Indian Journal of Experimental Biology Vol. 51, January 2013, pp. 88-96 In vitro seed germination of economically important edible bamboo Dendrocalamus membranaceus Munro† Jasmine Brar1, Manju Anand2 & Anil Sood1* 1Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology (IHBT), Palampur 176 061, India Received 18 April 2012; revised 19 September 2012 An in vitro propagation protocol using mature seeds of D. membranaceus was successfully established. Scarcity of seeds in bamboos because of their long flowering periods and irregular seed set resulting in low viability and germination potential, motivated us to undertake the present study. The effects of sterilants, light conditions, exogenous application of plant growth regulators and temperature in overcoming germination barriers in ageing seeds of bamboo were studied. It was found that HgCl2 (0.1%) along with bleach (15%) was more effective in raising aseptic cultures. Dark conditions, high temperatures around 30 °C and soaking of seeds in GA3 solution (50 ppm) overnight stimulated high percent of seed germination with corresponding increase in shoot length (2.7±0.7 mm) and number of sprouts (2.1±0.7) per explants during culture initiation. 6-benzylaminopurine acted synergistically with kinetin to give optimum germination rate of 70±13.9% as compared to 63.13% when used individually. For prolonged maintenance of cultures, 2% sucrose was found to be suitable for promoting photomixotrophic micropropagation. Following this procedure, about 65% survival of plantlets could be achieved during hardening. Biochemically seeds consume starchy endosperm for emergence of radicle which is taken as a sign of germination as also evident from the present study. Loss of viability and vigour after a year was confirmed by Tetrazolium chloride test. Micropropagation protocol developed here will ensure regeneration of large number of plants in a relatively short time. Conclusively, in vitro propagation protocol developed in D. membranaceus using mature seeds as an explants is reported for the first time. Keywords: Acclimatization, Dendrocalamus membranaceus, Germination, Gibberellic acid, Low viability, Seeds Bamboos have been in demand due to their management. Demographic characteristics of bamboo multifarious uses. India has abundant bamboo regeneration from seeds remain largely unknown2 and resources with about 24 genera and 138 species. there is a great variation in their capacity to remain About 2.5 billion people depend on or use bamboos to dormant and viable in soil. Intermast period being a value of US$7 billion per year and these figures very long3, it would be difficult to store large have gone much beyond that as these provide quantities of seeds for mass scale planting till next alternative source for meeting many of our needs1. flowering cycle is repeated. Seed viability is generally The current work focuses on the knowledge of seed very low in bamboos, as endogenous levels of auxins viability, germination pattern and seedling behaviour and abscisic acid (ABA) in seeds is found to be one of of bamboo in the sub-Himalayan zone. Though the major factors related to the loss in seed viability in bamboos are fast growing and early maturing, their stored bamboo seeds4. Bamboo seeds are short lived, peculiar monocarpic nature and the mass scale dying germinate within 3-7 days, lose viability within out after flowering has become a problem to the 1-2 months5 and germination potential being season managers for programming a sustainable dependent. Absence of seed set in bamboos due to cumulative effect of physical and physiological _________ factors6 have prompted researchers for devising * Correspondent author methods for artificial seed production for Telephone: 01894- 233 337 7 Fax: 01894 – 230 433 propagation . In some species as Bambusa tuldoides, E-mail: [email protected] viable seed production was reported for the first time [email protected] in Argentina8. Potential application of tissue culture [email protected] 2 for propagation using seeds in bamboos is well Department of Biotechnology and Enviornmental Sciences, 9,10 Thapar University, Patiala 147 004, India documented . Conventional methods of vegetative †IHBT publication no. 2309 propagation pose problems for a number of reasons as BRAR et al.: IN VITRO PROPAGATION OF DENDROCALAMUS 89 cuttings, offsets and rhizomes are bulky and not in list of plant families with seeds having physical available in desired numbers, are difficult to handle dormancy13,14. The seeds show natural ageing in other and transport and plantlet survival after natural species of Poaceae as well15. It has been seen several regeneration is usually very low. Most of the forest times that the seeds of significant number of species covers of bamboos especially of D. membranaceus are sold and distributed by commercial seed companies are fast declining because of poor harvesting methods, in a dead on delivery (DOD) state16, which is also lack of knowledge on propagation techniques, among the major problems encountered. By the present