Abstract
A protoplast-to-plant system was developed in Gentiana using a gellan gum-embedding culture. Viable protoplasts could be routinely isolated from in vitro-grown plantlets, and they were embedded in 0.2% gellan gum-solidified B5 medium containing 2 mg l-1 NAA, 0.1 mg l-1 TDZ, 0.1 M sucrose and 0.4 M mannitol. Weekly addition of fresh liquid medium was essential for preventing cell browning. Colony growth was promoted by lowering mannitol concentration of the culture media after one month, and visible colonies were produced after 2 months of culture. Shoot regeneration from protoplast-derived calluses was stimulated by 1 to 10 mg l-1 TDZ in combination with 0.1 mg l-1 NAA. Protoplast-derived plants were recovered following rooting of the shoots in plant growth regulator-free medium and they were successfully transferred to soil.
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Abbreviations
- BA:
-
benzylaminopurine
- FDA:
-
fluorescein diacetate
- FW:
-
fresh weight
- MES:
-
2-N-morpholinoethane sulfonic acid
- NAA:
-
α-naphthaleneacetic acid
- TDZ:
-
N-1,2,3-thiadiazol-5-yl-N′-phenylurea (also called thidiazuron)
References
Al-Atabee JS, Mulligan BJ & Power JB (1990) Interspecific somatic hybrids of Rudbeckia hirta and R. laciniata (Compositae). Plant Cell Rep. 8: 517–520
Dons JJM & Colijn-Hooymans CM (1989) Agarose plating of protoplasts and its applications. In: Bajaj YPS (Ed) Biotechnology in Agriculture and Forestry 8. Plant Protoplasts and Genetic Engineering I (pp 50–62). Springer-Verlag, Berlin
Dragnet KI, Myhre S, Skjak-Brk G & Ostegaard K (1988) Regeneration, cultivation, and differentiation of plant protoplasts immobilized in calcium-alginate beads. J. Plant Physiol. 132: 552–556
Ellis DD, Barczynska H, McCown BH & Nelson N (1991) A comparison of BA, zeatin and thidiazuron for adventitious bud formation from Picea glauca embryos and epicotyl explants. Plant Cell Tiss. Org. Cult. 27: 281–287
Gamborg OL, Miller RA & Ojima K (1968) Nutrient requirements of suspension culture of soybean root cells. Exp. Cell Res. 50: 151–158
Huetteman CA & Preece JE (1993) Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tiss. Org. Cult. 33: 105–119
Ichi T, Koda T, Asai I, Hatanaka A & Sekiya J (1986) Effect of gelling agents on in vitro culture of plant tissues. Agric. Biol. Chem. 50: 2397–2399
Isa T, Ogasawara T & Kaneko H (1990) Regeneration of saffron protoplasts immobilized in Ca-alginate beads. Jpn. J. Breed. 40: 153–157
Koda T, Ichi T, Yamagishi H & Yoshikawa H (1988) Effects of phytohormones and gelling agents on plant regeneration from protoplasts of red cabbage. Agric. Biol. Chem. 52: 2337–2340
Kunitake H & Mii M (1990) Plant regeneration from cell culture-derived protoplasts of statice (Limonium perezii Hubbard). Plant Sci. 70: 115–119
Lu C-Y (1993) The use of thidiazuron in tissue culture. In vitro Cell. Dev. Biol. 29P: 92–96
MacRae S & van Staden J (1990) In vitro culture of Eucalyptus grandis: effect of gelling agents on propagation. J. Plant Physiol. 137: 249–251
Meyer H & van Staden J (1988) In vitro multiplication of Ixia flexuosa. HortSci. 23: 1070–1071
Mii M, Zou Y-M, Sugiyama T, Yanagihara S & Iizuka M (1991) High-frequency callus formation from protoplasts of Vitis labruscana Bailey and Vitis thunbergii Sieb. et Zucc. by embedding in gellan gum. Sci. Hort. 46: 253–260
Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497
Nakano M & Mii M (1992) Protoplast culture and plant regeneration of several species in the genus Dianthus. Plant Cell Rep. 11: 225–228
Nakano M & Mii M (1993a) Somatic hybridization between Dianthus chinensis and D. barbatus through protoplast fusion. Theor. Appl. Genet. 86: 1–5
Nakano M & Mii M (1993b) Interspecific somatic hybridization in Dianthus: selection of hybrids by the use of iodoacetamide inactivation and regeneration ability. Plant Sci. 88: 203–208
Niedz RP (1993) Culturing embryogenic protoplasts of ‘Hamlin’ sweet orange in calcium alginate beads. Plant Cell Tiss. Org. Cult. 34: 19–25
Schlangstedt M, Hermans B, Zoglauer K & Schieder O (1992) Culture of sugar beet (Beta vulgaris L.) protoplasts in alginate-callus formation and root organogenesis. J. Plant Physiol. 140: 339–344
Shillito RD, Paszkowski J & Potrykus I (1983) Agarose plating and a bead type culture enable and stimulate development of protoplast-derived colonies in a number of plant species. Plant Cell Rep. 2: 244–247
Sihachakr D & Ducreux G (1987) Cultural behavior of protoplasts from different organs of eggplant (Solanum melongena L.), and plant regeneration. Plant Cell Tiss. Org. Cult. 11: 179–188
Takahata Y & Jomori H (1989) Plant regeneration from mesophyll protoplasts of gentiana (Gentiana scabra Bungei). Plant Tiss. Cult. Lett. 6: 19–21
Visser C, Qureshi JA, Gill R & Saxena PK (1992) Morphoregulatory role of thidiazuron: substitution of auxin and cytokinin requirement for the induction of somatic embryogenesis in geranium hypocotyl cultures. Plant Physiol. 99: 1704–1707
Widholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Tech. 47: 189–194
Woodson WR (1991) Biotechnology of floricultural crops. HortSci. 26: 1029–1033
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Nakano, M., Hosokawa, K., Oomiya, T. et al. Plant regeneration from protoplasts of Gentiana by embedding protoplasts in gellan gum. Plant Cell Tiss Organ Cult 41, 221–227 (1995). https://doi.org/10.1007/BF00045085
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DOI: https://doi.org/10.1007/BF00045085