Comparative Study Of The Regulatory Mechanism Between Direct And Indirect Shoot Regeneration

Under the treatment of appropriate exogenous hormones,higher plants can regenerate shoots through restablishing shoot apical meristem and subsequently produce new individuals.Reconstruction of shoot apical meristem not only exemplifies plant cell pluripotency,but also provides foundation for genetic transformation and in vitro propagation.It has been found that shoot meristem can be formed directly or indirectly.For indirect shoot regeneration,the callus is firstly derived from explants in an auxinriched callus-inducing medium(CIM),and then transferred into a cytokinin-riched shoot-inducing medium(SIM)for shoot induction.For direct shoot regeneration,lateral root primordia(LRP)transdifferentiates into shoot meristems without callus formation.The difference of regulatory mechanisms between the two regeneration types remains unknown.In this study,Arabidopsis seedlings 6 days after germination were used.Using fragements containing the hypocotyl and root as explants,we established a bidirectional system in which shoots regenerated directly from LRP and indirectly from hypocotyl-derived callus simultaneously.In this system,explants were firstly cultured on the medium containing NAA for 48 h,then transferred into the medium containing2-i P.Observation with confocal microscope revealed that structural changes were similar between direct and indirect shoot regeneration procedure.However,compared with those derived from LRP,the basal part of shoot meristems regenerated form callus was much wider,which thus gave rise to converting organs and shoot meristems with significantly larger size in the subsequent stages.q RT-PCR results show that transcriptional levels of hormone signal related genes AUXIN RESPONSE FACTOR 5and ARABIDOPSIS RESPONSR REGULATOR 1(ARR1),as well as stem cell regulating genes LATERAL ORGAN BOUNDARIES DOMAIN 16(LBD16),PLETHORA 1(PLT1),SCARECROW(SCR),WUSCHEL RELATED HOMEOBOX 5(WOX5)and WUSCHEL(WUS)were not significantly altered between the two regeneration types.Analysis using reporter lines demonstrate that the expression patterns of WOX5,WUS,SCR and ARR1 were similar between direct and indirect shoot regeneration procedures.However,expression pattern of WOX11 was different between the two regenerated types.During indirect regeneration,the number of founder cells expressing WOX11 was relatively more which gave rise to larger meristem,whereas less founder cells were produced during direct regeneration and generated smaller meristem.Further analysis showed that in the mutant with defect DOMAINS REARRANGED METHYLTRANSFERASE 1(DRM1),DRM2 and CHROMOMETHYLASE 3(CMT3),where in the non-CG DNA methylation almost completely lost,direct shoot regeneration was significantly promoted while indirect shoot regeneration was unaffected.Correspondingly,the transcriptional level of WOX11 was increased in the drm1 drm2 cmt3(ddc)mutant compared with that of wild type during direct regeneration,but was unchanged between ddc mutant and wild type during indirect regeneration.Bisulfite sequencing analysis showed that non-CG DNA methylation specifically regulated WOX11 transcription and promoted direct shoot regeneration,but had no influence on indirect regeneration.In this study,we demonstrate that the number of founder cells expressing WOX11 was tightly associated with regeneration types.Non-CG DNA methylation specifically regulated WOX11 transcription and promoted direct shoot regeneration,but had no influence on indirect regeneration.The result provides new insights for understanding the regulatory mechanisms of cell fate transition during de novo organogenesis.