Plant material, growth and grafting conditions
Two independent tomato transgenic lines, SP5 and SP12, in the genetic background of the wild-type (WT) cultivar Ailsa Craig (AC) (Thompsonet al. 2007b) were used in this study as rootstocks of the commercial cherry variety Sugar Drop (SD, Unigenia Semillas, Murcia, Spain). SP5 and SP12 transgenic rootstocks constitutively overexpress the SlNCED1 gene (Thompson et al. 2000), under the control of the Gelvin superpromoter (SP) and contain elevated ABA levels compared to WT, with SP5 accumulating more ABA than SP12 (Thompsonet al. 2007a b). Since germination rates differed between genotypes, different sowing dates were used to synchronise development of the three genotypes: SP12 and SP5 seeds were sown one and two weeks before the WT, respectively, as described previously (Martínez-Andújaret al. 2020b). Seeds of the scion SD were sown 5 days earlier than AC seeds (12 days earlier than SP12 and 19 days earlier than SP5) to ensure equal stem diameters at grafting. For all genotypes, seeds were sown in commercial vermiculite, watered with deionized water and kept at 26-28ºC and 80-90% relative humidity in the dark until germination. Grafting was performed using the splicing method at the two to three true leaf stages (3–4 weeks after sowing) where the scion was attached at the first node of the rootstock (Savvas et al. 2011). Grafting with the two transformants and the WT AC resulted in the following three graft combinations: SD/SP5, SD/SP12 and SD/AC.
One month later, when the grafted plants were well established, they were cultivated under commercial-like conventional plastic greenhouse conditions using a sand substrate during an autumn-winter season, in Almería area (Spain). Fertilizers and water were supplied by a drip fertigation. From 10 days after transplanting, a low salinity treatment with an electroconductivity (EC) of 3.5 dS m-1 was applied for a period of 200 days. Six plants per graft combination were randomly cultivated and distributed in blocks. After 130 days of salt treatment (DST), the second fully expanded mature leaf over the fourth truss (with actively growing fruits) of 6 plants per graft combination was assayed for various physiological parameters (described below), then detached to weigh and determine leaf area using an LI-3100AC area meter (LI-Cor, Lincoln, NE, USA). Plant stem diameter was also measured at the second node level using an electronic LCD digital vernier caliper (0-150 mm).
At the end of the experiment (200 DST), the shoot and root were detached and weighed to determine biomass. Young fully expanded leaves and young roots were immediately frozen in liquid nitrogen and stored at -80°C for hormonal and gene expression analysis.