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Plant Regeneration in Protoplast Cultures of Tylophora indica
Plant Regeneration in Protoplast Cultures of Tylophora indica MINAL MHATRE*),
v. A. BAPAT and P. S. RAo
Plant Morphogenesis and Tissue Culture Section, Bio-Organic Division, Bhabha Atomic Research Centre, Bombay 400085, India Received January 4, 1984 . Accepted March 20, 1984
Summary Protoplasts isolated from callus tissue derived from freshly cultured stem segments of Tylophora indica (Asclepiadaceae) rapidly divided and resulted in callus. Subsequent embryoid! shoot bud differentiation in callus was observed on MS medium supplemented with auxins and cytokinins. The embryoids!shoot buds on transfer to appropriate basal medium without growth substances grew into plandets. Protoplasts isolated from callus tissue maintained for over five years showed divisions and subsequent callus formation but failed to regenerate plants.
Key words: Tylophora, protoplasts, plant regeneration.
Introduction The demonstration of plant regeneration from protoplasts of several plant species (Vasil and Vasil, 1980; Rao, 1982) offers new options for the improvement of medicinal plants. Protoplast methodology would be useful for selecting protoclonal variants and mutants. Additionally, techniques for somatic hybridization experiments have become available (Schieder and Vasil, 1980) which could be used for the improvement of economically important plants. In order to utilize this technique effectively, efficient and reproducible methods for plant regeneration from protoplasts must be established first. In this communication we describe the isolation, and culture of protoplasts, and subsequent regeneration of a medicinal plant, Tylophora indica.
Materials and Methods Stem segments (ca. 5 mm) excised from young plants of Tylophora indica L. (Asclepiadaceae) growing at the Field Experiment Station, Trombay, Bombay, were surface-sterilized with 0.1 % HgCh for 10 min and rinsed 4 times in sterile distilled water. The sterilized explants were cultured on MS medium with 2,4-D (2 mg .1- 1) and KIN (0.2 mg .1- 1). The cultures were grown in continuous fluorescent light (1000 Ix) at 25 ±2 0c. The segments produced callus within 4 weeks. *) To whom correspondence and reprint requests should be addressed. Abbreviations: Ad, adenine; BAP, 6-benzylaminopurine; CH, casein hydrolysate; CM, coconut milk; 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indoleacetic acid; KIN kinetin; MS, Murashige and Skoog's (1962) basal nutrient medium.
J. Plant Physiol. Vol.
115. pp. 231-235 (1984)
232
MINAL MHATRE, V. A. BAPAT and P. S. RAo
For the isolation of protoplasts, 3-4 weeks old callus tissue derived from stem segments as well as stem callus which was periodically subcultured on MS+2,4-D (2mg·I- I ) + KIN (0.2 mg .1- 1) for 5 years were incubated in different combinations and concentrations of enzymes [cellulase R-10 Onozuka - 0.5%,1 %,2%; macerozyme R-10 (Kinki Yakult) - 0.5%, 1 %; and hemicellulase (Sigma) - 0.5 %, 1 %] for 4-7 h in light on a gyrotary shaker (80 rpm) with 0.6 M mannitol. The pH was adjusted to 5.5-5.8. The enzyme solutions were filter sterilized. The protoplasts were sieved through sieves of pore size 100 p,m and later washed with 0.6 M mannitol by centrifuging at 40 g for 5 mins. For purification of the preparation protoplasts were floated on 20.5 % sucrose solution and after 10 min of centrifugation at 40 g, pipetted off and washed again with 0.6 M mannitol. The protoplast suspension was plated as a thin layer in 6 cm diameter plastic or glass petri dishes with 2 ml of different liquid nutrient media such as MS (Murashige and Skoog, 1962), V-47 (Binding, 1974), VK-M (Binding and Nehls, 1977) supplemented with various growth adjuvants. Mannitol was used as an osmotic stabilizer (ca. 730mOsm). All the petri dishes were sealed with Parafilm. The cultures were kept either in dark or light, at 25±2 0c. As soon as cell colonies became visible the liquid medium was gelled with 2 ml of agar medium, having the same composition of nutrients as before and a final concentration of agar of 0.2 %. Colonies were subcultured on fresh medium of the same composition devoid of mannitol but containing 0.8 % Difco bacto agar. Some cultures were kept in continuous light and others in dark. To induce regeneration, pieces of protoplast derived callus were cultured on MS medium supplemented with various growth regulators.
