Tissue culture, characterization and evaluation of in vitro salt tolerance in Arizona 8601 --E. Lupotto, M. C. Lusardi and F. Locatelli
It is still rather controversial whether through tissue culture selection in the presence of salt, it is possible to regenerate individuals effectively salt tolerant in the environment, when grown as normal plants in the field. Although it is quite easy to select and isolate salt tolerant cell cultures in vitro and characterize them as effectively stable and tolerant, it is, however, not fully clear if there are common parameters which may be indicative of the selected material in vitro in respect to the behaviour in planta. A study performed on several Medicago species showed indeed that the most salt tolerant species, M. marina, capable of growing in salty environments, displays in vitro the most sensitive behaviour (McCoy, Plant Cell Rep. 6:31-34, 1987).

During our work on the isolation and characterization of salt tolerant embryogenic cultures of maize (Lupotto et al., MNL 62:30-31, 1988; MNL 63:31, 1989), the identification of parameters indicative of salt tolerance, both in vitro and in planta, was considered essential for the development of the work. Therefore, tissue culture studies on the behaviour of a maize germplasm derived for growth in salty soils was undertaken. Arizona 8601 (Day, Crop Sci. 27:1096, 1987) released by the Arizona Agricultural Experiment Station, Tucson, USA, can be grown in soils containing up to 6000-7000 ppm TDS (total soluble salts) and irrigated with saline water (2000-4000 ppm TDS): it represented a useful material for comparisons against salt-selected and salt-sensitive maize. Arizona 8601 can be established as embryogenic callus cultures of type 1 in vitro (Fig. 1a, b). By the use of N6I medium (Lupotto and Lusardi, Maydica 33:163-177, 1988) a callus induction frequency of about 70% of the explanted immature embryos (10-11 DAP) was obtained. Embryogenic callus cultures could easily be established and propagated on N6P medium with high efficiency (91.3% on the derived embryogenic cell lines). Growth of the callus cultures was differentially affected by the presence of 86 mM NaCl in N67P medium, a level representing the LD50 at which our STSC (salt tolerant somaclones) were originally derived from the salt sensitive material. Ten different cell lines were tested for their response to the presence of 86 mM NaCl in the medium, and the alterations recorded as loss/gain % in fresh weight after a 21 day subculture: this varied between -35 and +27% of the control on salt devoid medium. Comparisons were made with other salt sensitive genotypes (Fig. 2). Regenerative capability, expressed as total regenerated plantlets from 1 g fresh weight tissue, was unaffected by the presence of salt (18±6.5). The sensitivity of Arizona 8601 to increasing amounts of NaCl in the medium was tested in comparison to salt sensitive genotypes (W64A and A188) and salt selected clones (STSC10, 20 and 21), capable of growth on 129 mM NaCl. The LD50 for Arizona 8601 cell lines ranged between 171 and 250 mM NaCl comparable to STSC10 (158 mM), STSC20 and STSC21 (200 mM NaCl), whereas LD50 was around 86 mM for the salt sensitive material used for the subsequent isolation of the three STSCs.

Figure 1. Embryogenic callus cultures of Arizona 8601. a) Propagation on N6P medium, growth of the tissues at the end of a 21 day subculture; b) early stages of somatic embryos at the callus surface (48 X).

Figure 2. Behaviour of Arizona 8601 cell lines on 86 mM NaCl. Growth expressed as % of the control grown on salt devoid medium. Comparison is made with other maize salt sensitive genotypes.

It was also interesting to investigate whether the specific polypeptides detected in the STSCs grown in the presence of NaCl in the medium were also expressed in Arizona 8601. Callus cultures of Arizona 8601 were characterized by a protein pattern, analyzed through SDS-polyacrylamide gel electrophoresis, very similar to the pattern detected in the STSCs (strong increase of bands at 24-26 kDa), different from the pattern of salt sensitive material. The presence of augmented amounts of NaCl in the medium led to an overproduction of the b26 band with a corresponding decrease of a group of protein bands between 20 and 17 kDa. The same pattern was observed for the three STSCs considered. Interestingly, the permanence of Arizona 8601 on salt led to a gradual regression of the b26 polypeptide to the normal level, after 4 month subculture in vitro, whilst the STSCs retained this overproduction. This behaviour might be indicative of a different pathway of regulation of Arizona 8601 in respect to the STSCs in response to the salt stress.

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