Shoot Propagation of Tectona grandis L. f. by Tissue Culture

Universities Research Journal 2011, Vol. 4, No. 1 Shoot Propagation of Tectona grandis L. f. by Tissue Culture Yin Yin Waing Abstract An efficient study of teak propagation by tissue culture technique was done in Plant Tissue Culture Laboratory, Department of Botany, Dagon University. Teak (kyun) botanically known as Tectona grandis L. f. belonging to the family Verbenaceae is one of the most valuable and extremely durable timber yielding plant in Myanmar. In this study, the shoot tips were used as experimental explants. The shoot tips containing apical meristem were taken from the plants which were grown under shaded places. The explants were cultured on White basal medium (1954) and sub-cultured on Woody Plant Medium (WPM) which were supplemented with the combination of BAP (0.01, 1.0 mgl -1 ) and NAA (0.005, 0.5 mgl -1 ) for shoot multiplication respectively. Among these treatments, WPM medium supplemented with the combination of BAP (1.0 mgl -1 ) and NAA (0.005 mgl -1 ), the T 4 treatment showed the best shoot proliferation. Key words: tissue culture, propagation, multiplication Introduction Tectona grandis L. f. (Teak) is one of the most important timber yielding trees of Asian countries particularly India, Indonesia, Myanmar and Thailand. It is also the most popular, valuable and extremely durable wood of indigenous timber among all over the world. "Tectona and teak" are both derived from the Portuguese name "teca and tekton". In Greek it is known as "a carpenter". Whereas "grandis" is a Latin word means "large"(schubert, 1959). Teak grows widely as natural vegetation in Cambodia, India, northwest Laos, Myanmar, North Thailand and Vietnam. But it has been widely cultivated throughout its natural range since the fourteenth century. In Myanmar and India, it has been cultivated for timber production since at least 1840 (Schubert, 1959). In Myanmar, teak plays a dormant role in socioeconomic life of the people of the nation and is environmentally conserved. The vegetation of teak has been officially protected by government. The teak's plantations are Assistant Lecturer, Department of Botany, Dagon University

246 Universities Research Journal 2011, Vol. 4, No. 1 being managed only by the government. But nowadays, it can be done by public under government restriction. It is usually grown within tropical semi-evergreen forests, mixed deciduous forest and deciduous Dipterocarp forests. Under natural stand, teak is a large deciduous tree which is up to at least 20 m in height. It is one of the dominant trees in Upper Mixed Deciduous Forest (UMDF). It requires a high light intensity for its growth and development. It grows best in a warm, moist tropical climate (Schubert, 1959). The optimum ph range of the soil is from 6.5 to 7.5. The teak plant, a "calcicolous" species requires a relatively large amount of calcium in the soil for growth and development (White, 1991). Teak is found to grow where the precipitation ranges from 800 mm to 2500 mm (Kaosa-ard, 1986). Teak usually germinates by epigeal germination and is difficult to germinate evenly and adequately. Nearly all of the teak's seeds possess some degree of dormancy and need to pretreat to break its dormancy. Although the real cause of the seed dormancy is still unknown; however, there are three main factors which influence seed dormancy: seed structure, seed maturity and seed biochemistry (Kaosa-ard, 1986). The main cause of the delay of germination is due to its thick hard woody pericarp (Kadambi, 1972). The wood of the plants are used in constructions, building, furniture, crafts and instruments. The bark of roots and young leaves produce yellowish-brown or reddish substance for fabric dye, thus can be used to dye paper, clothes and matting. Teaks' sawdust is also useful in making incense (Soerianegara and Lemmens, 1994). The wood is very heavy, strong and durable, resists white-ants and contains oil with strong characteristic scent which preserves the timber. Some parts of the plant have been used in numerous medicines. The wood can also be used to alleviate headaches, dyspepsia and stomach complaints. A concoction from the bark and flowers can be prepared to relieve bronchitis. The teak plants can be propagated by vegetative propagation such as cutting, budding, grafting and air layering. However, the quantitative production was limited due to the poor rooting in cuttings collected from mature trees. The availability of stocks is also limited due to the intensive

