World Journal of Agricultural Sciences 10 (1): 09-13, 2014 ISSN 1817-3047 IDOSI Publications, 2014 DOI: 10.5829/idosi.wjas.2014.10.1.1755 Albino Regenerants Proliferation of Dendrocalamus Asper In vitro 1 2 Vijay Kumar and Madhuparna Banerjee 1 Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi-835215, Jharkhand India 2 College of Biotechnology, Birsa Agricultural University, Kanke, Ranchi-834006 Jharkhand, India Abstract: The present study deals with the refinement of micropropagation protocol of Dendrocalamus asper for increasing the rate of multiplication. Single nodes of lateral branches were used as explant. Bud breaking and shoot proliferation were induced in solid Murashige and Skoog (MS) basal medium supplemented with 6- benzyladenine (BA) and adenine sulphate (Ads) within 15 days. Results showed that the rate of multiplication increased by 5-10 times in liquid media supplemented with 6-benzyladenine BA (3.0 mg/l) and Ads (50 mg/l). Albino regenerants have been derived from long term cultures in multiplication. The albino regenerants maintained separately showed high rate of multiplication (21.40 ± 0.73). Maximum rooting (13.96 ± 1.52) as well as growth of green plantlets was observed in MS with 1.0 mg/l IBA. Rooted plantlets were hardened and after 15-20 days of primary hardening the plantlets were transferred to poly bags for secondary hardening. Abbreviations: Kn = Kinetin; PGRs = Plant growth regulators; MS = Murashige and Skoog; Ads = Adenine sulphate, BA = 6-benzyladenine; IAA = Indole acetic acid; NAA = napthalene acetic acid and IBA = Indole butyric acid. Key words: Dendrocalamus asper Mass multiplication Albino Adenine sulphate INTRODUCTION D. asper is commonly propagated through seeds and culm cuttings. Small viability period, long flowering cycle and Bamboo, the largest member of Poaceae family, large scale consumption of seed by wild animals are the perhaps is the only natural resource having so many major drawbacks of large scale propagation of bamboos varied uses. Bamboo is one of the most important woody through seed. Vegetative methods for propagation is also shrubs known to mankind. Presently over two million t unreliable due to limited availability, low survival rate and (tonnes) of bamboo shoots are consumed world over transportation problem [4]. For production of quality and every year, mostly in Asian countries [1]. Every year US large requirement planting material at an accelerated pace imports 30000 t of edible bamboo shoots from China, within a short period of time is necessary. Protocol for in Indonesia, Japan, Taiwan and Thailand [2]. Among the vitro propagation of different species of bamboo has several species of bamboo Dendrocalamus asper is an already been established. In vitro mass multiplication of economically and environmentally important ornamental D. asper was successfully established by few other plant. It is native to China and commonly found in Asian authors [5-10]. Albino regenerants are frequently countries. The mature culms of D. asper are consumed in observed in bamboo tissue culture [11, 12], but no reports the cooking, construction, handicraft, outriggers on till date is available for albino regenerants of D. asper. To fishing boats and these are suitable for pulp and paper meet the increasing demand much beyond availability, manufacture also. Indian Council of Forestry Research micropropagation offers a better method for rapid and Education, Dehradun, India has successfully production of this valued plant of agro-forestry interest. introduced this exotic bamboo to India in 1994 [3]. Therefore, in the present communication, we describe an Corresponding Author: Madhuparna Banerjee, College of Biotechnology, Birsa Agricultural University, Kanke, Ranchi-834006 Jharkhand, India. 9
efficient proliferation protocol in which albino regenerants Rooting and Acclimatization: In vitro regenerated shoots of D. asper can be maintained and induced to proliferate were excised from culture and transferred to liquid MS new albino shoots directly using single node of lateral medium supplemented with IAA (1.0mg/l, 2.0mg/l and branches in vitro. We also systematically examined the 3.0mg/l), IBA (1.0mg/l, 2.0mg/l and 3.0mg/l) and NAA effects of adenine sulphate (Ads) and with 6- (1.0mg/l, 2.0mg/l and 3.0mg/l) separately. The numbers of benzyladenine (BA) on long-term albino regenerants roots developed per shoot were recorded after 4 weeks. proliferation. Well rooted plantlets were removed from in vitro culture, washed properly with distilled water and transferred to MATERIALS AND METHODS portray containing cocopeat for primary hardening for 15 days. After that plantlets were transferred to polybags Plant Source and Culture Conditions: D. asper plantlets containing sand: soil: farmyard manure (1:1:1 v/v) for were brought from Indian