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In vitro-Selection and Regeneration of Hydroxyproline-Resistant Lines of Winter Wheat with Increased Proline Content and Increased Frost Tolerance
J. Plant Physiol. Vol.
142. pp. 222-225 (1993)
In vitro-Selection and Regeneration of HydroxyprolineResistant Lines of Winter Wheat with Increased Proline Content and Increased Frost Tolerance KARL DORFFLING, HELGA DORFFLING,
and GERDA
LESSELICH
Institut fiir Allgemeine Botanik und Botanischer Garten, Universitat Hamburg, Ohnhorststr. 18,22609 Hamburg, Germany Received February 2, 1993 . Accepted March 29, 1993
Summary
An embryogenic callus obtained from immature embryos of a Finnish winter wheat (Triticum aestivum L. cv. Jo 3063) was used for in vitro-selection of hydroxyproline (Hyp) resistant calli. Calli were plated on solid Gamborg B5 medium containing 10-20mM Hyp and 2mgL -1 2,4-D. From 6018 embryogenic calli exposed to Hyp in the course of three subcultures nine calli proved to be Hyp-resistant and remained viable and embryogenic. These were transferred to a regeneration medium and finally to Gamborg B5 medium before being transplanted to soil. The regenerated plants were grown at 18°C for 6 weeks and then cold hardened at 2°C for 18 weeks. The mean osmotic potential of the Hyp-resistant cold hardened regenerates was significantly lower than that of hardened controls. At the same time their mean proline content and their mean frost tolerance were significantly higher compared with regenerated controls.
Key words: In vitro-selection, Hydroxyproline, Winter wheat, Frost tolerance, Regeneration. Abbreviations: Hyp = hydroxyproline; 2,4-D = 2,4-dichlorophenoxyacetic acid; LTso = temperature for 50 % ion efflux.
Introduction
Breeding for tolerance against cold and drought by classical methods of selection and crossing is a time-consuming and often inefficient procedure. This is the case in winter wheat breeding with regard to frost tolerance. On the basis of somaclonal variation and by using biochemical markers as selection tools, in vitro-selection techniques may provide an alternative way to select new genotypes with improved properties. In previous studies we found a positive correlation between the frost tolerance of nine cold hardened winter wheat genotypes and the accumulation of free proline in their leaves (Dorffling et al., 1990). Similar observations have © 1993 by Gustav Fischer Verlag, Stuttgart
been reported by other authors for winter barley (Dobslaw and Bielka, 1988) and for potato (Van Swaaij et al., 1986). In fact, conventional breeding programs already use proline accumulation as a biochemical marker for increased frost tolerance in winter barley (Winkel, 1989). Thus, selection of genotypes may yield improved frost tolerance. Van Swaaij et al. (1986, 1987) were the first ones to succeed, by means of Hyp, with the in vitro-selection of potato plants with increased frost tolerance. Recently, we selected stable Hyp-resistant cell lines from a spring wheat cell culture that proved to be more frost tolerant than the wild type. These cell lines contained increased levels of free proline and had decreased osmotic potentials (T ant au and Dorffling, 1991). However, it has been impossible to regen-
In vitro-selection of winter wheat with increased frost tolerance erate the respective cell lines. Therefore, we continued the selection with cell cultures of a Finnish winter wheat variety named Jo 3063, which had a high capacity to regenerate.
223
of the osmotic potential, or freeze-dried for determination of the proline content.
Frost tolerance assay Materials and Methods
Callus induction and culture Immature embryos from field grown plants of the Finnish winter wheat (Triticum aestivum L.) variety Jo 3063 (Plant Breeding Station Jokioinen, Finland) were cultured under sterile conditions in 9 em diameter Petri dishes at 24 °C in the dark on solid Gamborg B5 medium (Flow Laboratories, Irvine, Scotland; Gamborg et al., 1968) supplemented with 9.1 J.LM 2,4-D (Serva, Heidelberg, Germany) and 58 mM sucrose (Merck, Darmstadt, Germany). Embryogenic calli were subcultured at intervals of 3 weeks on fresh medium (24°C, 16 h photoperiod) for several months.
