Somatic Embryo Induction And Its Transcriptome Analysis Of Rose

Rose（Rosa hybrida）is one of the most important flowers in my country and even in the world.Because of its rich colors,diverse flower types,rich floral fragrance and good meaning,it has been loved by everyone.However,due to unclear genetic relationship,narrow genetic background,incompatibility and other reasons,the regular breeding process of rose was inhibited.The development of genetic engineering technology is a new way to breed the rose.However,the high degree of genotype dependence and low induction rate severely restrict the perfection of somatic embryogenesis and breeding applications.In addition,there is also a lack of molecular mechanisms research based on somatic embryogenesis of rose leaves.Therefore,the establishment of a mature somatic embryo induction system and the exploration of its molecular mechanism are of great significance.In this study,the single bud stems of three kinds of roses were used as explants to explore the factors that affect the regeneration process of rose,and an in vitro culture system was established.And taking the common induced callus and somatic embryos as materials,transcriptome sequencing technology was used to study the gene expression patterns in the process of somatic embryogenesis,which could further explore the molecular mechanism of rose somatic embryogenesis.It can provide theoretical reference for exploring the external culture conditions and related molecular mechanisms of rose somatic embryogenesis on the future.The main findings are as follows:1.A tissue culture system of the three kinds of roses was established.Among them,the optimal starting medium for R.chinensis‘Old Blush’was MS+6-BA 1.0 mg/L+NAA0.5 mg/L,the optimal starting medium for‘Baisemeian’was MS+6-BA 1.0 mg/L+NAA0.1 mg/L,and the optimal starting medium for‘Tianshi’was MS+6-BA 1.0 or 1.5 mg/L+NAA 0.1 mg/L;the optimal serile seedling subculture medium for R.chinensis‘Old Blush’was MS+6-BA 1.0 or 1.5 mg/L+NAA 0.05 mg/L,the optimal serile seedling subculture medium for‘Baisemeian’and‘Tianshi’was MS+6-BA 1.0 mg/L+NAA 0.01mg/L.2.The most suitable medium for callus induction was selected by using the leaves of sterile seedlings as explants.The optimal medium of callus induction medium for the three kinds of rose was MS+2,4-D 1.0-3.0 mg/L.The induction rate of R.chinensis‘Old Blush’was above 94%,and the induction rate of‘Baisemeian’and‘Tianshi’were both above 98%.3.The differentiation of rose callus is affected by many factors,such as genotype,plant hormones,culture conditions and additives.The callus of R.chinensis‘Old Blush’didn’t differentiate after inducing.The callus of‘Baisemeian’and‘Tianshi’were differentiated into embryogenic callus or somatic embryo after 2-3 times of subculture on the medium MS+2,4-D 1.0-3.0 mg/L,the differentiation rates of callus were respectively8.30%and 10.62%.The callus of modern rose‘Baisemeian’and‘Tianshi’were differentiated into adventitious bud on the differentiation medium MS+TDZ 3.0 mg/L+6-BA 1.0 mg/L+NAA 0.1 mg/L+GA₃ 1.0 mg/L+proline 300 mg/L after dark culture about2-3 months,and the differentiation rates were respectively 6.4%and 4.5%.The callus of‘Baisemeian’and‘Tianshi’were differentiated into somatic embryo on the differentiation medium（MS+TDZ 3.0 mg/L+6-BA 1.0 mg/L+NAA 0.1 mg/L+GA₃ 0.1 mg/L+proline300 mg/L）after culture about 2-3 months,and the differentiation rates were respectively5.4%and 5.2%.4.Take samples of‘Baisemeian’common callus and somatic embryos to extract RNA.Using RNA-seq technology for transcriptome analysis,a total of 3539 differentially expressed genes were obtained,including 2195 up-regulated genes and 1,344 down-regulated genes.A large number of differentially expressed genes were found to be related to plant hormones,transcription factors and stress response.Genes related to auxin such as GHs,SAUR,and AUX,genes related to cytokinin such as CKX,IPT5,and ARR,genes related to stress such as GST and POD,and genes related to transcription factors including BBM2,AIL,WOX,are all up-regulated in somatic embryos.5.A total of 397 transcription factors were detected by transcriptional factor analysis of differentially expressed genes,among which 251 were up-regulated and 146 were down-regulated.These transcription factors mainly include b HLH,MYB,WRKY,AP2,NAC,C2H2 and C2C2-Dof and so on.GO enrichment shows that differential genes are mainly involved in metabolic processes and cellular processes in biological processes,membrane,cells and cell part in cellular component,and catalytic activities and conjugates in molecular functions;The results of KEGG showed that plant hormone signal transduction,amino acid,polysaccharide,and secondary metabolism played an important role in the formation of rose somatic embryos.The expression trends of 11genes differentially expressed in somatic embryos were consistent with the transcriptome sequencing results by the q RT-PCR verification,which proved the reliability of the results on sequencing.