Results and Discussion
Satisfactory yield of protoplasts was obtained in callus tissues incubated for 7 h in an enzyme mixture comprising of cellulase (2 %), macerozyne (1 %), and hemicellulase (1 %). A correlation was also observed between the yield of protoplasts and the source from which the tissues were derived. A good yield of viable protoplasts was obtained from callus derived from freshly cultured stem segments in contrast to callus tissues which had been maintained for 5 years as continuous cultures. One gram of the callus was enough to liberate a sufficient number of protoplasts. Freshly isolated protoplasts were spherical with prominent nuclei and showed good cytoplasmic streaming. Protoplasts of various sizes were observed, but large-sized ones were often vacuolated. Within 72 h, swelling of protoplasts took place, followed by budding and shape distortions indicating the synthesis of the new cell wall. First division generally occurred within a week or sometimes within two weeks. The density of the suspension was 105 protoplasts per ml medium in order to obtain a high frequency of division. The first division was rapidly followed by subsequent diTable 1: Response of protoplasts of Tylophora indica to various media. Medium
Frequency of division
MS+2,4-D (1)+BAP (1) MS+2,4-D (l)+CM (10%) MS+2,4-D (l)+Ad (10)+CM (10%)+CH (200) MS+2,4-D (1)+BAP (l)+CM (10%)+CH (200)
+++ ++ +++ ++++
Concentration of growth adjuvants: mg .1- 1• Frequency of division: + + moderate, + + + high, + + + + intense.
J. Plant Physiol. Vol.
115. pp. 231-235 (1984)
Plant regeneration from Tylophora protoplasts
233
Fig. 1 (A-B): Plantlet regeneration from protoplast culture in Tylophora indica. A: Protoplast-derived callus tissue showing embryogenesis. - B: Regenerated plantlets.
visions. A fairly good (60 %) rate of division occurred on MS medium supplemented with various growth adjuvants (Table 1). Of the total number of dividing protoplasts about 50-60 % showed sustained divisions leading to colonies. Protoplast derived cell colonies which were intensely green were transferred to MS media supplemented with numerous combinations of 2,4-D, NAA, BAP and KIN. MS containing 2,4-D (1 mg .1- 1) and KIN (0.2 mg .1- 1) stimulated the growth of the callus. To induce plant development, protoplast derived callus was subcultured on various media. Regeneration of embryoids (Fig. 1A) occurred on MS+CM (10%)+2,4-D
J. Plant Physiol.
Vol. 115. pp. 231-235 (1984)
234
MrnAL MHATRE, V. A. BAPAT and P. S. RAo
(1 mg .1- 1) whereas, initiation of shoot buds was observed on media containing low auxin and high cytokinin ratio (Table2). Occasionally some cultures exhibited simultaneous regeneration of embryoids as well as shoot buds. Shoot buds and embryoids were separated from the callus and were cultured on a fresh medium without growth substances to obtain plantlets (Fig. 1 B).
Table2: Morphogenetic responses of protoplast derived callus of Tylophora indica to different hormonal treatments. Medium MS+2,4-D (0.1) MS+2,4-D (0.5) MS+CM (10% v/v)+2,4-D (0.5) MS+CH (200)+2,4-D (0.5) MS+IAA (O.S)+BAP (1) MS+IAA (l)+KlN (1) MS+IAA (l)+BAP (l)+Ad (10) MS+NAA (O.5)+BAP (1) MS+2,4-D (O.l)+BAP (1) MS+2,4-D (O.l)+BAP (0.5) MS+2,4-D (O.l)+KlN (0.5) MS+2,4-D (0. 1) +KlN (1)
Shoot buds
Embryoids + + + +
+ + + + + + + +
+ + + +
- No regeneration, + regeneration. Concentration of growth substances in mg' 1- 1• A significant observation in the present study was the contrast in morphogenetic response between fresh callus and that from the five year old callus stock. Although protoplasts isolated from 5 year old callus showed repeated divisions and formed viable colonies, the callus developed from such colonies failed to undergo differentiation. On the other hand, protoplasts isolated from fresh callus underwent repeated divisions to form small colonies and callus leading to differentiation of embryoids and shoot buds. The failure of protoplast callus obtained from 5-year-old stock callus to regenerate shoots or embryoids may be due to repeated subcultures over a long period, during which the cells have progressively lost the capacity to undergo differentiation. The morphogenetic response of tissue cultures as well as alkaloid synthesis in tissue cultures and regenerated plants of Tylophora have been investigated (Benjamin and Mulchandani, 1973; Benjamin et al., 1979; Rao et al., 1970; Rao and Narayanaswamy, 1972). The present findings indicated the capacity of protoplasts to regenerate plants. It may be worthwhile to use protoplast fusion technology in this species to explore the possibilities of boosting the alkaloid yield. Acknowledgements We thank Dr. Otto Schieder, Max-Planck-Institut fUr Ziichtungsforschung, K61n, FRG for a critical review of the manuscript. We also thank Kernforschungsanlage Jiilich, FRG and Dr.
