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Abscisic acid induced somatic embryogenesis in immature embryo explants of coconut (Cocosnucifera L.)
Plant Science 151 (2000) 193 – 198 www.elsevier.com/locate/plantsci
Abscisic acid induced somatic embryogenesis in immature embryo explants of coconut (Cocos nucifera L.) S.C. Fernando *, C.K.A. Gamage Coconut Research Institute, Lunuwila 61150, Sri Lanka Received 20 July 1999; received in revised form 6 October 1999; accepted 21 October 1999
Abstract Somatic embryogenesis in coconut (Cocos nucifera L.) is generally induced by gradual reduction of auxin concentration in the culture medium and incorporation of cytokinins. Although plant regeneration through somatic embryogenesis is possible, the protocol is yet to be perfected. In this study, nodular callus was obtained from 7 – 9 months old immature zygotic embryos of coconut on a medium containing 24 mM 2,4-dichlorophenoxy acetic acid (2,4-D). As a novel approach, abscisic acid (ABA) at a concentration of 2.5–7.5 mM was incorporated into the culture medium for 3 – 7 weeks to induce somatic embryogenesis. Alternately, callus was subcultured at 5 weekly intervals on media containing gradually reducing concentrations of 2,4-D to induce somatic embryogenesis. Incorporation of ABA enhanced the production of somatic embryos. Application of 2.5 – 5 mM ABA for 5 weeks was found to be effective. A large number of somatic embryos developed on media containing ABA formed normal shoots and complete plants as compared to those produced in the media with low levels of 2,4-D. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Coconut; Cocos nucifera L.; Somatic embryogenesis; Abscisic acid
1. Introduction Development of a reliable vegetative propagation method for coconut would pave the way to rapid multiplication of elite palms. This would significantly improve the productivity and homogeneity of coconut plantations. Tissue culture would be the only approach for vegetative propagation of coconut. Tissue culture of coconut has a long history dating back to the 1970s. There are several reports on induction of callus or development of organized structures from different sources of explants, such as meristematic shoot and root tissue, inflorescence, leaf, zygotic embryo, endosperm tissue, anther and plumule. However, plant regeneration was reported only on a few occasions [1–8]. * Corresponding author. Tel.: + 94-31-57395; fax: +94-31-57395. E-mail address: [email protected] (S.C. Fernando)
According to the published material, callus initiation from coconut tissues requires the presence of an auxin (mainly 2,4-dichlorophenoxy acetic acid (2,4-D) and a-naphthalene acetic acid (NAA)) in the culture medium. Somatic embryogenesis in this callus was generally induced by gradual reduction of auxin concentration in the culture medium and addition of cytokinins. However, Verdeil et al. [5] reported the formation of isolated embryogenic cells and proembryos, when the callus was initially subcultured on a medium containing high levels of 2,4-D for 2–8 months. The proembryo maturation and plant regeneration was achieved by a gradual reduction of 2,4-D concentration in the culture medium and addition of 6-benzylaminopurine (BAP) into the culture medium. Even though there are reports on the production of clones of several coconut genotypes, the rate of regeneration remains low due to the lack of an optimum plant regeneration phase.
0168-9452/00/$ - see front matter © 2000 Publilshed by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 9 ) 0 0 2 1 8 - 6
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S.C. Fernando, C.K.A. Gamage / Plant Science 151 (2000) 193–198
Several studies have shown positive effects of abscisic acid (ABA) on induction of embryogenic calli and somatic embryos in different plant species [9 – 13]. However, very little work has been done on the effect of ABA on somatic embryogenesis in coconut. Therefore, the present study was undertaken to examine the possibility of using ABA to enhance somatic embryogenesis and plant regeneration of coconut.
2. Materials and methods
2.1. Callus initiation Nodular callus was initiated from 7–9 month old immature zygotic embryos of coconut (improved variety T X T) following the method described by Karunaratne and Periyapperuma [14]. The basal medium 72 (BM 72) (developed at Coconut Research Institute, Sri Lanka by following a broad spectrum trial [14]) supplemented with 24 mM 2,4-D and 0.25% (w/v) activated charcoal was used for callus initiation. Callus was separated from the explant after 2–3 months in culture and used for the regeneration experiments.
The concentration of ABA (2.5–7.5 mM) in RM1 medium and the duration (3–7 weeks) of ABA application varied with the treatment. The callus was then transferred to the medium BM 72 supplemented with 4% (w/v) sucrose, 0.8% (w/v) agar and 0.25% (w/v) activated charcoal (medium RM2) for further 4 weeks and kept in the dark. Finally, the differentiating callus was transferred to Eeuwens Y3 medium [15] supplemented with 0.1 mM 2,4-D, 5.0 mM BAP, 0.8% (w/v) agar and 0.25% (w/v) activated charcoal. The cultures were maintained by subculturing to fresh Y3 medium at 4–5 weekly intervals. Cultures were initially maintained in 28 ml screwcapped vials containing 10 ml medium and then in 100 ml flasks containing 50 ml medium. Depending on the somatic embryo development, the cultures were gradually exposed to light (16 h photoperiod).
