--E. Lupotto, M. C. Lusardi, D. Bartels* and M. Mongodi

*Max-Planck Institut, Cologne

The in vitro selection scheme followed for the isolation and regeneration of salt tolerant somaclones (STSC) has been previously reported (Lupotto et al., MNL 62:30, 1988). At the end of the selection, 18 embryogenic calli, perfectly regenerable and tolerant to 85mM NaCl in the culture medium, were obtained. A total of 142 regenerates were produced, 93 (65.5%) on MS (Murashige and Skoog, 1962) hormone-free medium without NaCl, and 49 (34.5%) on salt-containing medium. Plantlets developed in both cases and were transplanted to soil; 78 of them (54.9%) were grown to maturity in the greenhouse. Of 27 flowering plants, complete with tassels and ears, only four set seeds, three by selfing and one by crossing with pollen from anther regenerates. About fifty percent of the regenerates were plants which only developed ears and 29 could be outcrossed with pollen from control plants: four of them set seeds. Five regenerates only developed tassels which were sterile and seven plants showed evident phenotypic abnormalities and failed to develop. In Table 1 the breeding procedures followed for obtaining progenies of the regenerated plants which set seeds are reported. R2 populations are consistent for in planta analyses and comparisons with nonselected progenies, in order to evaluate if any trait conferring salt tolerance has been transmitted to the progenies of the regenerates.

Table 1. Salt tolerant regenerates-situation.

Some experiments have been developed in vitro for testing the nature of the trait of salt tolerance acquired by some STSC. A curve of sensitivity to NaCl drawn on non-selected embryogenic somaclone no. 10 (SC10) and the correspondent STSC10 tolerant to 85mM NaCl, showed that the STSC could easily tolerate higher doses of salt, the relative growth being reduced to 50% of the control at 230mM for STSC10 against 90mM NaCl for SC10. Furthermore, the effective embryogenesis of the callus culture resulting in plant regeneration in STSC10 disappeared around 220mM, while it was at 85mM in the case of SC10. As reported before, some STSC tolerant to 85mM NaCl also resulted in derivatives spontaneously tolerant to higher dosages of NaCl, and three clones, STSC10-II, STSC20-II and STSC21-II, are currently maintained on 128mM NaCl; their embryogeny and capability of plant regeneration remained intact. A test of stability of the character in vitro was performed by recording the growth curve as GI (growth index: increment in fresh weight during subculture with respect to the initial fresh weight) of STSC20-II and STSC21-II in the absence of salt, in the presence of 128mM NaCl, and in the presence of salt after a period of three months on 0mM NaCl. This was done for evaluating the possible maintenance of the trait of salt tolerance also after a rather long period of growth in absence of NaCl. Indeed, STSC20-II and STSC21-II grew better on NaCl-devoid medium, suggesting their independence from NaCl in the culture medium; in addition the curve relative to their growth on 128mM NaCl and on salt after a period on non-salt medium was practically identical, confirming the stability in vitro of the acquired trait. In each case calli were embryogenic and regenerable. However, in STSC10 and STSC10-II cultures grown respectively on 85 and 128mM NaCl, dependence on NaCl supplement in the medium was recorded when drawing the curves of sensitivity to a range of NaCl dosages. This might suggest, in the case of STSC10, an in vitro halophytic behaviour.

The appearance of specific proteins typically expressed by salt tolerant cell lines (e.g. Singh et al., Plant Physiol. 79:126, 1985) has been investigated on some STSC grown on 128mM NaCl by one- and two-dimensional gel electrophoresis analysis. While the appearance and disappearance of specific protein bands were difficult to interpret with one-dimensional SDS polyacrylamide gel electrophoresis, though revealing several evident differences between each SC and the corresponding STSC, a better analysis could be performed by 2D gel electrophoresis according to O'Farrell (J. Biol. Chem. 250:4007, 1975). Indeed 5 STSC stably growing on 128mM NaCl expressed four different peptides of deduced molecular weight respectively of 24, 26, 31 and 36.5kDa. These peptides were clearly detected and common to the STSC 20, 21, 16, 41 and 3 and absent in the non-selected counterparts. Peptides of 24-26kDa are clearly expressed in salt tolerant suspension cultures of Nicotiana tabacum and in the plant roots (King et al., Plant Mol.Biol. 7:441, 1986). Current investigations are focused on the analysis of the expression of these peptides and their role in salt tolerance; furthermore, studies on the detection of their eventual presence in the regenerated plants are also underway.

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