Establishment Of Regeneration And Genetic Transformation System Of Neolamarckia Cadamba And Transcriptome Analysis Involved In Embryogenic Callus Formation

Neolamarckia cadamba,affectionately known as miracle tree,is a perennial and deciduous tropical hardwood belongs to Rubiaceae family.As a fast growing tree with excellent morphological,anatomical and chemical characteristics,N.cadamba have tremendous ecological and economic value in landscape,furniture industry and pharmaceutical production.Previous researches on N.cadamba mainly focused on tranditional cultivation,medicinal component identification and pharmacological analysis,while it has no any reported on molecular breeding.Genetic transformation is a useful method for studying plant functional genes,and it is also a convenient way for shortening breeding cycle.However,plant regeneration and genetic transformation of N.cadamba have not yet been established.Fortunately,the technologies of plant tissue culture and transgene are becoming mature,these provide the preconditions for cloning plantlet with excellent agronomic trait and launching molecular breeding for N.cadamba.In this study,firstly,N.cadamba stems which contained endophytes were used as explant to test the inhibition effects of different antibiotics in endophyte contamination.Secondly,an efficient regeneration protocol was established firstly,and then the molecular mechanism of regulation involved in embryogenic callus formation in N.cadamba was analyzed.In addition,a reliable Agrobacterium-mediated transformation system was also established.Based on the regeneration and transformation protocol,cry1Ac-CBF1 genes was transferred into N.cadamba.The main results are as follows:1.Research on contamination control of endophytes.in order to find the optimum antibiotics and their used concentration for inhibition of endophytes in N.cadamba,the effects of different antibiotics were tested.The results showed that the contamination was caused by bacteria and fungi.The joint use of bacteria and fungi antibiotics performed the best effect on the inhibition of endophytes,especially in the formula contained 300 mg/L streptnomycin and 800 mg/L fluconazole,and the comtamination rate could reduce to 30%.In addition,the inhibited ability of antibiotics have certain timeliness.It is better to transfer the young leaf which germinate from the stem explant into the induction medium to induce adventitious shoots,and this process could prevents the endophytes from re-contamination.2.Establishment of an efficient regeneration protocol.Three types of callus were induced from leaf explants on MS medium with different plant growth regulators(PGRs).Among them,only type II callus could be differentiated into embryogenic callus and regenerated adventitious shoot.It was found that MS medium supplemented with 3 mg/L TDZ,0.1 mg/L 2,4-D and 0.05 mg/L NAA provided the most suitable medium for embryogenic callus induction,and delivering the highest number of shoot(8 shoots per explant).Meanwhile,the neomorph was also formed on the surface of calluses.The neomorph went into a cycle differentiation via two way:adventitious shoots and the new neomorphs in the medium supplemented with 0.5 mg/L 6-BA and 0.05 mg/L NAA.We observed that the developmental stage and inoculated orientation of the explants have impact on the regeneration frequency.The best leaves for regeneration is the 1?2 top leaves of a plantlet,and orienting the abaxial surface down towards the medium was much better than upwards.3.Transcriptome analysis of embryogenic callus formation.We constructed the transcriptomic profiles of the different samples during embryogenic callus differentiation stages using Illumina Hiseq X Ten platform.De novo assembly generated 74584 unigenes and a total of 18003 differentially expression genes(DEGs).Comparison of stage transcriptomes showed that a total of 9353 unigenes were differentially expression in the initiation of callus,among them,4,244 unigenes were up regulated.In subsequent development stage,9435 unigenes were differentially expression with 4084 unigenes up regulated.Besides,there were 5892 and 5974 DEGs specifically expressed during two stages,respectively,while 3461 unigenes were present during both developmental stages.KEGG annotation and homologous genes blast analysis indentified 34 genes related to auxin and cytokinin singnaling pathways,and 36 genes related to somatic embryogenesis formation played important roles in embryogenic callus induction.4.A reliable Agrobacterium-mediated transformation system of N.cadamba was established.Based on the regeneration protocol,some factors which affected transformation efficiency were standardized.Prior to transformation,the leaves were pre-culture 2 days.With the use of vacuum infiltration(0.1 Mpa,10 min)assisted Agrobacterium infection(OD₆₀₀=0.2?0.4)for 30 min,and then co-cultivation for 4 days significantly increased the transformation frequency.Genomic integration and transgene expression were confirmed by GUS assays,PCR,YFP detection and RT-PCR analysis.This transformation protocol could be helpful in genetic improvement of N.cadamba.5.Transgenic insect and cold resistant of N.cadamba.By modifying the pCAMBIA1305 vector,cry1Ac and At CBF1 genes were assembled into pCAMGWB2?cry1Ac?CBF1 vector.Based on the regeneration and transformation protocol,the double gene were transferred into N.cadamba.The analysis of PCR and Southern blot indicated that the exogenous gene was integrated into N.cadamba genome.The results of qRT-PCR showed that the expression pattern of exogenous gene in five transgenic plants was significantly difference,however,the expression trend of the two genes was similar in the same transgenic plant.