In vitro-selection ofHyp-resistant embryogenic lines For in vitro-selection, embryogenic calli (in total 12988) were exposed to Gamborg B5 medium containing 5, 10 or 20 mM hydroxyproline (Serva, Heidelberg, Germany). Hyp-resistant calli were detected by their continuous growth, white colour and embryogenic status. The selection procedure lasted 12 weeks with a transfer of the calli to fresh Hyp-containing medium at 3-week intervals. In the first experiments either 5, 10 or 20 mM Hyp-containing medium was used during the whole exposure of 12 weeks. However, exposure to 5 and 10 mM Hyp was found to be inefficient for the selection of pronounced Hyp-resistance and increased expression of the other parameters. In further experiments the embryogenic calli were therefore exposed to higher Hyp concentrations: A 10 mM Hyp medium during the first 3 weeks and a 20 mM Hyp medium during the following weeks. ,Controls> of embryogenic calli (in total 1362) were subcultured on Hyp-free Gamborg B5 medium.
Regeneration After 12 weeks the Hyp-resistant lines and the control lines on Hyp-free medium were transferred to a regeneration medium (Gamborg B5 supplemented with 1.0 gL -1 casein hydrolysate (Sigma, Deisenhofen, Germany), 20gL -1 sucrose, 6.4gL- 1 agar (Difco, Detroit, USA), 1 mgL -1 IAA (Merck, Darmstadt, Germany), 0.05mgL- 1 zeatin (Sigma, Deisenhofen, Germany) and 100 mL L -1 sterile coconut milk (Sigma, Deisenhofen, Germany)) for 3 - 6 weeks. After that time all regenerated lines were transferred to Gamborg B5 medium without 2,4-D. Two to three weeks later the plants were potted in small pots in standard soil and grown in growth chambers (18 °C day/night, photoperiod 16 h, 60% relative humidity, light intensity 350 J.Lmols- 1 m- 2 from Sylvania cool white fluorescent lamps). For 8 days the pots were covered with a plastic foil to prevent dessiccation of the regenerates. After a further 3 - 5 weeks the roots had developed well and the plants were potted in 14-cm diameter pots.
Cold hardening treatment Six to eight-week-old Hyp-resistant and control plants were exposed for 12-18 weeks to a 2°C day/night, 9h photoperiod with 300J.Lmols- 1 m- 2 light intensity and 60/95% (day/night) relative humidity. Before and during the cold hardening process young, fully developed leaves were collected and either used directly for the frost tolerance assay or frozen in liquid nitrogen for determination
Frost tolerance was determined according to Dexter et al. (1932) and as described by Dorffling et al. (1990). Freshly cut segments of 5 mm length obtained from young, fully grown leaves were placed on moist filter paper in ice-cooled Petri dishes and subsequently cooled overnight at -1 0c. Afterwards, the temperature was gradually reduced at a cooling rate of 3 °C/h down to -19 0c. At each interval one segment was removed, thawed and incubated in tubes with 3.5 mL pure water for 24 hours at 4 °C on a shaker. After that, the electrolytical conductivity was measured at 25 °C with a WTW LF 530 conductometer equipped with aLTA 01 sensor (WTW, Weilheim, Germany) and again after boiling for 30min and subsequent cooling to 25 0c. The percentage of injury was calculated according to the formula: conductivity after freezing x 100 conductivity after boiling. LTso values were calculated by pro bit analysis from the plots of temperature vs. per cent injury.
Determination of osmotic potential For determination of the osmotic potential two young, fully grown leaves were frozen, thawed and the cell sap pressed out; 25 J.LL of filtered cell sap were used for determination of the freezing point by means of a freezing point osmometer (Roebling, Berlin, Germany).