J. Plant Physiol.
VoL 115. pp. 231-235 (1984)
Plant regeneration from Tylophora protoplasts
235
Schieder for providing the enzymes and necessary equipment under the Indo-FRG Bilateral Agreement which made this study possible.
References BENJAMIN, B. D. and N. B. MULCHANDANI: Planta Med. 23, 393-397 (1973). BENJAMIN, B. D., M. R. HEBLE, and M. S. CHADHA: Z. Pflanzenphysio!. 92, 77-84 (1979). BINDING, H.: Z. Pflanzenphysio!. 74,327-356 (1974). BINDING, H. and R. NEHLS: Z. Pflanzenphysio!. 85, 279-280 (1977). MURASHIGE, T. and F. SKOOG: Physio!. Plant. 15, 473-497 (1962). RAo, P. S., S. NARAYANASWAMY, and B. D. BENJAMIN: Physio!' Plant. 23, 140-144 (1970). RAo, P. S. and S. NARAYANASWAMY: Physio!. Plant. 27, 271-276 (1972). RAo, P. S.: In: Experimental Embryology of Vascular Plants (B. M. JOHRl, ed. ), Springer-Verlag, Berlin, pp. 231-262 (1982). SCHIEDER, o. and 1. K. VASIL: In: Perspectives in Plant Cell and Tissue Culture (1. K. VASIL, ed.), Int. Rev. Cyto!. Supp!. 11 B, Acad. Press, New York, pp. 21-46 (1980). VASIL, 1. K. and V. VASIL: In: Perspectives in Plant Cell and Tissue Culture (I. K. VASIL, ed.), Int. Rev. Cyto!. Supp!. 11 B, Acad. Press, New York, pp. 1-19 (1980).
J. Plant Physiol. Vol.
115. pp. 231-235 {1984}
v. A. BAPAT and P. S. RAo
Plant Morphogenesis and Tissue Culture Section, Bio-Organic Division, Bhabha Atomic Research Centre, Bombay 400085, India Received January 4, 1984 . Accepted March 20, 1984
Summary Protoplasts isolated from callus tissue derived from freshly cultured stem segments of Tylophora indica (Asclepiadaceae) rapidly divided and resulted in callus. Subsequent embryoid! shoot bud differentiation in callus was observed on MS medium supplemented with auxins and cytokinins. The embryoids!shoot buds on transfer to appropriate basal medium without growth substances grew into plandets. Protoplasts isolated from callus tissue maintained for over five years showed divisions and subsequent callus formation but failed to regenerate plants.
Key words: Tylophora, protoplasts, plant regeneration.
Introduction The demonstration of plant regeneration from protoplasts of several plant species (Vasil and Vasil, 1980; Rao, 1982) offers new options for the improvement of medicinal plants. Protoplast methodology would be useful for selecting protoclonal variants and mutants. Additionally, techniques for somatic hybridization experiments have become available (Schieder and Vasil, 1980) which could be used for the improvement of economically important plants. In order to utilize this technique effectively, efficient and reproducible methods for plant regeneration from protoplasts must be established first. In this communication we describe the isolation, and culture of protoplasts, and subsequent regeneration of a medicinal plant, Tylophora indica.
Materials and Methods Stem segments (ca. 5 mm) excised from young plants of Tylophora indica L. (Asclepiadaceae) growing at the Field Experiment Station, Trombay, Bombay, were surface-sterilized with 0.1 % HgCh for 10 min and rinsed 4 times in sterile distilled water. The sterilized explants were cultured on MS medium with 2,4-D (2 mg .1- 1) and KIN (0.2 mg .1- 1). The cultures were grown in continuous fluorescent light (1000 Ix) at 25 ±2 0c. The segments produced callus within 4 weeks. *) To whom correspondence and reprint requests should be addressed. Abbreviations: Ad, adenine; BAP, 6-benzylaminopurine; CH, casein hydrolysate; CM, coconut milk; 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indoleacetic acid; KIN kinetin; MS, Murashige and Skoog's (1962) basal nutrient medium.