2.3. Rooting In cases where roots did not develop spontaneously, rooting was induced by an indole-3-acetic acid (IAA) pulse treatment [16].
3. Results
2.2. Plant regeneration 3.1. Callus initiation 2.2.1. Method 1 Nodular callus was subcultured at 5 weekly intervals to the same basal medium supplemented with gradually reducing concentrations of 2,4-D and 2 – 10 mM cytokinin (BAP, kinetin, zeatin). Cultures were initially maintained in 28 ml screwcapped vials containing 10 ml medium and then in 100 ml flasks containing 50 ml medium. Cultures were incubated at 30 9 1°C in the dark for 2 months. Then they were gradually exposed to light (16 h photoperiod). 2.2.2. Method 2 Nodular callus was either transferred directly to the medium BM 72 supplemented with 2.5– 7.5 mM ABA (filter-sterilized), 6% (w/v) sucrose, 0.8% (w/v) agar and 0.25% (w/v) activated charcoal (medium RM1) or transferred after subculturing to the same basal medium with gradually reducing levels of 2,4-D (16 and 8 mM). The cultures were kept in the dark at 30 91°C.
Nodular callus was initiated on more than 70% of cultured immature zygotic embryos within 2–3 weeks of culture. The callus (1A) was ready for regeneration experiments within 2–3 months of culture.
3.2. Plant regeneration 3.2.1. Method 1 Gradual reduction in 2,4-D level in the medium below 8 mM and the addition of cytokinin initiated somatic embryogenesis in about 50% callus. Most of the embryogenic structures formed were found to be incomplete or abnormal. Further development of these structures led to the formation of shoot like structures, fused shoots or other abnormal structures. Adventive root formation and haustorial development was also common. Regeneration of normal shoots which could develop into plantlets was sporadic.
S.C. Fernando, C.K.A. Gamage / Plant Science 151 (2000) 193–198
3.2.2. Method 2 In the present study, the effects of the drop in 2,4-D level in the callus maintenance medium prior to ABA application, the duration of ABA application and the concentration of ABA in the RM1 medium on somatic embryogenesis and shoot regeneration were assessed. The initial results showed that the rate of somatic embryogenesis and shoot regeneration depended on the level of 2,4-D in the callus maintenance medium prior to ABA application. When the three levels of 2,4-D (24, 16 and 8 mM) were compared, the callus maintained in the medium containing 16 mM 2,4D for 5 weeks followed by ABA (5 mM) applica-
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tion for 5 weeks resulted in an increased somatic embryogenesis (61.4%). Furthermore, the shoot regeneration was also higher (11.4%) when nodular callus was initially subcultured to 16 mM 2,4-D prior to culturing in RM1 medium (Table 1). With regard to the duration of ABA application, the results showed that ABA application for 5 weeks was better for induction of somatic embryogenesis and shoot regeneration than 3 or 7 weeks (Table 2). Out of the different concentrations of ABA tested (2.5–7.5 mM), 7.5 mM gave the highest percentage of somatic embryogenesis (73.7%). However, the somatic embryos formed by applica-
Fig. 1. Plant regeneration from coconut immature zygotic embryo-derived callus. (A) Embryogenic callus derived from an immature zygotic embryo after 2 months in culture (bar = 5.0 mm). (B) Embryogenic structures developed after application of ABA for 5 weeks (bar =5.7 mm). (C) Fused shoots formed from embryogenic structures (bar = 7.5 mm). (D) Shoot development from a somatic embryo cultured in the germination medium for 6 weeks (bar= 10.0 mm). (E) Complete plant developed from a somatic embryo (bar =22.5 mm).
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Table 1 Effect of the reduction in 2,4 D concentration in callus maintenance medium prior to ABA application on somatic embryogenesis and shoot regenerationa Reduction in 2,4-D level prior to ABA application (mM)
Somatic embryogenesis (%)
Shoot regeneration (%)
24 2416 24168
33.3 61.4 37.5
4.5 11.4 8.3
a
Five micromoles ABA was applied for 5 weeks. Callus was used for ABA treatments either directly from 24 mM 2,4-D containing medium or after subculturing on media with each reduced level of 2,4-D for 5 weeks.
