Induction Of Embryogenic Callus And Preliminary Study Of Empryogenesis-Related Gene PsSERK In Paeonia Suffruticosa

Tree peonies?Paeonia suffruticosa?are famous ornamental flowers frequently seen in gardens nearly all over the world.People enjoy their large,showy,colorful and fragrant flowers.In this study peony callus in vitro from petioles was used as the material for induction of embryogenic callus;cloning of SERK gene from callus,expression analysis of SERK gene by real-time quantitative PCR technology in P.suffruticosa.genetic transformation system of P.suffruticosa callus was constructed in this study.The main results are as follows:1.Induction of peony embryogenic callusIn this study the callus inducted by petioles of P.suffruticosa ‘Feng dan bai' and ‘Wu long peng sheng' were as explants to induce embryonic callus.Experiments were executed to evaluate the effect of different concentrations of Ca Cl₂,H₂O₂ and SNP?NO donor?on peony callus growth and differentiation.The results showed that the degree of browning in ‘Wu long peng sheng' was much more serious than in ‘Feng dan bai' through morphology and microscopic observation,the growth and multiplication of the callus were influenced in ‘Wu long peng sheng'.Under the different concentration of treatments a consistently occurring phase of response,within 30 d of culture,was the appearance of clear,loose,soft,raised and “snow-flake” callus tissue on the surface of the explants.These changes were related to the formation of the embryonic callus.In these three factors,the effects of SNP treatment were the most significant.The studies about the changes of antioxidase activity and endogenous phytohormones in P.suffruticosa callus during the different periods showed that embryonic callus was induced under CaCl₂ at above 1.6 mmol·L-1 after 10-20 d,H₂O₂ was not significant in the peony embryonic callus induction.SNP at low concentration level below?mol·L-1 promoted the formation of the peony embryonic callus and may be conducive to the peony somatic embryogenesis.2.Cloning and expression of SERK gene in P.suffruticosaIn this study,the full-length cDNA of SERK gene was cloned by homology cloning combined with RACE technology from the callus inducted by petioles of P.suffruticosa 'Feng dan bai',and named as Ps SERK?Gen Bank accession number KF876175?.Sequencing of the full-length cDNA got 2362 bp,contained 299 bp 5? untranslated region,179 bp 3?untranslated region and 1884 bp open reading frame,and encoding 627 amino acids.Nucleotide sequence alignment showed that the Ps SERK align had high homology more than 82% with of SERK genes from other species in Gen Bank.The results of bioinformatics analysis of Ps SERK showed that Ps SERK gene encoded 627 amino acids and was a kind of hydrophilic protein,mainly located in the plasma membrane,contained transmembrane domains,4 conserved structure domains,24 phosphorylation sites and a signal peptide sequence.The secondary structure of Ps SERK was made up mostly of alpha helix and random coil.When compared the Ps SERK with the other plant amino acid sequences,the identity percentage varied from 91% to 95%.Actin gene was used as the reference gene for Ps SERK gene expression analysis by real-time quantitative PCR technology in the leaves,petals and embryonic calluses of P.suffruticosa.The results showed that transcriptional expression levels of Ps SERK gene at different tissues were expressed at different degree,the expression level was the lowest at the petals;the next was at the leaves,and the highest was at the embryonic calluses,which suggested that Ps SERK gene expression played an important role during embryonic calluses morphogenesis in P.suffruticosa.3.Constructing an Agrobacterium-mediated genetic transformation system of P.suffruticosa callusThe optimization of Agrobacterium mediated genetic transformation system was studied in the peony callus.During the genetic transformation the strains were selected and at the same time the study optimized the various impact factors,the pre-cultured time,concentration of bacterium,infection time,co-cultured time.And Agrobacterium mediated genetic transformation system of peony callus had been established.The minimum lethal homomycin concentration of peony callus was 3 mg·L-1 and the minimum lethal cephalosporin concentration for growth of agrobacterium in the initial select medium was 200 mg·L-1,and that for the continuous medium was 100 mg·L-1.The system was no pre-culture,numerical OD600 0.6,20 minutes for infection,3 days for co-culture in darkness at 22?.And then the callus was rinsed and immersed using 200 mg·L-1 cephalosporin for 30 minutes.The callus treated was transferred to the liquid culture medium added 200 mg·L-1 cephalosporin for by shaking culture for 24 hours and then was transferred to the screening solid medium added 200 mg·L-1 cephalosporin and 3 mg·L-1 homomycin,and was done once 20 days.The peony callus after transformation was successfully tested by GUS expression at the staining temperature of 24?.