J. Plant Physiol. Vol.
115. pp. 231-235 (1984)
232
MINAL MHATRE, V. A. BAPAT and P. S. RAo
For the isolation of protoplasts, 3-4 weeks old callus tissue derived from stem segments as well as stem callus which was periodically subcultured on MS+2,4-D (2mg·I- I ) + KIN (0.2 mg .1- 1) for 5 years were incubated in different combinations and concentrations of enzymes [cellulase R-10 Onozuka - 0.5%,1 %,2%; macerozyme R-10 (Kinki Yakult) - 0.5%, 1 %; and hemicellulase (Sigma) - 0.5 %, 1 %] for 4-7 h in light on a gyrotary shaker (80 rpm) with 0.6 M mannitol. The pH was adjusted to 5.5-5.8. The enzyme solutions were filter sterilized. The protoplasts were sieved through sieves of pore size 100 p,m and later washed with 0.6 M mannitol by centrifuging at 40 g for 5 mins. For purification of the preparation protoplasts were floated on 20.5 % sucrose solution and after 10 min of centrifugation at 40 g, pipetted off and washed again with 0.6 M mannitol. The protoplast suspension was plated as a thin layer in 6 cm diameter plastic or glass petri dishes with 2 ml of different liquid nutrient media such as MS (Murashige and Skoog, 1962), V-47 (Binding, 1974), VK-M (Binding and Nehls, 1977) supplemented with various growth adjuvants. Mannitol was used as an osmotic stabilizer (ca. 730mOsm). All the petri dishes were sealed with Parafilm. The cultures were kept either in dark or light, at 25±2 0c. As soon as cell colonies became visible the liquid medium was gelled with 2 ml of agar medium, having the same composition of nutrients as before and a final concentration of agar of 0.2 %. Colonies were subcultured on fresh medium of the same composition devoid of mannitol but containing 0.8 % Difco bacto agar. Some cultures were kept in continuous light and others in dark. To induce regeneration, pieces of protoplast derived callus were cultured on MS medium supplemented with various growth regulators.
Results and Discussion
Satisfactory yield of protoplasts was obtained in callus tissues incubated for 7 h in an enzyme mixture comprising of cellulase (2 %), macerozyne (1 %), and hemicellulase (1 %). A correlation was also observed between the yield of protoplasts and the source from which the tissues were derived. A good yield of viable protoplasts was obtained from callus derived from freshly cultured stem segments in contrast to callus tissues which had been maintained for 5 years as continuous cultures. One gram of the callus was enough to liberate a sufficient number of protoplasts. Freshly isolated protoplasts were spherical with prominent nuclei and showed good cytoplasmic streaming. Protoplasts of various sizes were observed, but large-sized ones were often vacuolated. Within 72 h, swelling of protoplasts took place, followed by budding and shape distortions indicating the synthesis of the new cell wall. First division generally occurred within a week or sometimes within two weeks. The density of the suspension was 105 protoplasts per ml medium in order to obtain a high frequency of division. The first division was rapidly followed by subsequent diTable 1: Response of protoplasts of Tylophora indica to various media. Medium
Frequency of division
MS+2,4-D (1)+BAP (1) MS+2,4-D (l)+CM (10%) MS+2,4-D (l)+Ad (10)+CM (10%)+CH (200) MS+2,4-D (1)+BAP (l)+CM (10%)+CH (200)
+++ ++ +++ ++++
Concentration of growth adjuvants: mg .1- 1• Frequency of division: + + moderate, + + + high, + + + + intense.
J. Plant Physiol. Vol.
115. pp. 231-235 (1984)
Plant regeneration from Tylophora protoplasts
233
Fig. 1 (A-B): Plantlet regeneration from protoplast culture in Tylophora indica. A: Protoplast-derived callus tissue showing embryogenesis. - B: Regenerated plantlets.
visions. A fairly good (60 %) rate of division occurred on MS medium supplemented with various growth adjuvants (Table 1). Of the total number of dividing protoplasts about 50-60 % showed sustained divisions leading to colonies. Protoplast derived cell colonies which were intensely green were transferred to MS media supplemented with numerous combinations of 2,4-D, NAA, BAP and KIN. MS containing 2,4-D (1 mg .1- 1) and KIN (0.2 mg .1- 1) stimulated the growth of the callus. To induce plant development, protoplast derived callus was subcultured on various media. Regeneration of embryoids (Fig. 1A) occurred on MS+CM (10%)+2,4-D
J. Plant Physiol.
Vol. 115. pp. 231-235 (1984)
234
MrnAL MHATRE, V. A. BAPAT and P. S. RAo
(1 mg .1- 1) whereas, initiation of shoot buds was observed on media containing low auxin and high cytokinin ratio (Table2). Occasionally some cultures exhibited simultaneous regeneration of embryoids as well as shoot buds. Shoot buds and embryoids were separated from the callus and were cultured on a fresh medium without growth substances to obtain plantlets (Fig. 1 B).