Determination ofproline The content of free proline was measured according to Bates et al. (1973). Freeze-dried plant material (30 to 60mg DW) was extracted overnight with 10 mL 3 % sulphosalicylic acid at 4°C on a shaker and 1.0 - 2.0 mL were used for the acid ninhydrin reaction.
Results and Discussion
Hyp-resistant calli could be detected on the Hyp medium by their viability, continued embryogenic growth and their light colour. Non-resistant calli lost their embryogenic capacity, became brown and died. From 6018 embryogenic calli exposed to 10/20 mM Hyp, nine survived and regenerated. Thus, only 0.15 % of the embryogenic, Hyp-exposed calli produced regenerated plants. On the other hand, 58 % of the control calli (not exposed to Hyp) regenerated. The plants that had regenerated from Hyp-resistant calli (henceforth referred to as Hyp-resistant plants) and a comparable number of control plants were cold hardened at 2°C for up to 18 weeks. Afterwards, the proline content, frost tolerance and the osmotic potential of the young, fully developed leaves were determined. Proline contents were significantly (P < 0.01, t-test and D-test) higher in Hyp-resistant plants (Fig. 1) after 4 weeks of cold hardening. In unhardened plants proline levels were low, but Hyp-resistant plants had significantly higher levels than the controls (Table 1). Frost tolerance expressed as LTso value of unhardened regenerated Hyp-resistant plants, however, was not different from that of unhardened regenerated control plants (Table 1).
224
KARL DORFFLING, HELGA DORFFlING, and GERDA LESSEUCH Proline I mg·g OW-I)
LT5D(OCI
20~----------------------------------~
-16.------------------..,
- 2 OL--L-~_L_~
o Weeks at 2° C Fig. 1: Proline contents in the leaves of in vitro-regenerated plants of winter wheat cv. Jo 3063 during cold hardening at 2°C. Hyp = regenerates from hydroxyproline-containing medium; Control = regenerates from Hyp-free medium. Data from two independent experiments. Each point represents the mean of S-24 measurements ± SE. The difference between the proline contents of Hyp and control after 4 weeks is significant with P < 0.01 (t-test and U-test).
The LTso values of cold hardened controls and Hyp-resistant plants varied greatly after 4, 6 and 8 weeks. The LTso values of the cold hardened Hyp-resistant plants were significantly lower than those of the controls (Fig. 2). The difference after 4 weeks hardening was nearly 10°C. The mean LTso value for Hyp-resistant plants was -10 ± 0.7 °e, that of the controls - 5.8 ± 0.3 0C. This difference was significant (P < 0.01, t-test). Three of the nine Hyp-resistant lines proved to be especially frost tolerant. Their LTso values were lower than -19°C. The lowest LTso value observed was - 22°C. Plants raised from seeds and hardened for 8 weeks were not significantly different from control plants regenerated from tissue cultures with regard to their frost tolerance: the LTso value of seed-raised plants was - 8.4 ± 0.7 °e after 8 weeks cold hardening, that of regenerated control plants -7.8 ± O.S°C. Towards the end of hardening Hyp-resistant plants were easily detectable by their dark green colour and an absence of leaf senescence in older leaves. Besides increased proline contents and frost tolerance, the Hyp-resistant plants were characterized by a significant (P <0.05) decrease in the osmotic potential. After 8 weeks of cold hardening the mean osmotic potential of the leaves from Hyp-resistant plants was -2.30 ± 0.04 MPa, that of control plants - 2.19 ± 0.03 MPa. The results show that it is possible to select stable wheat variants with an increased frost tolerance by means of in
Table 1: Proline contents and frost tolerance in unhardened regenerates. Treatment Control regenerates Hyp-resistant regenerates
*=
(Ilg g- I DW)
Proline content
Frost tolerance (LTso, 0C)
177*± 11 297* ± 41
-3.68±0.SO - 3.68 ± 0.12
difference significant with P
__~__L_~_ __ L_ _L_~
10 12 Weeks at 20 C
14
16
18
20
Fig. 2: LTso values of in vitro-regenerated winter wheat plants cv. J 0 3063 during cold hardening at 2°C. H yp = regenerates from hydroxyproline-containing medium. Control = regenerates from Hyp-free medium. Data from two independent experiments. Each point represents the mean of S-27 measurements ± SE. The differences between the LTso values of Hyp and the controls after 4 and 6 weeks are significant with P <0.01 (t-test and U-test), after 8 weeks with P < O OS . (t-test).
