| Low frequency of rooting and the low root quality of plantlets in vitro of tree peony(Paeonia suffruticosa Andr.)have become a bottleneck in rapid propagation technologies,which caused by the the discontinuity between the vascular bundle and the xylem in the roots and resulted in low survival rate of transplant.Previous studies have shown that rooting tree peony plantlets in vitro is a similar process to rooting tree peony cuttings and scions.In cuttings,grafted scions,and plantlets in vitro,adventitious root formation follows similar physiological and biochemical processes,such as cell dedifferentiation and primordial root formation.Previously,our research group has been studied the anatomy and physiology and biochemical indexes during rooting of test-tube seedlings,and preliminary research and analysise on the changes of indexes of physiology and biochemical of cuttings and grafted scions has been done.However,the relationship about the changes of physiology and biochemical indexes between cuttings,grafted scions and test-tube seedlings during rooting has not been systematically elaborated.This study will further research the change rules about the anatomy and indexes of physiology and biochemical of cuttings and grafted scions during rooting,and determine the crucial period of rooting,and perform transcriptome analysis combining with the key period of rooting of seedling,and screen for relevant genes associating with adventitious rooting,and verify the expression levels of relating genes in critical period of rooting of cuttings and grafted scions by qRT-PCR,and screen further for key genes,and transfer them to Arabidopsis thaliana for overexpression analysis to determine theie function finally based on previous research The research above will be an in-depth study of the molecular mechanism and provide theoretical basis and technical support for solving the current situation of difficulty for adventitious roots of plantlets in vitro of tree peony.The main results of research were as follows:1.Determination the critical period during rooting of cuttings and grafting seedlings Three-year hard branches of tree peony 'Fengdanbai' were used as cuttings and grafted scions,and Paeonia lactiflora root as rootstock were used to observe the external morphology and anatomical structure during the rooting.The results showed that the rooting parts were all phloem,and there did't have the potential root primordia,and adventitious roots began to form from the formation layer,only differed in the timing of cuttings and grafted scions.The induction period of root primordium was 0-12 d,12-20 d was the germination of root primordium,and 2030 d was the period of expression and elongation of adventitious roots of cuttings.The induction period of root primordium was 0-20 d,the germination period of root primordia was 20-40 d,and the expression and elongation period of adventitious root were 40-60 d of grafted scions.2.Indicators measurement of physiological and biochemicalThe content of soluble sugar and protein,endogenous hormone(IAA,ABA,GA3 and ZT),and the activities of indoleacetate oxidase(IAAO),peroxidase(POD),polyphenol oxidase(PPO),and change of mineral elements(N,P,K,B,Cu and Zn)were measured and analyzed.The results as follows:(1)Changes of soluble sugar and protein:Soluble sugar content has been falling before the adventitious rooting of cuttings breaking through the epidermis,which indicated that cuttings consumed their own nutrients supplying energy for induction and germination of root primordium.The content of sugar increased after the adventitious roots breaking through the epidermis,which showed that the formation of adventitious roots enchancing the the capacity of absorption of roots,and the cuttings differentiating into a whole plant and the energy beginning to accumulation.The changes of soluble sugar content throughtout the rooting period indicated that it was closely related to the occurrence of adventitious roots.The content of soluble protein of grafted scions decreased during root induction and germination,and increased after adventitious roots breaking through the epidermis.The results showed that soluble protein had a certain promoting effect on the occurrence of adventitious roots,and the overall change was not as significant as soluble sugar.It indicated that soluble protein had a certain relationship with the occurrence of adventitious roots and it was not significant effect than of soluble sugar.Soluble sugar content of grafted scions has been decreasing during adventitious roots.It showed the occurrence of adventitious roots required the supply of energy and closely related to the occurrence of adventitious roots.The change of soluble protein content during the induction of root primordium was not significantly,and increased during the germination,which reflected the survival status of grafted seedlings and indicated strong physiological activities of grafted seedlings,and it also indicated that there was not directly relation between soluble protein and the occurrence of adventitious roots.The content of soluble sugar of rootstocks decreased significantly during rooting,which showed that rootstocks provided its own energy and promoted the occurrence of adventitious roots during rooting.The content of soluble protein did't change significantly during rooting,and it could be inferred that it was not significant effect on adventitious roots.