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Clonal propagation of Zizyphus species through tissue culture
Scientia Horticulturae, 51 (1992) 165-168
i65
Elsevier Science Publishers B.V., Amsterdam
Short Communication
Clonal propagation of Zizyphus species through tissue culture T.S. Rathore, R.P. Singh, N.S. Deora and N.S. Shekhawat Plant Biotechn~lo~, Laboratory, Department of Botany, University of Jodhpur, Jodhpur 342 OOl, India (Accepted 20 January 1992)
ABSTRACT Rathore, T.S., Singh, R.P., Deora, N.S. and Shekhawat, N.S., 1992. Clonal propagation of Zizyphus species through tissue culture. Scientia Hortic., 5 l" 165-168. Protocols for cloning of Zizyphus nummularia and Z. mauritiana, plants of arid horticulture and forestry, through tissue ctdture were developed. From a nodal stem explant ofZ. nummularia, five to seven shoots proliferated on Murashige and Skoog's (MS) medium containing 6-benzylaminopurine (BAP) 5.0 mg l- ~+indole-3-acetic acid (IAA) 0.05 mg l- ~. While on MS+ 7.5 mg l- ~BAP ~-0.l m g l i IAA, four to five shoots were produced from the nodal region ofexplant of Z. mauritiana. Shoots generated in vitro could be further multiplied on fresh medium. The isolated shoots were placed for rooting on a filter paper bridge in White's liquid medium conlaining 25 mg l-I IBA; after 48 h these shoots were transferred to agar-gelled hormone-free White's medium. Regenerated plants were hardened and transferred to pots. Keywords: clonal multiplication; plantlcts; shoot segment; Z,..~,tJhu~mauritiana; Zizyphus nummularia. Abbreviations: BAP = 6-benzylaminopurine; IAA = indole-3-acetic acid; IBA = indole-3-butyric acid; Kn = 6-furfurylaminopurine; MS = Murashige and Skoog's medium; NAA = naphthaleneacetic acid; NOA = 2-naphthoxyacetic acid; PVP = polyvinylpyrrolidone.
INTRODUCTION
Zizyphus nummularia (Burm. f. ) Wt. and Arn. and Zizyphus mauritiana Lamk. (family Rhamnaceae) are drought- and heat-resistant plants of arid horticulture and forestry. Both the plant species are ecologically and economically important. Natural propagation of these plants is through seed. These plants are out-breeding and a wide range of genetic variability exist~ in naCorrespondence to: N.S. Shekhawat, UGC-Research Scientist, Department of Botany, University of Jodhpur, Jodhpur 342 001, India.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4238/92/$05.00
166
T.S. RATHORE ET AL.
ture. Therefore selected germplasm cannot be maintained if propagated through seed. Plant tissue culture techniques offer promises for mass and clonal multiplication of woody plants (Dunstan and Thorpe, 1986; Cheliak and Rogers, 1990 ). These techniques were used previously for grafted Zizyphus sp. (Goyal and Arya, 1985 ). Tissue culture protocols for cloning of Z. nummul~ria and Z. mauritiana are described in the present communication. MATERIALS AND METHODS
Shoot segments (fifth to eighth node below the shoot apices) were harvested from mature and selected plants of Z. mauritiana and Z. nummularia during the months of March-April and July-August. These were cut into single node ( 1.0-1.5 cm × 0.2-0.3 cm) segments and surface sterilized with 0. 