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1 Available online at Life Science Archives (LSA) ISSN: Volume 1; Issue - 5; Year 2015; Page: Research Article In vitro STANDARDIZATION OF EXPLANT STERILIZATION AND SHOOT INITIATION IN THREE BAMBOO SPECIES Bambusa balcooa, Dendrocalamus stocksii AND Dendrocalamus asper S. Nasreen*, R. Ananthalakshmi, G. Simla, M. Muthu Kumari and J. Subhashini, Department of Biochemistry, Sacred Heart College (Autonomous), Tirupattur , Tamil Nadu, India. Abstract The present study was conducted on three important edible Bamboo species namely Bambusa balcooa, Dendrocalamus stocksii and Dendrocalamus asper with an aim to establish an efficient surface sterilization and initiation protocol for their large scale shoot initiation under in vitro conditions. Different explants like nodal explants were taken from precocious branches of field grown, healthy plants for initiating aseptic cultures in all the three species. Among the various bamboos explants tested, unexpanded nodes from primary and secondary branches were found to be the best for raising cultures as they responded favorably to different media combinations. Sterilization of nodal segments was carried out by using Mercuric chloride (HgCl 2 ) by varying their time of exposure. Highest percentage of explants survival was observed when explants were sterilized with 0.1 % HgCl 2 for 9 or 11 minutes. Likewise, larger explants had a direct effect on culture initiation and took least time for sprouting. An efficient procedure for shoot initiation from nodes was achieved on media supplemented with growth regulators. In Bambusa balcooa the best response for multiple shoots induction was achieved on Murashige and Skoog s medium (MS) medium containing 4 and 5 mg of 6-Benzylaminopurine (BAP) along with 0.1 % of Naphthalene Acetic Acid (NAA) and Thidiazuron (TDZ) forming shoots (23.8) respectively. In Dendrocalamus stocksii, highest shoot proliferation (23.0 shoots) was obtained when 5 mg of kinetin was used in conjunction with 0.1 % of NAA and TDZ. In Dendrocalamus asper shoots (24.6) were formed response to concentration of 5 mg of zeatin in combination with NAA and TDZ. Once the clusters of shoots were formed, small clumps of 3-4 shoots were excised and transferred onto fresh multiplication medium every 4 weeks for continuous shoot proliferation. Article History Received : Revised : Accepted : Introduction Bamboos belonging to family Poaceae which are considered as one of the most versatile multiutility forest tree grasses. Though, the distribution of bamboos is worldwide with over 1250 species, their presence is predominantly * Corresponding author: S. Nasreen E. mail: nazrinsyed281@gmail.com Key words: In vitro, Contamination, MS medium, Bambusa balcooa, Dendrocalamus stocksii and Dendrocalamus asper. found in South Asia (Nongdam and Leimapokpam Tikendra, 2014). Bamboo is an ancient grass which has celebrated as the High Emperor of all the Grasses. Bamboo is the fastest growing plant on the planet with a growth rate of up to 1.2 meters a day. Its roots can reduce soil erosion by up to 75 per cent. It can sequester carbon faster than similar other fast growing tree species. It is a

2 S. Nasreen /Life Science Archives (LSA), Volume 1, Issue 5, Page 287 to 292, useful resource for local economies and also as structural raw material, fodder and source of fiber for paper manufacture. As bamboos are fast growing plants, recently they are considered as a prime renewable resource for biomass production. The potential of micro propagation for mass scale propagation has raised high hopes and a lot of research has been focused on the development of protocols for large and rapid scale production (Pratibha Sharma and Sarma, 2011). In vitro propagation method has various stages: (i) Selection of explants; (ii) Aseptic culture establishment; (iii) Multiplication of propagules and (iv) Rooting and acclimatization of plantlets. But, the most important and challenging step is sterilization of explant for aseptic culture establishment. As early in 1985, in vitro propagation of bamboos was advocated for mass propagation. The technique of axillary bud proliferation leading to clonal propagation was standardized for many bamboo species (Parthiban et al., 2013). Sterilization is a process where explants are made as contamination free before establishment of culture. Explant contamination depends on the several plant and environmental related factors such as species, age, explant source and prevailing weather condition. In fact, according to losses due to contamination under in vitro conditions, the majority of which was caused by fungal, yeast and bacterial contaminant. Consequence leads to the waste of time, effort and material which if not mitigated can have serious economic problems (Nidhi Srivastava et al., 2010). Improvement of bamboo was