Table2: Morphogenetic responses of protoplast derived callus of Tylophora indica to different hormonal treatments. Medium MS+2,4-D (0.1) MS+2,4-D (0.5) MS+CM (10% v/v)+2,4-D (0.5) MS+CH (200)+2,4-D (0.5) MS+IAA (O.S)+BAP (1) MS+IAA (l)+KlN (1) MS+IAA (l)+BAP (l)+Ad (10) MS+NAA (O.5)+BAP (1) MS+2,4-D (O.l)+BAP (1) MS+2,4-D (O.l)+BAP (0.5) MS+2,4-D (O.l)+KlN (0.5) MS+2,4-D (0. 1) +KlN (1)
Shoot buds
Embryoids + + + +
+ + + + + + + +
+ + + +
- No regeneration, + regeneration. Concentration of growth substances in mg' 1- 1• A significant observation in the present study was the contrast in morphogenetic response between fresh callus and that from the five year old callus stock. Although protoplasts isolated from 5 year old callus showed repeated divisions and formed viable colonies, the callus developed from such colonies failed to undergo differentiation. On the other hand, protoplasts isolated from fresh callus underwent repeated divisions to form small colonies and callus leading to differentiation of embryoids and shoot buds. The failure of protoplast callus obtained from 5-year-old stock callus to regenerate shoots or embryoids may be due to repeated subcultures over a long period, during which the cells have progressively lost the capacity to undergo differentiation. The morphogenetic response of tissue cultures as well as alkaloid synthesis in tissue cultures and regenerated plants of Tylophora have been investigated (Benjamin and Mulchandani, 1973; Benjamin et al., 1979; Rao et al., 1970; Rao and Narayanaswamy, 1972). The present findings indicated the capacity of protoplasts to regenerate plants. It may be worthwhile to use protoplast fusion technology in this species to explore the possibilities of boosting the alkaloid yield. Acknowledgements We thank Dr. Otto Schieder, Max-Planck-Institut fUr Ziichtungsforschung, K61n, FRG for a critical review of the manuscript. We also thank Kernforschungsanlage Jiilich, FRG and Dr.
J. Plant Physiol.
VoL 115. pp. 231-235 (1984)
Plant regeneration from Tylophora protoplasts
235
Schieder for providing the enzymes and necessary equipment under the Indo-FRG Bilateral Agreement which made this study possible.
References BENJAMIN, B. D. and N. B. MULCHANDANI: Planta Med. 23, 393-397 (1973). BENJAMIN, B. D., M. R. HEBLE, and M. S. CHADHA: Z. Pflanzenphysio!. 92, 77-84 (1979). BINDING, H.: Z. Pflanzenphysio!. 74,327-356 (1974). BINDING, H. and R. NEHLS: Z. Pflanzenphysio!. 85, 279-280 (1977). MURASHIGE, T. and F. SKOOG: Physio!. Plant. 15, 473-497 (1962). RAo, P. S., S. NARAYANASWAMY, and B. D. BENJAMIN: Physio!' Plant. 23, 140-144 (1970). RAo, P. S. and S. NARAYANASWAMY: Physio!. Plant. 27, 271-276 (1972). RAo, P. S.: In: Experimental Embryology of Vascular Plants (B. M. JOHRl, ed. ), Springer-Verlag, Berlin, pp. 231-262 (1982). SCHIEDER, o. and 1. K. VASIL: In: Perspectives in Plant Cell and Tissue Culture (1. K. VASIL, ed.), Int. Rev. Cyto!. Supp!. 11 B, Acad. Press, New York, pp. 21-46 (1980). VASIL, 1. K. and V. VASIL: In: Perspectives in Plant Cell and Tissue Culture (I. K. VASIL, ed.), Int. Rev. Cyto!. Supp!. 11 B, Acad. Press, New York, pp. 1-19 (1980).
J. Plant Physiol. Vol.
115. pp. 231-235 {1984}