tion of 7.5 mM ABA for 5 weeks were not able to develop into shoots during the period of observation. In contrast, somatic embryos formed by application of 2.5 and 5 mM ABA for 5 weeks successfully germinated and produced shoots (Table 3). Further experiments are required to show the statistical significance of results obtained in different ABA treatments. Morphological observations showed that the callus subcultured in the medium RM1 containing ABA became more compact. At the end of the ABA application, whitish embryogenic structures were visible (1B). Upon culturing in the medium RM2 for 4 weeks, some of the embryogenic structures elongated and the others developed into haustorial tissues or fused shoots (1C). When cultured on the Y3 medium, elongated somatic embryos split open and produced shoots (1D). The number of plants regenerated per clone varied from one to three. Occasionally, it reached a value as high as 16 plants per clone. Table 2 Effect of the duration of ABA application on somatic embryogenesis and shoot regenerationa Duration (weeks)
Somatic embryogenesis (%)
Shoot regeneration (%)
3 5 7
45.5 57.6 35.5
3.6 8.5 3.8
a Callus was maintained in 16 mM 2,4-D containing medium for 5 weeks prior to ABA application. Five micromoles ABA was used for all the treatments.
Table 3 Effect of ABA concentration on somatic embryogenesis and shoot regenerationa ABA level (mM)
Somatic embryogenesis Shoot regenera(%) tion (%)
2.5 5.0 7.5
67.4 57.6 73.7
9.4 8.5 0.0
a Callus was maintained in 16 mM 2,4-D containing medium for 5 weeks prior to ABA application. The different levels of ABA were applied for 5 weeks.
3.3. Rooting Most of the regenerated shoots produced roots spontaneously and developed into complete plants (1E). When the rooting was suppressed, application of a pulse treatment with 500 mM IAA solution resulted in profuse rooting.
4. Discussion It has been accepted that endogenous growth regulators play a major role in the regulation of morphogenesis. Therefore, initiation of embryogenic structures from callus may be related to the establishment of a particular balance between different endogenous plant growth regulators [12]. In coconut, exogenic auxins play a predominant role in the development of embryogenic structures [17]. According to the published work, the essential factor in coconut embryogenesis is the gradual drop in 2,4-D concentration in relation to the initial level. If the drop in auxin level was too rapid, the maturation of the embryogenic structures usually led to incomplete or deviated forms [18]. This may be one of the reasons for obtaining shoots with suppressed growth, fused shoots, haustorial tissues and roots when the plant regeneration method 1 was used in this study. Furthermore, the use of different coconut genotypes and culture media may have also contributed to the varying results obtained by various research groups. In the present study, removal of auxin and application of ABA in the regeneration medium (RM1) resulted in the normal development of somatic embryos and plants of coconut. Similar results were obtained in He6ea brasiliensis either
S.C. Fernando, C.K.A. Gamage / Plant Science 151 (2000) 193–198
by reducing the auxin and cytokinin concentration and adding ABA into the medium [12] or by completely removing auxin and adding ABA into the culture medium [13]. Rajasekaran et al. [9] have demonstrated the positive effect of ABA in somatic embryogenesis in Napier grass (Pennisetum purpureum Schum) whereas Qureshi et al. [11] have shown the role of ABA in restored embryogenic capacity in late-stage wheat (Triticum aesti6um L.) embryos. The use of ABA for improved somatic embryo formation and their maturation in mature zygotic embryo-derived callus of coconut has been reported by Samosir et al. [19]. The work presented in this paper was conducted independently and the results were comparable to those of Samosir et al. [19] and showed that application of ABA increased the somatic embryo formation and plantlet regeneration in coconut immature zygotic embryo-derived callus. Samosir et al. [19] have used 45 mM ABA to induce somatic embryo formation and their maturation, whereas much lower levels of ABA (2.5–7.5 mM) were used in the present study. However, the efficiency of the two methods could not be compared as quantitative data from the study of Samosir et al.[19] was not available. In a previous study, Hornung [6] reported the use of 1.5 – 2.0% mannitol in the medium for plant regeneration from coconut plumule derived callus. Belefant and Fong [20] have shown an increase in the ABA levels in embryos plasmolyzed in increasing concentration of mannitol. Therefore, the effect of mannitol in plant regeneration reported by Hornung [6] might be a result of increased levels of endogenous ABA in coconut callus. Eventhough, there is enough evidence to show that ABA plays an important role in the expression of somatic embryogenesis in in vitro cultures, the mode of action is not established. However, there are reports which suggest that the favorable effect of ABA on somatic embryogenesis may be due to an increased production of storage reserves (e.g. proteins, triglycerides and lipids) [21,22] and improved sucrose uptake [23] or starch synthesis [24]. Buffard-Morel [18] reported that embryogenic cells of coconut contained starch and protein reserves. In a very recent study on nutritional re-
