ASIAN J. EXP. BIOL. SCI.

VOL 2(3) 2011: 498-501

Society of Applied Sciences

SHORT COMMUNICATION

Acclimatization and Macroproliferation of Micropropagated Plants of Bambusa tulda Roxb.

Yogeshwar Mishra*, Pradeep Patel and S.A.Ansari
Genetics and Plant Propagation Division Tropical Forest Research Institute, P.O.-R.F.R.C., Mandla Road, Jabalpur (M.P) 482021, India ABSTRACT In the present paper, a procedure for acclimatization and macroproliferation of in vitro plantlets raised from field grown clump of Bambusa tulda is described. The acclimatization procedure included gradual hardening from culture room to natural environment, taking 30 45 days. Rooted plantlets of about 2-3 cm height were subsequently transferred to different potting media for acclimatization. Plantlets grown in perlite showed 91% survival followed by soilrite with 66% of survival. The acclimatized plantlets were macroproliferated in a cycle of six months and provided with a uniform dose of 100 ppm urea. The proliferation rate was stabilized to be 3 plantlets per macroproliferation cycle in B.tulda. KEY WORDS: Hardening, MS inorganic salts, plantlets production, substratum

INTRODUCTION Micropropagation has been widely utilized for rapid and mass multiplication of many species including bamboos. However, its sample application is restricted often by the high rate of percentage of plant loss when transferred to ex vitro condition. This is due to regenerates get adjusted with many abnormalities in ex vitro environment like high level of irradiance, low humidity and water is limiting due to low hydraulic conductivity of roots and root-stem connections [1]. Acclimatization of regenerates will overcome these problems with gradual lowering in air humidity [2]. The development of successful hardening technique is prerequisite for micropropagation method. These days many computer based acclimatization units are available but at a high price [3]. Hardening accounts for about 60% of the total production cost [4]. Therefore, an efficient and cost-effective acclimatization technique is necessary for in vitro raised plantlets. So, we attempted to develop a new efficient, cost-effective and one step of in vitro hardening technique for in vitro raised plantlets of Bambusa tulda Roxb. It is a very versatile and rapidly growing bamboo of South East Asia but suffer from enormous reduction in their stocks due to over exploitation by ever-increasing population and colossal use in pulp and paper industries. B. tulda is a native to north and north-east India, Bangladesh, Myanmar and Thailand [5]. It has been reported as a reluctant-to-root species [6, 7]. Its culms are used for construction, scaffolding, furniture, handicrafts and paper pulp while young shoots are pickled [8]. Being rich in phytosterols, B. tulda shoots are excellent source for production of sterol drugs [9]. Various tissue culture protocols have been evolved for large scale production of the plantlets of B.tulda using seedling and field grown explants [1012]. However, hardening of in vitro plantlets remains a crucial step for success of tissue culture protocols of this species. No attempt has been made describing detailed hardening procedure of this species. In the present paper, the authors describe a method for acclimatization and successful hardening of the plantlets of Bambusa tulda raised through micropropagation. Following the pioneering work done by Banik [13] and Adarsh Kumar et al. [14], we have optimized the macroproliferation technique of hardened and acclimatized plantlets of B. tulda for further stock build up.

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MATERIALSAND METHODS Hardening experiment In vitro regeneration of B. tulda plantlets was achieved by the method described earlier [12]. The plantlets of 2-4 cm length were taken out from the culture flasks and washed in running tap water thoroughly so as to remove adhered medium from the surface of plantlets followed by washings with 0.1% (w/v) Bavistin for 2 min and tap water. The washed plantlets were transferred to root trainers comprising 25 cells each of 150cc filled with four different potting media viz., soilrite, perlite, vermiculite and compost for hardening (Fig 1a). The potting media were autoclaved at 0 -1 121 C of 1.05 kg cm for 1 hr before being used for hardening.

Fig. 1: Hardening and macroproliferation of in vitro raised B.tulda Roxb.: (a) transfer of plantlets in root trainer filled with different hardening substratum; (b) root trainer shifted to tray filled with MS solution and covered with perforated polythene sheet; (c) in vitro hardened plantlets ready for transfer to mist chamber; (d) macroproliferable plantlets after six month of transfer; (e-g) separation and planting of individual tillers in polythene bags (h) macroproliferated propagules maintained in shadehouse; (i) six month old propagule ready for macroproliferation.