1% mercuric chloride for 3-4 min and then washed 10-12 times (each wash 2-3 min ) with autoclaved water containing polyvinylpyrrolidone (PVP) (0.25%), ascorbic acid ( 100 mg l-z ) and citric acid (50 mg l- ~). The surface-sterilized explants were cultured on Murashige :,nd Skoog (MS) (Murashige and Skoog, 1962) medium. Ascorbic acid (50 mg l- ~), citric acid (25 mg l- ~) and adenine sulphate (25 mg l -z ) were incorporated into the culture medium. The culture medium for Z. mauritiana was supplemented wRh activated charcoal (500-2500 mg l -z ) and PVP (tool. wt. 40 000; 250-2500 mg l-Z). Various auxins, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and naphthaleneacetic acid (NAA); 0.01-1.0 mg l -~, and cytokinins, 6-furfurylaminopurine (Kn) and 6-benzylaminopurine (BAP), 0.l-10 mg l- ~, were incorporated into the culture media to find out the optimum concentration and combination of suitable growth regulators for each of the species. The cultures were incubated at temperatures ranging from 26_2°C to 33 ± 2 °C under 3000 Ix light intensity for 12 h day-~ photoperiod and 60% relative humidity. Differentiated shoots were multiplied on fresh cult~re medium. Tissue culture-derived shoots were first cultured on filter paper in White's liquid medium (White, 1943) containing IBA ( 10-50 mg 1-I ) for 12-72 h and subsequently transferred to hormone-free White's agar medium. Rooted plantlets (8-12 cm long) were removed from the culture vessels and washed carefully with water. The plantlets were transferred to clay pots containing a mixture of sand and vermiculite (4: l ). RESULTS AND DISCUSSION
Explants harvested during July and August were found to be the best for culture. In Z. mauritiana, exudation of mucilagenous substances was encountered, which could be overcome by repeated washing ( 10-12 times) with an autoclaved aqueous solution of PVP, ascorbic acid and citric acid. Addition of activated charcoal (1000 mg l -~ ) and PVP (500 mg l -~ ) to the culture
167
CLONAL PROPAGATION OF ZIZYPHUS THROUGH TISSUE CULTURE
medium checked browning ofexplants. Using MS medium supplemented with 0.05 rng 1-~ IAA + 5 mg 1-~ BAP, five to seven shoots were produced from the nodal region of the stem explant of Z. nummularia (Fig. i ), whereas four to five shoots per node proliferated from an explant of Z. mauritiana on MS medium with 0.1 mg 1-~ IAA + 7.5 mg 1-~ BAP. An increase in the auxin levels or replacement of IAA by NAA or IBA caused callusing of explants. The optimum temperature for proliferation of shoots from the explant was found to be 31 + 2 ° C. This is probably a reflection of the nature of these plants which survive and thrive in hot climates. The in vitro-produced shoots were cut into nodal segments ( 1.5-2.0 cm) and cultured. Each of these produced three to five shoots in Z. nummularia on MS + 0.01 mg 1- ~ IAA + 2.5 mg IBAP (Fig. 2) and two to three shoots in Z. mauritiana on MS + 0.05 mg IIAA +4.0 mg 1-~ BAP. The original explant of Z. nummularia could be repeatedly transferred (four times) using a culture interval of 20-25 days between transfers producing four to five shoots per explant each time. The in vitro-produced shoots were rooted by a two-step method; the shoots were first cultured on a filter paper bridge in White's liquid medium containing 25.0 mg 1-~ of IBA for 48 h followed by transfer to semi-solid medium. About 75-80% and 45-50% of Z. nummularia and Z. mauritiana shoots, respectively, rooted by this method (Fig. 3 ). Plantlets of Z. nummularia could be easily transplanted to pots (Fig. 4). After hardening in the culture room, the potted plants could be transferred to t
Figs. 1-4. In vitro clonai propagation of Z. nummularia. Fig. 1. Shoots induced from nodal shoot segment on MS medium + IAA, 0.05 mg 1- I + BAP, 5.0 mg 1- ~. Fig. 2. Subcultured shoot segment induced multiple shoots on MS + IAA, 0.01 mg !-I + BAP, 2.5 mg 1-~ (4-week-old culture). Fig. 3. Root induction from an isolated shoot by two-step method. First shoots were treated with IBA, 25 mg 1-~ in White's liquid medium with a filter paper bridge for 48 h and then treated shoots were cultured on hormone-free White's agar-gelled medium; 3-week-old culture. Fig. 4. Potted plants (6 weeks old ).