197
quirements of coconut calli during somatic embryogenesis, Magnaval et al. [25] observed a higher content of soluble sugars in calli in initial stages of embryogenesis induction. This indicates a higher requirement of carbohydrates by embryogenic cells of coconut as in other species [26,27]. Therefore, the positive effect of ABA observed in this study might be a result of increased production of storage reserves and uptake of sucrose from the medium. Histological observations and quantification of proteins and carbohydrates in cultures are necessary to confirm the hypothesis. This initial study showed that the application of ABA can improve clonal plant regeneration of coconut as compared to the application of gradually reducing concentrations of 2,4-D. The efficiencies of somatic embryogenesis and shoot regeneration seemed to depend on several factors, such as 2,4-D level in the callus maintenance medium, ABA concentration and duration of ABA application. Statistical analyses are required to select the best ABA treatment. Furthermore, in this study, the occurrence of somatic embryogenesis was assessed using the morphological observations. However, histological studies are needed to understand the problems related to the conversion of somatic embryos formed in different ABA levels into plantlets. This plant regeneration protocol was tested on callus derived from various explants of several coconut genotypes. Preliminary results showed that the clonal plant regeneration can be achieved (data is not given). However, more data is to be collected for comparison of the success rates between genotypes. The results of this initial study are encouraging as consistent plant regeneration is possible by this method. However, the number of plants regenerated still remains low. Further experiments are in progress to improve regeneration of plants. Acknowledgements We are most grateful to Dr Kaushalya Weerakoon for the very useful comments in the preparation of the manuscript. Thanks are also due to the staff of the Tissue Culture Division, Coconut Research Institute, Sri Lanka for the assistance in performing this investigation. We also gratefully acknowledge the assistance of P. Silva and A. Kumara, the photographers.
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References [1] R. Branton, J. Blake, A lovely clone of coconuts, New Sci. 26 (1983) 554–557. [2] C.K. Raju, P. Prakash Kumar, M. Chandramohan, R.D. Iyer, Coconut plantlets from leaf tissue cultures, J. Plant. Crops 12 (1984) 75–91. [3] N. Bhala-Sarin, S. Bagga, S.K. Sopory, S. GuhaMukhergee, Induction and differentiation of callus from embryos of Cocos nucifera L. by IAA conjugates, Plant Cell Rep. 5 (1986) 322–324. [4] A.G. Del Rosario, L.C. Malijan, A.P. Miniano, Plantlet regeneration from coconut callus cultures, Paper Presented at the 5th Scientific Meeting of Crop Scientists of the Philippines, Ioila city, 26–29 April, 1989. [5] J.L. Verdeil, C. Huet, F. Grosdemanger, J. BuffardMorel, Plant regeneration from cultured immature inflorescence of coconut (Cocos nucifera L.): evidence for somatic embryogenesis, Plant Cell Rep. 13 (1994) 218 – 221. [6] R. Hornung, Micropropagation of cocos nucifera L. from plumular tissues excised from mature zygotic embryos, Plantations, Res. Dev. 2 (1995) 38–41. [7] N.T. Thanh-Tuyen, D.I. Apurillo, Plant regeneration through somatic embryogenesis from cultured zygotic embryos of coconut, Philippine J. Coconut Stud. XVII (1995) 1–7. [8] J.L. Chan, L. Saenz, C. Talavera, R. Hornung, M. Robert, C. Oropeza, Regeneration of coconut (Cocos nucifera L.) from plumule explants through somatic embryogenesis, Plant Cell Rep. 17 (1998) 515–521. [9] K. Rajasekaran, M.B. Hein, I.K. Vasil, Endogenous abscisic acid and indole-3-acetic acid and somatic embryogenesis in cultured leaf explants of Pannisetum purpureum Schum, Plant Physiol. 84 (1987) 47–51. [10] C. Brown, F.J. Brooks, D. Pearson, R.J. Mathias, Control of embryogenesis and organogenesis in immature wheat embryo callus using increased medium osmolarity and abscisic acid, J. Plant Physiol. 133 (1989) 727 – 733. [11] J.A. Qureshi, K.K. Kartha, S.R. Abrams, L. Steinhauer, Modulation of somatic embryogenesis in early and late stage embryos of wheat (Triticum aesti6um L.) under the influence of (9 )-abscisic acid and its analogs, Plant Cell Tissue Organ Cult. 18 (1989) 55–69. [12] H. Etienne, B. Sotta, P. Montoro, E. Miginiac, M.P. Carron, Relations between exogenous growth regulators and endogenous indole-3-acetic acid and abscisic acid in the expression of somatic embryogenesis in He6ea brasiliensis Mull, Arg. Plant Sci. 88 (1993) 91–96. [13] P. Veisseire, L. Linossier, A. Coudret, Effect of abscisic acid and cytokinins on the development of somatic embryos in He6ea brasiliensis, Plant Cell Tissue Organ Cult. 39 (1994) 219–223. [14] S. Karunaratne, K. Periyapperuma, Culture of immature embryos of coconut, Cocos nucifera L: callus prolifera-
