Frost tolerance and leaf proline content were examined in a number of potato hybrids selected for frost tolerance and in the cv. Astarte before and after hardening. Cold hardening (2°C for 20 days) in a dry environment (50/90% relative humidity, day/night) resulted in decreased water content, increased proline content and increased frost tolerance of the leaves of all genotypes. Frost tolerance before and after hardening was positively related to leaf proline content, but not to leaf water content. Drought stress alone, imposed by wilting excised leaves for 4 days, resulted in an accumulation of proline comparable to that after hardening in a dry environment, but the increase in frost tolerance was smaller. Cold hardening in a humid environment (90% relative humidity continuously) only caused a minor accumulation of proline and a small increase in frost tolerance, but the increase in frost tolerance was high in relation to the amount of proline accumulated. Proline, exogenously applied to one of the genotypes, was accumulated in the leaves of shoot cultures, resulting in an increase in frost tolerance. A possible role of proline in frost tolerance is discussed.

Sixty‐seven hydroxyproline‐resistant (hypr) cell lines were selected from cell suspensions of a diploid potato (Solanum tuberosum L., clone H2578) after plating on 5 and 10 mM hydroxyproline (hyp). Resistant colonies were obtained with a spontaneous frequency of 2.9×10−6. No clear influence could be shown from treatment with N‐ethyl‐N‐nitrosourea (10 or 50 μM). Ninety % of the variant lines contained more proline than the wild type when cells were grown away from hyp for 1 month. Total free amino acid content was increased 2.2 to 6.8 times. When the lines were grown for another 2–5 months on non‐selective medium, the content of proline and other amino acids and hyp resistance decreased. After this period the values were, however, still substantially higher than in the wild type. When tested for growth on media with other amino acid analogues (azetidine‐2‐carboxylic acid and dehydroproline, analogues of proline; aminoethyl‐cysteine, analogue of lysine and 3‐fluorotyrosine, analogue of tyrosine) and on media with inhibitory concentrations of lysine + threonine. lines H4a and H4b4 were cross resistant to these compounds. When tested on media with inhibitory NaCl concentrations, variant lines H2a, H4a, and H6 showed better tolerance than the wild type. One variant cell line (H4a) was successfully regenerated into plants. Preliminary results showed an increased frost tolerance in the leaves of these plants (−4.5°C compared to −3°C for the wild type), accompanied by a higher leaf proline content. Callus initiated from leaves of the regenerated clones was more resistant to hyp than wild type callus, indicating that the variant trait might be due to a mutation.

Sohumtuberosum, potato, amino acids, amino acid analogue resistance, frost tolerance, plant regeneration, proline, somaclonal variation. SUMMARY A number of previously selected hydroxyproline (hyp) resistant cell lines of a diploid potato (Solunum tuberosum L., clone H2578, 2n=2x=24) could be regenerated into plants which were further analysed. Hyp resistance, although lower than in the originally selected calli, was still present in regenerated shoots and in callus initiated from these shoots and it was not lost upon (mini)tuber propagation. Regenerated shoots showed a wide range of phenotypic variation. The chromosome number, analysed in 4 clones, appeared to be hypotetraploid (44 or 45). Tuber-propagated regenerants generally showed increased frost tolerance both at the plant and the cell level. In leaves this seemed to be associated with increased levels of both proline and total amino acid content. However, in callus only the total amino acid content but not proline was still elevated. In the wild type the frost tolerance of the tubers appeared to be lower than that of the leaves. Between tubers of wild type and the hyp resistant regenerants no differences in frost tolerance were found, although proline and total amino acid content tended to be higher in tubers from the regenerants.

Different methods of freezing and of estimating frost damage in cell cultures of Solanum tuberosum L. and a number of wild Solanum species were compared. Frost‐killing temperatures (FKT, i.e. the temperature resulting in 50% of the maximum possible frost damage) in leaves of these species were ‐6°C (S. acaule), 5°C (S. me‐gistacrotobum), ‐4.5°C (S. commersonii) and ‐3°C (S. polytrichon and S. tubero‐sum) No appreciable species differences were found in FKT when cells were submerged in either buffer or medium and frozen. However, differences did exist when cells were frozen in a non‐submerged condition: S. acaule and S. commersonii callus were more sensitive to frost than suspensions, whereas suspensions of the other species were the most sensitive. Measurement of freezing damage by either electrolyte leakage or by 2,3,5‐triphenyltetrazolium chloride viability assays revealed similar FKT values. Cell cultures of S. acaule showed better frost tolerance than S. tuberosum (FKT values were ‐4.5 to ‐6°C and ‐2.5°C. respectively), however, frost tolerance of S. megistacrolobum and S. commersonii was only poorly expressed at the cell level (FKT values were between ‐2 and ‐3°C). Variant cell lines previously selected for resistance to the amino acid analogues hydroxyprotine, aminoethylcysteine and 5‐methyhryptophan appeared to be more tolerant to frost than the wild type S. tuberosum clone.

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