Adventitious Shoot Formation on Teak (Tectona grandis L.f.) Callus Cultures Derived from Internodal Segments Sri Nanan Widiyanto, Dwi Erytrina and Heni Rahmania Department of Biology, Institut Teknologi Bandung, Bandung - 40132 Indonesia Keywords: Compact-callus, micropropagation, shoot-organogenesis, thidiazuron Abstract A procedure was developed to induce shoot organogenesis of teak (Tectona grandis L.f.) callus cultures. Calli were induced from internodal segments on woody plant medium containing 1.0 µm thidiazuron in combination with 0.01 µm indole butyric acid. Results showed that compact callus, with white to light yellow nodular structures on its surface, were produced in four weeks. The most regenerative callus was noted as the compact callus with light yellow or light green color. Shoot organogenesis was induced on medium containing 10.0 µm benzyl adenine in combination with 1.0 µm gibberellic acid. Adventitious shoots proliferated from the surface of regenerative compact callus after three subcultures. Regenerated shoots were propagated on shoot multiplication medium with the addition of 10.0 µm BA. Most elongated shoots were rooted in a soil-sand mixture medium (1:1), and were established under greenhouse conditions. INTRODUCTION Teak (Tectona grandis L.f.) belongs to the family Verbenaceae, and is indigenous to Peninsular India, Myanmar, Thailand and Laos. In Indonesia, teak has been naturally distributed throughout Java, Muna and some small islands near Java. It has also been introduced to several countries in tropical and subtropical regions. Teak is famous worldwide for its high value timber (Mascarenhas et al., 1987). In vitro propagation has been successfully applied to teak, and became an alternative tool to overcome some problems occurring in sexual regeneration. Currently, mass propagation of selected teak clones is possible through in vitro multiple shoot production. Regenerative organs such as pre-existing shoots, meristem shoot-tips, nodal segments or seedling organs have been widely used as explants (Gupta et al., 1980; Mascarenhas et al., 1987; Apavatjrut et al., 1988; Devi et al., 1994). The development of an in vitro regeneration procedure is required not only for the propagation of superior genotypes, but also for the regeneration of genetic improved plants. Adventitious shoot formation from callus tissues has been proposed for the regeneration of genetically engineered tissues of many species (Siemens and Schieder, 1996; Tawfik and Noga, 2001). This paper reports the development of an in vitro regeneration procedure for teak through organogenesis on callus derived from internodal segments. MATERIALS AND METHODS Plant Material Internode segments of 5-8 mm were sampled from elongated shoots of 4-6 weeks, which were cultured in 200 ml glass culture-bottles with transparent plastic-lids containing 25-ml shoot multiplication medium. Shoot-buds were originally isolated from a 45 year-old teak tree grown in the university campus area of the Institut Teknologi Bandung, located in Bandung, West Java, Indonesia. Teak shoot-buds were collected during the period of rainy season in October-December 2000. All in vitro cultures were maintained in a culture room at 25±2ºC under continuous light with 40μmol m -2 s -1 emitted by 40W Philips cool white fluorescent tubes. Proc. IInd IS on Biotech. of Trop & Subtrop. Species Eds: W.-C. Chang and R. Drew Acta Hort 692, ISHS 2005 153
Media Composition The basic composition of the woody plant medium (Lloyd and McCown, 1981) was used in all in vitro experiments. Two different callus induction media, noted as CM-1 and CM-2 were used. The CM-1 medium was selected from preliminary experiments, with different concentrations of thidiazuron (TDZ), 0.1 to 10.0 µm, in combination with 0.01 µm indole butyric acid (IBA). The selected CM-1 was containing 1.0 µm TDZ and 0.01 µm IBA. The CM-2 medium was composed of 0.5 µm benzyl adenine (BA) in combination with 5.0 µm α-naphthalene acetic acid (NAA), based on our previous report (Widiyanto et al., 1999). Two different media were used for shoot induction (SM-1 and SM-2). SM-1 was supplemented with 10.0 µm BA and 1.0 µm gibberellic acid (GA 3 ), which basically refers to Harini et al. (1994). SM-2 contained 1.0 µm BA and 0.5 µm NAA (Widiyanto et al., 1999). As reference we also used the best CM medium in shoot induction experiments. For shoot multiplication, we used the basic medium supplemented with 10.0 μm BA (Widiyanto et al., 1999). All media were solidified with 0.8 % (w/v) agar (Sigma A-1296) and adjusted to ph 5.8 prior to autoclaving at 121 o C under a pressure of 1.2 kg cm -2 for 15 min. Callus Induction Internode segments were placed on CM-1 or CM-2 for callus induction. After three successive passages (four weeks per passage) on medium of the same composition, regenerative (RC) and non-regenerative callus (NRC) clumps were separated. The RCclumps were characterized as compact, highly organized callus, slow-growing with