vitro-selection of hydroxyproline-resistant cell lines. The increased frost tolerance is expressed on the cell level and at the plant level. Whether this trait is genetically transmittable is the objective of future work. Heritable somaclonal variation in frost resistance of wheat has been shown by Galiba and Sutka (1989), and Kendall et al. (1990) have shown that spring wheat plants with heritable increased freezing tolerance can be selected by in vitro-cryoselection. Further work is in progress for in vitro-selection of higher frost tolerance in winter barley. Acknowledgements
We wish to thank Dr. Hanny Tantau, Institut fur Allgemeine Botanik, Universitat Hamburg, Barbel Brettschneider and Dr. G. Melz, Institut fur Zuchtungsmethodik landwirtschaftlicher Kulturpflanzen, GraB Lusewitz, for their valuable suggestions. We are grateful to Mathias Bohn for linguistic improvement.
References BATES, L. S., R. P. WALDREN, and 1. D. TEARE: Rapid determination of free proline for water stress studies. Plant and Soil 39, 20S207 (1973). DEXTER, S. T., W. E. TOTTINGHAM, and L. F. GRABER: Investigations of the hardiness of plants by measurement of electrolytical conductivity. Plant Physio!. 7, 63 - 78 (1932). DOBSLAW, S. and S. BIELKA: Untersuchungen zur Ermittlung des Frosttoleranzgrades bei Wintergerste mittels Prolinakkumulation. 1. Mitt. Prlifung am Indikatorsortiment. Arch. Zuchtungsforsch. 18, 23S-240 (1988). DORFFLING, K., S. SCHULENBURG, G . LESSELlCH, and H. DORFFLlNG: Abscisic acid and proline levels in cold hardened winter wheat leaves in relation to variety-specific differences in freezing resistance.1- Agron. Crop Sci. 165, 230-239 (1990).
In vitro-selection of winter wheat with increased frost tolerance
GAL/BA, G. and J. SUTKA: Frost resistance of somaclones derived from Triticum aestivum L winter wheat calli. Plant Breeding 102, 101-104 (1989). GAMBORG, O. L., R. A. MILLER, and K. OJIMA: Nutrient require. ments of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151-158 (1968). KENDALL, E. J., j . A. QURESHI, K. K.KARTHA, N . LEUNG, N . CHEVRIER, K. CASWELL, and T. H. H. CHEN: Regeneration of freezingtolerant spring wheat (Triticum aestivum L.) plants from cryoselected callus. Plant Physio!. 94, 1756-1762 (1990). TANTAU, H . and K. DORFFLING: In vitro-selection of hydroxyproline-resistant cell lines of wheat (Triticum aestivum ): accumula-
225
tion of proline, decrease in osmotic potential, and increase in frost tolerance. Physio!. Plant. 82, 243-248 (1991). VAN SWAAlj, A. C, E. JACOBSEN, J. A. K.W. KJEL, and W. J. FEENSTRA: Selection, characterization and regeneration of hydroxyproline-resistant cell lines of Solanum tuberosum: tolerance to NaCl and freezing stress. Physio!. Plant. 68,359-366 (1986). VAN SWAAlj, A. C, H . NljDAM, E. JACOBSEN, and W. J. FEENSTRA: Increased frost tolerance and amino acid content in leaves, tubers and leaf callus of regenerated hydroxyproline resistant potato clones. Euphytica36, 369-380 (1987). WINKEL, A.: Breeding for drought tolerance in cereals. Vortrage Pflanzenziicht. 16,357 -368 (1989).