attempted through the selection of superior culms and their mass multiplication. For bamboo, different propagation techniques are available, such as seed propagation, clump division, rhizome and culm cuttings. Micropropagation using tissue culture allows much greater control and manipulation of the development of tissues within the culture tube than conventional methods (Parthiban et al., 2000). Tissue culture techniques help to mass multiply the trees species and the techniques have already revolutionized mass propagation of many hardwoods and softwoods (Bajaj, 1986; Tewari, 1994). Micropropagation is the method of choice for production of huge number of genetically identical plants or cloning of superior genotypes in shorter time span (Chelak and Rogers, 1990). Bambusa balcooa Roxb., is an indigenous widespread bamboo of North East India. It is tallest, strongest and highly durable and was utilized mostly for structural use and pulping. The species was also valued for its edible tender shoots, mainly for food and pickle industry (Pratibha Sharma and Sarma, 2011). Dendrocalamus stocksii is an important bamboo species with different end uses. It is a graceful mid-sized non-thorny bamboo species with loosely spaced solid erect culms ranging from mm diameter. This species is mainly found in Central Western Ghats from Kasargod in Kerala to Ratnagiri in Maharashtra. It is a valuable multipurpose bamboo species used as substitute for cane and rattan in bamboo based furniture industry. This species has a wide adaptability comes up well in tropical humid, sub-humid and semi-arid conditions under black and red soil conditions. Due to poor seed setting and nongregarious nature of this bamboo, genetic diversity could be highly restricted and continuous vegetative propagation from a narrow genetic base could have serious implication for conservation of the species (Viswanath et al., 2012) Dendrocalamus asper, a bamboo species, is valued for its edible tender shoots. The food industry based on these young shoots is well developed and expanding rapidly. However, the available methods for its propagation are slow and difficult for a number of reasons. Like other bamboo species, it is also known to be monocarpic, i.e. flowering once before culm death. Vegetative propagation is commonly practiced in bamboo cultivation but the plants developed will all be as old as their stock and will tend to flower and die simultaneously as the actual age is the same in every part of the bamboo. Vegetative propagation through cuttings and rhizomes was undependable due to the bulky size of the propagules and the non-availability of propagules in the required number. They are difficult to handle and transport, and plantlet survival in such cases was usually low (Ruchi KurapaShroti et al., 2012).The objective of this

3 S. Nasreen /Life Science Archives (LSA), Volume 1, Issue 5, Page 287 to 292, present research work is to study the selection of explant of Bambusa balcooa, Dendrocalamus asper, Dendrocalamus stocksii to perform surface sterilization of the selected explants and to estimate and compare the Initiation response of the selected explants. 2. Materials and Methods Explant Collection The explants of Bambusa balcoooa, Dendrocalamus stocksii, Dendrocalamus asper were collected from Genewin Biotech, Hosur, Tamil Nadu, India. The explant chosen for the selected species is nodes. The collected explants were brought to the production laboratory and washed thoroughly in running tap water for 10 min in order to eliminate the muddy particles from the explants. Nodal explants were excised of about cm with the help of secateur. Surface Sterilization Contamination of explants takes place by microbes which compete adversely with plant tissue cultures for nutrients. The presence of these microbes usually resulting increased culture mortality. Explants were subjected to repeated washing in distilled water. After that, the explants were treated with an antibiotic Streptomycin sulphate of 0.1 % concentration which acts as antibacterial agent and antifungal agent like Benzimidazole of 0.1 % concentration for 15 minutes to remove fungus and bacteria. Then, the explants were washed in distilled water 3 times repeatedly. The explants were then washed with liquid detergent Poly sorbate in 15 minutes and then washed properly with distilled water to remove the traces of detergent. Under sterile conditions in a laminar air flow cabinet, the explants were treated with Mercuric chloride solution (0.1 %) for 5, 7, 9, 11, 13, 15 minutes. After that, the explants were washed in distilled water thoroughly. Mortality rate was calculated for the explants by using the formula, % Mortality = Total number of explant dead/total number of explants 100 Initiation After the explants have been properly sterilized, the meristematic cells in the nodes will begin to