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Vernalization of in Vitro-Grown Clone Plants of Digitalis lanata
J PlantPhysiol. Vol. 135. pp. 378-379(1989)
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Vernalization of in Vitro-Grown Clone Plants of
Digitalis lanata H.
MERTINAT,
B. DIETTRICH, and M.
LUCKNER
University Halle, Section of Pharmacy, Weinbergweg 15, Halle, DDR-4010, GDR Received May 22, 1989 . Accepted July 10, 1989
Summary Juvenile Digitalis lanata clone plants became flower-induced upon cultivation at reduced temperature (vernalization). Flowers were formed if (a) 3-month old or older plants were kept for several weeks at 1-5 °C, (b) plants were illuminated with white light (lOW m- 2) during the cold treatment, and (c) coldtreated plants were further cultivated in the greenhouse at a photoperiod of 16 h d - I.
Key words: Digitalis lanata, Scrophulariaceae, clone plants, cold treatment, vernalization. Abbreviation: GA3 - gibberellin A3. Introduction
Digitalis lanata clone plants obtained by shoot tip culture in vitro were juvenile even if they were derived from flowering mother plants [Luckner et al. 1984, Diettrich et al. 1987]. They formed a rosette as did juvenile seed-born plants and flowered in the second year of vegetation if kept in the open ground during winter. Plants cultivated in a warm greenhouse, however, remained in the juvenile state for indefinite periods [Diettrich et al. 1987]. This indicated that it is growth at reduced temperature (vernalization) which induces flower formation in D. lanata. It was the aim of the experiments described in this paper to elucidate the appropriate conditions for the vernalization of D. lanata and to establish a routine method for flower induction during the whole year.
Materials and Methods D. lanata clone plants [Luckner et al. 1984, Diettrich et al. 1987] were used in the experiments. Before and after cold treatment the plants were cultivated in the greenhouse at IS-18°C (period of illumination 16 h per day). During the winter the plants were illuminated in the morning and evening hours with mercury high pressure lamps (intensity of illumination 8-12 W m - 2). The cold © 1989 by Gustav Fischer Vedag, Stuttgan
treatment of the plants was carried out at 3 - 5 °C with illumination (8 h per day) by fluorescent tubes, daylight 10 and lumoflor, in the ratio 1: 1 [Scheibner et al. 1987] (intensity of illumination 10Wm- 2).
Results and Discussion The D. lanata clone plants became flower-induced if cultivated at reduzced temperature (Table 1). They resembled in this respect D. purpurea plants [Lang 1965, Halevy 1985]. Positive results were obtained, however, only with relatively old D. lanata plants. The minimal age was 3 months after the transfer of the in vitro grown plants to the greenhouse. In agreement with results obtained with other plant species [Hansel 1953, Lang 1965, Pierik 1967] younger plant individuals were not vernalized under all experimental conditions tested. Cultivation of the plants at 1- 2 °C or 3 - 5 °C resulted in a similar degree of vernalization. The time period necessary was in both cases relatively long (at least 3 months with younger plants and at least 2 months with older plants). There was no influence of light intensity (between 4.5 and 19.0W m -2) or of the photoperiod (8 h illumination per day or continuous illumination) on vernalization. Use of lower
Vernalization of Digitalis in vitro Table 1: Vernalization of D. lanata clone plants. Size of the leaves Age Leaves Length 2 Width2 (months)1 (number) (cm) (cm) 4- 9 3.5- 7 0.5-1.2 1.5
