Planta (Berl.) 103, 278-280 (1972) 9 by Springer-Verlag 1972
Short Communication
Embryoid Formation by Fragments of Cotyledons and Hypoeotyls in Cucurbita pepo SIBILA JELASKA Institute of Botany of the University Zagreb, Yugoslavia Received December 2, 1971
Summary. After a prolonged culture on Murashige-Skoog medium the primary explants of hypoeotyls and cotyledons of the pumpkin develop embryoid-producing callus tissue. Ten separate strains of such tissue have been obtained and have now been in culture for more than one year, continuing to produce embryoids. E m b r y o i d f o r m a t i o n in higher p l a n t s from tissue grown from excised h y p o c o t y l s or stems has been r e p o r t e d b y K o n a r a n d N a t a r a j a (1964), W i l m a r a n d H e l l e n d o o m (1968), K a t o a n d T a k e u e h i (1966), a n d others. I n t h e p r e s e n t c o m m u n i c a t i o n t h e f o r m a t i o n of e m b r y o s b y in vitro eulture of excised f r a g m e n t s of h y p o e o t y l s a n d cotyledons of p u m p k i n (Cucurbita pepo L.) will be briefly described. So far we k n o w such embryogenesis in p u m p k i n e x p l a n t s has n o t y e t been r e p o r t e d . Seeds of pumpkin were sterilized with 3 % calcium hypochloride and germinated in test tubes in distilled water under light for 10 d under sterile conditions. Fragments, 1 em long, of cotyledons and hypoeotyls were cut and cultivated individually in test tubes (23 • 200 ram) on 25 ml of culture medium M-S complete (MurashigeSkoog, 1962) + 3% glucose + 0.9% agar + one of the following combinations of growth substances: a) 10 mg/1 fi-indolylbutyric acid (IBA), b) 1 rag/1 IBA, e) 0.3 rag/1 2,4-diehlorophenoxyacetic acid (2,4-D), d) 0.3 mg/l 2,4-D + 10 % autoclaved watermelon sap, e) 0.1 mg/l 2,4-D+10% autoclaved water-melon sap, f) 0.3rag/1 2,4-D + 2 g/1 yeast extract, g) 0.3 rag/1 2,4-D + 0. l mg/1 kinetin, h) 1 mg/1 e-naphthylacetie acid (NAA) + 13.5 rag/1 adenine (natural) 1, i) 1 mg/1 2,4-D + 300 rag/1 casein hydrolysate. The cultures were maintained at 26 ~: 1~ C under artificial light (485-~ 45 lux) from fluorescent lamps IPR 40 W, 220 V (8586~ K 16 h light and 8 h darkness daily). A small p e r e e n t a g e of explants, which d i d n o t i m m e d i a t e l y form callus or roots, p r o d u c e d a callus-like tissue on their cut surfaces after a long period with no g r o w t h a c t i v i t y (3 m o n t h s or more). This new tissue (Figs. 1, 2), t h o r o u g h l y different from t h e old one, grew q u i c k l y a n d d i f f e r e n t i a t e d into more or less n o r m a l e m b r y o s (Fig. 3) a n d y o u n g p l a n t l e t s , a n d into various a b n o r m a l shoots. The e x p l a n t s r e a e t e d a l w a y s 1 Adenin CHR natiirlich puriss. Fluka AG. Buehs SG.