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tion and somatic embryogenesis, Plant Sci. 62 (1989) 247 – 253. C.J. Eeuwens, Effects of organic nutrients and hormones on growth and development of tissue explants from coconut (Cocos nucifera) and date (Phoenix dactylifera) palms cultured in vitro, Physiol. Planta. 42 (1978) 173– 178. S.C. Fernando, C.K. Gamage, Clonal propagation of coconut: Improved culture conditions for rhizogenesis, Cocos 10 (1994 – 1995) 20 – 25. J.L. Verdeil, J. Buffard-Morel, C. Pannetier, Somatic embryogenesis of coconut (Cocos nucifera L.) from leaf and inflorescence tissue research findings and prospects, Oleagineux 44 (1989) 403 – 411. J. Buffard-Morel, J.L. Verdeil, S. Dussert, C. Magnaval, C. Huet, F. Grosdemange, Initiation of somatic embryogenesis in coconut (Cocos nucifera L.), in: C. Oropeza, et al. (Eds.), Lethal Yellowing: Research and Practical Aspects, Kluwer Academic Publishers, Netherlands, 1995, pp. 217 – 223. Y.M.S. Samosir, I.D. Godwin, S.W. Adkins, An improved method for the maturation of coconut (Cocos nucifera L.) somatic embryos, Paper Abstract International Symposium on Coconut Biotechnology, Merida, Yucata´n, Mexico, 1 – 5 December 1997. H. Belefant, F. Fong, Abscisic acid biosynthesis in Zea mays embryos: influence of tetcyclacis and regulation by osmotic potential, Plant Sci. 78 (1991) 19 – 25. R.P. Feirer, J.H. Conkey, S.A. Verhagen, Triglycerides in embryogenic conifer calli: a comparison with zygotic embryos, Plant Cell Rep. 8 (1989) 207 – 209. D.R. Roberts, B.S. Flinn, D.T. Webb, F.B. Webster, B.C.S. Sutton, Abscisic acid and indole-3-butric acid regulation of maturation and accumulation of storage proteins in somatic embryos of interior spruce, Physiol. Planta. 78 (1990) 355 – 360. R.A. Saftner, R.E. Wyse, Effect of plant hormones on sucrose uptake by beet root tissue discs, Plant Physiol. 74 (1984) 951 – 955. T.J.V. Higgings, J.V. Jacobsen, J.A. Zwar, Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers, Plant Mol. Biol. 1 (1982) 191 – 215. C. Magnaval, M. Noirot, J.L. Verdeil, A. Blattes, C. Huet, F. Grosdemange, T. Beule, J. Buffard-Morel, Specific nutritional requirements of coconut calli (Cocos nucifera L.) during somatic embryogenesis induction, J. Plant Physiol. 150 (1997) 719 – 728. P.V. Ammirato, Organizational events during somatic embryogenesis, in: A.R. Liss (Ed.), Plant Tissue and Cell Culture, Macmillan Inc, New York, 1987, pp. 57–81. S. Eapen, L. George, Influence of phytohormones, carbohydrates, amino acids, growth supplements and antibiotics on somatic embryogenesis and plant regeneration in finger millet, Plant Cell Tissue Organ Cult. 22 (1990) 87 – 93.
Abscisic acid induced somatic embryogenesis in immature embryo explants of coconut (Cocos nucifera L.) S.C. Fernando *, C.K.A. Gamage Coconut Research Institute, Lunuwila 61150, Sri Lanka Received 20 July 1999; received in revised form 6 October 1999; accepted 21 October 1999
Abstract Somatic embryogenesis in coconut (Cocos nucifera L.) is generally induced by gradual reduction of auxin concentration in the culture medium and incorporation of cytokinins. Although plant regeneration through somatic embryogenesis is possible, the protocol is yet to be perfected. In this study, nodular callus was obtained from 7 – 9 months old immature zygotic embryos of coconut on a medium containing 24 mM 2,4-dichlorophenoxy acetic acid (2,4-D). As a novel approach, abscisic acid (ABA) at a concentration of 2.5–7.5 mM was incorporated into the culture medium for 3 – 7 weeks to induce somatic embryogenesis. Alternately, callus was subcultured at 5 weekly intervals on media containing gradually reducing concentrations of 2,4-D to induce somatic embryogenesis. Incorporation of ABA enhanced the production of somatic embryos. Application of 2.5 – 5 mM ABA for 5 weeks was found to be effective. A large number of somatic embryos developed on media containing ABA formed normal shoots and complete plants as compared to those produced in the media with low levels of 2,4-D. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Coconut; Cocos nucifera L.; Somatic embryogenesis; Abscisic acid