grow. Surface sterilized nodes were cultured on MS basal medium containing 3 % (w/v) sucrose for culture initiation and served as explant sources for subsequent experiments in upright position. The ph of the medium was adjusted to 5.8 before gelling with 0.8 % (w/v) agar (Hi-media, Mumbai, India). The explants initially were implanted vertically on the culture medium in bottles and capped tightly and sealed with Klin wrap. Preparation on Murashige and Skoog (MS) media with different concentration of growth regulators M1 = MS + 3 % Sucrose + 6 BAP 1 to 5 mg/l + NAA 0.1 % M2 = MS + 3 % Sucrose + 6 BAP 1 to 5 mg/l + TDZ 0.1 % M3 = MS + 3 % Sucrose + Kinetin 1 to 5 mg/l + NAA 0.1 % M4 = MS + 3 % Sucrose + Kinetin 1 to 5 mg/l + TDZ 0.1 % M5 = MS + 3 % Sucrose + Zeatin 1 to 5 mg/l + NAA 0.1 % M6 = MS + 3 % Sucrose + Zeatin 1 to 5 mg/l + TDZ 0.1 % Culture Conditions All the established cultures were subjected to light intensity in the growth room for hours photoperiod provided by cool white fluorescent lamps of lux, temperature of about 25 ± 2 C and humidity of %. The observation for the shoot induction was recorded after 4-5 weeks. 3. Results and Discussion Effect of Surface Sterilization Surface sterilization of nodal segments of Bambusa vulgaris cv. Wamin (BVW) with 0.1 % mercuric chloride for 5 min and 70 % alcohol for 20 seconds reduced the bacterial contamination and yielded 95 % aseptic cultures. Sterilization of the explants with 0.1 % Bavistin for 5 min

4 S. Nasreen /Life Science Archives (LSA), Volume 1, Issue 5, Page 287 to 292, prevented fungal contamination. Mercuric chloride and 70 % ethanol have been used to disinfect the explants of Bambusa wamin, Bambusa balcooa and Dendrocalamus farinosus respectively were studied by Babitha Baskaran et al. (2014). Kalpataruduttamudoi et al. (2014) reported that the Tween 20 (5 %) % solution of Mancozeb (fungicide) + Gentamycin (antibiotic) + alcohol (70 %) containing treatment was the best for axenic culture establishment of Bambusa nutans. The surface sterilization was done for the Rhizome explants with different concentrations of mercuric chloride and mortality rate was calculated. An effective sterilization of nodal segments using sterilizing agent 0.1 % Mercuric Chloride (HgCl 2 ) by varying their time of exposure. The percentage of mortality rate were found by calculating number of explants dead. Highest percentage of explants survival when explants were sterilized with 0.1 % HgCl 2 at 9 min for Bambusa balcooa, 11 minutes for Dendrocalamus stocksii and also 9 % for Dendrocalamus asper. Initiation MS medium supplemented with different concentrations and combinations of Cytokinins and auxins showed variation in the regeneration percentage. MS medium with solidifying agent Gelrite supplemented with different concentrations of plant growth regulators like BAP, NAA, TDZ, Zeatin and Kinetin were prepared. The sterilized nodal explants collected were cultured in those media concentrations. After one to three weeks, axillary bud break was noticed but those, which did not sprout remained green for a longer period and dried up. Breaking of nodal buds and sprouting of shoot depend on the condition of explants, season of the year and culture condition. The BA (0.50 mg/l, 1.0 mg/l) was a suitable growth-hormone for shoot-induction which was concluded by Abha Jha et al. (2013). In vitro bud break on Bambusa bulcooa was achieved on basal MS media with 2 % sucrose in about 15 days. Best results for shoot multiplication were obtained on agarified MS medium supplemented with 4.4 μm of BAP and 0.53 μm of NAA where 19.8 ± 1.4 shoots per explant grew in about 30 days (Brar et al., 2014). Nirmala et al. (2011) described that in vitro multiple shoots of Dendrocalamus asper and D. membranaceus, proliferated for 6 8 months on MS medium supplemented with 7 mg/l BAP for Dendrocalamus asper and 5 mg/l BAP for Dendrocalamus membranaceus were used for rhizome induction. In vitro bud breaking of three bamboos (Bambusa balcooa, Dendrocalamus stocksii and Dendrocalamus asper) was studied. After the bud break, the elongated shoots were separated from nodes by sharp scalpel and transferred in the same fresh medium. Initially, the sprouted nodal buds produced thick shoots. Table 1: Effective surface sterilization - Exposure to HgCl 2 Species Concentration of HgCl 2 No. of Explant taken Exposure to HgCl 2 (min) No. of Explants dead Mortality rate (%) B. balcooa 0.1 % D. stocksii 0.1 % D. asper 0.1 % The clusters of shoots are of varied number. However, in the present study BAP 6 - Benzylaminopurine (4 and 5 mg -1 ) and 0.1 % of NAA and TDZ proved to be ideal for healthy shoot initiation in B. balcooa, followed by Kinetin (5 mg -1 ) and 0.1 % of NAA and TDZ gives optimum growth in D. stocksii and finally Zeatin (5 mg -1 ) and 0.1 % of NAA and TDZ give normal and healthy rise to the D. asper.