Forest productivity (ICFRE), better survival. Ranchi, Jharkhand India. The young, actively growing nodal segments (2-3 cm) from lateral branches were Statistical Analysis: The experiments were conducted collected and washed thoroughly under running tap water with a minimum of three replicates and all experiments and soaked in 0.2% Bavistin (systemic fungicide) solution were repeated three times. Data obtained from all followed with Tween 20, (5-6drops/100ml) solution for 20 experiments were presented as the mean ± SE of three min. Finally, the nodal explants were surface sterilized with replications. Statistically significant differences were 0.1% HgCl 2 w/v for about 20 min and thoroughly rinsed 4- determined by analysis of variance (ANOVA) and the 5 times with sterilized distilled water and both the ends Duncan multiple range test (DMRT) at a P < 0.05 level of were trimmed off. The whole procedure of surface significance. sterilization was performed under laminar air flow cabinet. Sterilized explants were cultured on Murashige and Skoog RESULTS AND DISCUSSION (MS) [13] medium supplemented with various PGRs such as BA, Kn, Ads with 3% sucrose (w/v) and 0.7% agar. In the present study, complete regeneration was Before autoclaving the medium for 16 min at 121 C, the ph successfully achieved from node segment of D. asper was adjusted to 5.8. Cultures were maintained in a growth (Fig. 1). Node segment collected from wild grown of D. chamber with a 16 h/8 h light/dark photoperiod at 24 ± 1 C asper produced varied number of multiple shoots in MS (day) and 20 ± 1 C (night). Light was supplied at intensity medium supplemented with PGRs such as BA (1.0, 2.0, 3.0, of 25 µmol m 2 s 1 by cool-white fluorescent lamps. 4.0 and 5.0 mg/l) and Kn alone and Ads in combination with BA (1-5 mg/l). The type and concentration of PGRs Multiple Shoot Induction and Shoot Proliferation: For influenced the differentiation and average number of multiple shoot induction and shoot proliferation after 4 shoots per explants as well as mean length of shoots. The weeks of incubation of nodal explants, the sprouted buds highest number (12.84 ± 0.44) of multiple shoots was were separated from culture and placed in MS medium obtained within 30 days of culture (Fig. 1b) when the with various PGRs. Five different concentration (1.0, 2.0, initiated buds were transferred to MS liquid medium 3.0, 4.0 and 5.0 mg/l) of BA and Kn were used for multiple supplemented with BA (3.0 mg/l) in combination of Ads shoot induction and Ads (50 mg/l) in combination with (50.0 mg/l). BA (1-5 mg/l) used to enhance the multiplication Different concentration of BA and Kn were used frequency. MS medium without any PGR served as a for the establishment of multiple shoot induction control. (Table 1.). BA alone proved to be more effective for induction of axillary shoot buds compare to Kn. Similar Production of Albino Variant Regenerants: Few cultures results were found on D. asper [2] and Gigantochloa of D. asper were kept in growth chamber in same medium atroviolaceae [14]. The explants did not respond well without any subculturing. Albino regenerants have been when Kn alone was used. Ads play a vital role for the derived from long term cultures in multiplication. These mass multiplication as PGR. The simulative role of Ads in albino regenerants maintained separately in MS liquid shoot multiplication has been emphasized from time to medium supplemented with BA (1.0, 2.0, 3.0, 4.0 and 5.0 time in various plants [15, 16]. Among all PGRs used, BA mg/l) in combination with Ads (50 mg/l) for high (3.0 mg/l) in combination with Ads (50 mg/l) was the most regeneration frequency. effective (12.84 ± 0.44) for multiple shoot induction. 10
Fig. 1: In vitro regeneration of D. asper from nodal explants (a) Shoot bud initiation from a single node explants taken from a field grown lateral branches on MS medium after 7 days (b) Multiple shoot proliferation after subculture in liquid MS medium supplemented with 3 mg/l BA in combination with 50 mg/l Ads after 30 days (c) Multiple albino shoots proliferation after 60 days (d) In vitro rooting in liquid MS supplemented with 1mg/l IBA after 28 days (e) primary acclimatization of D. asper in sand:soil:farmyard manure (1:1:1 v/v) Table 1: Effects of various concentrations of 6-benzyladenine (BA) and Kinetin (Kn) alone on percent response, mean number of shoots and mean shoot length of D. asper Hormone Concentrations (mg/l) Response (%) Mean Number of Shoots Mean Shoot Length (mm) BA 1.0 59.66 ± 0.37e 2.12 ± 0.76d 18.65 ± 0.65 bc 2.0 74.66 ± 0.44b 4.78 ± 0.55b 20.49 ± 0.30bc 3.0 79.33 ± 0.41a 6.14 ± 0.99a 40.66 ± 0.70a 4.0 68.33 ± 0.47c 3.79 ± 0.22c 32.33 ± 0.67ab 5.0 62.66 ± 0.31d 2.69 ± 0.39cd 22.93 ± 0.53bc KN 1.0 5.66 ± 0.61e 1.62 ± 0.55bc 8.60 ± 0.65bcd 2.0 12.33 ± 0.30d 2.42 ± 0.76ab 12.65 ± 0.67abc 3.0 16.66 ± 0.33c 1.00 ± 0.64c 19.93 ± 0.30a 4.0 24.33 ± 0.44b 1.24 ± 0.42c 14.33 ± 0.42ab 5.0 30.66 ± 0.68a 3.29 ± 0.84a 10.66 ± 0.88bcd In Table columns with different letters are significantly different at P<0.05 according to Duncan s multiple range test. 11
