The Mechanism Of Multiple Hormones On Promoting Rooting During Cutting Of Walnut Rejuvenation

The only way for walnut(Juglans L.)industrialization is to achieve improved variety asexual reproduction.Cutting is the main propagation method of woody plants,while it is difficult to root in walnut,especially when the cultivated varieties and mature plants as test materials adventitious root formation is extremely difficult.This phenomenon is universal for many species of non-wood forest.On the basis of previous studies,our laboratory,after many years of experiments,used grafting and burying,etiolation and other measures to rejuvenate mature cuttings,and successfully induced adventitious roots in many difficult to root woody plants,such as walnut.But it is lack of deep understanding for the rejuvenation treatment promoting adventitious root physiological mechanism,especially on the principle of key regulatory role in adventitious root formation factors,endogenous and exogenous plant growth regulators.In this study,the rejuvenated and mature soft shoots of walnut cultivars ‘Zhongningsheng’ were used as materials.A multi hormone immune colloidal gold localization system was established.The mechanism of adventitious root formation in walnut soft shoots which rejuvenated and non-rejuvenated was studied at the histological,physiological and molecular levels.The main results as follows:The paraffin section technique was improved to applied on the semi-woody cuttings of walnut and the immunohistochemical localization system was established.The specific improvement as follows: the semi-lignification walnut shoots were fixed by paraformaldehyde and glutaraldehyde;the sections were kept in low temperature long(40 ℃)to keep the section on and keep the antigen activity.In immunohistochemical reaction the dilution ratio of indole acetic acid(IAA)and abscisic acid(ABA)was primary antibody 50 ×,secondary antibody 50×,and gibberellin(GA3)and zeatin riboside(ZR)was primary antibody 100 ×,secondary antibody 50 ×;the silver development conditions at 26 ℃ for 15-20 minutes was best.Histologically,adventitious root primordia developed from the cross-sections of the cambium and phloem,in the direction of the pith rays.Based on the walnut cutting system with high rooting rate and consistent growth.The rooting rate of rejuvenated soft shoots of the walnut cultivar ‘Zhongningsheng’ attained 98.1% 20 days after cutting;non-rejuvenated plants did not root.After cutting 3 days,the cambium cells started to divide strongly.In day 4,the cambium thickened obviously,then the root meristem formed.Until cutting 7 days,the root primordia initial cells can be observed.In day 9,the root primordia were formed and growth outward.In day 15,the adventitious root developed completely.The rejuvenated treatment is an important measure to vary the level of endogenous hormones.We recorded the spatial and temporal distributions of endogenous indole-3-acetic acid,abscisic acid,gibberellin 3,and zeatin-riboside,as well as dynamic changes in these distributions during rhizogenesis.We performed immunohistochemical assays on serial histological sections and combined with HPLC-MS.When root induction commenced,the four phytohormones were principally confined to the phloem sieve and companion cells(S&Cs)at the bases of either rejuvenated or mature soft shoots.IAA levels were higher in rejuvenated shoots than in mature shoots,while ABA,GA3 and ZR levels were higher in mature shoots.The temporal and spatial distribution characteristics of multi-hormones in the process of rooting induction were found.During rooting induction,GA3 was the first hormone to be observed outside phloem S&Cs,followed by IAA,ABA,and ZR.In rejuvenating soft shoots,IAA accumulated in the cross-sectional areas of the cambium and phloem where root primordia were evident.In mature shoots,all four phytohormones remained in phloem S&Cs.The improvement in rooting ability evident after rejuvenation is explained by transformation of the plant to a juvenile form.The phytohormone levels in phloem S&Cs changed and all four phytohormones now appeared in tissues other than phloem S&Cs.In particular,IAA accumulated in the cross-sectional areas of the cambium and phloem.Thus,we suggest that the observed improvement in soft-shoot rooting of walnut cultivars can be explained by spatial and temporal rejuvenation triggered by phytohormones.Exogenous IBA induction was an important factor for controlling the adventitious roots formation.There is a significant effect on the rooting rate of walnut cuttings which treated with exogenous IBA.The endogenous hormones of cuttings induced by IBA were significantly changed during cutting.The IAA and GA3 increased first and reached the peak on the 5th day when root meristem formed,and then decreased.While,the ABA decreased first and then increased in 7 day,after that decreased again.The ZT did not changed significantly in the early stage,but as root primordia induction in 5 day the ZT level increased significantly.The relationship between RNA-seq expression profile and the change of hormone content in walnut cuttings was analyzed by bioinformatics correlation.Five genes of PIN6,ABP19,SAUR21,GA2 ox and GA20ox2 were found to relate with the change of hormone directly.GO and KEGG analyses showed that the main function of the five genes was to regulate auxin transport(PIN6),to respond to exogenous auxin inducing(SAUR21),auxin signaling receptor(ABP19)and gibberellin synthesis(GA2ox and GA20ox2).In this study,we have made innovations in the mechanisms of endogenous hormone interaction in walnut rejuvenation processing promoting root primordium at the histochemical,physiological,biochemical and molecular levels.