Development of NaCl-resistant callus culture and regenerants
--S. N. Larina*, Yu. I. Dolgykh* and Z. B. Shamina*

A rapid and specific procedure was developed to screen salt-resistant cell cultures and to obtain plants with salt-tolerant phenotype.

Cell cultures were initiated from immature embryos of A188 inbred 10-11 days after pollination, cultivated in 1% NaCl-supplemented MS growth medium, and subcultured every 30 days.

NaCl considerably decreased callus viability (Table), and by the 6th subculture salt-sensitive explants were virtually eliminated. Viable cells were salt-resistant.
Subcultures 1 2 3 4 5 6
Viability, % growing calli 13.0 2.95 0.97 0.28 0.04  0.004 

NaCl-resistant embryogenic callus from every subculture was used for regeneration. Eight regenerants were produced after one, 23 after two, and 4 after three months of cultivation. Regenerants were weaker and did not differ substantially from the initial plants in their morphology. About half of the regenerants were branched and had 2-4 shoots.

After three subcultures, NaCl-resistant calli were analyzed for their content of osmoticals. Resistant and sensitive cell lines were cultivated in the standard and 1% NaCl-supplemented MS medium for 7 days and extracted for determination of sugar and monovalent ion concentrations. Sensitive cells contained more soluble sugars (Fig. 1), both in the standard and the selective media, however in the resistant cells salt stress stimulated sugar accumulation by 25%. Na content differed inconsiderably in two cell lines when grown in the absence of NaCl (Fig. 2), the salt stress increased Na content in both cell lines, however, the resistant line accumulated 1.3 times more Na than the sensitive line. Two lines did not differ substantially in their K content under salt stress. 

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