Selection in vitro on NaCl containing medium seems a promising approach for selecting cell lines which tolerate salt in their nutritional environment. Despite the possibly complex nature of the salt tolerance expressed by plant cells, many examples are reported in literature, on the in vitro isolation of salt tolerant cell lines with eventual recovery of regenerates displaying acquired traits of tolerance at the whole plant level (C. P Meredith, in: J.P Gustafson (ed) Gene Manipulation in Plant Improvement. Plenum Press, New York, 1984; Croughan et al., Env. Expt. Bot. 21:317-324, 1981). Maize is classified among the cereals which are rather sensitive to the presence of salt. Exposure of maize calli to salt stress results in drastic changes in growth and protein synthesis (Ramagopal, Plant Cell Rep. 5:430-434, 1986). Therefore, studies on in vitro screening for, obtaining NaCl tolerant embryogenic lines might help in elucidating the genetic and physiological aspects of the tolerance, and possibly the recovery of the acquired trait in the regenerates.
We developed a selection scheme mainly considering the following points: a) choice of genotypes which can develop in vitro long-term highly embryogenic cultures; b) choice of material in which genetic recombination was occurring, in order to have the chance to meet ab initio an advantageous genetic set; c) use of somaclonal variation as a source of further genetic variability; d) the choice of a level of selection not drastically hampering the embryogenic capability of the selected cell lines in order to test the acquired tolerance in the progenies of the regenerates.
Embryogenic cultures were derived from W64A X A188 F2 immature embryos and explanted at 12 DAP on N6 basic medium (C.L. Armstrong and C.E. Green, Planta 164:207, 1985). Each cell lineage derived from an initial zygotic embryo, which stably performed as embryogenic culture along the first three months of life, was considered a somaclone, in that both its initial genetic set and the variation eventually originated during the period of culture, might account for the specific results in each case. A pool of the original material was first subjected to growth in the presence of various amounts of NaCI (0, 50, 100, 148, 198, 222 and 291 mM). Growth was seriously affected at 100 mM NaCl with a drastic effect on the somatic embryogenesis of the calli. Therefore selection was performed at 85 mM NaCl (0.5% NaCl) in which some perfectly embryogenic outgrows could be rescued on the calli at each passage and subcultured in the same conditions. Thirty-nine original somaclones were subjected to the scheme of selection described in Figure 1. Each step lasted 21 days. At the end of each subculture, embryogenic outgrows were subcultured on fresh medium. At the end of the 3rd step on 0.5% NaCl, 18 of 39 somaclones gave resulting tolerant derivatives. All the tolerant cultures were perfectly embryogenic calli of type 1. Out of these latter ones, 8 performed particularly well and were subcultured on higher dosage (128 mM NaCI), to test whether they might be tolerant to higher levels of NaCl. Indeed, 2 clones (STSC 20 and 21) grew as much as the control maintaining a highly embryogenic phenotype. These latter ones are now being evaluated for regeneration and growth on higher levels of salt (1% NaCI). Seventeen tolerant somaclones were transferred to Murashige and Skoog (1962) hormone-free medium for plant regeneration. Regenerative conditions were both in the presence and in absence of the selective level of NaCl. Final results are summarized in Table 1. At the end of the 3rd step of selection, all the somaclones were embryogenic calli of type 1 capable of regeneration in various degrees. Twelve somaclones produced plantlets on both selective and non-selective conditions: the plantlets produced were equally efficient. Four somaclones (STSC 19, 23, 37, 41) produced plants only in non-selective conditions, while STSC 27 regenerated only in NaCl containing medium. Over the total number of regenerates obtained, a good percentage have been successfully transplanted into soil. The efficiency of establishment in pots was not apparently correlated to the conditions of regeneration. Although observations on the morphology of direct regenerates are strongly affected by epigenetic changes due to the in vitro culture itself, however, a larger root system was observed in the regenerates on NaCl containing medium, with more than 4- to 20-fold difference compared to the control. No phenotypic aberration or noticeable variation could be observed on the aerial parts, except that a few regenerates showed intense tillering. This was noticed also in regenerates from the control. Further investigations will be carried out on the progenies of these regenerates.
Figure 1 - Scheme of selection.
Table 1 - Regenerates obtained in salt tolerant somaclones in selective and non-selective conditions.
E. Lupotto, M. Mongodi and M.C. Lusardi
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