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Regeneration of Coryphantha elephantidens (Lem.) Lem. (Cactaceae) from root explants
Scientia Horticulturae 81 (1999) 337±344
Regeneration of Coryphantha elephantidens (Lem.) Lem. (Cactaceae) from root explants Brijmohan Singh Bhau Plant Tissue Culture Lab., Department of Botany, University of Jammu, Jammu-180006, India Accepted 14 December 1998
Abstract Regeneration of Coryphantha elephantidens (Lem.) Lem. from callus cultures was obtained from (5±20 mm long) root explants of in vitro rooted shoots cultivated on the Murashige±Skoog medium, supplemented with different concentrations of growth regulators. Callus proliferation and shoot formation was optimal on a medium supplemented with 9 mM 2,4-D and 4.6 mM Kn. Explant size and sucrose concentration in the medium influenced the regeneration potential of the callus cultures significantly. Callus obtained from 10 mm long explant when cultured on a medium supplemented with 9 mM 2,4-D, 4.6 mM Kn and 7% sucrose induced maximum shoot differentiation. Regenerants were rooted on growth-regulator-free Murashige±Skoog medium and then acclimatized in a greenhouse. Regenerated plants showed 100% survival and performed better than seedlings. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Cactus; Coryphantha elephantidens; Callus; Organogenesis
1. Introduction Coryphantha elephantidens (Lem.) Lem., commonly called elephant-tusk cactus, is an ornamental species valued for its large size and beautiful pink to red flowers (Slaba, 1992). The conventional methods of propagation of the species are inadequate to meet the commercial demand on account of low seed germination, slow growth, and low rate of offshoot production. Although in vitro propagation is a potential alternative for production of cacti for commercial purposes and also for conservation of rare cacti (Clayton et al., 1990), not many tissue culture studies have been conducted on this species. To the best of my knowledge, the present report is the very first on C. elephantidens. In cacti, 0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 0 5 - 9
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different explant sources, namely shoot-tip explants from seedlings, (Smith et al., 1991; Martinez-Vazquez and Rubluo, 1989; Ault and Blackmon, 1987), pith excised from stem tissue of plants growing in nature (Johnson and Emino, 1979) and shoot tips of mature plants have been used for in vitro studies (Clayton et al., 1990). King (1957) has tried different explants, including roots; the latter proved highly recalcitrant although many species of plants have been regenerated from root cultures (Son and Hall, 1990; Komai et al., 1996; Nef-Campa et al., 1996), but there is no report in cacti. Root explants from in vitro grown plants offer several advantages, such as easy manipulation, availability, sterilization and no oxidation of secondary metabolites in the medium; in comparison with the destructive sampling for explant preparation, especially vital for rare plants, small plant size and slow growth of the seedling derived explants of cacti (Hubstenberger et al., 1992). The objective of the present study was to determine the type and optimum concentration of the growth regulators for rapid plantlet regeneration from root explants of C. elephantidens. The paper describes an efficient protocol for plant regeneration from callus derived from root explants of C. elephantidens and successful establishment of the regenerated plantlets in field. 2. Materials and methods The plant material (tubercles 2 1.5 cm2) used for this study was obtained from the Jammu University Botanical Garden. The tubercles were washed thoroughly with running tap water, surface-sterilized first in 70% ethanol for 45 s, and then in 0.2% HgCl2 for 3 min and rinsed with sterile distilled water five times. These sterilized tubercles were cultured on MS (Murashige and Skoog, 1962) medium containing 3% sucrose and 6.6 mM 8-benzylaminopurine (BAP). The multiple shoots formed on these tubercles were rooted on MS basal medium. Root tip explants (5, 10, 15 mm, in length) were collected from four-week old rooted plantlets of C. elephantidens growing on MS basal medium. For callus initiation and organogenesis, the explants were cultured on MS medium, containing 3% sucrose and fortified with different concentrations of auxins (2,4dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1-naphthaleneacetic acid (NAA)), and cytokinins (BAP, kinetin (Kn)) alone or in combination with each other. The pH of the medium was adjusted at 5.8 and gelled with 0.8% bacteriological grade agar (Ranbaxy, India) prior to autoclaving at 1.02 kg cm2 at 1218C for 15 min. Cultures were incubated at 25 28C and 50 5% relative humidity in a culture room. Bajaj fluorescent tubes (40 W) provided 30 mmol mÿ2 sÿ1 cool white light for a 16-h photoperiod, unless otherwise stated. For callus initiation, five explants (5, 10, 15 mm in length) were cultured per treatment with five replicates in Petri plates. The material was first grown in
