Effects of different indole-3-butyric (IBA) concentrations (0, 0.5, 1.0, 2.0, 3.0 and 5.0 mg/l), two light regimes of in vitro rooting and acclimatization on in vitro teak (Tectona grandis L.f) were investigated. Shoots incubated in the light produced higher mean number of roots (2.0) and mean root length of 15.0 and 4.5 mm when supplemented with low IBA concentrations of 0.5 and 1.0 mg/l, respectively. Conversely, when shoots were incubated in the dark, higher mean number of roots (8.0 and 3.0) and mean root lengths (14.0 and 8.8 mm) were produced when supplemented with higher IBA concentrations of 2.0 and 3.0 mg/l, respectively. IBA was found necessary for root induction and growth in light and dark where 2.0 mg/l was optimum as it recorded high mean number of root (6.0 and 8.0) with mean root lengths of 11.8 and 14.0 mm, respectively. High survival percentage of 80% was recorded forthose plantlets raised in “jiffy-7” but survival reduced to 40% when grown in the autoclaved soil after one week

Goh, BL

Nor Aini, AS

Ridzuan, R

English

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African Journal of Biotechnology Vol. 8 (22), pp. 6158-6161, 16 November, 2009     Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2009 Academic Journals      Full Length Research Paper  The effects of different indole-3-butyric acid (IBA) concentrations, two light regimes of in vitro rooting and acclimatization of in vitro teak (Tectona grandis L.f) plantlets  Nor Aini, A. S.1,2*, Goh, B. L.2 and Ridzuan, R.1  1Laboratory of Sustainable of Bioresources Management, Institute of Tropical Forestry and Forests Products (INTROP), Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. 2Department of Forest Production, Faculty of Forestry, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.  Accepted 15 June, 2009  Effects of different indole-3-butyric (IBA) concentrations (0, 0.5, 1.0, 2.0, 3.0 and 5.0 mg/l), two light regimes of in vitro rooting and acclimatization on in vitro teak (Tectona grandis L.f) were investigated. Shoots incubated in the light produced higher mean number of roots (2.0) and mean root length of 15.0 and 4.5 mm when supplemented with low IBA concentrations of 0.5 and 1.0 mg/l, respectively. Conversely, when shoots were incubated in the dark, higher mean number of roots (8.0 and 3.0) and mean root lengths (14.0 and 8.8 mm) were produced when supplemented with higher IBA concentrations of 2.0 and 3.0 mg/l, respectively. IBA was found necessary for root induction and growth in light and dark where 2.0 mg/l was optimum as it recorded high mean number of root (6.0 and 8.0) with mean root lengths of 11.8 and 14.0 mm, respectively. High survival percentage of 80% was recorded for those plantlets raised in “jiffy-7” but survival reduced to 40% when grown in the autoclaved soil after one week.      Key words: Teak (Tectona grandis) plantlets, indole-3-butyric acid, light regime, in vitro rooting, acclimatization.    INTRODUCTION  Tropical deforestation and degradation of forests in the world are negatively affecting the availability of forest goods and services (FAO, 2005). Recently, global trend towards greater reliance on plantation as a source of industrial wood has been adopted as an effort to reduce timber harvesting from natural forest. One of the current recommended high quality timber species, identified to be potential for this purpose in Malaysia is teak (Tectona grandis). Teak is listed as one of nine selected species for forest plantation in Malaysia (MTIB, 2007) and ranked among the top five tropical hardwood species in term of plantation area established worldwide (Dah and Baw, 2001). In addition, teak wood is considered to be one of the best general utility timber with worldwide  repu-    *Corresponding author. E-mail: w.ridzuan85@yahoo.com. tation, being extensively used for shipbuilding, interior and exterior luxury furnishing, bridges and wharves, rail-way carriages and wagons, ordnance, shingles, wheels, carvings and general carpentry (Mendoza de Gyves et al., 2007; Appanah and Weinland, 1993). Establishment of teak plantation programme is limited to its seed problems. Besides having low seed quality and late seed production, it also has hard seed coat which further contributes to poor germination rate (Bonal and Monteuuis, 1997). It also shows irregular seed bearing habit (Lee and Rao, 1981) and teak raised from seeds show wide phenotypic variation on its growth which becomes disadvantages to commercial propa-gation and plantation (Krishnapillay et al., 1998). Thus, vegetative propagation can facilitate the possibilities to have constant supply of quality seedlings or plantlets of teak with desired growth performance. Tissue culture offers a practical  afforestation  program-       Figure 1. The elongated shoots used for in vitro rooting experiment. Shoots used were healthy thick shoots, with average length of 70 mm (Note: white bar is equal to 1 cm).    