The planted root trainers shifted to plastic tray filled with half strength of iron free MS salts covered with perforated 0 2 transparent polythene sheet and maintained at 24 1 C under normal laboratory light condition (40 mol /m /sec.) provided with fluorescent tube and 12/12 h (light/dark) photoperiod for in vitro hardening (Fig 1b). The observations were recorded for survival percentage and height of the plants after 8 weeks of transfer.After an acclimatization period of 8 weeks when new leaves had emerged, the plantlets were transferred to shadehouse and maintained for 2 weeks for ex vitro hardening (Fig 1c). Finally, the hardened and acclimatized plantlets were transplanted in polythene bags containing soil: sand: farmyard manure (1:1:1) in the shadehouse condition for 1 to 2 months receiving irrigation on
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alternate days and spray of MS solution once in 15 days. In the hardening experiment, a total of 25 plants (in a root trainer) were taken for each potting medium and regarded as one replication. A total of three replications were used in this experiment. The data representing means of three replications were analysed with the SX statistical package according to a completely randomize design (CRD) using one way analysis of variance. Least Significant Differences (LSD) values were calculated at p= 0.05 for comparing means of the treatments. Macroproliferation experiment The hardened and acclimatized plantlets were planted in polybags of 24x18 cm size in the month of October 2006. Six months old hardened plantlets of B.tulda having tillers, rhizomes and roots were carefully removed from the polybags (Fig 1d). Each proliferated tiller along with some rhizome and roots was separated to act as a propagule (Fig 1e, f) and planted in fresh polythene bags filled with soil: sand: farmyard manure (1:1:1) (Fig 1g) in the month of March and initially supplied with 100 ppm urea and maintained in shadehouse (Fig 1h). The data on average diameter of rhizomes, average root length, average root number, average tiller diameter, average number of nodes, average tiller length and average number of tillers were recorded after six months of transfer (Fig 1i). A total of 30 tillers were taken and regarded as one replication. A total of three replications were used. The data representing means of three replications were analysed with the SX statistical package according to a completely randomize design (CRD) using one-way analysis of variance. Least Significant Differences (LSD) values were calculated at p= 0.05 for comparing means of the treatments. RESULTSAND DISCUSSION The data recorded on the survival percentage and height of the plants during hardening is presented in Table 1. In the hardening experiment, perlite resulted in maximum survival (91%) of plantlets of B.tulda, which was statistically higher than all other substrates. In accordance with this finding, Ndiaye et al. [15] and Lin and Chang [16] were also found perlite as one of the most suitable potting media for hardening of bamboo species. Macroproliferation is a simple and effective procedure of vegetative propagation recommended when bamboo plants are available. This technique was successfully used by Banik [17], Adarsh Kumar et al. [18] and Adarsh Kumar et al. [19] and attempted at Tropical Forest Research Institute, Jabalpur. In this method, the tillers (individual thin culms along with rhizome) of a proliferated seedling were separated (by rhizome division) and planted as individual propagules. New tillers arose from these propagules in a few months. When they reached a 34 tiller stage, they were again subjected to tiller separation and planting. The in vitro regenerated plantlets subjected to macroproliferation produced 3 and 2.44 number of tillers in B.tulda in March August and May-October, respectively (Table 2). The data revealed that there is significant variation in number of tillers in this species in two seasons. The relatively low number in May-October may be possibly due to the onset of manson and comparatively low temperature. Whereas there was no dormant phase between March- August and the growth of propagule was steady and continuous. Accordingly, each propagule became suitable to give a maximum of 3 numbers of propagules in the month of March-August. Similarly, other parameters like, diameter of rhizome, root length, root number, tiller diameter and number of nodes are also significantly higher in March-August season in B.tulda as compared to May-October. In earlier study Banik [13] obtained 3 numbers of tillers after 9 month in macroproliferated seedling of Bambusa tulda. However, obtaining 3 numbers of tillers in a shorter span of 6 months is an added advantage of this study, which may be attributed to the use of clonal materials for macroproliferation. The vegetative propagation of B.tulda through macroproliferation has been found as one of the most dependable techniques. The multiplied tillers, thus produced, remain smaller in size due to continuous rhizome separation. This is additional advantage as such propagules are easy to handle and transport. This procedure can be successfully applied to self incompatible bamboo particular B.tulda that has inherent difficulty in generating plants through vegetative methods in general.
Table 1: Performance of in vitro raised plants of B.tulda under different hardening substratum. Observation recorded after 8 weeks
P o tting m ed ia S urv ival % S o ilrite P erlite V erm ic ulite C o m p o st L S D (0 .0 5 ) 6 6 .6 (5 6 .6 ) 9 1 .0 (8 0 .0 ) 5 8 .0 (5 0 .0 ) 2 0 .0 (2 6 .5 6 ) 2 2 .6 7 P ara m eters H eig ht (c m ) N e w lea f fo rm atio n (% ) 1 6 .2 76 2 2 .3 3 2 1 .3 3 1 9 .6 7 6 .7 1 89 65 15 1 9 .2 3 D a ys req uired leaf fo rm atio n 12 9 14 16 fo r