Period of cold Flowering treatment plants (%)4 (months)3 1 0 0 2 0 3 0 7-10 2.5 6 -11 0.9-1.7 1 0 2 0 3 0 4 OS 3.0 14-23 10 -20 1.4-2.4 1 OS 2 60 3 80 4 100 8 OS 4.0 15-35 20 -26 1.8-2.8 1 2 10 80 3 90 4 100 8 100 7.0 15 -20 2.1-3.4 2 20-40 100 3 I After transfer of the in vitro grown clone plants to the green house 2 Mean of the 3 largest leaves 3 Temperature 3-5°C, illumination 10 W m -28 h per day 4 n - 30 S The plants produced elongated leaves, but showed neither the formation of elongated axes nor that of flowers temperatures for shorter periods (5 d at -4°C after 1 month at 1 0C) did not result in the formation of flowering plants. The artificially vernalized plants formed 1- 2 inflorescences with 30-40 flowers each. The flowers showed normal morphology and contained vivid pollen and ovules. They developed seeds germinating with high rates (> 90 %). Pollen and ovules were used successfully in the establishment of haploid and homozygous diploid cell cultures as well as in cross-breeding experiments (unpublished results). There was no difference between the plants vernalized in the laboratory and plants cold-treated «naturally» in the open ground during the winter. Treatment with GA3 (application of up to 1000 J.tg per plant by spraying the surface of the leaves with an aqueous solution of GA 3) caused elongation of leaves and stems, but no induction of flowering. This is in contrast to the results of
379
Wittwer and Bukovac [1957] with D. purpurea. GA3 also did not reduce the period of cold treatment necessary for flower induction. During further cultivation without GA3 in the greenhouse the gibberellin-treated plants formed a new rosette at the top of the elongated axis. Flowering could also not be induced in vitro (in axenic D. lanata shoots or plants) under many different conditions shown to be suitable for other plant species [Nitsch and Nitsch 1967, Tanimoto and Harada 1981, Chaouat 1983, Fujioka et al. 1983, van den Ende et al. 1984], e.g., increased sugar content of the medium, different concentrations of auxins and cytokinins, administration of GA 3, benzoic acid, orotic acid, and adenine. Even a period of cultivation of up to 12 months in vitro at 3 °C did not cause vernalization and no flowers were formed if the treated shoots or plants were transferred afterwards to higher temperatures.
References CHAOUAT, L.: Bul!. Soc. bot. Fr., Lettres bot. 130 (1),9-14 (1983). DIETTRICH, B., H. MERTINAT, M. LUCKNER, K. BREUEL, and C. DAUTH: Wiss. Z. Univ. Halle XXXVI'87, H. 5, 90-102 (1987). FUJIOKA, S., I. YAMAGUCHI, N. MUROFUSHI, N. TAKAHASHI, S. KAIHARA, and A. T AKIMOTO: Plam Cell Physio!. 24, 241- 246 (1983). HALEVY, H. A. (ed.): CRC Handbook of Flowering, Vol. 2. CRC Press, Boca Raton (1985). HANSEL, H.: Z. Pflanzenziichtung 32,233-274 (1953). LANG, A.: In: RUHLAND, W. (ed.): Handbuch der Pflanzenphysiologie, Vo!' XV/I, pp. 1380-1536, Springer Verlag, Berlin (1965). LUCKNER, M., B. DIETTRICH, M. SPRINGER, K. BREUEL, and C. OERTEL: In: Methoden und Verfahren der Ziichtung, des Anbaus, der Sammlung und der industriellen Verarbeitung von Arznei- und Gewiirzpflanzen, Artern 18.-22. Juni 1984, Vortragstexte, Teil 1, pp. 113 -127, Pharmazeutisches Werk Halle, Halle (1984). NITSCH, C. and J. P. NITSCH: Planta 72, 371-384 (1967). PIERIK, R. L. M.: Z. Pflanzenphysio!. 56, 141-152 (1967). SCHEIBNER, H., L. BJORK, U. SCHULZ, B. DIETTRICH, and M. LUCKNER: J. Plam Physio!. 130, 211-219 (1987). TANIMOTO, S. and H. HARADA: Plant Cell Physio!. 22, 533 - 541 (1981). VAN DEN ENDE, G.: J. Exp. Bot. 35 (161), 1853-1859 (1984). WITTWER, S. H. and M. J. BUKOVAC: Science 126, 30-31 (1957).
