Molecular Mechanism Of Plantlet Regeneration From Rhizome In Cymbidium

Chinese Cymbidium is a kind of traditional and precious flower with important ornamental,economical,and cultural values and broad market prospect,however,the seedling production still depends on division propagation and can’t satisfy the demand of market due to the recalcitrant regeneration of rhizome.In order to clarify the molecular mechanism of plantlet regeneration from rhizome in Cymbidium,in this paper,the rhizome intermediate propagules from Cymbidium aloifolium,C.sinense‘Xiaofeng’ and Cymbidium.sinense ‘Qijianbaimo’ were employed to systematically analyzes the micropropagation characteristics of different genotype Cymbidium.After that,the genes related to easy to micropropagation of rhizome from C.aloifolium were identified through de novo transcriptome assembly.The molecular mechanism of YUCCAs underlying plantlet regeneration are illustrated with the aid of an auxin biosynthesis inhibitor,yucasin,which specifically inhibits the function of YUC proteins.The genes and promoter sequence of YUCCA8 from different genotype Cymbidium were cloned and analysized.The main results were as follows:1.When cultured on MS medium without additive hormones in light,the rhizomes of C.aloifolium were regenerated with very high efficiency,going through five sequential stage,namely,light response,induction of shoot primordium,bud differentiation,induction of root primordium and root regeneration.However,plantlets were completely unable to regenerate from those cultured under darkness.2.The rhizomes cultured on MS medium without additive hormones under darkness were significantly different in micropropagation characteristics between C.aloifolium and C.sinense ‘Qijianbaimo’.The rhizomes of C.aloifolium had sheath and leaf,and rapidly differentiate into plantlets under light,while the rhizomes grew slowly and were difficult to differentiate due to the lack of sheath and leaf formed under darkness.3.After exposured to light,there were remarkable difference in dynamic changes of endogenous phytohomone of rhizomes formed on MS medium without additive hormones between C.aloifolium and C.sinense ‘Qijianbaimo’.4.Rhizomes of C.aloifolium belong to the light response,induction of shoot primordium,bud differentiation,induction of root primordium and root regeneration stages respectively were subjected for RNA-seq analysis on Illumina Hiseq2500 platform.227,012 unigenes were obtained,and most of them were enriched to translation,carbohydrate metabolism,folding,sorting and degradation,amino acid metabolism,transport and catabolism.Differential expressed genes(DEGs)analysis demonstrated that there were 2,654 DEGs at light response stage,and among them were enriched to protein processing in endoplasmic reticulum,photosynthesi,circadian rhythm and plant hormone signal transduction pathways,821 DEGs at induction of shoot primordium stage,and among them were enriched to carbon metabolism,biosynthesis of amino acids,carbon fixation in photosynthetic organisms and phenylpropanoid biosynthesis pathways,893 DEGs at bud differentiation stage,and among them were enriched to carbon metabolism,carbon fixation in photosynthetic organisms and phenylpropanoid biosynthesis pathways,1,484 DEGs at root primordium stage,and among them were enriched to carbon metabolism,biosynthesis of amino acids,phenylpropanoid biosynthesis and cell cycle pathways pathways,and 1,148 DEGs at root regeneration stage,and among them were enriched to carbon metabolism,photosynthesis,phenylpropanoid biosynthesis and diterpenoid biosynthesis pathways,respectively.5.From the transcriptome of C.aloifolium,13 light response related genes(CRY1α、CRY1β、CRY2、CRY3、PHOT1、PHOT2、PHYA,PHYB、PHYC、UVR8、PIF3、COP1、HY5),10 auxin synthesis related genes(2 TAA genes and 8 YUCCA genes),22 auxin polarity transports related genes(2 