Universities Research Journal 2011, Vol. 4, No. 1 247 maintenance of stock plants. Moreover, some of the other major limiting factors in vegetative propagation depend on season and slow multiplication rate. Although, propagation could be achieved through seeds germination, there are certain disadvantages like low fruit production, abortive seeds and low germination rate. Besides, it can be infected with latent diseases. Although propagation by vegetative cutting is a predominant technique in teak but it does not ensure healthy and disease-free plants. Therefore, scientists started an alternative method of plant propagation, the tissue culture method; called micropropagation method which could overcome the disadvantages of the vegetative propagation. It involves the culture of whole organism from cells or tissues or plant parts via in vitro on a defined medium under aseptic conditions. With such technique, the enormous multiplication capacity could be available to generate propagules around the year. High uniformity may help to ease the management of the teak plantations. The tissue cultured teak plant can be pre-selected to propagate from the mother plants which have straight with no branching (Ahloowalia, 1996 and Yasodha et al., 2004). The aim of this present paper was to investigate the proper concentration and combination of selected plant growth regulators (BAP and NAA), to increase the number of teak plantations and thereby to establish the forests cover which are under rapid depletion caused by over exploitations. Therefore, in vitro clonal propagation technique may hope to offer an alternative technique for superior trees. Materials and Methods The study of Tectona grandis L. f. was conducted at Plant Tissue Culture Laboratory in Department of Botany, Dagon University during 2009-2010. It involved two stages: initial stage and multiplication stage for the production of shoot-lets from the shoot tips of teak plant under aseptic condition. A. Initial Stage for shoot tips culture of teak (In vitro) (1) Collection of stock plants The teak seeds (Figure 1) were collected from Mawbe Township, Yangon Region. The seeds were alternatively soaked in water and dried

248 Universities Research Journal 2011, Vol. 4, No. 1 under sunlight for 24 hours. The process was done repeatedly at three or four times. Fig.1. Collected seeds of teak The seeds were propagated in the polyethylene bags containing the mixture of sand and burnt rice husk. Then watering and proper care was done regularly. After 4 weeks of propagation, the two cotyledons emerged from the seeds (Figure 2). Later, the first true leaves emerged from the seedlings (Figure 3). The germinated seedlings were transferred to the polyethylene bags containing the mixture of sand, burnt rice husk and compost (Figure 4). The seedlings were placed under shaded place. After 2 weeks cultivation, the plants were placed under sunlight (Figure 5). Fig. 2. Two cotyledons emerged from teak seeds

Universities Research Journal 2011, Vol. 4, No. 1 249 Fig. 3. First true leaves emerged from seedling Fig. 4. Germinated seedling (2) Source of explants Fig. 5. Seedlings were propagated in polyethylene bags The shoot tips containing apical meristem of teak were used as experimental explants. The explants were taken from selected and sterilized shot tips (Figure 6).

250 Universities Research Journal 2011, Vol. 4, No. 1 Fig. 6. Mother plants of Teak (3) Preparation of stock solution According to White basal medium (1954), the stock solutions were prepared. The stock solutions consisted of chemicals groups such as inorganic salts and vitamins. This solution was divided into four groups. The sterilized glass bottles containing stock solutions were kept in refrigenerator and in volumes sufficient to make 1 to 5 liter media at a time. (4) Preparation of culture medium White basal medium was used for shoot multiplication. Sucrose solution 20 gl -1 in distilled water was prepared in beakers. The prepared sucrose solution was then added to 50 mll -1 of stock solution A, B, C and 10 mll -1 of stock solutions D respectively and mixed thoroughly with glass rod. Before the gelling agent (agar) was added, the prepared solutions, were adjusted to ph 5.6 by using either 1N NaOH or 1N HCl. (5) Culture bottles Glass bottles with a capacity of 100 ml were used. Before the preparation of the media, the bottles were washed with detergent solution and sterilized with Clorox solution. Then, the bottles were cleaned with tap water and being autoclaved at 121 º C for 1½ hours and dried in oven at 110 º C for 30 minutes (Fig. 7 and 8).