142. pp. 222-225 (1993)
In vitro-Selection and Regeneration of HydroxyprolineResistant Lines of Winter Wheat with Increased Proline Content and Increased Frost Tolerance KARL DORFFLING, HELGA DORFFLING,
and GERDA
LESSELICH
Institut fiir Allgemeine Botanik und Botanischer Garten, Universitat Hamburg, Ohnhorststr. 18,22609 Hamburg, Germany Received February 2, 1993 . Accepted March 29, 1993
Summary
An embryogenic callus obtained from immature embryos of a Finnish winter wheat (Triticum aestivum L. cv. Jo 3063) was used for in vitro-selection of hydroxyproline (Hyp) resistant calli. Calli were plated on solid Gamborg B5 medium containing 10-20mM Hyp and 2mgL -1 2,4-D. From 6018 embryogenic calli exposed to Hyp in the course of three subcultures nine calli proved to be Hyp-resistant and remained viable and embryogenic. These were transferred to a regeneration medium and finally to Gamborg B5 medium before being transplanted to soil. The regenerated plants were grown at 18°C for 6 weeks and then cold hardened at 2°C for 18 weeks. The mean osmotic potential of the Hyp-resistant cold hardened regenerates was significantly lower than that of hardened controls. At the same time their mean proline content and their mean frost tolerance were significantly higher compared with regenerated controls.
Key words: In vitro-selection, Hydroxyproline, Winter wheat, Frost tolerance, Regeneration. Abbreviations: Hyp = hydroxyproline; 2,4-D = 2,4-dichlorophenoxyacetic acid; LTso = temperature for 50 % ion efflux.
Introduction
Breeding for tolerance against cold and drought by classical methods of selection and crossing is a time-consuming and often inefficient procedure. This is the case in winter wheat breeding with regard to frost tolerance. On the basis of somaclonal variation and by using biochemical markers as selection tools, in vitro-selection techniques may provide an alternative way to select new genotypes with improved properties. In previous studies we found a positive correlation between the frost tolerance of nine cold hardened winter wheat genotypes and the accumulation of free proline in their leaves (Dorffling et al., 1990). Similar observations have © 1993 by Gustav Fischer Verlag, Stuttgart
been reported by other authors for winter barley (Dobslaw and Bielka, 1988) and for potato (Van Swaaij et al., 1986). In fact, conventional breeding programs already use proline accumulation as a biochemical marker for increased frost tolerance in winter barley (Winkel, 1989). Thus, selection of
In vitro-selection of winter wheat with increased frost tolerance erate the respective cell lines. Therefore, we continued the selection with cell cultures of a Finnish winter wheat variety named Jo 3063, which had a high capacity to regenerate.
223
of the osmotic potential, or freeze-dried for determination of the proline content.
Frost tolerance assay Materials and Methods
Callus induction and culture Immature embryos from field grown plants of the Finnish winter wheat (Triticum aestivum L.) variety Jo 3063 (Plant Breeding Station Jokioinen, Finland) were cultured under sterile conditions in 9 em diameter Petri dishes at 24 °C in the dark on solid Gamborg B5 medium (Flow Laboratories, Irvine, Scotland; Gamborg et al., 1968) supplemented with 9.1 J.LM 2,4-D (Serva, Heidelberg, Germany) and 58 mM sucrose (Merck, Darmstadt, Germany). Embryogenic calli were subcultured at intervals of 3 weeks on fresh medium (24°C, 16 h photoperiod) for several months.