Short COll1111U11 icatioll
Vernalization of in Vitro-Grown Clone Plants of
Digitalis lanata H.
MERTINAT,
B. DIETTRICH, and M.
LUCKNER
University Halle, Section of Pharmacy, Weinbergweg 15, Halle, DDR-4010, GDR Received May 22, 1989 . Accepted July 10, 1989
Summary Juvenile Digitalis lanata clone plants became flower-induced upon cultivation at reduced temperature (vernalization). Flowers were formed if (a) 3-month old or older plants were kept for several weeks at 1-5 °C, (b) plants were illuminated with white light (lOW m- 2) during the cold treatment, and (c) coldtreated plants were further cultivated in the greenhouse at a photoperiod of 16 h d - I.
Key words: Digitalis lanata, Scrophulariaceae, clone plants, cold treatment, vernalization. Abbreviation: GA3 - gibberellin A3. Introduction
Digitalis lanata clone plants obtained by shoot tip culture in vitro were juvenile even if they were derived from flowering mother plants [Luckner et al. 1984, Diettrich et al. 1987]. They formed a rosette as did juvenile seed-born plants and flowered in the second year of vegetation if kept in the open ground during winter. Plants cultivated in a warm greenhouse, however, remained in the juvenile state for indefinite periods [Diettrich et al. 1987]. This indicated that it is growth at reduced temperature (vernalization) which induces flower formation in D. lanata. It was the aim of the experiments described in this paper to elucidate the appropriate conditions for the vernalization of D. lanata and to establish a routine method for flower induction during the whole year.
Materials and Methods D. lanata clone plants [Luckner et al. 1984, Diettrich et al. 1987] were used in the experiments. Before and after cold treatment the plants were cultivated in the greenhouse at IS-18°C (period of illumination 16 h per day). During the winter the plants were illuminated in the morning and evening hours with mercury high pressure lamps (intensity of illumination 8-12 W m - 2). The cold © 1989 by Gustav Fischer Vedag, Stuttgan
treatment of the plants was carried out at 3 - 5 °C with illumination (8 h per day) by fluorescent tubes, daylight 10 and lumoflor, in the ratio 1: 1 [Scheibner et al. 1987] (intensity of illumination 10Wm- 2).
Results and Discussion The D. lanata clone plants became flower-induced if cultivated at reduzced temperature (Table 1). They resembled in this respect D. purpurea plants [Lang 1965, Halevy 1985]. Positive results were obtained, however, only with relatively old D. lanata plants. The minimal age was 3 months after the transfer of the in vitro grown plants to the greenhouse. In agreement with results obtained with other plant species [Hansel 1953, Lang 1965, Pierik 1967] younger plant individuals were not vernalized under all experimental conditions tested. Cultivation of the plants at 1- 2 °C or 3 - 5 °C resulted in a similar degree of vernalization. The time period necessary was in both cases relatively long (at least 3 months with younger plants and at least 2 months with older plants). There was no influence of light intensity (between 4.5 and 19.0W m -2) or of the photoperiod (8 h illumination per day or continuous illumination) on vernalization. Use of lower
Vernalization of Digitalis in vitro Table 1: Vernalization of D. lanata clone plants. Size of the leaves Age Leaves Length 2 Width2 (months)1 (number) (cm) (cm) 4- 9 3.5- 7 0.5-1.2 1.5