Fig. 1. Callus and plantlets growing on medium with 1 mg/1 IBA Fig. 2. Differentiation of plantlets and roots on medium with 0.3 mg/1 2,4-D + 10 % water-melon sap Fig. 3a-e. Embryo-like bodies (embryoids) grown on the medium with 1 mg/1 IBA. a I-Ieart-shaped and torpedo-like bodies; b Embryo with developing cotyledons; e Mature embryos with well developed cotyledons Fig. 4. Adventitious embryo arising from a giant club-like body. Medium with 1 mg/l IBA. The scale in Fig. 4 (1 ram) refers to Figs. 3 and 4
280
S. Jelaska: Embryoid Formation in Cucurbita pepo
in the same way, independently of the growth substances present. This indicates that the embryo formation in pumpkin is mainly determined by the physiological conditions of the tissue, the age of the culture, and the composition of the medium, rather than by a specific, exogenous growth-regulating substance. Ten different strains of such pumpkin callus tissue have now been cultivated over 1 year, being subcultured every 2 months and continuing to form callus, embryos and plantlets. The embryos grow inside the callus as well as adventitiously on the leaflets and hypocotyls of plantlets which are formed by the callus tissue as found earlier in other plants (Konar and Nataraja, 1964; Konar and Oberoi, 1965; ttom~s and Guillaume, 1967)--or on embryo-like bodies (Fig. 4). Details on the morphology of the induced embryos, their development, and aberrations which were found depending on growth substances added to the medium will be published elsewhere.
References t/om~s, J.L.A., Guillaume, M.: Ph~nom~nes d'organogen~se dans des cultures in vitro de tissus de carotte (Daucus carota L.). Bull. Soc. roy. Bot. Belg. 100, 239-258 (1967). Kato, H., Takeuchi, M. : Embryogenesis from the epidermal cells of carrot hypocotyl. Sci. Papers Coll. Gen. Educ. Univ. Tokyo 16, 245-254 (1966). Konar, R.N., Nataraja, K. : I n vitro control of floral morphogenesis in Ranunculus sceleratus L. PhytomorphoIogy 14, 558-563 (1964). - - Oberoi, u P. : I n vitro developments of embryoids on the cotyledons of Biota orientalis. Phytomorphology l~, 137-140 (1965). Murashige, T., Skoog, F. : A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantarum (Cph.) 15, 473497 (1962). Wilmar, C., Hellendoom, M.: Growth and morphogenesis of Asparagus cells cultured in vitro. Nature (Lond.) 217, 369-370 (1968). (Mrs.) S. Jelaska BotaniSki zavod PMF l~ooseveltov trg 6/III, p.p. 933 YU-41001 Zagreb, Yugoslavia
Short Communication
Embryoid Formation by Fragments of Cotyledons and Hypoeotyls in Cucurbita pepo SIBILA JELASKA Institute of Botany of the University Zagreb, Yugoslavia Received December 2, 1971
Summary. After a prolonged culture on Murashige-Skoog medium the primary explants of hypoeotyls and cotyledons of the pumpkin develop embryoid-producing callus tissue. Ten separate strains of such tissue have been obtained and have now been in culture for more than one year, continuing to produce embryoids. E m b r y o i d f o r m a t i o n in higher p l a n t s from tissue grown from excised h y p o c o t y l s or stems has been r e p o r t e d b y K o n a r a n d N a t a r a j a (1964), W i l m a r a n d H e l l e n d o o m (1968), K a t o a n d T a k e u e h i (1966), a n d others. I n t h e p r e s e n t c o m m u n i c a t i o n t h e f o r m a t i o n of e m b r y o s b y in vitro eulture of excised f r a g m e n t s of h y p o e o t y l s a n d cotyledons of p u m p k i n (Cucurbita pepo L.) will be briefly described. So far we k n o w such embryogenesis in p u m p k i n e x p l a n t s has n o t y e t been r e p o r t e d . Seeds of pumpkin were sterilized with 3 % calcium hypochloride and germinated in test tubes in distilled water under light for 10 d under sterile conditions. Fragments, 1 em long, of cotyledons and hypoeotyls were cut and cultivated