In vitro-selection ofHyp-resistant embryogenic lines For in vitro-selection, embryogenic calli (in total 12988) were exposed to Gamborg B5 medium containing 5, 10 or 20 mM hydroxyproline (Serva, Heidelberg, Germany). Hyp-resistant calli were detected by their continuous growth, white colour and embryogenic status. The selection procedure lasted 12 weeks with a transfer of the calli to fresh Hyp-containing medium at 3-week intervals. In the first experiments either 5, 10 or 20 mM Hyp-containing medium was used during the whole exposure of 12 weeks. However, exposure to 5 and 10 mM Hyp was found to be inefficient for the selection of pronounced Hyp-resistance and increased expression of the other parameters. In further experiments the embryogenic calli were therefore exposed to higher Hyp concentrations: A 10 mM Hyp medium during the first 3 weeks and a 20 mM Hyp medium during the following weeks. ,Controls> of embryogenic calli (in total 1362) were subcultured on Hyp-free Gamborg B5 medium.
Regeneration After 12 weeks the Hyp-resistant lines and the control lines on Hyp-free medium were transferred to a regeneration medium (Gamborg B5 supplemented with 1.0 gL -1 casein hydrolysate (Sigma, Deisenhofen, Germany), 20gL -1 sucrose, 6.4gL- 1 agar (Difco, Detroit, USA), 1 mgL -1 IAA (Merck, Darmstadt, Germany), 0.05mgL- 1 zeatin (Sigma, Deisenhofen, Germany) and 100 mL L -1 sterile coconut milk (Sigma, Deisenhofen, Germany)) for 3 - 6 weeks. After that time all regenerated lines were transferred to Gamborg B5 medium without 2,4-D. Two to three weeks later the plants were potted in small pots in standard soil and grown in growth chambers (18 °C day/night, photoperiod 16 h, 60% relative humidity, light intensity 350 J.Lmols- 1 m- 2 from Sylvania cool white fluorescent lamps). For 8 days the pots were covered with a plastic foil to prevent dessiccation of the regenerates. After a further 3 - 5 weeks the roots had developed well and the plants were potted in 14-cm diameter pots.
Cold hardening treatment Six to eight-week-old Hyp-resistant and control plants were exposed for 12-18 weeks to a 2°C day/night, 9h photoperiod with 300J.Lmols- 1 m- 2 light intensity and 60/95% (day/night) relative humidity. Before and during the cold hardening process young, fully developed leaves were collected and either used directly for the frost tolerance assay or frozen in liquid nitrogen for determination
Frost tolerance was determined according to Dexter et al. (1932) and as described by Dorffling et al. (1990). Freshly cut segments of 5 mm length obtained from young, fully grown leaves were placed on moist filter paper in ice-cooled Petri dishes and subsequently cooled overnight at -1 0c. Afterwards, the temperature was gradually reduced at a cooling rate of 3 °C/h down to -19 0c. At each interval one segment was removed, thawed and incubated in tubes with 3.5 mL pure water for 24 hours at 4 °C on a shaker. After that, the electrolytical conductivity was measured at 25 °C with a WTW LF 530 conductometer equipped with aLTA 01 sensor (WTW, Weilheim, Germany) and again after boiling for 30min and subsequent cooling to 25 0c. The percentage of injury was calculated according to the formula: conductivity after freezing x 100 conductivity after boiling. LTso values were calculated by pro bit analysis from the plots of temperature vs. per cent injury.
Determination of osmotic potential For determination of the osmotic potential two young, fully grown leaves were frozen, thawed and the cell sap pressed out; 25 J.LL of filtered cell sap were used for determination of the freezing point by means of a freezing point osmometer (Roebling, Berlin, Germany).
Determination ofproline The content of free proline was measured according to Bates et al. (1973). Freeze-dried plant material (30 to 60mg DW) was extracted overnight with 10 mL 3 % sulphosalicylic acid at 4°C on a shaker and 1.0 - 2.0 mL were used for the acid ninhydrin reaction.
