Molecular And Physiological Mechanisms Of Grafting Compatibility Mechanisms In Different Polyploid Watermelon(Citrullus Lanatus)

Watermelon(Citrullus lanatus)is an important horticultural crop.Polyploid watermelon produces few seeds(or seedless)than diploid,and has high yield,high sugar content,long shelf-life and better transportability,high resistance to disease and stress.High-quality traits have been developed rapidly all over the world.As the female parent for producing triploid watermelon,tetraploid watermelon has a low seed number,low germination rate,and high breeding cost.Triploid seedless watermelon has a low germination rate,a low seedling rate during grafting and production processes.The problem of low survival rate is particularly important to find a more economical and effective way to improve the quality of triploid and tetraploid watermelon seeds and seedlings.Grafting is a common method of asexual reproduction in cucurbitaceous crops,especially widely used in watermelons,but not all grafting methods are suitable for seeded and seedless watermelons.The grafting compatibility mechanism of different ploidy watermelon seedlings is still unclear.To study the graft compatibility mechanism of different ploidy watermelon seedlings,we selected the three common grafting methods(splice,hole,and tongue)for polyploid watermelon and investigated their survival rate,physiological and biochemical changes,cell structure differences,and gene expression changes.Further,by comparing the compatibility mechanism of different ploidy watermelon grafted by branches,it provided a new technical and theoretical support for watermelon grafting and breeding.The main research results are as follows.There are significant differences in grafting efficiency and survival rate between different ploidy watermelon seedlings when grafted using hole and splice methods.In the diploid,the hole grafting method is more efficient and gave a higher survival rate.In contrast,in the tetraploid,the splice grafting method has a higher grafting efficiency and higher survival rate.The triploid seedless watermelon showed high grafting efficiency and grafting survival rate when grafted using splice and hole methods.No significant differences were found in different ploidy watermelon seedlings by tongue grafting.By comparing the differences in physiological and biochemical changes of grafting union of different ploidy watermelon seedlings,we found that the hole method showed a high survival rate and high content for hormones(IAA and ZR),sucrose and starch,and AOX activities in diploid than tetraploid.Also,the increment rates were higher during the grafting process,quite the opposite when the splice method was used with a tetraploid.Through observing the changes in the structure of the healing graft union through tissue sections,it was found that the adhesion area of the tetraploid grafted seedlings using the splice method was longer than that of the hole method.And since tetraploid hypocotyls are flat(not round),the hole method is not suitable for grafting.In the diploid,the microstructures showed that the adhesion area in the hole method is bigger and longer than the splice;this may explain the higher survival rate with the hole than the splice method.Likewise,the splice's adhesion area was bigger than the hole method with triploid,explaining the splice's increased survival rate more than the hole.Comparing the transcriptome levels of the graft union 0,3,and 15 days after grafting between diploid and tetraploid watermelon seedlings grafted by the splice method,GO analysis found that these genes are mainly involved in biological processes,cellular components,and molecular function.KEGG results showed that the increase of chromosome ploidy significantly affected the expression of related genes in the IAA and ZR signal transduction pathways and AOX biosynthesis pathway in the grafted seedlings and then regulated the hormone levels signal pathways,which promoted plant survival.Moreover,by using the RNA-Seq data set,we performed weighted gene co-expression analysis(WGCNA)and identified hub/candidate genes related to hormones,AOX,and carbohydrates biosynthesis.Three different kinds of branches of diploid,triploid,and tetraploid plants are used as scions,including apical meristem(AM),branch with one node(1N),and two nodes(2N),grafted on the squash rootstock,and the survival rate of different ploidy watermelon branch grafting was compared.The results showed a higher survival rate when AM was used as scion followed by 2N in diploid,triploid,and tetraploid.While using 1N as a scion showed significant differences between polypoid watermelon,tetraploid gave higher survival rate,higher contents of hormones,carbohydrates,and AOX activities than diploid,which may be the possible reasons for high compatibility in tetraploid and degrading the grafting zone in diploid.RT-q PCR results confirm that the expression of genes linked to compatibility is consistent with the carbohydrates,hormonal and AOX activities.This study provides an alternative and economical approach to producing more tetraploid and triploid plants for breeding and seed production by using branches as scions.Furthermore,it provides insights into more understanding of graft compatibility mechanism underlying different polyploidy watermelon scions with grafting methods.