Improved frost tolerance and winter survival in winter barley (Hordeum vulgare L.) by in vitro selection of proline overaccumulating lines

Embryogenic calli derived from anther cultures of the two-rowed winter barley cultivar ‘Igri’ were plated on solid L3 medium containing the proline analogue hydroxyproline (Hyp), 10–20 mmol l⁻¹. Exposure to Hyp caused severe degeneration of most of the calli. Hyp resistant calli, distinguishable by their lighter colour and higher growth rate, and control calli not exposed to Hyp were plated on L3 regeneration medium. From 22,500 anthers exposed to Hyp 46 Hyp resistant regenerates were obtained, which were transferred to soil. After cultivation for 5–10 weeks at normal growth conditions they were cold hardened at 2 ∘C under short day conditions together with control regenerates. Frost tolerance assays with segments of fully grown leaves of unhardened and cold hardened plants revealed that Hyp resistant regenerants were significantly more frost tolerant than the control regenerants. Improved frost tolerance was found also in the progenies R₁ to R₉, and genotypic segregation in the R₁ generation in a 1:2:1 ratio was indicated. Increased proline content was observed in the R₂ generation and in subsequent generations and was significantly (P ≤ 0.001) correlated with increased frost tolerance in the Hyp lines. Comparative studies of R₉ progenies from homozygous R₂ plants with the wild type ‘Igri’ under field conditions in winter at three locations in Europe as well as crossing experiments confirmed the heritable improvement of frost tolerance and winter survival, respectively, in the Hyp lines. The results support the hypothesis that proline accumulation in cold acclimated winter barley plants is causally related to the acquisition of frost tolerance. Moreover, the described biotechnological procedure may be applicable in breeding programs for improved winter hardiness and possibly also for other stress tolerances.