Results and Discussion
Hyp-resistant calli could be detected on the Hyp medium by their viability, continued embryogenic growth and their light colour. Non-resistant calli lost their embryogenic capacity, became brown and died. From 6018 embryogenic calli exposed to 10/20 mM Hyp, nine survived and regenerated. Thus, only 0.15 % of the embryogenic, Hyp-exposed calli produced regenerated plants. On the other hand, 58 % of the control calli (not exposed to Hyp) regenerated. The plants that had regenerated from Hyp-resistant calli (henceforth referred to as Hyp-resistant plants) and a comparable number of control plants were cold hardened at 2°C for up to 18 weeks. Afterwards, the proline content, frost tolerance and the osmotic potential of the young, fully developed leaves were determined. Proline contents were significantly (P < 0.01, t-test and D-test) higher in Hyp-resistant plants (Fig. 1) after 4 weeks of cold hardening. In unhardened plants proline levels were low, but Hyp-resistant plants had significantly higher levels than the controls (Table 1). Frost tolerance expressed as LTso value of unhardened regenerated Hyp-resistant plants, however, was not different from that of unhardened regenerated control plants (Table 1).
224
KARL DORFFLING, HELGA DORFFlING, and GERDA LESSEUCH Proline I mg·g OW-I)
LT5D(OCI
20~----------------------------------~
-16.------------------..,
- 2 OL--L-~_L_~
o Weeks at 2° C Fig. 1: Proline contents in the leaves of in vitro-regenerated plants of winter wheat cv. Jo 3063 during cold hardening at 2°C. Hyp = regenerates from hydroxyproline-containing medium; Control = regenerates from Hyp-free medium. Data from two independent experiments. Each point represents the mean of S-24 measurements ± SE. The difference between the proline contents of Hyp and control after 4 weeks is significant with P < 0.01 (t-test and U-test).
The LTso values of cold hardened controls and Hyp-resistant plants varied greatly after 4, 6 and 8 weeks. The LTso values of the cold hardened Hyp-resistant plants were significantly lower than those of the controls (Fig. 2). The difference after 4 weeks hardening was nearly 10°C. The mean LTso value for Hyp-resistant plants was -10 ± 0.7 °e, that of the controls - 5.8 ± 0.3 0C. This difference was significant (P < 0.01, t-test). Three of the nine Hyp-resistant lines proved to be especially frost tolerant. Their LTso values were lower than -19°C. The lowest LTso value observed was - 22°C. Plants raised from seeds and hardened for 8 weeks were not significantly different from control plants regenerated from tissue cultures with regard to their frost tolerance: the LTso value of seed-raised plants was - 8.4 ± 0.7 °e after 8 weeks cold hardening, that of regenerated control plants -7.8 ± O.S°C. Towards the end of hardening Hyp-resistant plants were easily detectable by their dark green colour and an absence of leaf senescence in older leaves. Besides increased proline contents and frost tolerance, the Hyp-resistant plants were characterized by a significant (P <0.05) decrease in the osmotic potential. After 8 weeks of cold hardening the mean osmotic potential of the leaves from Hyp-resistant plants was -2.30 ± 0.04 MPa, that of control plants - 2.19 ± 0.03 MPa. The results show that it is possible to select stable wheat variants with an increased frost tolerance by means of in
Table 1: Proline contents and frost tolerance in unhardened regenerates. Treatment Control regenerates Hyp-resistant regenerates
*=
(Ilg g- I DW)
Proline content
Frost tolerance (LTso, 0C)
177*± 11 297* ± 41
-3.68±0.SO - 3.68 ± 0.12
difference significant with P
__~__L_~_ __ L_ _L_~
10 12 Weeks at 20 C
14
16
18
20
Fig. 2: LTso values of in vitro-regenerated winter wheat plants cv. J 0 3063 during cold hardening at 2°C. H yp = regenerates from hydroxyproline-containing medium. Control = regenerates from Hyp-free medium. Data from two independent experiments. Each point represents the mean of S-27 measurements ± SE. The differences between the LTso values of Hyp and the controls after 4 and 6 weeks are significant with P <0.01 (t-test and U-test), after 8 weeks with P < O OS . (t-test).