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darkness for one week and then transferred to light conditions. Pieces of 12-week old calli raised from explants of different sizes were transferred to fresh medium for inducing shoot differentiation; one callus piece (250 mg fresh weight) was cultured per Petri plate. Ten replicates were used per treatment. The effect of sucrose level (0±10% in the medium) on shoot differentiation from callus raised from 10 mm long explants, was investigated. The regenerated shoots were transferred to basal medium for rooting. Rooted plantlets were transferred to pots containing equal quantities of sand and vermiculite, covered with transparent polythene and watered every alternate day for two weeks. Hardened plants were finally transferred to pots containing garden soil, sand and vermiculite (1 : 1 : 1) for further growth. All experiments were repeated. Data were recorded after four weeks of culture initiation. Growth regulator treatment, explant size and sucrose concentration were arranged as factorial in a completely randomized design. Response variables were callus induction, plant regeneration and final plant production. Data on the percentage of explants forming calli were subjected to arcsin transformation for proportions before analysis and then converted back to percentage for presentation in tables (Snedecor and Cochran, 1968). Data were analyzed by ANOVA (p 0.05) and means were compared using Duncan's new multiple range test (Duncan, 1955). 3. Results Calli were initiated from 10 mm long root explants within 10±15 days of inoculation on MS basal medium supplemented with different concentrations of auxins (2,4-D, IAA, IBA, NAA) with kinetin and BAP alone or in combination (Table 1). Proximal root tip portion appeared swollen (possibly due to dissolution of cortical tissue) in the first week of culture. Within 10±15 days of inoculation, whitish and granular calli developed at the cut ends which subsequently covered the entire explant. Callus formation did not occur on explants cultured in media not supplemented with growth regulators. The medium containing 9.06 mM 2,4-D or IBA with 4.6 mM Kn promoted rapid callus growth on explants. Higher concentrations of 2,4-D promoted a high rate of friable callus formation. Calli derived from IAA showed relatively slow rate of growth. The growth rate of callus was faster during the first four weeks of culture after which it declined. The callus was sensitive to manipulation and often became necrotic following subculturing. 3.1. Differentiation of shoots from callus After four weeks in the callus induction medium, the callus raised from 10 mm long explants was subcultured on the same medium for further proliferation. The
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Table 1 Effect of combination of auxins and Kn on callus development and shoot regeneration from 10 mm long root explants of Coryphantha elephantidens after 4 weeks of culture Auxina 2,4-D
IAA
NAA
IBA
Explants with callusb (%)
Callus with shootsb (%)
Number of shoots per explantb
0 2.2 4.5 9.0
0 28ef 84a 88a
0f 42.8c 47.1c 100a
0c 0.3b 0.4b 1.6a
2.8 5.7 11.4
24f 32e 32e
0f 25d 12d
0c 0.1b 0.2b
2.6 5.3 10.7
24f 44d 52c
66.7b 54.5b 15.4c
0.1b 0.3b 0.1b
2.4 4.9 9.8
20f 40f 60b
0f 30d 6.7e
0c 0.1b 0.1b
(mM)
a
Plus 4.6 mM Kn. Values with the same letters are not different from each other at a 5% level by Duncan's new multiple range test. b
calli differentiated into green compact nodular structures that differentiated into green shoots with prominent tubercles and white spines. Maximum number of shoots (1.6 shoots) were formed on MS medium supplemented with 2.2 mM 2,4D and 4.6 mM Kn (Table 1). All calli that were cultured formed shoots on this medium. The shoots were healthy and attained average height of 3.4 cm within four weeks. Kn or BAP alone did not induce any morphogenetic response. Regeneration of shoot buds also occurred in low concentrations of Kn, but less frequently. The nature of auxin employed has significant influence on regeneration of shoots from callus. BAP proved less potent in shoot organogenesis than Kn in combination with different auxins (data not presented). Shoot regeneration from callus (100%) with 1.74 shoots per explant was recorded from callus raised from root explants of 10 mm length (Table 2). Following six subsequent subcultures at four-week intervals on MS medium supplemented with 2.2 mM 2,4-D and 4.6 mM Kn, the regeneration potential and number of shoot buds per culture remained fairly constant.
