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.