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Table 2: Propagule production and growth data of in vitro raised plantlets of B.tulda Parameters Diameter rhizome (cm) Root length (cm) Root number (cm) Tiller dia. (cm) Number of nodes Tiller length (cm) Number of tiller March-Aug 1.24 34.33 28.22 0.85 10.66 110.89 3 Number of propagule May-Oct LSD (0.05) 0.90 0.313 23.33 10.82 20.44 3.91 0.49 0.179 7.22 3.13 76.66 14.83 2.44 0.48

CONCLUSION The one step hardening technique described herein proves to be cost effective and efficient alternative to conventional hardening technique for acclimatization of in vitro raised plantlets of B.tulda. Production of plants in shorter period (6 months) through macroproliferation procedure is an added advantage to produce clonal planting materials of B.tulda raised through micropropagation. REFERENCES
[1]. Fila, G., Ghashghaie, J. and Cornic, G. (1998). Photosynthesis, leaf conductance and water relation of in vitro cultured grapevine rootstock in relation to acclimatization, Physiol. Plant., 102: 411-418. [2]. Lavanya, M., Venkateshwarlu, B. and Devi, B.P. (2009). Acclimatization of neem microshoots adaptable to semi sterile conditions, Ind. J. Biotechnol., 8:218-222. [3]. Pospisilova, J., Ticha, I., Kadleaeek, P., Haisel, D. and Plazkova, S. (1999). Acclimatization of micropropagated plants to ex vitro conditions. Biol. Plant., 42: 481-497. [4]. Hazarika, B.N. (2003).Acclimatization of in vitro raised plants, Curr. Sci., 85: 1704-1712. [5]. Gamble, J. S. (1896). Bambusae of British India.Ann. Bot. Garden Calcutta, 3:32-33. [6]. McClure, F.A. and Kennard, W.C. (1955). Propagation studies (with bamboo). Report of Federal Experimental Station Puerto Rico. [7]. Rao, I.V.R., Rao, U.I. and Roohi, F.N. (1992). Bamboo propagation through conventional and in vitro techniques (Ed. Baker, F.W.G.) Rapid Propagation of Fast-growing Woody Species, CASAFAReport Series No. 3: 41-56. [8]. Seethalakshmi, K.K. and Kumar, M.S.M. (1998). Bamboo of India. Bamboo Information Centre-India, KFRI, Peechi. [9]. Srivastava, R. C. (1990). Bamboo, new raw material for phytosterols. Curr. Sci., 59: 1333-1334. [10]. Saxena, S. (1990). In vitro propagation of the bamboo (Bambusa tulda Roxb.) through shoot proliferation, Plant Cell Rep., 9: 431434. [11]. Pal, A. and Das, M. (2005). Clonal propagation and production of genetically uniform regenerants from axillary meristems of adult bamboo. J. Plant Biochem. and Biotech., 14: 185-188. [12]. Mishra, Y., Patel, P., Yadav, S., Shirin, F. and Ansari, S.A. (2008) A micropropagation system for cloning of Bambusa tulda Roxb. Sci. Horti., 115: 315-318. [13]. Banik, R.L. (1987). Techniques of bamboo propagation with special reference to pre-rooted and pre-rhizomed branch cuttings and tissue culture (Ed. Rao,A. N.), Recent Research on Bamboos IDRC, Canada, pp. 160169. [14]. Adarsh, Kumar., Gupta, B.B. and Negi, D.S. (1988). Vegetative propagation of Dendrocalamus strictus through macroproliferation. Ind. For., 114: 564568. [15]. Ndiaye, A., Diallo, M.S., Niang, D. and Gassama-dia, Y.K. (2006). In vitro regeneration of adult trees of Bambusa vulgaris, African J. Biotech., 5: 1245-1248. [16]. Lin, C.S. and Chang, W.C. (1998) Micropropagation of Bambusa edulis through nodal explants of field-grown culms and flowering of regenerated plantlets, Plant Cell Rep., 17: 617-620. [17]. Banik, R. L. (1995). A manual for vegetative propagation of Bamboos, INBAR Technical Report No. 6, INBAR, FORTIP and Bangladesh Forest Research Institute, pp. 166. [18]. Adarsh Kumar, Gupta, B.B. and Negi, D.S. (1991). Vegetative propagation of Dendrocalamus strictus through macroproliferation II. Ind. For.,117: 621623. [19]. Adarsh Kumar, Pal, M. and Kumar, S. (1992). Mass production of field planting stock of Dendrocalamus hamiltonii vegetatively through macrorpoliferation. Ind. For., 118: 638645.

Corresponding author: Yogeshwar Mishra , Genetics and Plant Propagation Division, Tropical Forest Research P.O. -R.F.R.C., Mandla Road, Jabalpur (M.P) 482021, India, e -mail: m_yogiraj@yahoo.com

Institute,

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