1. Introduction Development of a reliable vegetative propagation method for coconut would pave the way to rapid multiplication of elite palms. This would significantly improve the productivity and homogeneity of coconut plantations. Tissue culture would be the only approach for vegetative propagation of coconut. Tissue culture of coconut has a long history dating back to the 1970s. There are several reports on induction of callus or development of organized structures from different sources of explants, such as meristematic shoot and root tissue, inflorescence, leaf, zygotic embryo, endosperm tissue, anther and plumule. However, plant regeneration was reported only on a few occasions [1–8]. * Corresponding author. Tel.: + 94-31-57395; fax: +94-31-57395. E-mail address: [email protected] (S.C. Fernando)
According to the published material, callus initiation from coconut tissues requires the presence of an auxin (mainly 2,4-dichlorophenoxy acetic acid (2,4-D) and a-naphthalene acetic acid (NAA)) in the culture medium. Somatic embryogenesis in this callus was generally induced by gradual reduction of auxin concentration in the culture medium and addition of cytokinins. However, Verdeil et al. [5] reported the formation of isolated embryogenic cells and proembryos, when the callus was initially subcultured on a medium containing high levels of 2,4-D for 2–8 months. The proembryo maturation and plant regeneration was achieved by a gradual reduction of 2,4-D concentration in the culture medium and addition of 6-benzylaminopurine (BAP) into the culture medium. Even though there are reports on the production of clones of several coconut genotypes, the rate of regeneration remains low due to the lack of an optimum plant regeneration phase.
0168-9452/00/$ - see front matter © 2000 Publilshed by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 9 ) 0 0 2 1 8 - 6
194
S.C. Fernando, C.K.A. Gamage / Plant Science 151 (2000) 193–198
Several studies have shown positive effects of abscisic acid (ABA) on induction of embryogenic calli and somatic embryos in different plant species [9 – 13]. However, very little work has been done on the effect of ABA on somatic embryogenesis in coconut. Therefore, the present study was undertaken to examine the possibility of using ABA to enhance somatic embryogenesis and plant regeneration of coconut.
2. Materials and methods
2.1. Callus initiation Nodular callus was initiated from 7–9 month old immature zygotic embryos of coconut (improved variety T X T) following the method described by Karunaratne and Periyapperuma [14]. The basal medium 72 (BM 72) (developed at Coconut Research Institute, Sri Lanka by following a broad spectrum trial [14]) supplemented with 24 mM 2,4-D and 0.25% (w/v) activated charcoal was used for callus initiation. Callus was separated from the explant after 2–3 months in culture and used for the regeneration experiments.
The concentration of ABA (2.5–7.5 mM) in RM1 medium and the duration (3–7 weeks) of ABA application varied with the treatment. The callus was then transferred to the medium BM 72 supplemented with 4% (w/v) sucrose, 0.8% (w/v) agar and 0.25% (w/v) activated charcoal (medium RM2) for further 4 weeks and kept in the dark. Finally, the differentiating callus was transferred to Eeuwens Y3 medium [15] supplemented with 0.1 mM 2,4-D, 5.0 mM BAP, 0.8% (w/v) agar and 0.25% (w/v) activated charcoal. The cultures were maintained by subculturing to fresh Y3 medium at 4–5 weekly intervals. Cultures were initially maintained in 28 ml screwcapped vials containing 10 ml medium and then in 100 ml flasks containing 50 ml medium. Depending on the somatic embryo development, the cultures were gradually exposed to light (16 h photoperiod).
2.3. Rooting In cases where roots did not develop spontaneously, rooting was induced by an indole-3-acetic acid (IAA) pulse treatment [16].
3. Results
2.2. Plant regeneration 3.1. Callus initiation 2.2.1. Method 1 Nodular callus was subcultured at 5 weekly intervals to the same basal medium supplemented with gradually reducing concentrations of 2,4-D and 2 – 10 mM cytokinin (BAP, kinetin, zeatin). Cultures were initially maintained in 28 ml screwcapped vials containing 10 ml medium and then in 100 ml flasks containing 50 ml medium. Cultures were incubated at 30 9 1°C in the dark for 2 months. Then they were gradually exposed to light (16 h photoperiod). 2.2.2. Method 2 Nodular callus was either transferred directly to the medium BM 72 supplemented with 2.5– 7.5 mM ABA (filter-sterilized), 6% (w/v) sucrose, 0.8% (w/v) agar and 0.25% (w/v) activated charcoal (medium RM1) or transferred after subculturing to the same basal medium with gradually reducing levels of 2,4-D (16 and 8 mM). The cultures were kept in the dark at 30 91°C.
Nodular callus was initiated on more than 70% of cultured immature zygotic embryos within 2–3 weeks of culture. The callus (1A) was ready for regeneration experiments within 2–3 months of culture.
