Plant Cell Reports
Plant Cell Reports (1989) 7:626-627
© Springer-Verlag1989
In vitro plant regeneration from leaf and cotyledon explants of Cucumis melo L. Rob Dirks and Marjan van Buggenum Nunhems Zaden BV, Voort 6, 6083 AC Nunhem, The Netherlands Received February 17, 1988 - Communicated by N. Amrhein
ABSTRACT Five different genotypes from in vitro as well as greenhouse grown melon plants were shown to b e highly responsive for in vitro shoot formation from leaf explants when placed on basic MS medium supplemented with 1 mg/l 6-benzylaminoDurine. In addition, a very suitable regeneration system was obtained when cotyledon pieces of mature seeds were incubated on the same culture medium. In this case, the first shoots already appeared after 10 days of incubation, and hundreds of shoots were formed on the cut surface 3 to 4 weeks later. Explants from mature cotyledons derived from seedlings did not lead to any shoot formation. Abbreviations MS: Murashige and Skoog; BA: 6-benzylaminopurine
IAA: 3-indoleacetic
acid;
INTRODUCTION Introduction of new characters into crop plants by means of ~enetic manipulation requires efficient regeneration procedures from either protoplasts or exDlant cultures. In melon (Cucumis melo L.), plant regeneration from callus cultures has been described by Moreno et al. (1985), Bouabdallah and Branchard (1986), Oridate and Oosawa (1986) and Orts et al. (1987). Plant regeneration from protoplasts was described by Moreno et al. (1984,1986) and Roig et al. (1986). In this paper we describe an efficient method for shoot regeneration from leaf explants and cotelydon pieces dissected out of mature seeds. MATERIALS AND METHODS Seeds of melon cultivars respectively Accent, Galia, 4215, Preco and Viva (all Nunhems F1 hybrids; the latter two are designated as Charentais types; 4215 is a Diamex type) were used in regeneration studies. a) explants from mature seeds: seeds were sterilized in a commercial chlorine solution (5%) for 25 minutes and subsequently washed three times in sterile distilled water. Embryos were dissected out of their seed coats and the cotyl~dons were cut into 2 square pieces of about 0.4 cm " The pieces were placed on culture media and incubated in a
Offprint requests to: R. Dirks
Fig I. Shoot regeneration from leaf explants (A) and mature cotyledon dissected out of the seed coat (B) (Pictures were taken I month after incubation.) culture room at 25°C with a light intensity of 3000 lux ( cool white fluorescent lamps). b) leaf and cotyledon explants from in vitro grown plants: seeds were sterilized as described above, and sown on MS medium (Murashige and Skoog 1962) containing 20 g/l sucrose. After germination, seedlings were transferred into plastic jars containing the same medium. Explants from leaves and mature cotyledons were taken when they were fully expanded. Incubation was carried out as described above.
627 All media were autoclaved
at 120 C ~ for 15 minutes.
RESULTS AND DISCUSSION In all experiments basic MS (Murashige and Skoog 1962) medium was used ,containin~ MS salts and vitamins and 30 g/l sucrose. The effects of BA concentrations at respectively i, 2.5 and 5 m~/l in combination with IAA at 0, 0.1, 0.25 or i mg/l were investigated on leaf explants. Best results were obtained at BA of 1 mg/l without IAA. For leaf explants no clear differences were observed between the different cultivars and about 20 to 50 shoots appeared on every leaf piece in four weeks time (see Fig IA). Substitution of zeatine for BA did not lead to noticable improvements. Explants from mature cotyledons only sporadically formed shoot buds and were usually overgrown with callus. On the other hand when cotyledon pieces were isolated when still encapsulated in the seed coat, over a hundred shoots appeared within 3 to 4 weeks (see Fig IB), the first shoots already formed as early as 10 days after bein~ placed on medium. In this case there was a clear cut effect of the genotype: cultivars Galia, Accent and Preco gave far better results than ~iva and 4215 although in the last cases at least 10 shoots were formed on every explant. Addition of IAA in these experiments only stimulated the formation of callus, and here also the best BA concentration proved to be 1 m~/l. Shoots that were transplanted on MS medium with 20 g/l of sucrose grew out further a n d r o o t i ~ was obtained on the same medium after one more round of subculture. Rooted Dlantlets were succesfully transferred into the greenhouse.