168
T.S. R A T H O R E ET AL.
a net house. More than 60% of the field-transferred plants of Z. nummularia survived. The survival percentage in Z. mauritiana was 40%. An earlier report (David and Vasaikar, 1980) on tissue culture of Zizyphus xylopyra dealt with seedling-derived explants, and therefore may not be relevant for cloning of mature plants. Goyal and Arya (1985) described methods for multiplication of Z. mauritiana (cultivars, 'Apple' and 'Gola' ). The number of shoots induced was less than in the present study. This was probably because they did not incorporate the required auxins and cytokinins in the shoot-induction medium. The procedure described here is reproducible and efficient and can be used for cloning mature Zizyphus plants. ACKNOWLEDGEMENTS
We are grateful to Professor D.No Sen, Head of the Department of Botany, University of Jodhpur, Jodhpur, for providing facilities and support for this research.
REFERENCES
Cheliak, W.M. and Rogers, D.L., 1990. Integrating biotechnology into tree improvement programs. Can. J. For. Res., 20: 452-463. David, S.B. and Vasaikar, S.M., 1980. Rapid multiplication of economically important plants by tissue culture and organ culture. In: P.S. Rao, M.R. Heble and M.S. Chadha (Editors), Plant Tissue Culture, Genetic Manipulation and Somatic Hybridization &Plant Cells. BARC, Bombay, pp. 285-289. Dunstan, D.I. and Thorpe, T.A., 1986. Regeneration in forest trees. Ill: I.K. Vasil (Editor), Cell Culture and Somatic Cell Genetics of Plants, Voi. 3. Academic Press, New York, pp. 223241. Goyal, Y. and Arya, H.C., 1985. Tissue culture of desert trees' II. Clonal multiplication of Zi. zyphus in vitro. J. Plant Physiol., 119: 399-404. Murashig¢, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassa~s with tobacco tissue cultures. Physiol. Plant., 15: 473-497. White, P.R., 1943. A Handbook of Plant Tissue Culture, Jaques Catell Press, Lancaster, p. 77.
i65
Elsevier Science Publishers B.V., Amsterdam
Short Communication
Clonal propagation of Zizyphus species through tissue culture T.S. Rathore, R.P. Singh, N.S. Deora and N.S. Shekhawat Plant Biotechn~lo~, Laboratory, Department of Botany, University of Jodhpur, Jodhpur 342 OOl, India (Accepted 20 January 1992)
ABSTRACT Rathore, T.S., Singh, R.P., Deora, N.S. and Shekhawat, N.S., 1992. Clonal propagation of Zizyphus species through tissue culture. Scientia Hortic., 5 l" 165-168. Protocols for cloning of Zizyphus nummularia and Z. mauritiana, plants of arid horticulture and forestry, through tissue ctdture were developed. From a nodal stem explant ofZ. nummularia, five to seven shoots proliferated on Murashige and Skoog's (MS) medium containing 6-benzylaminopurine (BAP) 5.0 mg l- ~+indole-3-acetic acid (IAA) 0.05 mg l- ~. While on MS+ 7.5 mg l- ~BAP ~-0.l m g l i IAA, four to five shoots were produced from the nodal region ofexplant of Z. mauritiana. Shoots generated in vitro could be further multiplied on fresh medium. The isolated shoots were placed for rooting on a filter paper bridge in White's liquid medium conlaining 25 mg l-I IBA; after 48 h these shoots were transferred to agar-gelled hormone-free White's medium. Regenerated plants were hardened and transferred to pots. Keywords: clonal multiplication; plantlcts; shoot segment; Z,..~,tJhu~mauritiana; Zizyphus nummularia. Abbreviations: BAP = 6-benzylaminopurine; IAA = indole-3-acetic acid; IBA = indole-3-butyric acid; Kn = 6-furfurylaminopurine; MS = Murashige and Skoog's medium; NAA = naphthaleneacetic acid; NOA = 2-naphthoxyacetic acid; PVP = polyvinylpyrrolidone.