AUX1/LAX genes,10 PIN/PILS genes and 10ABCB/PGP genes),64 auxin signal transduction pathway related genes(17 Aux/IAA genes,26 SAUR genes,6 GH3 genes and 15 ARF genes),13 cytokinin metabolism-related gene(3IPT genes,2 CYP735 A genes,4 LOG genes and 4 CKX genes),and 20 cytokinin signal transduction pathway related genes(6 CaHK genes,2 CaHP genes and 12 CaRR genes)were identified.Forthermore,31 genes(CRY1β,COP1,YUC2,4,5,7,8,6,LAX2,PIN3,4,ABCB7,8,9,IAA3,4,5,6,9,GH3.4,GH3.6,SAUR3,ARF12,13,LOG1,CaHK1,2,4,CaRR1,2and CaPRR95)related to rapid micropropagation of the rhizomes in C.aloifolium were obtained by qRT-PCR,and among them,YUC2,4,8,6,PIN3,4,ABCB8,IAA5,6,LOG1,CaHK1,2,CaRR1,2 and CaPRR95 were candidate genes related to light response,CRY1βand COP1 were candidate genes related to shoot primordium,YUC7,LAX2,PIN3,ABCB7,9,SAUR3,IAA6,ARF12,CaHK4 and CaRR2 were candidate genes related to bud differentiation,CRY1β,COP1,IAA3,9,GH3.4 and LOG1 were candidate genes related to root primordium,YUC5,PIN3,PIN4,IAA3,4,9,GH3.4,GH3.6,SAUR3,ARF13,CaRR2 and CaPRR95 were candidate genes related to root regeneration,respectively.6.Yucasin inhibited the plantlet regeneration of rhizomes in C.aloifolium by altering the endogenous phytohomone microenvironment.Besides,the inhibition of yucasin on root regeneration can be removed by adding exogenous auxin,however,exogenous auxin treatment can not completely eliminated this inhibition effect on shoot regeneration.7.The shoot differentiation ability of rhizomes in C.sinense ‘Qijianbaimo’ was significantly enhanced under the endogenous phytohomone microenvironment with a high concentration of DHZ+DHZR、BRs,JA-Me and low concentration of IAA,ABA,GA3,GA4,which can be achieved by the joint use of 50 μM yucasin and 1.0 mg/L 6-BA.8.During the process of shoot differentiation of rhizomes in C.sinense ‘Qijianbaimo’,the expression level of 15 genes(YUCCA7,8,LAX2,PIN3,ABCB7,9,IAA3,4,6,9,GH3.4,6,ARF13,CaRR2 and LOG1)were significantly upregulated.9.Full length of YUCCA8 cDNA of C.aloifolium,C.sinense ‘Qijianbaimo’ and C.sinense ‘Xiaofeng’ were cloned by using the SMARTer RACE and homologous cloning methods.According to sequence blast analysis,CDSs of this gene from these three species were completely consistent in sequence.The length of YUCCA8 was 1497 bp,with 3 exons and 2 introns.10.By using the Tail-PCR method,promotor sequence of YUCCA8 in C.aloifolium,C.sinense ‘Xiaofeng’,C.sinense ‘Qijianbaimo’ and Cymbidium ‘Xiaofeng’ were obtained,which was 2930 bp,1557 bp and 1621 bp in length,respectively.Although the transcriptional regulatory elements in the promotor of YUCCA8 was relatively conservative,the structural basis of transcription initiation was different among these three species.11.Overexpression and CRISPR/Cas9 vectors of YUCCA8 were constructed,and transformation was carried out via Agrobacterium-mediated transformation method using rhizomes of C.sinense ‘Xiaofeng’ as receptor material.Unfortunately,no transgenic plants were obtained.In conclusion,this study was preliminarily dissected the molecular mechanisms underlying plantlet regeneration from rhizome in Cymbidium,as well as the growth time nodes of light response,induction of shoot primordium,bud differentiation,induction of root primordium and root regeneration from rhizome in C.aloifolium.Besides.the differences in the morphological structure of rhizomes and the endogenous hormone microenvironment between C.aloifolium and C.sinense ‘Qijianbaimo’ were clarified.Some genes related to rapid propagation of rhizome in Cymbidium was also determined,among them,YUCCA8 gene was cloned and its structure basis which include promotor was analyzed.Finally,overexpression and CRISPR/Cas9 vectors of YUCCA8 gene were constructed.This study could lay a solid foundation for the improvement of micropropagation efficiency and new cultivars in Chinese Cymbidium.