vitro-selection of hydroxyproline-resistant cell lines. The increased frost tolerance is expressed on the cell level and at the plant level. Whether this trait is genetically transmittable is the objective of future work. Heritable somaclonal variation in frost resistance of wheat has been shown by Galiba and Sutka (1989), and Kendall et al. (1990) have shown that spring wheat plants with heritable increased freezing tolerance can be selected by in vitro-cryoselection. Further work is in progress for in vitro-selection of higher frost tolerance in winter barley. Acknowledgements
We wish to thank Dr. Hanny Tantau, Institut fur Allgemeine Botanik, Universitat Hamburg, Barbel Brettschneider and Dr. G. Melz, Institut fur Zuchtungsmethodik landwirtschaftlicher Kulturpflanzen, GraB Lusewitz, for their valuable suggestions. We are grateful to Mathias Bohn for linguistic improvement.
References BATES, L. S., R. P. WALDREN, and 1. D. TEARE: Rapid determination of free proline for water stress studies. Plant and Soil 39, 20S207 (1973). DEXTER, S. T., W. E. TOTTINGHAM, and L. F. GRABER: Investigations of the hardiness of plants by measurement of electrolytical conductivity. Plant Physio!. 7, 63 - 78 (1932). DOBSLAW, S. and S. BIELKA: Untersuchungen zur Ermittlung des Frosttoleranzgrades bei Wintergerste mittels Prolinakkumulation. 1. Mitt. Prlifung am Indikatorsortiment. Arch. Zuchtungsforsch. 18, 23S-240 (1988). DORFFLING, K., S. SCHULENBURG, G . LESSELlCH, and H. DORFFLlNG: Abscisic acid and proline levels in cold hardened winter wheat leaves in relation to variety-specific differences in freezing resistance.1- Agron. Crop Sci. 165, 230-239 (1990).
In vitro-selection of winter wheat with increased frost tolerance
GAL/BA, G. and J. SUTKA: Frost resistance of somaclones derived from Triticum aestivum L winter wheat calli. Plant Breeding 102, 101-104 (1989). GAMBORG, O. L., R. A. MILLER, and K. OJIMA: Nutrient require. ments of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151-158 (1968). KENDALL, E. J., j . A. QURESHI, K. K.KARTHA, N . LEUNG, N . CHEVRIER, K. CASWELL, and T. H. H. CHEN: Regeneration of freezingtolerant spring wheat (Triticum aestivum L.) plants from cryoselected callus. Plant Physio!. 94, 1756-1762 (1990). TANTAU, H . and K. DORFFLING: In vitro-selection of hydroxyproline-resistant cell lines of wheat (Triticum aestivum ): accumula-
225
tion of proline, decrease in osmotic potential, and increase in frost tolerance. Physio!. Plant. 82, 243-248 (1991). VAN SWAAlj, A. C, E. JACOBSEN, J. A. K.W. KJEL, and W. J. FEENSTRA: Selection, characterization and regeneration of hydroxyproline-resistant cell lines of Solanum tuberosum: tolerance to NaCl and freezing stress. Physio!. Plant. 68,359-366 (1986). VAN SWAAlj, A. C, H . NljDAM, E. JACOBSEN, and W. J. FEENSTRA: Increased frost tolerance and amino acid content in leaves, tubers and leaf callus of regenerated hydroxyproline resistant potato clones. Euphytica36, 369-380 (1987). WINKEL, A.: Breeding for drought tolerance in cereals. Vortrage Pflanzenziicht. 16,357 -368 (1989).