mes by producing uniform plants on a large scale and in a short period of time (Pandey and Brown, 2000). Al-though, in vitro research has been conducted on T. grandis, little work has been reported on acclimatization of micro propagated plantlets onto soil or other suitable rooting media. The practical prospects of micro propaga-tion are largely dependent on the success of the acclima-tization process and the capability of the acclimatized plantlets to resume growth (Bonal and Monteuuis, 1997).  Therefore, this study was carried out to investigate the effects of light and dark treatment on the rooting and different rooting media on the hardening of in vitro plantlets of T. grandis.   MATERIALS AND METHODS  In vitro rooting utilized elongated shoots which was initially cultured on MS medium supplemented with 0.2 mg/l of kinetin. They were raised for four weeks to allow the plantlets to produce thick shoots with an average length of 70 mm (Figure 1). The sources of the elongated shoots were provided by the Tropbio Research Sdn. Bhd., a private company specializing in plant tissue culture and molecular biology work.  The in vitro rooting experiments adopted a complete randomized design utilizing those elongated shoots cultured on white medium treated with six IBA concentrations; 0 mg/l (control), 0.5, 1.0, 2.0, 3.0 and 5.0 mg/l (15 replicates per treatment) for the dark  and  light  Nor Aini et al.         6159      Figure 2. Good root growth with mean number = 8.0 when supplemented with 5.0 mg/l IBA (Note: White bar is equal to 1 cm).    conditions. Aluminum foils were cut in the size of 10 x 6 cm and were then wrapped around the borosilicate test tubes to create the dark effect. Observation was made for a period of six weeks and data on percentage of shoot rooted, mean number of root and mean length of root were evaluated at the end of the sixth week.  The in vitro grown plantlets with developed root system obtained from rooting stage were later transplanted into jiffy “7” and sterile medium of 3:3:1 sand, soil and compost mixture. Evaluation was based on the survival percentage on rooting media.  In addition, the effects of in vivo rooting were compared using T-test. Data was analyzed using the Statistical Analysis System (SAS) computer package. The Duncan’s Multiple Range Test (DMRT) was used to determine the significant differences among treatment means only when the F test was significant. Differences at the pro-bability level of P>0.05 was only considered significant.     RESULTS AND DISCUSSION  Result indicated that the in vitro rooting of teak was signi-ficantly affected by different concentration of IBA used. Despite no root growth in IBA free medium, there was sign of swollen callus at the cut end. Generally, the per-centage of rooted shootlets was low when treated in dif-ferent concentrations of IBA. Almost all those rooted shootlets showed callus growth when treated with dif-ferent IBA. Gyves et al. (2007) also reported that callus would normally form at the cut end with an increase of IBA concentration.  Root growth in terms of mean number and length showed significantly differences in different IBA (Table 1). Those supplemented with 2.0 and 5.0 mg/l produced reasonably many roots with mean of 6.0 and 8.0 respec-tively (Figure 2).   Roots initiation and development were also found to  be   6160         Afr. J. Biotechnol.    Table 1. Effects of various concentration of IBA on root formation of in vitro shootlets.   IBA (mg/l) Percentage of rooted shoots (%) Mean number of roots Mean length of roots (mm) 0 0 0e 0e 0.5 15 2.0cd 15.5a 1 20 2.0d 4.5d 2 22.5 6.0b 11.8b 3 20 3.0c 3.8d 5 22.5 8.0a 8.0c  *Values having the same superscripts are not significantly different at P<0.05 based on Duncan’s Multiple Range Test.    Table 2. T-test of rootability of shoots incubated in the dark and light of different concentrations of IBA.  IBA  concentrations  No of Root Root Length  Dark  Light Dark  Light 0.5 mg/l Mean 1.5  2.3 3.0  15.5 Std Deviation 1.0  0.9 0.9  0.8 Observed T value  1.4ns   1.5*  1.0 mg/l Mean 2.6  1.8 1.8  4.5 Std Deviation 0.5  0.5 0.3  0.2 Observed T value  2.7*   7.6*  2.0 mg/l Mean 7.5  6.3 14.0  11.8 Std Deviation 1.2  0.8 2.4  1.9 Observed T value  1.9ns   0.9ns  3.0 mg/l Mean 3.3  3.1 8.8  3.8 Std Deviation 0.5  1.2 2.2  0.7 Observed T value  0.3ns   5.2*  5.0 mg/l  Mean 3.8  7.6 4.5  8.0 Std Deviation 1.0  1.2 1.0  0.9 Observed T value  5.4*   6.2*   nsNon significant at p<0.05, *Significance different at P<0.05.    