STUDY OF GRAPEVINE ROOTSTOCK RESPONSE TO WATER STRESS

More than 80% of vineyards around the world use grafted plants: a scion of Vitis vinifera grafted onto a rootstock of single or interspecific hybrids of American Vitis species, resistant or partially resistant to Phylloxera (Daktulosphaira vitifoliae (Fitch, 1856)). The genetic variability of grapevine rootstocks plays a fundamental role in their adaptation to the environment (Serra et al., 2013). In the climate change scenario, predicting an increase of aridity in the near future (Dai, 2013), the more frequent and severe drought events may represent the major constrain for the future of viticulture (IPCC, 2018; Schultz, 2000). Therefore, the selection of new rootstocks able to cope with unfavourable environmental condition is a key asset, as well as a strategy to improve crop yield/vegetative growth balance on scion behaviour (Corso and Bonghi, 2014). So far, the influence of rootstock on scion physiological performance during water stress has always aroused great interest. On the contrary, the scion impact on rootstock response is still less debated. Therefore, the effect of grafting on rootstock behaviour have been investigated. Phenotypical and large-scale whole transcriptome analyses on two genotypes, a drought-susceptible (101-14) and a drought-tolerant (1103 P), own-rooted and grafted with Cabernet Sauvignon, subjected to a gradual water shortage in semi-controlled environmental conditions have been performed. The ungrafted condition affected photosynthesis and transpiration, meaning the decisive role of scion in modulation of gas exchanges and in general in plant adaptation. Molecular evidence highlighted that the scion delays the stimulus perception and rootstock reactivity to drought. Since 1985, the DiSAA research group operating at the University of Milan is carrying on a rootstock crossbreeding program which has led to the release of four genotypes: M1, M2, M3 and M4. They show from moderate to high tolerance to drought (M4 > M1 = M3 > M2). In order to characterize their performance during water stress, their physiological (gas exchanges and stem water potential) and transcriptome response (genes involved in ABA-synthesis and ABA-mediated responses to drought) under well-watered and water stress conditions were examined. The behaviour of M-rootstocks (M1, M2 and M3) was compared with that of other commercial genotypes largely used in viticulture, either tolerant (140 Ru, 41 B, 110 R, 1103 P), less tolerant (SO 4, K 5BB) and susceptible (420 A and Schwarzman). Discriminant analysis (DA) showed that when water availability starts to decrease, rootstocks firstly perceives the stress activating a transcriptome response, consequently physiological changes have been observed. It also demonstrated that the three M-rootstocks were clearly discriminated: M4 was grouped with the most tolerant genotypes while M3 with the less tolerant or susceptible ones from a physiological standpoint, confirming their different attitude to tolerate water stress. M4 has proven to be a promising rootstock due to its ability to adapt to drought conditions. Considering the constant great demand for vine planting materials, the obtainment of genetically homogeneous populations (i.e. clones) from elite individuals through micropropagation represents a rapid alternative to conventional multiplication. For this reason, an efficient high-throughput protocol for M4 in vitro propagation was set up. Its attitude to shooting, root development and callus proliferation was compared to that of other rootstocks largely used in viticulture (K5BB, 1103P, 101-14 and 3309C). Moreover, pro-embryogenic and embryogenic callus from bud explants were also produced, representing a cellular material manipulable with the genetic engineering techniques. In water scarcity condition, among the mechanisms activated by M4, the great ability to scavenge ROS, related to the increased accumulation of stilbenes and flavonoids, may be such as to give it tolerance to the stress. In particular, the higher levels of trans-resveratrol were correlated with the up-regulation of some stilbene synthase genes, mainly VvSTS16, VvSTS18, VvSTS27 and VvSTS29. The over expression of these genes was linked to a structural variation in their promoter region. To confirm that VvSTSs genes may be considered putative factors of M4 better adaptation to water stress, a genome editing protocol based on the CRISPR/Cas9 system, aimed at knock-out the genes, was performed. For testing the gRNAs functionality, a transient assay on in vitro micropropagated plantlets of M4 and 101-14 was performed. The positive results obtained by this experiment will lead to the transformation of somatic embryos and regeneration of whole-edited plants using the vectors developed.