Universities Research Journal 2011, Vol. 4, No. 1 251 Fig. 7. Autoclave Fig. 8. Storing the culture bottles in dried sterilizer (6) Surface sterilization of the shoot tips The shoot tips were thoroughly washed under running tap water and again washed with double distilled water. The selected shoot tips were dipped in 70% of ethanol solution for 5 minutes and rinsed with double distilled water (DDW) at three times. Then, the shoots were soaked in 0.5 gl -1 of homai (fungicide) solution for 10 minutes and again rinsed with double distilled water (DDW) at three times. In the last step of surface sterilization procedure, the shoot tips were immersed in 2% of Clorox solution. Finally, they were rinsed in double distilled water (DDW) for 5 times. (7) Inoculation and Incubation of explants The explants were extracted under laminar flow cabinet from the sterilized shoot tips and then cultured on modified White basal solidified media without plant growth regulators. All the cultured bottles were incubated at 27±1 º C, light intensity 1000-1200 Lux, relative humidity 30-50% and then the culture bottles were kept alternatively under fluorescent tube light to obtain 16 hours photoperiod and 8 hours dark period respectively. B. Multiplication Stage for shoot tip culture of teak (in vitro) (1) Preparation of culture medium Woody Plant Medium (WPM) (1981) was supplemented with cytokinin (BAP) and auxin (NAA) in different concentrations and

252 Universities Research Journal 2011, Vol. 4, No. 1 combinations were used for the growth and multiplication of axillary shoot. The ph of the medium was adjusted to 5.8 with either 1N NaOH or 1 N HCl. Then the media were sterilized by using autoclave at 121 º C under 1.2 kg/cm 2 pressure for 30 minutes (Fig. 7). (2) Inoculation and Incubation of explants In the laminar flow cabinet, the sterilized shoot tips were cut about 0.2 cm in length. They were transferred into 100 ml bottle containing fresh solid WPM basal medium with different concentrations and combination of selected PGR as shown in Table 1. After inoculation, all the cultures were incubated at 27±1 º C, under light intensity 1000-1200 Lux and relative humidity 30-50%. Then, the culture bottles were kept alternatively under 16 hours photoperiod by fluorescent tube light and 8 hours dark period. The explants were sub-cultured again after every three weeks intervals. Table 1 Concentrations and combinations of selected plant growth regulators in WPM medium for multiplication stage Treatment WPM medium selected plant growth regulators (mgl -1 ) Remark T 1 WPM basal media Each treatment had T 2 WPM media + (0.01) BAP + (0.005) NAA 10 replications and each T 3 WPM media + (0.01) BAP + (0.5) NAA replicate had a explant. WPM media + (1.0) BAP + (0.005) NAA T 4 T 5 WPM media + (1.0) BAP + (0.5) NAA Results A. Initial Stage for shoot tips culture It was observed that the type of germination of teak was epigeal germination. Moreover, the germination rate of teak was delayed due to its hard pericarp and seed dormancy. The basic principle of surface sterilization is required to kill the microbes with minimal damage to the explant. Most of the microbial contaminants (bacterial and fungal) could be killed with sodium hypochloride or mecuric chloride affectively or with calcium hypochloride