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Table 2 Effect of root explant size on callus formation and shoot regeneration in Coryphantha elephantidens on MS medium supplemented with 2.2 mM 2,4-D and 4.6 mM Kn after 4 weeks of culture Explant size (mm)
Explants with callusa (%)
Callus with shootsa (%)
Number of shoots per explanta
5 10 15 20
12c 90a 64b 56b
33.3bc 100a 40b 26c
0.04c 1.7a 0.6b 0.5bc
a
Values with the same letters are not different from each other at a 5% level by Duncan's new multiple range test.
3.2. Effect of explant size Root segment length had significant influence on callus induction and subsequent shoot regeneration from the callus (Table 2). Maximum callus induction (100%) was obtained from root segments of 10 mm length. As the explant size was reduced (5 mm) or increased (15 mm), the rate of regeneration after callogenesis and the number of shoots formed per explant decreased significantly. 3.3. Effect of sucrose Sucrose level in the medium influenced the regeneration rate significantly (Table 3). Maximum number of shoots (3.3) per piece of callus was produced when sucrose concentration in the medium was 7%. Other levels of sucrose (0, 1.5, 3.0 or 10) lowered the number of shoots formed per callus piece and induced formation of stunted shoots. Elimination of sucrose resulted in the death of callus. Sucrose level also has marked influence on shoot length; maximum shoot length is attained with 5±7% sucrose concentration. 3.4. Induction of rooting and field establishment All the isolated shoots rooted when transferred to MS medium free of growth regulators. Root initiation from the shoot was recorded two weeks after inoculation. Roots formed were thick, with an average length of 2.2 cm. Rooted plantlets transferred to pots containing sand and vermiculite showed 100% survival. Hardened plants transferred to pots containing mixture of garden soil, sand and vermiculite showed luxurious growth.
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Table 3 Effect of sucrose concentration on shoot development of Coryphantha elephantidens explants after 12 weeks of culture Sucrosea (%)
Average no. of shoots/explantb
Shoot lengthb (mm)
0 1.0 3.0 5.0 7.0 10
0c 0.2c 1.8b 2.4ab 3.2a 1.4bc
0d 18bc 34b 42a 40a 10c
a
Plus 4.6 mM Kn and 2.2 mM 2,4-D. Means with the same letters in a column are not significantly different from each other at a 5% level by Duncan's new multiple range test. b
4. Discussion The study reveals the capacity of C. elephantidens,, Cactaceae, to regenerate plantlets from root explants. This is the first report for the family. Root explant, used in the present study, has an advantage over the other more widely used explants, such as young seedlings, pith tissue from the stem and tubercle tissue (Johnson and Emino, 1979; Oliveira et al., 1995; Smith et al., 1991; Kolar et al., 1976; Steinhart, 1962) in that they can be employed even in multiplying without killing the source plants. Unlike the non-organogenic callus which can develop from various explants, formation of organogenic callus seems to be controlled by auxin cytokinin ratio of the medium. In case of C. elephantidens, Kn has proved most suitable for organogenesis, which is in agreement with the findings of Oliveira et al. (1995). In case of C. macromeris, BAP proved to be a more potent cytokinin for organogenesis (Smith et al., 1991). The difference in requirement of specific cytokinin may either be on account of genotypic difference or the nature of the explant used (Minocha and Mehra, 1974; Hubstenberger et al., 1992).. Explant size seems to be an important factor for C. elephantidens