Period of cold Flowering treatment plants (%)4 (months)3 1 0 0 2 0 3 0 7-10 2.5 6 -11 0.9-1.7 1 0 2 0 3 0 4 OS 3.0 14-23 10 -20 1.4-2.4 1 OS 2 60 3 80 4 100 8 OS 4.0 15-35 20 -26 1.8-2.8 1 2 10 80 3 90 4 100 8 100 7.0 15 -20 2.1-3.4 2 20-40 100 3 I After transfer of the in vitro grown clone plants to the green house 2 Mean of the 3 largest leaves 3 Temperature 3-5°C, illumination 10 W m -28 h per day 4 n - 30 S The plants produced elongated leaves, but showed neither the formation of elongated axes nor that of flowers temperatures for shorter periods (5 d at -4°C after 1 month at 1 0C) did not result in the formation of flowering plants. The artificially vernalized plants formed 1- 2 inflorescences with 30-40 flowers each. The flowers showed normal morphology and contained vivid pollen and ovules. They developed seeds germinating with high rates (> 90 %). Pollen and ovules were used successfully in the establishment of haploid and homozygous diploid cell cultures as well as in cross-breeding experiments (unpublished results). There was no difference between the plants vernalized in the laboratory and plants cold-treated «naturally» in the open ground during the winter. Treatment with GA3 (application of up to 1000 J.tg per plant by spraying the surface of the leaves with an aqueous solution of GA 3) caused elongation of leaves and stems, but no induction of flowering. This is in contrast to the results of
379
Wittwer and Bukovac [1957] with D. purpurea. GA3 also did not reduce the period of cold treatment necessary for flower induction. During further cultivation without GA3 in the greenhouse the gibberellin-treated plants formed a new rosette at the top of the elongated axis. Flowering could also not be induced in vitro (in axenic D. lanata shoots or plants) under many different conditions shown to be suitable for other plant species [Nitsch and Nitsch 1967, Tanimoto and Harada 1981, Chaouat 1983, Fujioka et al. 1983, van den Ende et al. 1984], e.g., increased sugar content of the medium, different concentrations of auxins and cytokinins, administration of GA 3, benzoic acid, orotic acid, and adenine. Even a period of cultivation of up to 12 months in vitro at 3 °C did not cause vernalization and no flowers were formed if the treated shoots or plants were transferred afterwards to higher temperatures.
References CHAOUAT, L.: Bul!. Soc. bot. Fr., Lettres bot. 130 (1),9-14 (1983). DIETTRICH, B., H. MERTINAT, M. LUCKNER, K. BREUEL, and C. DAUTH: Wiss. Z. Univ. Halle XXXVI'87, H. 5, 90-102 (1987). FUJIOKA, S., I. YAMAGUCHI, N. MUROFUSHI, N. TAKAHASHI, S. KAIHARA, and A. T AKIMOTO: Plam Cell Physio!. 24, 241- 246 (1983). HALEVY, H. A. (ed.): CRC Handbook of Flowering, Vol. 2. CRC Press, Boca Raton (1985). HANSEL, H.: Z. Pflanzenziichtung 32,233-274 (1953). LANG, A.: In: RUHLAND, W. (ed.): Handbuch der Pflanzenphysiologie, Vo!' XV/I, pp. 1380-1536, Springer Verlag, Berlin (1965). LUCKNER, M., B. DIETTRICH, M. SPRINGER, K. BREUEL, and C. OERTEL: In: Methoden und Verfahren der Ziichtung, des Anbaus, der Sammlung und der industriellen Verarbeitung von Arznei- und Gewiirzpflanzen, Artern 18.-22. Juni 1984, Vortragstexte, Teil 1, pp. 113 -127, Pharmazeutisches Werk Halle, Halle (1984). NITSCH, C. and J. P. NITSCH: Planta 72, 371-384 (1967). PIERIK, R. L. M.: Z. Pflanzenphysio!. 56, 141-152 (1967). SCHEIBNER, H., L. BJORK, U. SCHULZ, B. DIETTRICH, and M. LUCKNER: J. Plam Physio!. 130, 211-219 (1987). TANIMOTO, S. and H. HARADA: Plant Cell Physio!. 22, 533 - 541 (1981). VAN DEN ENDE, G.: J. Exp. Bot. 35 (161), 1853-1859 (1984). WITTWER, S. H. and M. J. BUKOVAC: Science 126, 30-31 (1957).