INTRODUCTION
Zizyphus nummularia (Burm. f. ) Wt. and Arn. and Zizyphus mauritiana Lamk. (family Rhamnaceae) are drought- and heat-resistant plants of arid horticulture and forestry. Both the plant species are ecologically and economically important. Natural propagation of these plants is through seed. These plants are out-breeding and a wide range of genetic variability exist~ in naCorrespondence to: N.S. Shekhawat, UGC-Research Scientist, Department of Botany, University of Jodhpur, Jodhpur 342 001, India.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4238/92/$05.00
166
T.S. RATHORE ET AL.
ture. Therefore selected germplasm cannot be maintained if propagated through seed. Plant tissue culture techniques offer promises for mass and clonal multiplication of woody plants (Dunstan and Thorpe, 1986; Cheliak and Rogers, 1990 ). These techniques were used previously for grafted Zizyphus sp. (Goyal and Arya, 1985 ). Tissue culture protocols for cloning of Z. nummul~ria and Z. mauritiana are described in the present communication. MATERIALS AND METHODS
Shoot segments (fifth to eighth node below the shoot apices) were harvested from mature and selected plants of Z. mauritiana and Z. nummularia during the months of March-April and July-August. These were cut into single node ( 1.0-1.5 cm × 0.2-0.3 cm) segments and surface sterilized with 0. 1% mercuric chloride for 3-4 min and then washed 10-12 times (each wash 2-3 min ) with autoclaved water containing polyvinylpyrrolidone (PVP) (0.25%), ascorbic acid ( 100 mg l-z ) and citric acid (50 mg l- ~). The surface-sterilized explants were cultured on Murashige :,nd Skoog (MS) (Murashige and Skoog, 1962) medium. Ascorbic acid (50 mg l- ~), citric acid (25 mg l- ~) and adenine sulphate (25 mg l -z ) were incorporated into the culture medium. The culture medium for Z. mauritiana was supplemented wRh activated charcoal (500-2500 mg l -z ) and PVP (tool. wt. 40 000; 250-2500 mg l-Z). Various auxins, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and naphthaleneacetic acid (NAA); 0.01-1.0 mg l -~, and cytokinins, 6-furfurylaminopurine (Kn) and 6-benzylaminopurine (BAP), 0.l-10 mg l- ~, were incorporated into the culture media to find out the optimum concentration and combination of suitable growth regulators for each of the species. The cultures were incubated at temperatures ranging from 26_2°C to 33 ± 2 °C under 3000 Ix light intensity for 12 h day-~ photoperiod and 60% relative humidity. Differentiated shoots were multiplied on fresh cult~re medium. Tissue culture-derived shoots were first cultured on filter paper in White's liquid medium (White, 1943) containing IBA ( 10-50 mg 1-I ) for 12-72 h and subsequently transferred to hormone-free White's agar medium. Rooted plantlets (8-12 cm long) were removed from the culture vessels and washed carefully with water. The plantlets were transferred to clay pots containing a mixture of sand and vermiculite (4: l ). RESULTS AND DISCUSSION
Explants harvested during July and August were found to be the best for culture. In Z. mauritiana, exudation of mucilagenous substances was encountered, which could be overcome by repeated washing ( 10-12 times) with an autoclaved aqueous solution of PVP, ascorbic acid and citric acid. Addition of activated charcoal (1000 mg l -~ ) and PVP (500 mg l -~ ) to the culture
167
CLONAL PROPAGATION OF ZIZYPHUS THROUGH TISSUE CULTURE
