| Walnut(Juglans regia L.)is an important economic forest species.China’s walnut industry is facing the prominent problem of large-scale and commoditization process,and the improved asexual reproduction,especially the efficient bud grafting technology,is the effective way to solve this problem.Therefore,the grafting and breeding of walnut varieties has farreaching significance for improving the quality and efficiency of China’s walnut industry.In the practice of walnut bud grafting,people used physiology as a guide to try to change the traditional germination period and mode of fruit trees,which made the technology gain important progress.However,there are still problems in the economic forest tree species to survive and survive.Therefore,it is necessary to use walnut as a test material to analyze the mechanism and provide theoretical guidance for the improvement and improvement of the economical forest and even the germination and rapid propagation technology of horticultural crops.In this study,the walnut cultivar `Qingxiang’ was used as rootstock and `Xiangling’ as scion to carry out bud grafting experiment.Based on the analysis of the key external factors affecting the survival of bud grafting,the tissue and cell characteristic maps were established for the critical period of bud healing,and the genome transcription mechanism was analyzed.The key regulatory genes were revealed at the molecular level,and the walnut bud grafting was deeply understood from the core physiological process.The main results are as follows:(1)The developmental state of the rootstock and scion,the buding period and the integrity of the scion "Huyarou" are the main factors affecting the survival of the walnut bud grafting.Experiments on bud grafting of stock-scion combinations at different developmental stages showed that the combination of the central bud and the middle part of the stock in the semi-lignified state had the highest survival rate of 93.6%;The peak survival rate of bud grafting occurred in the grafting at 45 days and 105 days after germination.The survival rates were 94.5% and 66.7%,respectively,the difference was extremely significant(P<0.01);The irrigation on the 0th and 3rd days after the bud grafting will seriously inhibit the survival,andthe irrigation after the 6th and 9th days will have little effect on the grafting survival rate.Through the comparative observation of the external morphology,tissue sections and scanning electron microscopy,it was found that the "Huyarou" belonged to the mature tissue of the stem tip of the leaf bud,when taking the scion bud,the "Huyarou" should be well preserved,otherwise the grafting would not germinate and the grafting would not survive.(2)It overcomes the problem of uneven soft and hard structure and easy to fall off in various parts of the organization,and establishes a paraffin sectioning method suitable for heterogeneous tissues such as walnut.The specific process is: using the modified fixative(70%t-butanol: 35%-40% formaldehyde: propionic acid: glycerol = 85:5:5:5,v/v/v/v)to fix the walnut budding stem material;softened: hydrogen peroxide-glacial acetic acid mixed solution(1:1,v/v),treated for 3 hours,transferred to 70% t-butanol-glycerol mixed solution(1:1,v/v),50℃,treatment for 2 days;using t-butanol gradient dehydration method,due to the dual action of dehydration and transparency of t-butanol,the transparent step can be omitted;dip wax:replacing xylene with t-butanol The material is transferred to pure paraffin.(3)The healing mode of stock and scion induced by cambium cell is the best way to the survival of bud grafting,and a new healing mode induced by xylem ray cell is found.According to the key period of bud grafting found in previous studies,the histological study on the healing process of walnut grafts 40-47 days,70-77 days and 100-107 days after germination was carried out by using the established method of slicing walnut grafts.It was found that the healing modes were as follows: healing between cambium of rootstock and scion(mode 1),healing between stock xylem and scion cambium(mode 2),and healing between xylem of rootstock and scion(mode 3).Mode 3 is a new method for bud grafting healing of walnut.The callus is dedifferentiated from xylem ray cells,and the rootstock isolation layer is dissolved before the scion.The time required for the healing was 12-15 days,15-18 days and 25-30 days,respectively.The difference of healing modes led to the different time needed to complete the healing between stock and scion,and the shorter the healing time,it is more beneficial for the survival of grafting.There are three reasons for the failure of thebud graft: the proliferation of callus is inhibited;the callus fails to further differentiation;and the necrotic layer prevents the connection of rays between the stock and scion.(4)Based on histological studies to determine the key stage of bud graft healing for accurate samplingand transcriptome analysis.Through analyzing the differential gene expression of transcriptome during graft healing,2011 differential genes were screened out.The results of functional enrichment showed that these differential functions mainly involved catalytic activity,oxidoreductase activity,and secondary metabolic processes.Furthermore,the weighted co-expression network analysis method was used to identify that NCED1,AFP2,PIR18 and PYL8 genes involved in plant hormone homeostasis and signal transduction pathway,TBL38 gene involved in plant cell wall synthesis pathway,MYB44 gene acting as an important transcription factor gene and UPF3 gene involved in plant traumatic signal response are functional genes plays a pivotal role of regulation during the process of bud graft healing.(5)Based on the results of the weighted co-expression network of differentially expressed genes,the temporal and spatial distribution of endogenous hormones IAA,ABA and ZR during walnut bud healing and the dynamic changes of related enzyme activities were further studied.The dynamic changes of IAA and ZR were consistent.During the rapid proliferation of callus,the hormone content increased and accumulated in the interface cambium and the surface of the stock.After the callus entered the differentiated state,the content decreased rapidly and distributed in the callus.There was no significant change in the content and distribution of ABA in the rapid proliferation of callus.After the callus entered the differentiated state,its content increased sharply and concentrated in the scion cambium and callus.A spatiotemporal distribution model of endogenous hormones during budding healing was then proposed.The dynamic trends of IAA/ABA,IAA/ZR and ZR/ABA ratios are “S” type,“M” type and “N”type,respectively.The activity of PPO increased rapidly during the formation of callus;the activity of PAL was always on the rise;the antioxidant enzymes(SOD,POD and CAT)showed a trend of increased first and then decreased. |
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