3.2. Plant regeneration 3.2.1. Method 1 Gradual reduction in 2,4-D level in the medium below 8 mM and the addition of cytokinin initiated somatic embryogenesis in about 50% callus. Most of the embryogenic structures formed were found to be incomplete or abnormal. Further development of these structures led to the formation of shoot like structures, fused shoots or other abnormal structures. Adventive root formation and haustorial development was also common. Regeneration of normal shoots which could develop into plantlets was sporadic.
S.C. Fernando, C.K.A. Gamage / Plant Science 151 (2000) 193–198
3.2.2. Method 2 In the present study, the effects of the drop in 2,4-D level in the callus maintenance medium prior to ABA application, the duration of ABA application and the concentration of ABA in the RM1 medium on somatic embryogenesis and shoot regeneration were assessed. The initial results showed that the rate of somatic embryogenesis and shoot regeneration depended on the level of 2,4-D in the callus maintenance medium prior to ABA application. When the three levels of 2,4-D (24, 16 and 8 mM) were compared, the callus maintained in the medium containing 16 mM 2,4D for 5 weeks followed by ABA (5 mM) applica-
195
tion for 5 weeks resulted in an increased somatic embryogenesis (61.4%). Furthermore, the shoot regeneration was also higher (11.4%) when nodular callus was initially subcultured to 16 mM 2,4-D prior to culturing in RM1 medium (Table 1). With regard to the duration of ABA application, the results showed that ABA application for 5 weeks was better for induction of somatic embryogenesis and shoot regeneration than 3 or 7 weeks (Table 2). Out of the different concentrations of ABA tested (2.5–7.5 mM), 7.5 mM gave the highest percentage of somatic embryogenesis (73.7%). However, the somatic embryos formed by applica-
Fig. 1. Plant regeneration from coconut immature zygotic embryo-derived callus. (A) Embryogenic callus derived from an immature zygotic embryo after 2 months in culture (bar = 5.0 mm). (B) Embryogenic structures developed after application of ABA for 5 weeks (bar =5.7 mm). (C) Fused shoots formed from embryogenic structures (bar = 7.5 mm). (D) Shoot development from a somatic embryo cultured in the germination medium for 6 weeks (bar= 10.0 mm). (E) Complete plant developed from a somatic embryo (bar =22.5 mm).
S.C. Fernando, C.K.A. Gamage / Plant Science 151 (2000) 193–198
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Table 1 Effect of the reduction in 2,4 D concentration in callus maintenance medium prior to ABA application on somatic embryogenesis and shoot regenerationa Reduction in 2,4-D level prior to ABA application (mM)
Somatic embryogenesis (%)
Shoot regeneration (%)
24 2416 24168
33.3 61.4 37.5
4.5 11.4 8.3
a
Five micromoles ABA was applied for 5 weeks. Callus was used for ABA treatments either directly from 24 mM 2,4-D containing medium or after subculturing on media with each reduced level of 2,4-D for 5 weeks.
tion of 7.5 mM ABA for 5 weeks were not able to develop into shoots during the period of observation. In contrast, somatic embryos formed by application of 2.5 and 5 mM ABA for 5 weeks successfully germinated and produced shoots (Table 3). Further experiments are required to show the statistical significance of results obtained in different ABA treatments. Morphological observations showed that the callus subcultured in the medium RM1 containing ABA became more compact. At the end of the ABA application, whitish embryogenic structures were visible (1B). Upon culturing in the medium RM2 for 4 weeks, some of the embryogenic structures elongated and the others developed into haustorial tissues or fused shoots (1C). When cultured on the Y3 medium, elongated somatic embryos split open and produced shoots (1D). The number of plants regenerated per clone varied from one to three. Occasionally, it reached a value as high as 16 plants per clone. Table 2 Effect of the duration of ABA application on somatic embryogenesis and shoot regenerationa Duration (weeks)
Somatic embryogenesis (%)
Shoot regeneration (%)
3 5 7
45.5 57.6 35.5
3.6 8.5 3.8
a Callus was maintained in 16 mM 2,4-D containing medium for 5 weeks prior to ABA application. Five micromoles ABA was used for all the treatments.
Table 3 Effect of ABA concentration on somatic embryogenesis and shoot regenerationa ABA level (mM)
Somatic embryogenesis Shoot regenera(%) tion (%)
2.5 5.0 7.5
67.4 57.6 73.7
9.4 8.5 0.0
a Callus was maintained in 16 mM 2,4-D containing medium for 5 weeks prior to ABA application. The different levels of ABA were applied for 5 weeks.
3.3. Rooting Most of the regenerated shoots produced roots spontaneously and developed into complete plants (1E). When the rooting was suppressed, application of a pulse treatment with 500 mM IAA solution resulted in profuse rooting.