regeneration from callus. Very small explants do not survive well in culture and large explants are difficult to manipulate due to their physiological status. This has also been reported earlier by Minocha and Mehra, 1974 and Oliveira et al., 1995. The optimal size of the explant has also been identified as a requirement for regeneration of Petunia (Beck and Camper, 1991), Populus alba P. grandidentata hybrids (Son and Hall, 1990) and Aeschynomena sensitiva (Nef-Campa et al., 1996). A high concentration of sucrose (5±7%) increased shoot regeneration capacity in C. elephantidens. This is the characteristic feature of many cacti (Lassocinski, 1985; Escobar et al., 1986), and may be the result of
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highly negative osmotic potential caused by high sucrose concentration. A high sucrose concentration may prove beneficial for such plants. In the absence of sucrose, organogenesis does not occur in callus cultures which highlights the vital role of sucrose in shoot differentiation, at least in the species under discussion and some other plants (Brown and Thorpe, 1980; Damiano et al., 1987). The investigation has demonstrated the capacity of root segment explants of C. elephantidens for regeneration. The protocol standardized for this species will prove very useful for regenerating critically endangered and rare cacti, represented by a limited number of individuals. Acknowledgements The author is grateful to Dr. A.K. Wakhlu for his constant encouragement and support, the Head, Department of Botany, for providing necessary laboratory facilities and the UGC for the award of NET fellowship. References Ault, J.R., Blackmon, W.J., 1987. In vitro propagation of Ferocactus acanthodes (Cactaceae). Hort. Sci. 22, 126±127. Beck, M.J., Camper, N.D., 1991. Shoot regeneration from petunia leaf disc as a function of explant size, configuration and benzyladenine exposure. Plant Cell Tiss. Org. Cult. 26, 101±106. Brown, D.C.W., Thorpe, T.A., 1980. Changes in water potential and its components during shoot formation in tobacco callus. Physiol. Plant 49, 83±87. Clayton, P.W., Hubstenberger, J.F., Phillips, G.C., Butler-Nance, S.A., 1990. Micropropagation of members of the Cactaceae subtribe Cactinae. J. Am. Soc. Hortic Sci. 115, 337±343. Damiano, C., Curir, P., Cosmi, T., 1987. Short note on effects of sugar on the growth of Eucalyptus gunnii in vitro. Acta Hort. 212, 553±556. Duncan, D.B., 1955. Multiple range and multiple F-tests. Biometrics 11, 1±42. Escobar, H.A., Villalobos, V.M., Villegas, A., 1986. Opuntia micropropagation by axillary proliferation. Plant Cell Tiss. Org. Cult. 7, 269±277. Hubstenberger, J.F., Clayton, P.W., Phillips, G.C., 1992. Micropropagation of cacti (Cactaceae). In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry, vol. 20, Springer Verlag, Berlin, pp. 49±68. Johnson, J.L., Emino, E.R., 1979. In vitro propagation of Mammillaria elongata. Hort. Sci. 14, 605± 606. King, M.R., 1957. Studies in the tissue culture of cacti. Cactus Succ. J. (US) 29, 102±104. Kolar, Z., Bartek, J., Vyskot, B., 1976. Vegetative propagation of the cactus Mammillaria woodsii Craig through tissue cultures. Experientia 32, 668±669. Komai, F., Okuse, I., Harada, T., 1996. Somatic embryogenesis and plant regeneration in culture of root segments of spinach (Spinacia oleracea L.). Plant. Sci. 113, 203±208. Lassocinski, W., 1985. Chlorophyll-deficient cacti in tissue cultures. Acta Hort. 167, 287±293. Martinez-Vazquez, O., Rubluo, A., 1989. In vitro mass propagation of the near-extinct Mammillaria san-angelensis Sanchez-Mejorada. J. Hort. Sci. 64, 99±105.