(2)Changes of endogenous hormones:IAA of cuttings decreased during the induction of the root primordium,which was not benefical to the induction of root primordium.It increased in the process of the germination of the root primordium,which was good to the germination of root primordium.ABA of cuttings increased during root primordium induction,which inhibited the occurrence of root primordium,and present a trading off and taking turns with IAA.GA3 of cuttings increased in the process of induction of root primordium and formation of adventitious roots,which was conducive to rooting.ZT of cuttings has been on the rise and had the promoting effect on rooting.IAA of grafted scions increased during the induction and germination of root primordium,which indicated that it had a positive correlation with the occurrence of adventitious roots.ABA of grafted scions increased first and then decreased during rooting,indicating that it inhibited the occurrence of adventitious roots.GA3 content of grafted scions was on the decreasing,which indicated that it inhibited the healing between scions and rootstocks and was unfavorable for the occurrence of adventitious roots.ZT of grafted scions increased in the induction process of root primordium and promoted root primordium induction.In short,IAA and ZT played an active role in the different stages of the occurrence of adventitious roots of cuttings and grafted scions.ABA inhibited rooting.GA3 was beneficial to rooting of cuttings and inhibited the rooting of grafted scions.Change trends of IAA/ABA and IAA/ZT during the rooting of cuttings and grafted showed that they were negatively correlation,but they promoted the formation of adventitious roots,and the change of ratio of IAA/ABA was a greater impact on the occurrence of adventitious roots.(3)Changes of enzyme activities:IAAO activity in cuttings showed a high level throughout the rooting stage and kept going up during the induction and formation of adventitious root primordia,which showed that high level of IAAO activities was not conducive to rooting induction and cuttings of tree peony were more difficult to root.The activity of IAAO in grafted scions increased during the induction of root primordium,which was unfavorable to root primordium induction.It decreased in the process of root primordium germination and was beneficial to the germination of root primordium.IAAO of grafted scions increased overall and was beneficial to adventitious roots.The change tendency of POD of grafted scions was similar to that of cuttings,which decreased first and then rose during root primordium induction,and decreased during root germination and adventitious roots elongation,and increased gradually during the late root induction and adventitious root expression,it showed that POD had a negative correlation with the occurrence of adventitious roots.POD of rootstocks had intense changes,and showed an upward trend,which played a positive role in the occurrence of adventitious roots.PPO of cuttings increased during root primordium induction and was beneficial to root primordium induction.It decreased during the formation of root primordium and promoted the occurrence of adventitious roots,indicating that PPO was positively correlation with the occurrence of adventitious roots.PPO of grafted scions was similar to that of cuttings,and it also showed positive correlation with the occurrence of adventitious roots.PPO of rootstocks has been rising before the end of root primordium induction,and decreased after adventitious roots breaking through the epidermis,and increased again during the elongation of adventitious roots,which promoted the occurrence of adventitious roots.The results above indicated that three antioxidant enzymes had a certain relationship with the occurrence of adventitious roots,but the level of correlation was different on different enzyme.(4)Changes of mineral elements:Changes of total nitrogen of cuttings,grafted scions and rootstocks of tree peony during rooting were about the same and the overall trendency was on the rise,indicating that it was beneficial to the adventitious roots.P and K elements increased during root primordium induction,they participated in a series of metabolic activities of cuttings during rooting,and they promoted the induction of root primordium.Cu element of cuttings increased during germination of root primordium and expression,which promoted the germination of root primordium and the elongation of adventitious roots.B element of cuttings had a positive effect on the induction of root primordium.Zn element played an active role in root primordium induction,germination and adventitious roots expression,showing that it was beneficial to the occurrence of adventitious roots.P element of grafted scions has been on the rise during adventitious rootings and increased significantly during induction and germination of root primordium,indicating that it conducive to theoccurrence of adventitious roots.K element increased overall during rooting and had a positive effect on the induction of root primordium and promoted the germination of root primordium.B element increased first and then decreased during root primordium induction,and increased during root primordium germination,showing that there was not obvious effect on induction and could promote the germination of root primordium.Zn element increased in the process of root primordium induction,it rose first and then declined during root primordium germination,which showed that it was not