It is clear that in view of the economical importance of melon, this regeneration procedure could render melon a very attractive crop plant to be used for introduction of new genetic traits by transformation with A~robacterium.
REFERENCES Bouabdallah L, Branchard M (1986) Z.Pflanzenz~chtg. 96:82-85 Kathal R, Bhatnager SP, Bhojawani SS (1986) J.Plant Physiol. 1 2 6 : 5 9 - 6 2 Moreno V, Garcia-Sogo M, Granell I, Garcia-Sogo B, Roi~ LA (1985) Plant Cell Tissue Organ Culture 5: 139-146 Moreno V, Zubeldia L, Garcia-Sogo B, Nuez F, Roi~ LA (1986) In: Horn W, Jensen CJ, Odenbach W, Schieder O (ed) Genetic Manipulation in Pl~nt Breeding, Proceedings of the International EucarDia Symposium. September 8-13, 1985. Walter de Gruyter, Berlin New York. pp491-493. Moreno V, Znbeldia L, Roig LA (1984) Plant Sci. letters 3 4 : 1 9 5 - 2 0 1 Murashige T, Skoog F (1962) Physiol. Plant. 15: 473497 Oridate T, Oosawa K (1986) Japan J. Breed. 36: 424428 Orts MC, Garcia-Sogo B, Roche MV, Roi~ LA, Moreno V (1987) HortScience 2 2 : 6 6 6 Roig LA, Zubeldia L, Orts MC, Roche MV, Moreno V (1986) Curbitits Genetic cooperative 9 : 7 4 - 7 7
Plant Cell Reports (1989) 7:626-627
© Springer-Verlag1989
In vitro plant regeneration from leaf and cotyledon explants of Cucumis melo L. Rob Dirks and Marjan van Buggenum Nunhems Zaden BV, Voort 6, 6083 AC Nunhem, The Netherlands Received February 17, 1988 - Communicated by N. Amrhein
ABSTRACT Five different genotypes from in vitro as well as greenhouse grown melon plants were shown to b e highly responsive for in vitro shoot formation from leaf explants when placed on basic MS medium supplemented with 1 mg/l 6-benzylaminoDurine. In addition, a very suitable regeneration system was obtained when cotyledon pieces of mature seeds were incubated on the same culture medium. In this case, the first shoots already appeared after 10 days of incubation, and hundreds of shoots were formed on the cut surface 3 to 4 weeks later. Explants from mature cotyledons derived from seedlings did not lead to any shoot formation. Abbreviations MS: Murashige and Skoog; BA: 6-benzylaminopurine
IAA: 3-indoleacetic
acid;
INTRODUCTION Introduction of new characters into crop plants by means of ~enetic manipulation requires efficient regeneration procedures from either protoplasts or exDlant cultures. In melon (Cucumis melo L.), plant regeneration from callus cultures has been described by Moreno et al. (1985), Bouabdallah and Branchard (1986), Oridate and Oosawa (1986) and Orts et al. (1987). Plant regeneration from protoplasts was described by Moreno et al. (1984,1986) and Roig et al. (1986). In this paper we describe an efficient method for shoot regeneration from leaf explants and cotelydon pieces dissected out of mature seeds. MATERIALS AND METHODS Seeds of melon cultivars respectively Accent, Galia, 4215, Preco and Viva (all Nunhems F1 hybrids; the latter two are designated as Charentais types; 4215 is a Diamex type) were used in regeneration studies. a) explants from mature seeds: seeds were sterilized in a commercial chlorine solution (5%) for 25 minutes and subsequently washed three times in sterile distilled water. Embryos were dissected out of their seed coats and the cotyl~dons were cut into 2 square pieces of about 0.4 cm " The pieces were placed on culture media and incubated in a
Offprint requests to: R. Dirks
Fig I. Shoot regeneration from leaf explants (A) and mature cotyledon dissected out of the seed coat (B) (Pictures were taken I month after incubation.) culture room at 25°C with a light intensity of 3000 lux ( cool white fluorescent lamps). b) leaf and cotyledon explants from in vitro grown plants: seeds were sterilized as described above, and sown on MS medium (Murashige and Skoog 1962) containing 20 g/l sucrose. After germination, seedlings were transferred into plastic jars containing the same medium. Explants from leaves and mature cotyledons were taken when they were fully expanded. Incubation was carried out as described above.
