Functions Of PtPLT1 And PtWOX5B In Regulating Shoot Regeneration Of Poplar

Under appropriate in vitro culture conditions,plant tissues and organs can regenerate shoots from callus and give rise to complete new individuals.Plant regeneration is the prerequisite for in vitro rapid propagation and genetic engineering.Generation of callus with regenerative component is the key step for shoot regeneration.Previous studies on the model plant Arabidopsis have found that callus has the similar cellular properties with those of root meristems.Expressing key regulatory genes in root meristems such as PLETHORA 1(PLT1)and WUSCHEL-RELATED HOMEOBOX 5(WOX5)is necessary for obtaining regeneration ability.However,it is unclear whether the above genes play similar roles in forest trees.In this study,PtPLT1 and PtWOX5 B homologous of Arabidopsis PLT1 and WOX5,were isolated from 84K(Populus alba×Populus gllandulosa)poplar based on sequence homology.The results of evolutionary tree analysis showed that PLT1 and WOX5 B genes were conserved in the evolution of different plant species.The results of qRT-PCR showed that both PtPLT1 and PtWOX5 B showed higher expression levels in roots compared with stems and leaves.In situhybridization results showed that the expression signals of PtPLT1 were concentrated in the root stem cell niche.Subcellular localization analysis showed that both PtPLT1 and PtWOX5 B proteins were located in the nucleus,which was same as those of PLT1 and WOX5.We constructed two estrogen-induced expression vectors pER8::PtPLT1 and pER8::PtWOX5B,and transformed them into 84 K poplar.The results of qRT-PCR showed that the expression levels of PtPLT1 and PtWOX5 B were increased significantly after 6 days of estrogen induction.pER8::PtPLT1 explants treated with estrogen for 2 weeks and transferred to the common differentiation medium for 7 days induced the formation of adventitious roots.The results indicate that the 2-week estrogen treatment time is too long for inducing regenerated shoot.It was indicated that PtPLT1 functions in determining the cellular properties of root meristem.Further analysis revealed that pER8::PtPLT1 explants treated with estrogen for 6 days,and transferred to ordinary differentiation medium for 35 days exhibited significantly increased shoot regeneration frequency and the number of regenerated shoots on each explant.However,pER8::PtWOX5B explants strain did not show similar phenotypes.The above results indicate that the enhanced expression of PtPLT1 promoted shoot regeneration in poplar.The results of this study provided new information for understanding the molecular mechanism of shoot regeneration in forest trees.