white to light yellow or light green nodular structures on its surface. NRC-clumps were soft, friable and fast-growing. The percentage of RC and NRC produced was calculated after the 3 rd passage. Both RC and NRC clumps were used in further experiments. All experiments had 20-30 replications, and were repeated twice. Data were statistically analysed using the LSD test (p=0.05). Shoot Induction, Multiplication and Elongation The RC-clumps, produced from the best callus induction medium, were collected and used for the shoot induction experiments. As a comparison, NRC-clumps were also transferred and subcultured on the SM-media. Regeneration capacity was defined as the percentage of callus clumps that produced visible shoots. Excisable shoots with 2-3 pinnate leaves were detached from clusters and subcultured individually on shoot multiplication medium. Elongated shoots were transferred to a soil-sand (1:1) mixture substrate for root induction and acclimatization in a growth chamber (33±2 o C; RH 90%). After a 4-week period of acclimatization, teak plantlets were transferred to a greenhouse under natural light conditions. RESULTS AND DISCUSSION Callus Induction During the first 2-weeks in culture, swollen callus tissue developed at the two cut ends of the internode segments. After 4 weeks, further callus formation indicated the development of white-yellowish callus clumps, 10-12 mm in size. All internode segments produced callus on CM-1 and on CM-2. At the end of the second passage, two distinguishable callus types could be observed. The first callus type was hard, compact, highly organized and slow-growing, and is referred to as regenerative callus (RC). The RC-clumps had nodular structures on the surface, which were light yellow to green in color. The second callus type was soft, friable and fast-growing, and is called hereafter non-regenerative callus (NRC). It is well documented that compact calli with nodular structures are the best material to regenerate plantlets. Leupin et al. (2000) showed that compact calli of vetiver resembled embryogenic tissues, and proved that it was highly regenerative. Similar results were also reported for some Acacia species (Vengadesan et al., 2000; Xie and Hong, 154
2001). In the latter reference it was shown with histological sections, that the nodular structures contained meristematic cell clusters. On CM-1, 80% of the calli were RC and 20% NRC (Table 1). CM-1, with 1.0 µm TDZ and 0.01 µm IBA, was the best for the induction of RC. The effectiveness of TDZ in stimulating the formation of compact callus is well documented, either alone or in combination with auxins, such as IBA, NAA or indole acetic acid (IAA). Xie and Hong (2001) showed that the combination of TDZ and IAA highly promoted the formation of regenerative compact callus in A. mangium. Guohua (1998) also found that TDZ was highly effective in inducing regenerative callus in cassava. CM-2 also produced RC, but to a lower extent (only 60%). Our results showed that the combination of BA and NAA stimulated the formation of regenerative callus. The presence of BA, either alone or in combination with other growth regulators, promoted the formation of compact callus in some other species, such as in A. sinuata (Vengadesan et al., 2000), vetiver (Leupin et al., 2000) and cumin (Tawfik and Noga, 2001). Shoot Formation No shoot formation was observed during the first and second passage on SMmedia. Shoot formation was visible as the development of pinnate leaves that developed from RC-clumps. Shoot initials were visible after 3-4 weeks in the third passage on regeneration medium. No shoot regeneration response was observed on callus induction medium (CM-1). The regeneration capacity of compact teak callus to develop shoots seemed to be low, and several subcultures were required. Similarly, Harini et al. (1994) reported that in medium containing BA alone or in combination with GA 3, plantlets were obtained after six successive passages on the same medium. In some cases, maintaining regenerative tissues on the same medium for different subcultures stimulated and enhanced the regenerative potential of callus tissues (Guohua, 1998; Leupin et al., 2000). Fifty percent of the RC-clumps cultured on SM-1 regenerated shoots, with an average of 2.2 shoots per clump (Table 2); for SM-2 this was 40% and 1.25 shoots respectively. Therefore, we can conclude that the combination of 10.0 µm BA with 1.0 µm GA 3 was more effective than 1.0 µm BA with 0.5 µm NAA in promoting shoot formation on teak callus. However, since both combinations contained BA, it seems likely that BA is necessary for shoot induction. The effectiveness of BA to induce shoot organogenesis has also been documented in other species (Leupin et al., 2000; Vengadesan et al., 2000). Further observation showed that some teak RC-clumps had green leaf-like structures, but no further development occurred and no shoot formation was observed. In fact, shoot initiation on teak RC-clumps appeared