Abstract
A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO4), and a protectant (CaCl2 2H2O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.
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References
Bellincampi D and Morpugo G 1987 Conditioning factor affecting growth in plant cells in culture;Plant Sci. 51 83–91
Butt A D 1985 A general method for the high-yielding isolation of mesophyll protoplasts from deciduous tree species;Plant Sci. 42 55–59
Cocking E C 1960 A method for the isolation of plant protoplasts and vacuoles;Nature (London) 187 927–929
d’Utra Vaz F B, Slamet I H, Khatum A, Cocking E C and Power J B 1992 Protoplast culture in high molecular oxygen atmosphere;Plant Cell Rep. 11 416–418
Hedrich R, Raschke K and Stitt M 1985 A role for Fructose 2,6-Bisphosphate in regulating carbohydrate metabolism in guard cells;Plant Physiol. 79 977–982
Kao K N and Michayluk M K 1975 Nutritional requirements for growth ofVinca hajastana cells and protoplasts at a very low population density in liquid media;Planta 126 105–110
Kyozuka J, Hayashi Y and Shinomoto K 1987 High frequency plant regeneration from rice protoplasts by novel nurse culture methods;Mol. Gen. Genet. 206 408–413
Lee L, Schroll R E, Grimes H D and Hodges T K 1990 Plant regeneration from indica rice (Oryza sativa L.) protoplasts;Planta 178 325–333
Maheshwari S C, Gill R, Maheshwari N and Gharyal P K 1986 The isolation and culture of protoplasts; inDifferentiation of protoplasts and transformed plant cells (eds) J Reinert and Binding (New York: Springer-Verlag) pp 20–48
Mei-Lei M C T, Reitveld E M, Van Marrewijk G A M and Kool A J 1987 Regeneration of leaf mesophyll protoplasts of tomato cultivars (L. esculentum): factors important for efficient protoplast culture and plant regeneration;Plant Cell Rep. 6 172–175
Moore B D, Monson R K, Ku M S B and Edwards G E 1988 Activities of principal photosynthetic and photorespiratory enzymes in leaf mesophyll and bundle sheath protoplasts from the C3–C4 intermediateFlavaria ramosissima;Plant Cell Physiol. 29 999–1006
Niedz R P, Rutter S M, Handley L W and Sink K C 1985 Plant regeneration from leaf protoplasts of six tomato cultivars;Plant Sci. 39 199–204
Sankara Rao K and Srikantha T 1986 A simple method for the preparation of mesophyll protoplasts; inProceedings of the VI International Congress of Plant Tissue and Cell Culture, Minnesota, p 37
Sankara Rao K and Gunasekari K 1991 Control of morphogenesis in tobacco protoplast cultures: Organogenesis Vs. embryogenesis;Indian J. Biochem. Biophys. 28 467–471
Shahin E A 1985 Totipotency of tomato protoplasts;Theor. Appl. Genet. 69 235–240
Thirumala Devi M, Vani T, Malla Reddy M and Raghavendra A S 1992 Rapid isolation of mesophyll and guard cell protoplasts from pea and maize leaves;Indian J. Exp. Biol. 30 424–428
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Rao, K.S., Prakash, A.H. A simple method for the isolation of plant protoplasts. J. Biosci. 20, 645–655 (1995). https://doi.org/10.1007/BF02703304
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DOI: https://doi.org/10.1007/BF02703304