affected by light regime. Roots started to develop after 10 days in culture dark incubation. Though root formation ini-tiated earlier when incubated in the dark (after 10 days in culture), most cultures showed sign of defoliation and browning effects. Almost all the leaves turned brown and were detached from the stem after 30 days in culture. Mean number of roots and root length were significantly different between light regime after 42 days (Table 2). Generally, cultures which were supplemented with 2 mg/l IBA exhibited good root growth either when incuba-ted in the light and dark treatments which were not signi-ficantly different (Table 2). In fact, higher mean number of roots of 8.0 and length of roots of 14.0 mm were obtained when incubated in the dark (Figure 3).   Result indicated that both IBA and light regime affected root formation of in vitro shootlets. This is in accordance with those reported by Druart et al. (1982), who stated that low level of light and low temperature were neces-sary to stimulate shoot proliferation and rooting. In fact, Durga and Metha (1993) and Rahman et al. (2004) recommended complete darkness to be given at the beginning of the rooting stage in Cammiphora wightii and Elaeocarpus robustus, respectively. The browning and the defoliation to the plantlets incubated in dark could be due to reaction of peroxidase and phenolic activities of adventitious shoots. This phenomenon was also shown in Malus domestica when they were kept in the dark (Druart et al., 1982). When light become the limiting factor, more auxin is required for rooting signified by shoots incubated in the dark gave better rooting especially for those being supplemented with higher auxins. Such responses were also shown by many woody species whereby at higher photon fluxed caused inhibition to rooting (Rugini et al., 1998). To date, the effect of dark  treatment  in  rooting  is        Figure 3. Good root growth (mean numbers) when supplemented with 2.0 mg/l IBA and incubated in the dark (Note: white bar is equal to 1 cm).    Table 3. T-test on the number of plantlets survival on “jiffy-7” and Unautoclave soil.  Parameter ‘’Jiffy-7  Autoclaved soil mixture N 20  20 Std Deviation 0.41  0.50 Observed T value  2.75*  Percentage of survival 80%  40%  ns Not significant at 0.05%, *Significant difference at P<0.05.    still not fully understood.   Acclimatization is the determining key for the survival of the in vitro raised plantlets since they are not suited for in vivo conditions (Baksha et al., 2007). The survival rate of teak plantlets transferred to ex vitro acclimatization treat-ments showed that there was significant between rooting media that is 80% for “jiffy 7” and 40% for autoclaved soil mixture after 35 days (Table 3). Jiffy 7 was found to be a better rooting medium and Chan et al. (2009) reported 100 % of survival rate for Gynura procumbens when raised on the same medium.    ACKNOWLEDGEMENTS  We thank Tropbio Research Sdn Bhd for providing us the elongated shoots and allowing us to carried out the root-ing experiment in their tissue culture laboratory. Deepest gratitude also goes to the all the staffs of Faculty of Forestry, UPM for the assistance and cooperation.  Nor Aini et al.         6161    REFERENCES  Appanah S, Weinland G (1993). Planting Quality Timber Trees in Peninsular Malaysia. Malaysia Forest Records No. 38. Chan HT (ed.) A Review Forest Research Institute Malaysia. pp. 97-99. Baksha R, Jahan MAA, Khatun R, Munshi JL (2007). In vitro Rapid clonal propagation of Rauvolfia serpentine (Linn.) Benth. Bangladesh J. Sci. Ind. Res. 42(1): 37-44. Bonal D, Monteuuis O (1997). Ex vitro survival, rooting and initial development of in vitro rooted vs. unrooted microshoots from juvenile and mature Tectona grandis genotypes. Silvae Genet. 46: 301-306. Chan LK, Lim SY, Pan LP (2009). Micropropagation of Gynura procumbens ( Lour.) Merr. an important medicinal plants. J. Med. Plants Res. 3(3): 105-111.   Dah US, Baw US (2001). Regional teak marketing and trade, Proceedings of third regional seminar on teak, Indonesia. pp. 1-14.  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The effects of different indole-3-butyric acid (IBA) concentrations, two light regimes of in vitro rooting and acclimatization of in vitro teak (Tectona grandis L.f) plantlets

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The effects of different indole-3-butyric acid (IBA) concentrations, two light regimes of in vitro rooting and acclimatization of in vitro teak (Tectona grandis L.f) plantlets

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