only in some area of the nodular tissues. Tawfik and Noga (2001) found similar results on compact callus of cumin. It was suggested that a compact callus contains both competent and noncompetent cells. Multiplication and Elongation Shoots, regenerated from both SM-1 and SM-2 (Table 3), were able to proliferate on multiplication medium containing 10.0 μm BA. During the first 4-week period, the averages of 2.25 and 2.13 shoots were produced from a single transplanted shoot, respectively (the difference is not significant, Table 3). However, in the second subculture, shoot multiplication capacity increased significantly, and the averages of 4.50 and 4.38 shoots produced from a single shoot. Regenerated shoots grew and elongated (5-7 cm in length) during the third passage. Plantlets survived and developed normally under the natural conditions of a greenhouse. CONCLUSIONS We developed a workable protocol for regeneration of teak. The efficiency is still low, therefore further research is required to optimize the protocol. 155
ACKNOWLEDGEMENTS This research was fully funded by a Research Grant (2001-2002) from the Cellular and Molecular Laboratory, Department of Biology-Institut Teknologi Bandung, Bandung, Indonesia. Literature Cited Apavatjrut, P., Kaosa-Ard, A. and Paratasiplin, T. 1988. Current research on teak (Tectona grandis Linn. f.) tissue culture in Thailand. Biotrop. Spec. Publ. 35:107-115. Devi, Y.S., Mukherjee, B.B. and Gupta, S. 1994. Rapid cloning of elite teak (Tectona grandis Linn.) by in vitro multiple shoot production. Ind. J. Exp. Biol. 32: 668-671. Gupta, P.K., Nadgir, A.L., Mascarenhas, A.F. and Jagannathan, V. 1980. Tissue culture of forest trees: Clonal multiplication of Tectona grandis L. (teak) by tissue culture. Plant Sci. Lett. 17:259-268. Guohua, M. 1998. Effects of cytokinins and auxins on cassava shoot organogenesis and somatic embryogenesis from somatic embryo explants. Plant Cell Tiss. Org. Cult. 54:1-7 Harini, I., Arun-Nair, C., Subramani, J., Gopinathan, M.C. and Krishnamurthi, M. 1994. In vitro regeneration and multiplication of teak (Tectona grandis L.). Abstr.8 th.intl. Congr. Plant Tiss. Cell Cult. Firenze, Italy. June 12-17. p. 6. Leupin, R.E., Leupin, M., Ehret, C., Erismann, K.H. and Witholt, B. 2000. Compact callus induction and plant regeneration of a non-flowering vetiver from Java. Plant Cell Tiss. Org. Cult. 62:115-123. Lloyd, G. and McCown, B. 1981. Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot tip culture. Int. Plant Prop. Soc. Proc. 30:421-427. Mascarenhas, A.F., Kendurkar, S.V., Gupta, P.K., Khuspe, S.S. and Agrawal, D.C. 1987. Teak. p.300-315. In: Cell and Tissue Culture in Forestry, Vol. 3. Martinus Nijhoff Publ. Dordrecht. Boston. Lancaster. Siemens, J. and Schieder, O. 1996. Transgenic plants: genetic transformation - recent developments and the state of the art. Plant Tiss. Cult. Biotech. 2:66-75. Tawfik, A.A. and Noga, G. 2001 Adventitious shoot proliferation from hypocotyl and internodal stem explants of cumin. Plant Cell Tiss. Org. Cult. 66:141-147. Vengadesan, G., Ganapathi, A., Anand R.P. and Anbazhagan, V.R. 2000. In vitro organogenesis and plant formation in Acacia sinuata. Plant Cell Tiss. Org. Cult. 61:23-28. Widiyanto, S.N., Sisunandar, S. and Riani, E. 1999. Growth recovery and regeneration ability on post cold-storage of shoot-tip cultures of teak (Tectona grandis L.f.) Abstr. Forest Biotechnology Conf., Oxford, U.K. July 11-16. Xie, D. and Hong, Y. 2001. In vitro regeneration of Acacia mangium via organogenesis. Plant Cell Tiss. Org. Cult. 66:167-173. 156
Tables Table 1. Effects of different combinations of growth regulators on the formation of compact callus clumps derived from internode segments after 3 successive passages of subculture. Growth regulators (μm) Frequency of callus formation (%) Medium codes BA NAA IBA TDZ Explants forming calli Total RC-clumps Total NRC-clumps CM-1 - - 0.01 1.0 100 80 20 CM-2 0.5 5.0 - - 100 60 40 Table 2. Effects of different combinations of growth regulators on shoot formation by regenerative (RC) and non-regenerative (NRC) compact callus clumps after 3 successive subcultures. Growth regulators (μm) Shoot formation Medium codes BA NAA GA 3 Type of callus RC clumps producing shoots (%) Total no. shoots produced Mean shoot no. per clump 1) SM-1 10.0-1.0 RC 50 22 2.20 ± 1.03 a NRC 0 0 0 SM-2 1.0 0.5 - RC 40 10 1.25 ± 0.46 b NRC 0 0 0 1) Means ± SD of 20 replicates per treatment in two repeated experiments. Means followed by different letter in the column are significantly difference by LSD test (p=0.05). Table 3. Multiplication capacity of shoots regenerated from regenerative compact callus clumps. Number of shoots per subculture (SC) Shoot origin 1 st SC 2 nd SC RC from SM-1 2.25 ± 0.50 a 4.38 ± 1.70 b RC from SM-2 2.13 ± 0.70 a 4.50 ± 1.43 b Means ± SD of 20 replicates per treatment in two repeated experiments. Means followed by different letter in the column are not significantly difference by LSD test (p=0.05). 157