Universities Research Journal 2011, Vol. 4, No. 1 253 or benzyalkonium chloride which was being effected for highly contaminated tissues. In this study, Clorox solution (sodium hypochloride) was used for surface sterilization of explants. After one week, some of the explants were found to survive in all replications except the contamination ones. The result was consistent with George (1993). The presented outline of sterilization procedures were employed throughout the experiments. Sodium hypochloride was used, for it was easily available and found to be less expensive. The survived flesh explants were found to be observed in White basal medium. The survived shoot tips with initiated callus at the base of the cut surface of two weeks old were then cultured for multiplication stage (Figure 11). B. Multiplication Stage for shoot tips culture The survived shoot tips were transferred to the WPM medium supplemented with different concentrations and combinations of BAP (0.01 mgl -1, 1.0 mgl -1 ) and NAA (0.005 mgl -1, 0.5 mgl -1 ) for shoot multiplication. BAP and NAA were used to calculate the proper balance between cytokinin and auxin for proliferation of shoots. After six weeks, the shoot initiation was found to be occurred in T 2 (WPM medium supplemented with the combination of 0.01 mgl -1 BAP + 0.005 mgl -1 NAA), T 4 (1.0 mgl -1 BAP + 0.005 mgl -1 NAA) and T 5 (1.0 mgl -1 BAP + 0.5 mgl -1 NAA) except in T 1 (WPM basal medium) and T 3 (0.01 mgl -1 BAP + 0.5 mgl -1 NAA). However slightly large callus formation were observed in T 3 (0.01 mgl -1 BAP + 0.5 mgl -1 NAA). The survived shoot-lets and callus in all treatments were transferred to the respective fresh medium for further development. After two weeks, the transferred ones were found to grow axillary shoots with well developed in T 2 (0.01 mgl -1 BAP + 0.005 mgl -1 NAA), T 4 (1.0mgL -1 BAP + 0.005mgL -1 NAA) and T 5 (1.0mgL -1 BAP + 0.5mgL -1 NAA). Initiation of shoots from the callus found to arose in T 3 (0.01 mgl -1 BAP + 0.5 mgl -1 NAA). These were shown in Fig. 12, 13, 14, 15 and 16. But in T 1 (WPM basal medium) was found to develop a more large callus formation.

254 Universities Research Journal 2011, Vol. 4, No. 1 After four weeks, the cultures showed more proliferation of shoots in T 4 (1.0 mgl -1 BAP + 0.005 mgl -1 IBA) and followed by T 5 and T 2. The poor proliferation of shoots was found in T 1 and T 3. After six weeks, the transferred cultures about 3 4 cm in length of the survived shoot-lets in all treatments were cut and again transferred to the respective fresh medium for the proliferation of axillary shoots. After two weeks sub-cultured, T 4 (WPM medium supplemented with the combination of 1.0 mgl -1 BAP + 0.005 mgl -1 NAA) showed the best proliferation of shoots than the other treatments (T 1, T 2, T 3 and T 5 ) and was shown in Fig. 17. In these treatments, T 2 and T 5 showed more shoots proliferation than T 1 and T 3. When compared the treatments T 2 and T 5, the more shoots proliferation were observed in T 5 (WPM medium supplemented with the combination of 1.0 mgl -1 BAP and 0.5 mgl -1 NAA). Then, the well developed shoots were sub-cultured on the best medium, T 4 (WPM medium supplemented with the combination of 1.0 mgl -1 BAP and 0.005 mgl -1 NAA) and was shown in Fig. 18. It was chosen as the best medium to obtain well developed and vigorous shoots to proceed further sub-culture. The shoots were sub-cultured after every four weeks of intervals. The correlation of shoots length, number of shoots and used medium supplemented with plant growth regular (PGR) such as BAP and NAA were calculated in this experiment. It was observed that they were correlated with each other. The R 2 values of the shoot length and this treatment was 0.6723 and that of number of shoots was 0.6606 (Figure 9 and 10).

Universities Research Journal 2011, Vol. 4, No. 1 255 R 2 = 0.6723 Shoot length 20 Shoot Length (cm) 15 10 5 T2 T1 T3 0 0 1 2 3 4 5 Tre atm e n t Fig. 9 Correlation of the various concentrations and combinations of BAP and NAA, whereas shoot length from teak under aseptic condition T4 T5 8 R 2 = 0.6606 No. of shoot T4 6 Number of shoot 4 2 T2 T3 T5 T1 0 0 1 2 3 4 5 Treatment Fig. 10 Correlation of the various concentrations and combinations of BAP and NAA, whereas number of shoot from teak under aseptic condition