5 S. Nasreen /Life Science Archives (LSA), Volume 1, Issue 5, Page 287 to 292, Table 2: Effect of different growth regulators in MS medium on shoot initiation S. No Media (Liter) 1 MS + Sucrose 3 % 6 BAP 5 mg + NAA 0.1 % 2 MS + Sucrose 3 % 6 BAP - 4 & 5 mg + TDZ 0.1 % 3 MS + Sucrose 3 % Kinetin - 5 mg + NAA 0.1 % 4 MS + Sucrose 3 % Kinetin - 5 mg + TDZ % 5 MS + Sucrose 3 % Zeatin 5 mg + NAA 0.1 % 6 MS + Sucrose 3 % Zeatin 5 mg + TDZ % No. of survived explants No. of explants raised shoots Response (%) The clusters of shoots are of varied number. However, in the present study BAP 6 - Benzylaminopurine (4 and 5 mg -1 ) and 0.1 % of NAA and TDZ proved to be ideal for healthy shoot initiation in B. balcooa, followed by Kinetin (5 mg -1 ) and 0.1 % of NAA and TDZ gives optimum growth in D. stocksii and finally Zeatin (5 mg -1 ) and 0.1 % of NAA and TDZ give normal and healthy rise to the D. asper. 4. Conclusion In the present study, an effective sterilization of nodal segments was achieved using Mercuric Chloride (HgCl 2 ) as a sterilizing agent by varying their time of exposure and the effect of phytohormones was studied on the explants and this culture has been established at the laboratory. The well grown propagules show successful results hence, the experiment concluded with best growth pattern observed in media containing auxins like NAA and cytokinins like BAP, Kinetin, Thiadiazuron and Zeatin. The leftover cultures of this species would be sub- cultured and the developed propagules would be shifted to rooting media. Bamboo shoots have immense potential of being used as important health food as they have high content of useful proteins, amino acids, carbohydrates and many important minerals and vitamins and very low fat. Bamboo shoots are consumed predominantly in Asiatic countries. The usefulness of bamboo shoots as health food is not largely known by general public due to ignorance of their high nutritional values. It is important that to create awareness among the people about their nutritional health benefits so that they are widely accepted. Bamboos occupy a very significant position in everyday life of indigenous people of North - East India due to their enormous utility as traditional food, house construction materials and raw materials for production of useful domestic and other handicraft items. Intervention of modern micro propagation techniques was also essential to control the falling population of bamboos and to meet the needs. 5. Reference 1) Abha Jha De novo organogenesis in the form of rhizome in Dendrocalamus asper and D. membranaceus. Current Science, 100 (4): ) Babitha Baskaran In vitro propagation of Bambusa vulgaris cv. Wamin by axillary shoot proliferation. International Journal of Fundamental and Applied Sciences, 3 (4): ) Brar, J In vitro Propagation, biochemical studies and assessment of clonal fidelity through molecular markers in Bambusa balcooa. Journal of Tropical Forest Science, 26 (1): ) Kalpataru Dutta Mudoi Effect of nodal positions, seasonal variations, shoot clump and growth regulators on micropropagation of commercially important Bamboo. Bambusa

6 S. Nasreen /Life Science Archives (LSA), Volume 1, Issue 5, Page 287 to 292, nutans. Academic Journals,13 (19): ) Nidhi Srivastava Standardization of Sterilization Protocol for Micropropagation of Aconitum heterophyllum - An Endangered Medicinal Herb. Academic Arena, 2 (6): ) Nongdam, P and Leimapokpam Tikendra The Nutritional Facts of Bamboo Shoots and Their Usage as Important Traditional Foods of Northeast India, Hindawi Publishing Corporation, Volume ) Pratibha Sharma and K. P. Sarma International Conference on Advances in Biotechnology and Pharmaceutical Sciences (ICABPS). 8) Ruchi Kurapa Shroti Micropagation of Dendrocalamus asper through intermodal segments. Bulletin of Environment, Pharmacology and Life Sciences, 1 (3): ) Sharma Pratibha and K. P. Sarma. In vitro propagation of Bambusa nutan In Commercial Scale In Assam, India. Journal of Environmental Research And Development, 9 (2): ) Viswanath, S Dendrocalamus stocksii (Munro.): A potential multipurpose bamboo species for Peninsular India. A Publication of Institute of Wood Science & Technology, Bangalore.