In vitro‐selection of hydroxyproline‐resistant cell lines of wheat (Triticum aestivum): accumulation of proline, decrease in osmotic potential, and increase in frost tolerance

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Abstract
Five hundred hydroxyproline‐resistant cell lines were selected from cell cultures of wheat (Triticum aestivum L. cv. Koga II) after plating on 10 to 30 mM hydroxyproline (Hyp) containing solid Gamborg B 5 medium. All selected cell lines from 30 mM Hyp‐medium contained increased (up to 17‐fold) levels of free proline. Seventy‐four cell lines were transferred to Hyp‐free medium and subcultivated 25 times, for 12 months altogether, after which 80% still had increased proline levels. Fourteen cell lines with increased proline levels were further investigated in liquid media with regard to their frost tolerance, which was measured by means of electrolyte leakage. Ten of them showed increased fros tolerance, with LT 50 values as low as 2.7°C below that of the wild type (‐4.7°C). Besides increased proline levels and increased percentage dry weight, the Hyp‐resistant cell lines had lower osmotic potentials. Osmotic potentials correlated better than levels of free proline with the increase in frost tolerance.