256 Universities Research Journal 2011, Vol. 4, No. 1 Fig. 11 Formation of callus from the explants (shoot tip) after two weeks cultured Fig. 12 Well developed shoot initiation in T 1 after six weeks sub-cultured Fig. 13 Well developed shoot in T 2 Fig. 14 Well developed shoot in T 3 Fig. 15 Well developed shoot in T 4 Fig. 16 Well developed shoot in T 5

Universities Research Journal 2011, Vol. 4, No. 1 257 Fig. 17 Proliferation of shoots in T 4 after eight weeks cultured Fig. 18 Proliferation of shoots in T 4 after ten weeks cultured Discussion and Conclusion Teak is a large deciduous tree, with a tall straight stem. It stands out the most valuable of all known timbers for its extraordinary durability. Once seasoned, teak timber does not split, crack, shrink or alter its shape. It is not very hard and heavy but can give a beautiful appearance after being polished. With such preferences it has been over exploited for many centuries. Thus, teak plantation must be established and propagated rapidly to replenish the natural stand of those used areas. In order to meet those urgent demands, both extensive and intensive cultivation of teak is being undertaken. A major problem of teak plantation is that the seeds in general have very low germination rates (30 60%) due to difficulties to remove seed dormancy. Seed dormancy is an important characteristic of teak. In this paper, in vitro propagation, using shoot tips containing apical meristem has been a reliable method of mass clonal propagation of teak. In

258 Universities Research Journal 2011, Vol. 4, No. 1 this technique, node or shoot apices were induced to produce multiple shoots, followed by root induction under in vitro. In this research, the shoot tips were cultured on White basal medium and sub-cultured on Woody Plant Medium (WPM) supplemented with the combination of BAP (0.01 mgl -1, 1.0 mgl -1 ) and NAA (0.005 mgl -1, 0.5 mgl -1 ) for shoot multiplication respectively. It can be concluded that the best shoot proliferation is observed in WPM medium supplemented with the combination of BAP (1.0 mgl -1 ) and NAA (0.005 mgl -1 ), T 4 treatment. The beneficial impact of using biotechnology in forestry would be greater than most agronomic or horticultural species. Research on micropropagation; over a reasonable period, is needed to enhance the realization of the benefits to tree improvement. Acknowledgements My special thanks are due to Dr. Than Than Htay, Professor and Head of the Department of Botany, Dagon University, for her advices and encouragement in this work. I am also grateful to Daw Cho Mar Lynn, Associate Professor, Department of Botany, Dagon University, for her generous help. References Ahloowalia, B.S. (1996). A New System of Plant Micropropagation. BioLink 2:17. Waston module. Georage, E.F. (1993). Plant Propagation by Tissue Culture. Part 1. The Technology : Exegetics Ltd., Edington. Gomez, K.A., A.A. Gomez (1984). Statistical Procedures for Agricultural Research. New York: John Wiley and Sons, Inc., pp. 458-477. Kadambi, K. (1972). Siliviculture and Management of Teak. Bulletin 24 School of Forestry Stephen F. Austin State University Nacogdoches, Texas. Kaosa-ard, A. (1986). Seed leaflet No. 4A- Teak, Tectona grandis Nursery Techniques. (Danida Forest Seed Centre). Malana Impex Pvt. Ltd. (2009). The Teak Timber Trading Company Technology And Productivity of teak. Schubert, T.H. (1959). Teak Plantations in the Republic of Honduras. Ph.D. thesis, pp. 135. Harvard Univ., Cambridge, Mass. (Unpublished).

Universities Research Journal 2011, Vol. 4, No. 1 259 Soerianegra, I. and R.H.M.J. Lemmens (1994). Plant Resources of South-East Asia 5(1). Timber trees: Major commercial timbers. Wageningen: Pudoc Scientific Publishers. pp. 610. White, K.J. (1991). Teak. Some aspects of Research and Development. FAO Regional Office for Asia and the Pacific (RAPA) Bangkok. Yasodha, R., R. Sumathi and K. Gurumurthi (2004). Micropropagation for Quality Propagule Production in Plantation Forestry. Indian Journal of Biotechnology, vol. 3, pp. 159-170.