627 All media were autoclaved
at 120 C ~ for 15 minutes.
RESULTS AND DISCUSSION In all experiments basic MS (Murashige and Skoog 1962) medium was used ,containin~ MS salts and vitamins and 30 g/l sucrose. The effects of BA concentrations at respectively i, 2.5 and 5 m~/l in combination with IAA at 0, 0.1, 0.25 or i mg/l were investigated on leaf explants. Best results were obtained at BA of 1 mg/l without IAA. For leaf explants no clear differences were observed between the different cultivars and about 20 to 50 shoots appeared on every leaf piece in four weeks time (see Fig IA). Substitution of zeatine for BA did not lead to noticable improvements. Explants from mature cotyledons only sporadically formed shoot buds and were usually overgrown with callus. On the other hand when cotyledon pieces were isolated when still encapsulated in the seed coat, over a hundred shoots appeared within 3 to 4 weeks (see Fig IB), the first shoots already formed as early as 10 days after bein~ placed on medium. In this case there was a clear cut effect of the genotype: cultivars Galia, Accent and Preco gave far better results than ~iva and 4215 although in the last cases at least 10 shoots were formed on every explant. Addition of IAA in these experiments only stimulated the formation of callus, and here also the best BA concentration proved to be 1 m~/l. Shoots that were transplanted on MS medium with 20 g/l of sucrose grew out further a n d r o o t i ~ was obtained on the same medium after one more round of subculture. Rooted Dlantlets were succesfully transferred into the greenhouse.
It is clear that in view of the economical importance of melon, this regeneration procedure could render melon a very attractive crop plant to be used for introduction of new genetic traits by transformation with A~robacterium.
REFERENCES Bouabdallah L, Branchard M (1986) Z.Pflanzenz~chtg. 96:82-85 Kathal R, Bhatnager SP, Bhojawani SS (1986) J.Plant Physiol. 1 2 6 : 5 9 - 6 2 Moreno V, Garcia-Sogo M, Granell I, Garcia-Sogo B, Roi~ LA (1985) Plant Cell Tissue Organ Culture 5: 139-146 Moreno V, Zubeldia L, Garcia-Sogo B, Nuez F, Roi~ LA (1986) In: Horn W, Jensen CJ, Odenbach W, Schieder O (ed) Genetic Manipulation in Pl~nt Breeding, Proceedings of the International EucarDia Symposium. September 8-13, 1985. Walter de Gruyter, Berlin New York. pp491-493. Moreno V, Znbeldia L, Roig LA (1984) Plant Sci. letters 3 4 : 1 9 5 - 2 0 1 Murashige T, Skoog F (1962) Physiol. Plant. 15: 473497 Oridate T, Oosawa K (1986) Japan J. Breed. 36: 424428 Orts MC, Garcia-Sogo B, Roche MV, Roi~ LA, Moreno V (1987) HortScience 2 2 : 6 6 6 Roig LA, Zubeldia L, Orts MC, Roche MV, Moreno V (1986) Curbitits Genetic cooperative 9 : 7 4 - 7 7
