Techniques to study graft union formation in grapevine

Abstract

Context and purpose of the study – Grapevines are grown grafted in most viticultural regions. Grapevine rootstocks are either hybrids or pure species of different American Vitis spp. (particularly V. berlandieri, V. rupestris and V. riparia), which were primarily used to provide root resistance to the insect pest Phylloxera. In addition to Phylloxera resistance, grapevine rootstocks were also selected in relation their resistance to various abiotic stress conditions. Future rootstocks should have the potential to adapt viticulture to climate change without changing the characteristics of the harvested product. However, high grafting success rates are an essential prerequisite to be able to use them with all the varieties. The objective of this work is to develop quantitative techniques to characterize graft union formation in grapevine.

Material and methods – The development of grafts of different scion/rootstocks of grapevine was studied during the first few months after grafting. The quantity of callus produced (fresh and dry mass) and the mechanical strength of the graft union were quantified in five scion/rootstock combinations 5-6 weeks after grafting. The scion/rootstock combinations studied were homografts of the typical grapevine scion genotype, Vitis vinifera cv. Pinot Noir (PN), and homografts of two rootstocks, V. riparia cv. Gloire de Montpellier (RGM) and the V. berlandieri x V. rupestris cv. 140 Ruggeri, and two hetero-grafts, PN/RGM and PN/140Ru (n=27). We also used x-ray tomography to study functional xylem vessels by labelling functional vessels with the contrast agent Iohexol. This protocol was optimised in three omega grafts of V. vinifera cv. Tempranillo grafted onto the rootstock V. berlandieri x V. rupestris cv. 110 Richter. Grafts with solid and resistant graft unions were selected after one year of growth in a nursery and grown in a greenhouse until approximately 10 leaves had appeared to drive the movement of iohexol in the xylem. Scans were analyzed with the computer programs Fiji/ImageJ and Imaris.

Results – Equipment to quantify the mechanical strength of the graft union was developed and tested on different scion/rootstock combinations to determine the suitability of this technique to quantify graft union development. The quantity of callus produced at the graft interface is different between the tested genotypes and was not necessarily related to the mechanical strength of the graft union. Three-dimensional reconstruction of x-ray tomography images allowed us to visualize the vessel connections between the scion and rootstock, and this knowledge will be used to develop protocols to quantify xylem vessel connections using high-throughput methods.                   

Significance of the study – Difficulties in quantitatively phenotyping the different steps of the graft union formation have considerably delayed the identification of the genetic determinants of grafting success in all the plant species. In this study, we are developing various quantitative methods to overcome this bottleneck with the objective to be able to characterize the genetic mechanisms involved in graft union development in grapevine.