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Minocha, S.C., Mehra, P.N., 1974. Nutritional and morphogenetic investigation on callus cultures of Neomammillaria prolifera Miller (Cactaceae). Am. J. Bot. 61, 168±173. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473±497. Nef-Campa, C., Chaintreuil-Dongmo, C., Dreyfus, B.L., 1996. Regeneration of the tropical legume Aeschynomene sensitiva Sw. from root explants. Plant Cell. Tiss. Org. Cult. 44, 149±154. Oliveira, S.A., Machado, M.F.P.S., Prioli, A.J., Mangolin, C.A., 1995. In vitro propagation of Cereus peruvianus Mill (Cactaceae). In vitro Cell. Dev. Biol. Plant 31, 47±50. Slaba, R., 1992. The Illustrated Guide to Cacti. Chancellor Press, London, pp. 40±41. Smith, R.H., Burdick, P.J., Anthony, J., Reilley, A.A., 1991. In vitro propagation of Coryphantha macromeris. Hort. Sci. 26, 315. Snedecor, G.W., Cochran, W.G., 1968. Statistical Methods. Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi, Bombay, Calcutta. Son, S.H., Hall, R.B., 1990. Multiple shoot regeneration from root organ cultures of Populus alba P. grandidentata. Plant Cell Tiss. Org. Cult. 20, 53±57. Steinhart, C.E., 1962. Tissue culture of a Cactus. Science 137, 545±546.
Regeneration of Coryphantha elephantidens (Lem.) Lem. (Cactaceae) from root explants Brijmohan Singh Bhau Plant Tissue Culture Lab., Department of Botany, University of Jammu, Jammu-180006, India Accepted 14 December 1998
Abstract Regeneration of Coryphantha elephantidens (Lem.) Lem. from callus cultures was obtained from (5±20 mm long) root explants of in vitro rooted shoots cultivated on the Murashige±Skoog medium, supplemented with different concentrations of growth regulators. Callus proliferation and shoot formation was optimal on a medium supplemented with 9 mM 2,4-D and 4.6 mM Kn. Explant size and sucrose concentration in the medium influenced the regeneration potential of the callus cultures significantly. Callus obtained from 10 mm long explant when cultured on a medium supplemented with 9 mM 2,4-D, 4.6 mM Kn and 7% sucrose induced maximum shoot differentiation. Regenerants were rooted on growth-regulator-free Murashige±Skoog medium and then acclimatized in a greenhouse. Regenerated plants showed 100% survival and performed better than seedlings. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Cactus; Coryphantha elephantidens; Callus; Organogenesis
1. Introduction Coryphantha elephantidens (Lem.) Lem., commonly called elephant-tusk cactus, is an ornamental species valued for its large size and beautiful pink to red flowers (Slaba, 1992). The conventional methods of propagation of the species are inadequate to meet the commercial demand on account of low seed germination, slow growth, and low rate of offshoot production. Although in vitro propagation is a potential alternative for production of cacti for commercial purposes and also for conservation of rare cacti (Clayton et al., 1990), not many tissue culture studies have been conducted on this species. To the best of my knowledge, the present report is the very first on C. elephantidens. In cacti, 0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 0 5 - 9
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different explant sources, namely shoot-tip explants from seedlings, (Smith et al., 1991; Martinez-Vazquez and Rubluo, 1989; Ault and Blackmon, 1987), pith excised from stem tissue of plants growing in nature (Johnson and Emino, 1979) and shoot tips of mature plants have been used for in vitro studies (Clayton et al., 1990). King (1957) has tried different explants, including roots; the latter proved highly recalcitrant although many species of plants have been regenerated from root cultures (Son and Hall, 1990; Komai et al., 1996; Nef-Campa et al., 1996), but there is no report in cacti. Root explants from in vitro grown plants offer several advantages, such as easy manipulation, availability, sterilization and no oxidation of secondary metabolites in the medium; in comparison with the destructive sampling for explant preparation, especially vital for rare plants, small plant size and slow growth of the seedling derived explants of cacti (Hubstenberger et al., 1992). The objective of the present study was to determine the type and optimum concentration of the growth regulators for rapid plantlet regeneration from root explants of C. elephantidens. The paper describes an efficient protocol for plant regeneration from callus derived from root explants of C. elephantidens and successful establishment of the regenerated plantlets in field. 2. Materials and methods The plant material (tubercles 2 1.5 cm2) used for this study was obtained from the Jammu University Botanical Garden. The tubercles were washed thoroughly with running tap water, surface-sterilized first in 70% ethanol for 45 s, and then in 0.2% HgCl2 for 3 min and rinsed with sterile distilled water five times. These sterilized tubercles were cultured on MS (Murashige and Skoog, 1962) medium containing 3% sucrose and 6.6 mM 8-benzylaminopurine (BAP). The multiple shoots formed on these tubercles were rooted on MS basal medium. Root tip explants (5, 10, 15 mm, in length) were collected from four-week old rooted plantlets of C. elephantidens growing on MS basal medium. For callus initiation and organogenesis, the explants were cultured on MS medium, containing 3% sucrose and fortified with different concentrations of auxins (2,4dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1-naphthaleneacetic acid (NAA)), and cytokinins (BAP, kinetin (Kn)) alone or in combination with each other. The pH of the medium was adjusted at 5.8 and gelled with 0.8% bacteriological grade agar (Ranbaxy, India) prior to autoclaving at 1.02 kg cm2 at 1218C for 15 min. Cultures were incubated at 25 28C and 50 5% relative humidity in a culture room. Bajaj fluorescent tubes (40 W) provided 30 mmol mÿ2 sÿ1 cool white light for a 16-h photoperiod, unless otherwise stated. For callus initiation, five explants (5, 10, 15 mm in length) were cultured per treatment with five replicates in Petri plates. The material was first grown in
