Grafting Compatibility Of Arabidopsis/Nicotina Benthamiana Hetero-grafting System

Grafting technique plays an important role in agricultural production and horticultural research.It is also a key point to explore the long-distance signal of biological macromolecules between scions and rootstocks.In recent years,grafting technique is widely used in the same species of different genotypes or the different species with close relatives.However,homo-grafting with a similar genetic background can not clearly analyze the movement of macromolecules between scions and rootstocks,and hetero-grafting is easy to form incompatibility,which eventually leads to grafting failure.Therefore,how successfully construct hetero-grafts is critical for grafting technology.The successful construction of distant grafts can more clearly explain the mechanism of vascular re-connection and the movement of macromolecules.In this study,At was used as scion grafted onto Nb rootstock.Through the graft phenotype,paraffin section of the graft union,the microscope of vascular bundle reconnection,SEM of tracheary elements between scions and rootstocks,small RNA sequencing,RT-q PCR,RT-PCR,and subcellular localization,the mechanism of At / Nb hetero-grafting and the movement of small RNA molecules between scions and rootstocks were revealed.The main results are as follows:1.Using micrografting technology to graft At onto Nb rootstock and observe the phenotypic growth,it is found that the grafts mainly form three types: the mild-stressed,the chlorotic and the retarded grafts.The first two are grafted compatible plants,and the last is grafted incompatible plants.It shows that hetero-grafts can be constructed successfully,but the mechanism of forming compatibility and incompatibility at the same time is not clear.Further through paraffin section and staining,it was found that the xylem of graft incompatibility did not complete the reconnection,but grew laterally to form a spiral structure.Subsequently,At-35S-GFP and At-Suc2-GFP were grafted with Nb rootstock respectively.Observing the graft union of longitudinal hand-cut sections found that the green fluorescence signal of compatible plants could be detected in rootstocks,while the green fluorescence signal of incompatible plants accumulated in the spiral structure of scions.It is suggested that the formation of spiral structure between scions and rootstocks is the cause of graft incompatibility and protein can move across the graft union,and protein can move across the graft union in the compatibility graft.2.Loading the grafts at different stages with CFDA and acid fuchsin,it was found that the grafts completed the connection of phloem at 5 DAG(day after grafting).Xylem reconnection began at 5 DAG and completed the reconnection of the xylem at 7 DAG.At30 DAG,the CF signal of graft incompatibility and acid fuchsin accumulated on the contact surface of graft union,which further showed that the vascular bundle was not reconnected,resulting in graft failure.Then,the tracheary element of grafts was observed by SEM.It was first found that grafting healing needs three stages: during the stage I,the vascular strands at the grafting interface undergo segmentation and deformation to fill the gap between the scion and rootstock.During the stage II,the graft union is covered with membrane-like cellular deposits.Finally,the homogeneous tracheary element between scions and rootstocks overlap each other and grow vertically to complete the connection to form an affinity graft,but the non-homogeneous tracheary element repels each other and grow laterally to form a spiral structure,resulting in graft failure.These results show that the formation of spiral structure between scions and rootstocks is the cause of graft incompatibility.It provides a theoretical basis for the study of the mechanism of distant grafting.3.At and Nb have different genomes.The successful construction of distant grafts is conducive to the analysis of small RNA molecular mobility between scions and rootstocks.Small RNA sequencing was At scion and Nb rootstock respectively.The scion was compared with the Nb genome,and the rootstock was compared with At genome.Identification 82 mobility At mi RNAs move to the rootstock,and 6 mobilities Nb mi RNA move to the scions,they were Nb-mi R395-1,Nb-mi R395-2,Nb-mi R397 v,Nb-mi R156 v,and Nb-mi R164 v.Selection of seven At mi RNAs and six Nb mi RNAs to conform the mobility using RT-q PCR.It was found that all the expression levels were very low.In this study,mainly verified the upward moving mi RNAs molecules.Mi R156 sequencings can move in two directions.The remaining five mi RNAs molecules were overexpressed and observed the phenotypes of overexpressed plants.It was found that Nb-mi R395-1,Nbmi R395-2,Nb-mi R397 v,and Nb-mi R1446 v overexpressed plants had no phenotypic changes.They were used as rootstock grafted with wild-type At scions,they were no phenotypic changes in the At scions.But the leaves of Nb-mi R164 v overexpression plants will form defects,cup-shaped or heart-shaped.Phenotypic plants are selected as rootstocks to be grafted with wild-type At scions.At 45 DAG,one of the leaves of the scion changes phenotype,forming a similar cup-shaped structure and concave structure.It shows that Nb-mi R164 v can move upward and cause a change in the scion phenotype.Through RT-q PCR,RT-PCR,and subcellular localization detection to detect the mobility of Nb-mi R395-1,Nb-mi R395-2,and Nb-mi R164 v,it was found that their mature mi RNAs molecules and some precursor sequences containing mature mi RNAs molecules could move.In this study,construction of At / Nb distant graft was found two types of graft(graft compatibility and incompatibility).The paraffin section and SEM found that the graft incompatibility was caused by the failure of the xylem connection and the formation of a spiral structure.At the same time,SEM found that the healing process between scions and rootstocks has three stages during the formation of grafts.During the stage I,the vascular strands at the grafting interface undergo segmentation and deformation to fill the gap between the scion and rootstock.During the stage II,the graft union is covered with membrane-like cellular deposits.Finally,the homogeneous tracheary element between scions and rootstocks overlap each other and grow vertically to complete the connection to form an affinity graft,but the non-homogeneous tracheary element repels each other and grow laterally to form a spiral structure,resulting in graft failure.It provides a new vision for graft failure caused by distant graft incompatibility.In addition,combined with small RNA sequencing technology,the upward moving Nb mi RNAs molecules were obtained through sequencing and verification,in which Nb-mi R164 v can cause phenotypic changes,which laid a theoretical foundation for further study on the movement pathway of upward-moving mi RNAs molecules and the causes of phenotypic changes.