In vitro leaf-shoot regeneration and somaclone selection for sodium chloride tolerance in quince and pear

Abstract

SummaryThe aims of this research were to set up in vitro procedures to obtain NaCl-tolerant somaclones of the quince rootstock ‘BA 29’ and pear cvs. ‘Conference’ and ‘Abbé Fetel’, and to evaluate the selection-pressure effect of NaCl in the regeneration medium. Shoot regeneration was induced in leaf-explants on media enriched with 5.4 µM -naphthalene acetic acid (NAA), 4.5 µM thidiazuron (TDZ) and variable concentrations of NaCl (0, 3 or 5 g l–1 for pear; 0, 5 or 10 g l–1 for quince). Regeneration was strongly negatively affected by the presence of salt in the culture media. It was inhibited by 5 g l–1 NaCl, and completely prevented by 10 g l–1 NaCl in ‘BA 29’. No regeneration was obtained from ‘Abbé Fetel’ leaf explants, even at 5 g l–1 NaCl. Moreover, the few ‘Abbé Fetel’ and ‘Conference’ shoots regenerated at 3 and 5 g l–1 NaCl, respectively, could not survive transplantation. All shoots derived from each adventitious shoot were referred to as a somaclone and labelled with two numbers, the first referring to the NaCl concentration and the second being a progressive number. All were sub-cultured repeatedly on a standard medium before testing their salt tolerance in vitro. Somaclones and controls (shoots obtained by conventional micropropagation) were compared for proliferation, growth and rooting on standard and NaCl-enriched media (5 and 10 g l–1 NaCl for proliferation; 5 and 7.5 g l–1 NaCl for rooting). Shoot proliferation rate (PR), shoot relative growth rate [RGR = (final weight – initial weight)/initial weight], and proportion (%) of rooting of somaclones and controls were significantly reduced by salt in ‘BA 29’ and in the two pear cultivars, although great variability was found between somaclones regenerated at each NaCl concentration. No shoot proliferation occurred at the highest NaCl concentration. Most somaclones of ‘BA 29’ had higher shoot PR and RGR than controls on standard medium, and the presence of 5 g l–1 NaCl in the regeneration (induction, expression) media increased proliferation and growth across all NaCl concentrations. Moreover, a few somaclones regenerated at 5 g l–1 NaCl showed better root development on salt-enriched media. The most promising ‘BA 29’ somaclone seemed to be 5-3, whose RGR remained high in up to10 g l–1 NaCl, and which showed a high proportion of rooting (75%) and root development at 5 g l–1 NaCl. Also notable for its slightly higher shoot PR, RGR and better rooting on 5 g l–1 NaCl-enriched media, was somaclone 5-4. Among the pears, most ‘Abbè Fetel’ somaclones (all from regeneration media lacking NaCl) grew and proliferated more than the controls across all NaCl concentrations. These differences were significant for somaclone 0–8. Some somaclones produced more and longer roots than controls on standard medium, while only a few very short roots were found in a few somaclones and none in the controls on NaCl-enriched media. Many ‘Conference’ somaclones had lower shoot PR than the controls up to 5 g l–1 NaCl, especially those regenerated in the absence of salt. Greater proliferation and growth were observed for somaclone 3–5, and better growth for somaclone 3–10 in the presence of salt. Moreover, some other somaclones regenerated in the presence of salt (i.e., 3-3, 3-5, 3-6 and, especially, 3–8) showed interesting rooting behaviour on NaCl-enriched media. Across all combinations, ‘Conference’ somaclone 3–5 seemed the most tolerant, with good proliferation, growth and rooting on NaCl-enriched media. Thus, the present results suggest that the regeneration-selection protocols described here can be useful for inducing somaclonal variation and NaCl-tolerance in pear and quince, and indicate a positive NaCl selection-pressure effect in obtaining in vitro NaCl-tolerant variants of ‘BA 29’ and of ‘Conference’ pear.