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darkness for one week and then transferred to light conditions. Pieces of 12-week old calli raised from explants of different sizes were transferred to fresh medium for inducing shoot differentiation; one callus piece (250 mg fresh weight) was cultured per Petri plate. Ten replicates were used per treatment. The effect of sucrose level (0±10% in the medium) on shoot differentiation from callus raised from 10 mm long explants, was investigated. The regenerated shoots were transferred to basal medium for rooting. Rooted plantlets were transferred to pots containing equal quantities of sand and vermiculite, covered with transparent polythene and watered every alternate day for two weeks. Hardened plants were finally transferred to pots containing garden soil, sand and vermiculite (1 : 1 : 1) for further growth. All experiments were repeated. Data were recorded after four weeks of culture initiation. Growth regulator treatment, explant size and sucrose concentration were arranged as factorial in a completely randomized design. Response variables were callus induction, plant regeneration and final plant production. Data on the percentage of explants forming calli were subjected to arcsin transformation for proportions before analysis and then converted back to percentage for presentation in tables (Snedecor and Cochran, 1968). Data were analyzed by ANOVA (p 0.05) and means were compared using Duncan's new multiple range test (Duncan, 1955). 3. Results Calli were initiated from 10 mm long root explants within 10±15 days of inoculation on MS basal medium supplemented with different concentrations of auxins (2,4-D, IAA, IBA, NAA) with kinetin and BAP alone or in combination (Table 1). Proximal root tip portion appeared swollen (possibly due to dissolution of cortical tissue) in the first week of culture. Within 10±15 days of inoculation, whitish and granular calli developed at the cut ends which subsequently covered the entire explant. Callus formation did not occur on explants cultured in media not supplemented with growth regulators. The medium containing 9.06 mM 2,4-D or IBA with 4.6 mM Kn promoted rapid callus growth on explants. Higher concentrations of 2,4-D promoted a high rate of friable callus formation. Calli derived from IAA showed relatively slow rate of growth. The growth rate of callus was faster during the first four weeks of culture after which it declined. The callus was sensitive to manipulation and often became necrotic following subculturing. 3.1. Differentiation of shoots from callus After four weeks in the callus induction medium, the callus raised from 10 mm long explants was subcultured on the same medium for further proliferation. The
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Table 1 Effect of combination of auxins and Kn on callus development and shoot regeneration from 10 mm long root explants of Coryphantha elephantidens after 4 weeks of culture Auxina 2,4-D
IAA
NAA
IBA
Explants with callusb (%)
Callus with shootsb (%)
Number of shoots per explantb
0 2.2 4.5 9.0
0 28ef 84a 88a
0f 42.8c 47.1c 100a
0c 0.3b 0.4b 1.6a
2.8 5.7 11.4
24f 32e 32e
0f 25d 12d
0c 0.1b 0.2b
2.6 5.3 10.7
24f 44d 52c
66.7b 54.5b 15.4c
0.1b 0.3b 0.1b
2.4 4.9 9.8
20f 40f 60b
0f 30d 6.7e
0c 0.1b 0.1b
(mM)
a
Plus 4.6 mM Kn. Values with the same letters are not different from each other at a 5% level by Duncan's new multiple range test. b
calli differentiated into green compact nodular structures that differentiated into green shoots with prominent tubercles and white spines. Maximum number of shoots (1.6 shoots) were formed on MS medium supplemented with 2.2 mM 2,4D and 4.6 mM Kn (Table 1). All calli that were cultured formed shoots on this medium. The shoots were healthy and attained average height of 3.4 cm within four weeks. Kn or BAP alone did not induce any morphogenetic response. Regeneration of shoot buds also occurred in low concentrations of Kn, but less frequently. The nature of auxin employed has significant influence on regeneration of shoots from callus. BAP proved less potent in shoot organogenesis than Kn in combination with different auxins (data not presented). Shoot regeneration from callus (100%) with 1.74 shoots per explant was recorded from callus raised from root explants of 10 mm length (Table 2). Following six subsequent subcultures at four-week intervals on MS medium supplemented with 2.2 mM 2,4-D and 4.6 mM Kn, the regeneration potential and number of shoot buds per culture remained fairly constant.
