Molecular Mechanism Investigation Of Protocorm-like Bodies Morphogenesis By Thidiazuron Introduction In Rosa Canina L.

Regeneration of Rosa in vitro is inefficient and time-consuming,seriously limiting the application of transgenic engineering to this ornamental.PLBs is an efficient and rapid regeneration system that formed by TDZ induction.However the mechanism of PLB induction remains relatively unclear.In this research,PLBs regeneration system of Rosa canina and RcWUS transgenic plant lines weres used to studied the molecular mechanism of early stage of PLBs formation that induced by TDZ.The results was showed as follow:1.Analysis of transcript profiles of PLBs formation showed that:In total,97,336 unigenes were generated and 4,177,2,154,and 1,627 unigenes were detected between rizhoid and single meristematic centers stage,single meristematic center stage and mixtures stage,mixtures stage and mature PLBs stage,respectively.In the single meristem stage of PLBs,TDZ mainly acted as a stress signaling to promote photosynthesis system regeneration,promote communication of cells and biosynthesis of zeatin.Especially,some pattern special expressed genes were activated in single meristem that suggested that cells fate had finished conversation.Analysis of DEG showed that RcABP19a was significantly up-expressed and would be studied later.2.Expression of RcABP19a was significantly up-expressed by TDZ induction during the whole process of PLBs morphogensis in Rosa canina.L.Subcelluar location showed that RcABP19a was located on plastic membrane and ER-membrane.In situ hybridization of RcABP19a showed that RcABP19a was expressed in vascular,root stem nich cells and embryonic callus.The clone and analysis of RcABP19a promtor showed that this gene mainly expressed in root QC,vascular of root and basic leaves.Together with the results of In situ hybridization of RcABP19a,we conduced that it played an important role on cell fate conversion.Overexpression of RcABP19s showed more branches in Arabidopsis and fast growth in vegetable development.3.The study on the mechanism of PLBs formation by TDZ induction showed that:TDZ promoted accumulation of endogenous IAA and ZR while depressed the endogenous iPA accumulated in callue-rhizoids.The signaling of auxin and cytokinin were acted by up-expression RcARF1,4,RcRR1 and the active of endogenous cytokinin was improved by up-expression RcLOG1 genes in tip of rhizoids.As the auxin transport protein RcPINl was repressed while RcPIN2,3 were promoted in tip of rhizoids,the maximum content gradient of auxin was disturbed.Together with the results of NAA,KT,NPA treatment,we concluded that TDZ acted mainly as cytokinin and triggered PLBs formation by breaking the balance of auxin and cytokinin.The expression of root stem cell related genes RcWOX5,RcPLT1,RcPLT2,shoot stem cell related genes RcWUS,RcSERK1,RcCLV1 showed that the lateral root formation related genes RcWOX5,RcPLTl,RcPLT2 and embyonic marker genes RcSERKl were continue up-expression suggested that dedifferentiation pathway was established and cells fate was conversion by TDZ induction.RcWUS genes was transiently up-regulated in columella cells,epidermis and then decreased later,which suggested that RcWUS cann't promote callus formation during early stage of PLBs formation.Above all,we concluded that TDZ triggered PLBs formation through disturbed the balance of auxin and cytokinin in tip of rhizoids,and then cause the dedifferentiation pathway established directly or indirectly.More studies needed to be done about PLBs morphogenesis by TDZ induction in Rosa canina L.