The Effect Of Subculture On The Differentiation And Regeneration Ability Of Embryogenic Callus In Catalpa Bungei

Catalpa bungei C.A.Mey is a deciduous tree,known as the "king of trees" since ancient times.It is a unique and precious timber tree species in China,with medicinal and ornamental value.Its characteristics of cross pollination,low natural fruiting,small seeds,and difficult reproduction greatly limit the promotion and utilization of this timber tree species.Somatic cell embryo regeneration system has the advantages of few chimeras,easy regeneration,good homogeneity,and large number of individuals.Its reproduction speed is far beyond the conventional method,and it is widely used for the reproduction of rare or excellent new varieties of plants.The practicality of the somatic embryogenesis system is related to whether embryonic callus tissue with long-term subculture can maintain good differentiation and regeneration ability.By analyzing the non embryonic callus,primary embryonic callus and embryonic callus continuously cultivated for 7 years of C.bungei.,the differences in cytology,physiology and biochemistry,transcriptome sequencing,and endogenous hormone levels,this study explored the mechanism of differentiation and regeneration of embryonic callus of C.bungei.under different subcultures,so as to provide genetic stability The long-term stable maintenance of in vitro regeneration ability provides theoretical basis and technical support,and also provides reference for the decline and improvement measures of regeneration ability in other plants during long-term in vitro culture.The main results are as follows:(1)The chromosome counting method showed that the chromosome number of the primary embryogenic callus of C.bungei.was still in the state of diploid,2n=40,while the chromosome number of the embryogenic callus after continuous subculture for 7 years doubled,ranging from 80-162,with a large number of aneuploid somatic cell cells mixed;further validation using flow cytometry revealed that the DNA content of embryogenic callus cultured continuously for 7 years increased by 3 times compared to the seedlings of C.bungei.,and its cell ploidy was hexaploid;It is speculated that the phenomenon of chromosome doubling and mixed doubling may be the cytological mechanism that leads to a decrease in differentiation and regeneration.(2)The soluble protein content of embryonic callus that has been continuously subcultured for 7 years is significantly higher than that of newly induced embryonic and non embryonic callus tissues;The soluble sugar content of embryonic callus tissue that has been subcultured for 7 years is not different from that of newly induced embryonic callus,but significantly different from that of non embryonic callus.This may be an important reason for their vigorous cell division,proliferation,and growth.The activities of superoxide dismutase(SOD)and peroxidase(POD)of embryonic callus subcultured for 7 years were significantly lower than those of newly induced embryonic callus;This indicates that its low level of scavenging reactive oxygen species may be an important factor in the decline of cell differentiation ability.(3)The transcriptome of newly induced embryogenic calli and 7-year continuous embryogenic calli of C.bungei.were sequenced and 82396 Unigene were assembled.67727 Unigene were annotated in the seven databases of NR,NT,GO,KOG,KEGG,Swissprot and Intersection,accounting for 82.20% of the total;through GO and KEGG enrichment analysis,6149 differentially expressed genes(DEGs)were obtained.These DEGs were mainly concentrated in plant hormone activation signaling pathways,transcriptional regulation,MAPK signaling pathways,lipid transport,regulation of development processes,and biosynthesis of various secondary metabolites in plants.Compared to newly induced embryonic callus,2397 differentially upregulated genes were found in embryonic callus that had been continuously subcultured for 7 years,3752 differentially down regulated expression genes,of which response to auxin signal transduction SAUR(Small auxin up RNAs),ARF(Auxin response factors),AUX/IAA(Auxin/indole-3-acoustic acid),GH3(Green hagen 3),PYR/PYL(Abscisic acid receptor PYR/PYL family),extracellular protein AGP(Arabian protein),and transcription factor ABI3(Abscisic acid sensitive 3)promote somatic embryogenesis Gene expression was down regulated,The expression of the gene ETO1(Ethylene synthesis inhibitory factor),which inhibits ethylene synthesis,is upregulated.The abnormal expression of these genes may be the molecular basis for their reduced differentiation and regeneration ability.(4)The levels of endogenous hormones have a significant regulatory effect on the differentiation and regeneration of in vitro plants.The endogenous hormone levels of GA3(Gibberellin A3)and CTK(Cytokinin)in various callus tissues were detected for 7 consecutive years of subculture and newly induced embryogenic and non embryogenic callus of C.bungei.The levels of IAA(Indole acid)and ABA(Abscisic acid)remained high,and the newly induced embryogenic callus tissues had significantly higher levels of IAA and ABA than other tissues,High levels of IAA and ABA may be the key to regulate somatic cell embryogenesis.The content of IAA and ABA in embryogenic calli subcultured for 7 years was significantly lower than that in other tissues.Combined with transcriptome sequencing,the down-regulation expression of SAUR,ARF,AUX/IAA,GH3,PYR/PYL,key genes in the signal transduction pathway of auxin and abscisic acid,in embryogenic calli subcultured for 7 years,may be the reason for the low level of IAA and ABA activity,which is not conducive to the differentiation of somatic cell embryos.