flowering control and proper storage of seeds. Hence, study it is intended to improve the germination inducing seed germination in a sterile environment in percentage by subjecting seeds to different physico- presence of appropriate phytohormones leads to an chemical environments under in vitro and ex vitro enhancement of productivity and also for conditions so as to enable selection of plants which conservation of this important species. Therefore, use show better phenotypic traits (elite plants) and further of in vitro methods seems to be a good alternative to micropropagate them to produce plants for large scale counter this problem. plantation. The protocol for in vitro propagation in The present research intrest in D. membranaceus bamboos provides a good alternative for propagation Munro (Poaceae), native of Myanmar was due to the which is highly desirable due to its high demand and fact that it has been listed among 18 additional taxa depletion in the natural forests due to unscientific large under ‘Important’ category (www.unep-wcmc.org; scale extraction by the rural population for meeting www.inbar.int). Dendrocalamus membranaceus is their increased requirements. considered an ecologically important species as it has an extensive root system which prevents soil erosion. Materials and Methods Major threats to this species include logging of Plant material—Poaceae have caryopses as rainforests in which this bamboo resides, propagation units which are single seeded and where unsustainable land use by practices like slash – burn testa is fused with thin pericarp. Seeds for the present and shifting agriculture and harvesting for experimentation were procured from M/s Allen Green international exportation. The major threat Seeds, Wuyang province, China. The seeds were documented for this species is the beetle approximately more than 6 months old i.e. ageing Crytotrachelus longimanus which is a major forest seeds. Seeds are broadly ovate, rounded at base with a invasive species occurring mainly in Thailand which pointed end, 5-7.5 mm long and dark brown in colour. proves detrimental to the natural population of this The seeds were germinated and also preserved in the species. Hence the methods of conservation like seed bank of the Institute. Once the plants grew big, micropropagation through seeds, nodal explants or these were got identified by the scientists of IHBT somatic embryogenesis hold greater significance. It is Herbarium. The mother plants are been grown and fast becoming a high value crop. Besides innumerable preserved in the campus. other uses, it is edible, widely used for construction Culture establishment—The explants (glumed purposes in Myanmar and Thailand and one of the seeds) were prepared carefully by floating in water. most promising species for pulp. After studying 27 Debris and empty seeds were separated this way. The bamboos belonging to 10 genera for the edibility of de-husked seeds were rinsed in 0.01% (v/v) Tween 20 shoots, this bamboo was considered to be excellent for 10 min followed by antifungal treatment with from processing point of view, as the young shoots bavistin 0.25% (w/v) and a broad spectrum antibiotic are smooth and easy to handle11. In China, it is used streptomycin sulphate 0.25% (w/v) for 45 min by for making chopsticks, shreds and paper. Therefore, gentle shaking. Final steps of surface sterilization devising a protocol for their propagation by tissue were performed in laminar flow with 15% sodium culture is highly desirable and selection at the hypochlorite for 10 min and 0.1% (w/v) HgCl2 for seedling stage is quite useful in multiplying only the 10 min. Each treatment was followed by repeated fast growing ones as plus plants. washings for a minimum of 3 times in autoclaved Conservation and propagation of the bamboos need distilled water. attention as stressed upon by UNDEP, FAO, INBAR Culture establishment and germination—Explants 17 and the programs like FORTIP12. Chenopodiaceae, were inoculated on sterile MS media medium Liliaceae, Poaceae and Solanaceae often are included (pH 5.6-5.8) and supplemented with various 90 INDIAN J EXP BIOL, JANUARY 2013 concentrations and combinations of plant growth Out of the various combinations of microbial regulators like 6-benzylaminopurine (BAP) and disinfectants tried, treatment of seeds with 0.1% kinetin (KN). Depending upon the requirement for HgCl2 and 15% sodium hypochlorite (NaOCl) for initiation media or proliferation media 2.0-3.0% (w/v) 10 min each gave the best results with 77.8±9.6 sucrose was added as a carbon source with 0.75% aseptic cultures establishment (Fig. 1). A combination (w/v) agar. Three replicates were taken for each of two sterilants at a moderate concentration for a treatment having 10 seeds each. long duration during the treatment was effective Growth conditions—Explants were maintained at rather than high doses for a short time. 25±2 °C at photoperiod of 16 h light and 8 h dark Effect of light—Seeds were incubated in continuous under light intensity of 70 µmol m-2 s -1 with 75% RH.