic embryogenesis receptor-like kinases that play an important role in induce the somatic embryogenesis from plant tissue culture.SERK3/BAK1 is a member of the SERK family,which is involved in the brassinosteroid(BR)signal transduction pathway and floral organ shedding signal pathway.In this study,the safflower tissue culture system was established by comparing different cultures experiments of different safflower explants with different culture medium.The fulllength cDNA and protein sequences of BAK1 gene in safflower were obtained by bioinformatics techniques,and the BAK1-RNAi vector was constructed.The expression of BAK1 gene in safflower leaves under different hormone treatments was analyzed by real-time PCR.The research results are as follows:1.Germfree seedling culture : The plump seeds of Anhui safflower were selected for germfree seedling culture.The seeds were surface sterilized by soaking in 75% ethanol for 30 s and 0.1% mercury dichloride for 20 min.The sterilized seeds were transferred to MS medium and cultured for 7 to 9 days at a temperature of 23 �C to 25 �C and an illumination intensity of 3000 to 5000 lx,and then the explants were excised.2.Callus induction : Nine induction mediums were designed as HY1~HY9.Among them,HY1,HY2 and HY3 were added with different concentrations of NAA and 6BA to induce callus.HY4 was not added any hormone.HY5~HY9 were added with certain concentrations of NAA,6BA,GA3,ABA,and BR to induce callus,respectively.The roots,stems and leaf explants of the germfree seedlings were cut down and cultured in different callus induction media.The results showed that HY2 had the best effect in induced callus among the nine media.The callus induction rates from root,stem and leaf explants were 100%,84.61% and 88.63%,respectively.HY3 was also good and the callus induction rates from root,stem and leaf explants were 100%,60%,and 85.71%,respectively.In HY4,HY6 and HY7,only root explants had callus formation,but no callus formation from stem and leaf explants.In HY5,callus was formed from all of the root,stem and leaf explants,furthermore,new roots were found in the callus formed from leaf explants.In HY8 and HY9,the root explants had less callus formation than HY4,even the stem and leaf explants had no callus formation.3.Formation of tissue culture seedlings : Each leaf-induced callus was transferred into a shoot differentiation medium for seedlings induction.Among HM1 to HM9 culture media,HM1 had germination rate of 100% and emergence rate of 47.5%,respectively,which was the optimal differentiation medium.HM2 had few differentiated seedlings formation but a large number of callus production,which showed strong ability of expand reproduction.HM5 was easier to form glass seedlings.HM4,HM6 and HM9 had low germination rates,and less capable of shoot differentiation.4.Root induction : The differentiated shoots were transferred into a rooting medium for continue culturing.The rooting rates induced by HG1 and HG2 were 84.6% and 77.8%,respectively.Both of them had good effects of root induction,but HG2 could induce larger roots system.5.Bioinformatics Analysis of CtbBAK1 Gene : The SERK3/BAK1 of the SERK family was acquired from the safflower transcriptome data.Its ORF length is 1782 bp.According analysis of amino acid sequence alignment,it was named as CtbBAK1.The amino acid physicochemical properties were analyzed by ProtParam software,and showed with an isoelectric point of 5.40 and a molecular weight of 65.87 kDa.TMpred analysis revealed its two protein transmembrane regions.SWISS-MODEL analysis revealed its protein tertiary structure model.Align analysis with 12 other species' BAK1 genes displayed a phylogenetic tree,which showed that CtbBAK1 is most close to BAK1 genes of Helianthus annuus,Lactuca sativa and Artemisia annua.6.Expression analysis of CtbBAK1 gene: Fluorescence real-time quantitative PCR was used to analyze the expression of CtbBAK1 in roots,stems and leaves of safflower.At the five time points of 0h,3h,6h,12 h and 24 h,the leaves treated with HY4~HY9(five hormones)were used to analyze the expression of CtbBAK1.The results showed that the expression level orders of CtbBAK1 in safflower were root>stem>leaf.When treated with NAA and 6BA,the expression of CtbBAK1 was up-regulated,but not changed when treated with other hormones.