significant effect on the induction and germination of root primordium.Cu element increased during the induction and germination of root primordium,indicating that it was beneficial to rooting.K and P elements of rootstocks increased during the induction and germination of root primordium,and significantly during induction rooting,indicating that they played a positive role in the root primordium induction,and had a certain impact on the germination of root primordium.Zn element was not significantly changes during induction and germination of root primordium.Cu increased during root induction and decreased during root germination,B element elevated steadily during the induction and germination of root primordium,which indicating that Zn element had no obvious effect on the occurrence of adventitious roots,Cu element had a certain promoting effect on the induction of root primordium,and B element had a positive effect on the occurrence of adventitious roots.It could be seen from the above that total nitrogen and K elements all were beneficial to rooting of cuttings and grafted scions.3.Transcriptome analysisTranscriptome and sequencing analysis of the root cortex during the not germination(MDZC),germination(MDMF)and swelling(MDPD)of root prioridum of tree peony seedlings were performed,and 73613916,71441408,and 65814178 high quality reads of different developmental stages were identified respectively.8232 differentially expressed genes were identified during MDMF/MDZC,6,906 during MDPD/MDZC,and 10,686 during MDPD/MDZC.By performing GO functional annotation to differentially expressed genes and comparison analysis for the KEGG database,it was found that they mainly perform the functions of carbohydrate metabolism,transcriptome regulation,defense responses,cell membrane organic composition,nuclear components,intracellular components,adenosine triphosphate binding agents and DNA binding agents et al during rooting of tree peony seedlings other functions,and participated in mainly metabolic pathways involving in plant-pathogen interaction,RNA transport,ribosome,endoplasmic reticulum protein processing,plant signal transduction and et al.4.qT-PCR for related rooting genesFive genes of Unigene13430_All,Unigene14234_All,CL14445.Contig4_All L10096.Contig1_All and CL14666.Contig2_All were screened out from auxin pathwany from by analyzing the results of transcriptome sequencing,and detected them by qT-PCR to find the changes of expression during rooting of cuttings and grafted scions of tree peony.The results showed that the expression of gene Unigene 13430_All was the strongest in cuttings,and down-regulated expression in seedlings,and it was the least in grafted scions.The increase rate of seedlings and cutting seedlings was larger than that of grafted seedlings.The expression of CL14666.Contig2_All all increased during different rooting of the three propagation methods,the growth rate of seedlings and cuttings was larger than that of grafted scions.The expression of Unigene14234_All varied at different stages of different reproductive modes,up-regulated expression in cuttings,and down-regulated in grafted scions,and down-regulated first and then up-regulated in seedlings.The expression of CL14445.Contig4_All did not change significantly in grafted scions,it up-regulated expression in seedlings and cuttings,and was most abundant in seedlings.The expression of CL10096.Contig1_All in three propagation modes was All up-regulated,and the expression abundance of seedlings had the most change.Through the analysis of the expression levels of these five genes,combining with the possible attributations of the GO functional annotations on these five genes,the genes of CL10096.Contig1_All.CL14666.Contig2_All?and Unigene13430_All are possible associating with adventitious roots.CL10096.Contig1_All and CL14666.Contig2_All may be related with the formation of adventitious roots of tree peony,Unigene13430_All may affect the rooting of auxin during the adventitious root formation.We confirmed that Unigene13430_All and CL10096.Contig1_All are closely associated with rooting by verifying transgenosis into Arabidopsis transgenic.In onclusion,critical rooting period of cuttings and grafted scions of tree peony were determined by the obversation of the external morphology and anatomical structure in this study,and it was found that it was difficult to rooting of tree peony,especially cuttings.There were many relevant factors affecting the rooting of tree peony by analysis of relevant physiological indicators of cuttings and grafted scions of tree peony,for example the carbohydrate content,IAA content,PPO and POD activities was the most important factors,and the mineral elements N,P,K,B,Zn and Cu elements had affected the induction of root primordium and germination of adventitious roots.Transcriptome analyse of seedling phloem of different rooting stages further demonstrated that it was a complex process controlled by various factors such as endogenous substances and exogenous environment.Five genes related to possible rooting were screened on the auxin pathway and qR-PCR was performed.It was proved that they were associated with adventitious roots of tree peony.It was verified that Unigene13430_All and CL10096.Contig1_All were related to adventitious roots.The results laid the foundation for the further determination of the influencing factors associated with peony rooting. |
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