4. Discussion It has been accepted that endogenous growth regulators play a major role in the regulation of morphogenesis. Therefore, initiation of embryogenic structures from callus may be related to the establishment of a particular balance between different endogenous plant growth regulators [12]. In coconut, exogenic auxins play a predominant role in the development of embryogenic structures [17]. According to the published work, the essential factor in coconut embryogenesis is the gradual drop in 2,4-D concentration in relation to the initial level. If the drop in auxin level was too rapid, the maturation of the embryogenic structures usually led to incomplete or deviated forms [18]. This may be one of the reasons for obtaining shoots with suppressed growth, fused shoots, haustorial tissues and roots when the plant regeneration method 1 was used in this study. Furthermore, the use of different coconut genotypes and culture media may have also contributed to the varying results obtained by various research groups. In the present study, removal of auxin and application of ABA in the regeneration medium (RM1) resulted in the normal development of somatic embryos and plants of coconut. Similar results were obtained in He6ea brasiliensis either
S.C. Fernando, C.K.A. Gamage / Plant Science 151 (2000) 193–198
by reducing the auxin and cytokinin concentration and adding ABA into the medium [12] or by completely removing auxin and adding ABA into the culture medium [13]. Rajasekaran et al. [9] have demonstrated the positive effect of ABA in somatic embryogenesis in Napier grass (Pennisetum purpureum Schum) whereas Qureshi et al. [11] have shown the role of ABA in restored embryogenic capacity in late-stage wheat (Triticum aesti6um L.) embryos. The use of ABA for improved somatic embryo formation and their maturation in mature zygotic embryo-derived callus of coconut has been reported by Samosir et al. [19]. The work presented in this paper was conducted independently and the results were comparable to those of Samosir et al. [19] and showed that application of ABA increased the somatic embryo formation and plantlet regeneration in coconut immature zygotic embryo-derived callus. Samosir et al. [19] have used 45 mM ABA to induce somatic embryo formation and their maturation, whereas much lower levels of ABA (2.5–7.5 mM) were used in the present study. However, the efficiency of the two methods could not be compared as quantitative data from the study of Samosir et al.[19] was not available. In a previous study, Hornung [6] reported the use of 1.5 – 2.0% mannitol in the medium for plant regeneration from coconut plumule derived callus. Belefant and Fong [20] have shown an increase in the ABA levels in embryos plasmolyzed in increasing concentration of mannitol. Therefore, the effect of mannitol in plant regeneration reported by Hornung [6] might be a result of increased levels of endogenous ABA in coconut callus. Eventhough, there is enough evidence to show that ABA plays an important role in the expression of somatic embryogenesis in in vitro cultures, the mode of action is not established. However, there are reports which suggest that the favorable effect of ABA on somatic embryogenesis may be due to an increased production of storage reserves (e.g. proteins, triglycerides and lipids) [21,22] and improved sucrose uptake [23] or starch synthesis [24]. Buffard-Morel [18] reported that embryogenic cells of coconut contained starch and protein reserves. In a very recent study on nutritional re-
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quirements of coconut calli during somatic embryogenesis, Magnaval et al. [25] observed a higher content of soluble sugars in calli in initial stages of embryogenesis induction. This indicates a higher requirement of carbohydrates by embryogenic cells of coconut as in other species [26,27]. Therefore, the positive effect of ABA observed in this study might be a result of increased production of storage reserves and uptake of sucrose from the medium. Histological observations and quantification of proteins and carbohydrates in cultures are necessary to confirm the hypothesis. This initial study showed that the application of ABA can improve clonal plant regeneration of coconut as compared to the application of gradually reducing concentrations of 2,4-D. The efficiencies of somatic embryogenesis and shoot regeneration seemed to depend on several factors, such as 2,4-D level in the callus maintenance medium, ABA concentration and duration of ABA application. Statistical analyses are required to select the best ABA treatment. Furthermore, in this study, the occurrence of somatic embryogenesis was assessed using the morphological observations. However, histological studies are needed to understand the problems related to the conversion of somatic embryos formed in different ABA levels into plantlets. This plant regeneration protocol was tested on callus derived from various explants of several coconut genotypes. Preliminary results showed that the clonal plant regeneration can be achieved (data is not given). However, more data is to be collected for comparison of the success rates between genotypes. The results of this initial study are encouraging as consistent plant regeneration is possible by this method. However, the number of plants regenerated still remains low. Further experiments are in progress to improve regeneration of plants. Acknowledgements We are most grateful to Dr Kaushalya Weerakoon for the very useful comments in the preparation of the manuscript. Thanks are also due to the staff of the Tissue Culture Division, Coconut Research Institute, Sri Lanka for the assistance in performing this investigation. We also gratefully acknowledge the assistance of P. Silva and A. Kumara, the photographers.
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