Study On Shoot Rooting Technology In Vitro And PLT Gene Expression Analysis During Root Development Of Fraxinus Mandshurica

Fraxinus mandshurica Rupr.belongs to the family Oleaceae.It is a precious broad-leaved tree species in Northeast China,and it also has extremely high economic value and ornamental value.Exploring the rooting technology and rooting mechanism of the shoots of Fraxinus mandshurica could lay a foundation for the large-scale reproduction of the shoots and expand the development of new resources.The adventitious shoots induced by the apical buds and axillary buds of the Fraxinus mandhurica were used as the material to carry out the rooting treatment of the shoot.The influence of various treatments on the rooting of the shoots was studied,and the rooting system of the shoots of Fraxinus mandshurica was established.Meamwhile,researched the content of endogenous hormones and the tendency of expression levels in the auxin/PLT(PLETHORA)pathway to discuss the rooting mechanism,which is a key pathway to control the morphology of root meristem.The results showed that:(1)WPM medium can effectively promote the growth of adventitious shoots,and the mature semi-lignified adventitious shoots have the best growth,which is beneficial to the next rooting treatment(2)Comparing the effects of five factors on rooting,such as auxin,macroelement,presence or absence of bud,darkness,and month.It was found that the addition of IB A and NAA alone affected the rooting effect of adventitious shoots,and the optimal hormone combination was 1.4mg/L IBA+0.7mg/L NAA;Reducing the concentration of macroelements would affect the survival of adventitious shoots,in order to ensure the subsequent transplanting,the proportion of marocoelements in WPM medium should be kept unchanged;Comparing with the treatments with top buds and no buds,the rooting effect of the top buds is the best;Darkness affects the rooting effect of adventitious shoots,so it is not recommended to carry out darkness when rooting;The rooting effect was the best in April,and the worst in January.(3)Rooting technology suitable for transplanting was established:the robust semi-lignified adventitious shoots with top buds were connected to rooting medium(WPM+1.4mg/L IBA+0.7mg/L NAA+sucrose 20g/L),16 hours light per day,the light intensity was 80?mol·m2·s-1,the time of rooting was about 20 days,the rooting rate was 80.45%(April),the average number of roots was 7.55,the average root length was 1.4cm,and the transplanting survival rate was 81.2%.(4)After observing the external morphology of the rooting process of the shoots,it was found that adventitious roots occurred at 10 days of rooting treatment in Apirl,and the lateral roots occurred at 21 days.The anatomy of the stem base showed that most of the root primordium are differentiated from the cells at the cambium,and it was also observed that particular root primordium are derived from the cortex.(5)we cloned two PLT transcription factors from Fraxinus mandshurica and named them FmPLT2 and FmPLT3 by homologous alignment,encoding 532 and 497 amino acid residues,respectively.They have the highest expression in roots,the lowest expression in leaves,and certain tissue specificity.Phylogenetic analysis showed that FmPLT2 and FmPLT3 proteins had the highest homology with the OePLT2 and OePLT3 proteins of the Oleaceae respectively.(6)In the rooting process,the peak expression of the key genes of rooting(FmPINl,FmTPST,FmRGF and FmPLT2)in the PLT pathway coincided with the time of rooting,indicating that these genes were involved in the formation of adventitious roots;FmPLT2 is consistent with the expression pattern of FmTPST and FmRGF in the PLT pathway,while the FmPLT3 gene is different;The expression level of key gene in the PLT pathway in the optimal hormone combination for rooting(1.4 mg/L IBA+0.7 mg/L NAA)was higher than that in the poor rooting with the single hormone(1.8 mg/L IBA)on the whole,indicating different hormone combinations can control the rooting effect of shoots by affecting the expression of key genes in root development.(7)The ratio of endogenous hormone IAA to ABA peaked at 21 days during rooting,which was consistent with the peak of rooting genes in the PLT pathway and the time of the formation of adventitious roots,indicating that the ratio of IAA to ABA plays an important role in rooting.