medium checked browning ofexplants. Using MS medium supplemented with 0.05 rng 1-~ IAA + 5 mg 1-~ BAP, five to seven shoots were produced from the nodal region of the stem explant of Z. nummularia (Fig. i ), whereas four to five shoots per node proliferated from an explant of Z. mauritiana on MS medium with 0.1 mg 1-~ IAA + 7.5 mg 1-~ BAP. An increase in the auxin levels or replacement of IAA by NAA or IBA caused callusing of explants. The optimum temperature for proliferation of shoots from the explant was found to be 31 + 2 ° C. This is probably a reflection of the nature of these plants which survive and thrive in hot climates. The in vitro-produced shoots were cut into nodal segments ( 1.5-2.0 cm) and cultured. Each of these produced three to five shoots in Z. nummularia on MS + 0.01 mg 1- ~ IAA + 2.5 mg IBAP (Fig. 2) and two to three shoots in Z. mauritiana on MS + 0.05 mg IIAA +4.0 mg 1-~ BAP. The original explant of Z. nummularia could be repeatedly transferred (four times) using a culture interval of 20-25 days between transfers producing four to five shoots per explant each time. The in vitro-produced shoots were rooted by a two-step method; the shoots were first cultured on a filter paper bridge in White's liquid medium containing 25.0 mg 1-~ of IBA for 48 h followed by transfer to semi-solid medium. About 75-80% and 45-50% of Z. nummularia and Z. mauritiana shoots, respectively, rooted by this method (Fig. 3 ). Plantlets of Z. nummularia could be easily transplanted to pots (Fig. 4). After hardening in the culture room, the potted plants could be transferred to t
Figs. 1-4. In vitro clonai propagation of Z. nummularia. Fig. 1. Shoots induced from nodal shoot segment on MS medium + IAA, 0.05 mg 1- I + BAP, 5.0 mg 1- ~. Fig. 2. Subcultured shoot segment induced multiple shoots on MS + IAA, 0.01 mg !-I + BAP, 2.5 mg 1-~ (4-week-old culture). Fig. 3. Root induction from an isolated shoot by two-step method. First shoots were treated with IBA, 25 mg 1-~ in White's liquid medium with a filter paper bridge for 48 h and then treated shoots were cultured on hormone-free White's agar-gelled medium; 3-week-old culture. Fig. 4. Potted plants (6 weeks old ).
168
T.S. R A T H O R E ET AL.
a net house. More than 60% of the field-transferred plants of Z. nummularia survived. The survival percentage in Z. mauritiana was 40%. An earlier report (David and Vasaikar, 1980) on tissue culture of Zizyphus xylopyra dealt with seedling-derived explants, and therefore may not be relevant for cloning of mature plants. Goyal and Arya (1985) described methods for multiplication of Z. mauritiana (cultivars, 'Apple' and 'Gola' ). The number of shoots induced was less than in the present study. This was probably because they did not incorporate the required auxins and cytokinins in the shoot-induction medium. The procedure described here is reproducible and efficient and can be used for cloning mature Zizyphus plants. ACKNOWLEDGEMENTS
We are grateful to Professor D.No Sen, Head of the Department of Botany, University of Jodhpur, Jodhpur, for providing facilities and support for this research.
REFERENCES
Cheliak, W.M. and Rogers, D.L., 1990. Integrating biotechnology into tree improvement programs. Can. J. For. Res., 20: 452-463. David, S.B. and Vasaikar, S.M., 1980. Rapid multiplication of economically important plants by tissue culture and organ culture. In: P.S. Rao, M.R. Heble and M.S. Chadha (Editors), Plant Tissue Culture, Genetic Manipulation and Somatic Hybridization &Plant Cells. BARC, Bombay, pp. 285-289. Dunstan, D.I. and Thorpe, T.A., 1986. Regeneration in forest trees. Ill: I.K. Vasil (Editor), Cell Culture and Somatic Cell Genetics of Plants, Voi. 3. Academic Press, New York, pp. 223241. Goyal, Y. and Arya, H.C., 1985. Tissue culture of desert trees' II. Clonal multiplication of Zi. zyphus in vitro. J. Plant Physiol., 119: 399-404. Murashig¢, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassa~s with tobacco tissue cultures. Physiol. Plant., 15: 473-497. White, P.R., 1943. A Handbook of Plant Tissue Culture, Jaques Catell Press, Lancaster, p. 77.