Table 2: Effects of various concentrations and combination of 6-benzyladenine (BA) and Adenine sulphate (Ads) on percent response, mean number of shoots, mean number of albino shoots after 60 days and mean shoot length of regenerated D. asper Hormone Concentrations (mg/l) Response (%) Mean Number of Shoots Mean Number of Albino Shoots after 60 Days Mean Shoot Length (mm) BA + Ads 1.0 + 50 70.12 ± 0.81e 3.88 ± 0.44e 7.06 ± 0.38e 23.47 ± 0.63d 2.0 + 50 82.00 ± 0.98b 7.06 ± 0.38d 14.18 ± 0.67c 30.49 ± 0.30c 3.0 + 50 92.12 ± 0.33a 12.84 ± 0.44a 21.40 ± 0.73a 52.93 ± 0.67a 4.0 + 50 78.33 ± 0.30c 10.18 ± 0.42b 16.44 ± 0.58b 48.66 ± 0.88ab 5.0 + 50 74.66 ± 0.47d 8.16 ± 0.31c 11.77 ± 0.23d 35.33 ± 0.47c In Table columns with different letters are significantly different at P<0.05 according to Duncan s multiple range test. Few cultures of D. asper were kept as same condition and in same growth medium in a growth chamber. Albino regenerants of D. asper were found after long term of culture in 60 days of time (Fig. 1c). These albino regenerants were transferred to MS liquid medium supplemented with BA (1-5 mg/l) in combination with Ads (50 mg/l). The rate of multiple shoot induction of D. asper is significantly high with yield a cluster of highest numbers of shoots (21.40 ± 0.73) per explants with a maximum length (52.93 ± 0.67 mm) (Table 2). Using the method described here, we were able to obtain a maximum number of albino D. asper regenerants in vitro, which will serve as experimental material for future studies. In contrast to previous studies [11, 12, 17], few albinomutants were obtained among the regenerated shoots. Albino inflorescence proliferation in Dendrocalamus latiflorus is also reported by Lin et al. [18]. Banerjee et al. [2] also reported 14 shoots per explants when axillary buds were transferred to liquid MS medium with BA (5.0 mg/l) in combination with Ads (40 mg/l). But in our study decreased concentration of BA in combination with increased concentration of Ads showed maximum multiple shoot induction. According to Singh et al. [19] maximum number of shoots were obtained in MS medium supplemented with 10µM BA and 75µM Ads. Three different auxins (indole acetic acid (IAA), indole butyric acid (IBA) and napthalene acetic acid (NAA) were used to study the effect of root induction in D. asper. Among the three auxins tested IBA (1mg/l) was found to be more effective for root induction. The highest number of roots (13.96 ± 1.52) per shoot (Table 3) was produced on liquid MS medium containing IBA (1.0 mg/l) in 4 weeks of time. In the present experiment, 1mg/l auxin (IAA, NAA or IBA) was found to be sufficient to induce rooting. Banerjee et al. [2] reported maximum 7 roots per explants in same concentration of IBA. MS medium with IBA (10mg/l) was found to be best (10-20) for rooting [7]. Comparatively in our study least concentration of IBA (1mg/l) was found to be optimal for best rooting. Table 3: Effects of different auxins (IAA, IBA and NAA) on number of root per shoot of D. asper Hormone Concentrations (mg/l) --------------------------------------------------- IAA IBA NAA Mean Number of Roots 1 0 0 5.43 ± 0.80d 2 0 0 3.06 ± 0.49e 3 0 0 2.80 ± 0.98e 0 1 0 13.96 ± 1.52a 0 2 0 8.43 ± 0.80b 0 3 0 7.66 ± 0.35bc 0 0 1 8.30 ± 0.86b 0 0 2 6.43 ± 0.41bc 0 0 3 4.56 ± 0.67d In Table columns with different letters are significantly different at P<0.05 according to Duncan s multiple range test. After 4 weeks of root induction, the plantlets with fully expanded leaflets were successfully transferred to cocopeat and hardened in green house under shade net for 15 days. After that plantlets were transferred to poly bags containing potting mix sand: soil: farmyard manure (1:1:1 v/v) for secondary hardening. Plantlets exhibited up to 95% survival rate after 30 days. In conclusion, we report here a protocol of establishment of an in vitro proliferation system for producing albino regenerants in D. asper. The present study is efficient protocol which can be adopted commercially for mass multiplication of edible bamboo, D. asper. Our protocol is simple and reliable and can be used to produce a maximum number of albino shoots of D. asper within a short period of time and raising them on a mass scale for plantations and forestation purposes. The significance of this mass multiplication from albino variant regenerants studies becomes crucial as they provide maximum number of shoots and also fulfil the demand in global market of these economically important plants. 12
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