individually in test tubes (23 • 200 ram) on 25 ml of culture medium M-S complete (MurashigeSkoog, 1962) + 3% glucose + 0.9% agar + one of the following combinations of growth substances: a) 10 mg/1 fi-indolylbutyric acid (IBA), b) 1 rag/1 IBA, e) 0.3 rag/1 2,4-diehlorophenoxyacetic acid (2,4-D), d) 0.3 mg/l 2,4-D + 10 % autoclaved watermelon sap, e) 0.1 mg/l 2,4-D+10% autoclaved water-melon sap, f) 0.3rag/1 2,4-D + 2 g/1 yeast extract, g) 0.3 rag/1 2,4-D + 0. l mg/1 kinetin, h) 1 mg/1 e-naphthylacetie acid (NAA) + 13.5 rag/1 adenine (natural) 1, i) 1 mg/1 2,4-D + 300 rag/1 casein hydrolysate. The cultures were maintained at 26 ~: 1~ C under artificial light (485-~ 45 lux) from fluorescent lamps IPR 40 W, 220 V (8586~ K 16 h light and 8 h darkness daily). A small p e r e e n t a g e of explants, which d i d n o t i m m e d i a t e l y form callus or roots, p r o d u c e d a callus-like tissue on their cut surfaces after a long period with no g r o w t h a c t i v i t y (3 m o n t h s or more). This new tissue (Figs. 1, 2), t h o r o u g h l y different from t h e old one, grew q u i c k l y a n d d i f f e r e n t i a t e d into more or less n o r m a l e m b r y o s (Fig. 3) a n d y o u n g p l a n t l e t s , a n d into various a b n o r m a l shoots. The e x p l a n t s r e a e t e d a l w a y s 1 Adenin CHR natiirlich puriss. Fluka AG. Buehs SG.
Fig. 1. Callus and plantlets growing on medium with 1 mg/1 IBA Fig. 2. Differentiation of plantlets and roots on medium with 0.3 mg/1 2,4-D + 10 % water-melon sap Fig. 3a-e. Embryo-like bodies (embryoids) grown on the medium with 1 mg/1 IBA. a I-Ieart-shaped and torpedo-like bodies; b Embryo with developing cotyledons; e Mature embryos with well developed cotyledons Fig. 4. Adventitious embryo arising from a giant club-like body. Medium with 1 mg/l IBA. The scale in Fig. 4 (1 ram) refers to Figs. 3 and 4
280
S. Jelaska: Embryoid Formation in Cucurbita pepo
in the same way, independently of the growth substances present. This indicates that the embryo formation in pumpkin is mainly determined by the physiological conditions of the tissue, the age of the culture, and the composition of the medium, rather than by a specific, exogenous growth-regulating substance. Ten different strains of such pumpkin callus tissue have now been cultivated over 1 year, being subcultured every 2 months and continuing to form callus, embryos and plantlets. The embryos grow inside the callus as well as adventitiously on the leaflets and hypocotyls of plantlets which are formed by the callus tissue as found earlier in other plants (Konar and Nataraja, 1964; Konar and Oberoi, 1965; ttom~s and Guillaume, 1967)--or on embryo-like bodies (Fig. 4). Details on the morphology of the induced embryos, their development, and aberrations which were found depending on growth substances added to the medium will be published elsewhere.
References t/om~s, J.L.A., Guillaume, M.: Ph~nom~nes d'organogen~se dans des cultures in vitro de tissus de carotte (Daucus carota L.). Bull. Soc. roy. Bot. Belg. 100, 239-258 (1967). Kato, H., Takeuchi, M. : Embryogenesis from the epidermal cells of carrot hypocotyl. Sci. Papers Coll. Gen. Educ. Univ. Tokyo 16, 245-254 (1966). Konar, R.N., Nataraja, K. : I n vitro control of floral morphogenesis in Ranunculus sceleratus L. PhytomorphoIogy 14, 558-563 (1964). - - Oberoi, u P. : I n vitro developments of embryoids on the cotyledons of Biota orientalis. Phytomorphology l~, 137-140 (1965). Murashige, T., Skoog, F. : A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantarum (Cph.) 15, 473497 (1962). Wilmar, C., Hellendoom, M.: Growth and morphogenesis of Asparagus cells cultured in vitro. Nature (Lond.) 217, 369-370 (1968). (Mrs.) S. Jelaska BotaniSki zavod PMF l~ooseveltov trg 6/III, p.p. 933 YU-41001 Zagreb, Yugoslavia