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Table 2 Effect of root explant size on callus formation and shoot regeneration in Coryphantha elephantidens on MS medium supplemented with 2.2 mM 2,4-D and 4.6 mM Kn after 4 weeks of culture Explant size (mm)
Explants with callusa (%)
Callus with shootsa (%)
Number of shoots per explanta
5 10 15 20
12c 90a 64b 56b
33.3bc 100a 40b 26c
0.04c 1.7a 0.6b 0.5bc
a
Values with the same letters are not different from each other at a 5% level by Duncan's new multiple range test.
3.2. Effect of explant size Root segment length had significant influence on callus induction and subsequent shoot regeneration from the callus (Table 2). Maximum callus induction (100%) was obtained from root segments of 10 mm length. As the explant size was reduced (5 mm) or increased (15 mm), the rate of regeneration after callogenesis and the number of shoots formed per explant decreased significantly. 3.3. Effect of sucrose Sucrose level in the medium influenced the regeneration rate significantly (Table 3). Maximum number of shoots (3.3) per piece of callus was produced when sucrose concentration in the medium was 7%. Other levels of sucrose (0, 1.5, 3.0 or 10) lowered the number of shoots formed per callus piece and induced formation of stunted shoots. Elimination of sucrose resulted in the death of callus. Sucrose level also has marked influence on shoot length; maximum shoot length is attained with 5±7% sucrose concentration. 3.4. Induction of rooting and field establishment All the isolated shoots rooted when transferred to MS medium free of growth regulators. Root initiation from the shoot was recorded two weeks after inoculation. Roots formed were thick, with an average length of 2.2 cm. Rooted plantlets transferred to pots containing sand and vermiculite showed 100% survival. Hardened plants transferred to pots containing mixture of garden soil, sand and vermiculite showed luxurious growth.
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Table 3 Effect of sucrose concentration on shoot development of Coryphantha elephantidens explants after 12 weeks of culture Sucrosea (%)
Average no. of shoots/explantb
Shoot lengthb (mm)
0 1.0 3.0 5.0 7.0 10
0c 0.2c 1.8b 2.4ab 3.2a 1.4bc
0d 18bc 34b 42a 40a 10c
a
Plus 4.6 mM Kn and 2.2 mM 2,4-D. Means with the same letters in a column are not significantly different from each other at a 5% level by Duncan's new multiple range test. b
4. Discussion The study reveals the capacity of C. elephantidens,, Cactaceae, to regenerate plantlets from root explants. This is the first report for the family. Root explant, used in the present study, has an advantage over the other more widely used explants, such as young seedlings, pith tissue from the stem and tubercle tissue (Johnson and Emino, 1979; Oliveira et al., 1995; Smith et al., 1991; Kolar et al., 1976; Steinhart, 1962) in that they can be employed even in multiplying without killing the source plants. Unlike the non-organogenic callus which can develop from various explants, formation of organogenic callus seems to be controlled by auxin cytokinin ratio of the medium. In case of C. elephantidens, Kn has proved most suitable for organogenesis, which is in agreement with the findings of Oliveira et al. (1995). In case of C. macromeris, BAP proved to be a more potent cytokinin for organogenesis (Smith et al., 1991). The difference in requirement of specific cytokinin may either be on account of genotypic difference or the nature of the explant used (Minocha and Mehra, 1974; Hubstenberger et al., 1992).. Explant size seems to be an important factor for C. elephantidens regeneration from callus. Very small explants do not survive well in culture and large explants are difficult to manipulate due to their physiological status. This has also been reported earlier by Minocha and Mehra, 1974 and Oliveira et al., 1995. The optimal size of the explant has also been identified as a requirement for regeneration of Petunia (Beck and Camper, 1991), Populus alba P. grandidentata hybrids (Son and Hall, 1990) and Aeschynomena sensitiva (Nef-Campa et al., 1996). A high concentration of sucrose (5±7%) increased shoot regeneration capacity in C. elephantidens. This is the characteristic feature of many cacti (Lassocinski, 1985; Escobar et al., 1986), and may be the result of
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highly negative osmotic potential caused by high sucrose concentration. A high sucrose concentration may prove beneficial for such plants. In the absence of sucrose, organogenesis does not occur in callus cultures which highlights the vital role of sucrose in shoot differentiation, at least in the species under discussion and some other plants (Brown and Thorpe, 1980; Damiano et al., 1987). The investigation has demonstrated the capacity of root segment explants of C. elephantidens for regeneration. The protocol standardized for this species will prove very useful for regenerating critically endangered and rare cacti, represented by a limited number of individuals. Acknowledgements The author is grateful to Dr. A.K. Wakhlu for his constant encouragement and support, the Head, Department of Botany, for providing necessary laboratory facilities and the UGC for the award of NET fellowship. References Ault, J.R., Blackmon, W.J., 1987. In vitro propagation of Ferocactus acanthodes (Cactaceae). Hort. Sci. 22, 126±127. Beck, M.J., Camper, N.D., 1991. Shoot regeneration from petunia leaf disc as a function of explant size, configuration and benzyladenine exposure. Plant Cell Tiss. Org. Cult. 26, 101±106. Brown, D.C.W., Thorpe, T.A., 1980. Changes in water potential and its components during shoot formation in tobacco callus. Physiol. Plant 49, 83±87. Clayton, P.W., Hubstenberger, J.F., Phillips, G.C., Butler-Nance, S.A., 1990. Micropropagation of members of the Cactaceae subtribe Cactinae. J. Am. Soc. Hortic Sci. 115, 337±343. Damiano, C., Curir, P., Cosmi, T., 1987. Short note on effects of sugar on the growth of Eucalyptus gunnii in vitro. Acta Hort. 212, 553±556. Duncan, D.B., 1955. Multiple range and multiple F-tests. Biometrics 11, 1±42. Escobar, H.A., Villalobos, V.M., Villegas, A., 1986. Opuntia micropropagation by axillary proliferation. Plant Cell Tiss. Org. Cult. 7, 269±277. Hubstenberger, J.F., Clayton, P.W., Phillips, G.C., 1992. Micropropagation of cacti (Cactaceae). In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry, vol. 20, Springer Verlag, Berlin, pp. 49±68. Johnson, J.L., Emino, E.R., 1979. In vitro propagation of Mammillaria elongata. Hort. Sci. 14, 605± 606. King, M.R., 1957. Studies in the tissue culture of cacti. Cactus Succ. J. (US) 29, 102±104. Kolar, Z., Bartek, J., Vyskot, B., 1976. Vegetative propagation of the cactus Mammillaria woodsii Craig through tissue cultures. Experientia 32, 668±669. Komai, F., Okuse, I., Harada, T., 1996. Somatic embryogenesis and plant regeneration in culture of root segments of spinach (Spinacia oleracea L.). Plant. Sci. 113, 203±208. Lassocinski, W., 1985. Chlorophyll-deficient cacti in tissue cultures. Acta Hort. 167, 287±293. Martinez-Vazquez, O., Rubluo, A., 1989. In vitro mass propagation of the near-extinct Mammillaria san-angelensis Sanchez-Mejorada. J. Hort. Sci. 64, 99±105.
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