Nusrat Sultana
Silviculture Genetics Division, Bangladesh Forest Research Institute, P.O. Box No-273, Chattogram 4000, Bangladesh
Mohammad Azhar Uddin
Institute of Forestry and Environmental Sciences, Chattogram University. Chattogram – 4000, Bangladesh.
Saiful Alam Md. Tareq
Silviculture Genetics Division, Bangladesh Forest Research Institute, P.O. Box No-273, Chattogram 4000, Bangladesh
Waheeda Parvin
Silviculture Genetics Division, Bangladesh Forest Research Institute, P.O. Box No-273, Chattogram 4000, Bangladesh
Md. Aktar Hossain
Institute of Forestry and Environmental Sciences, Chattogram University. Chattogram – 4000, Bangladesh.
Md. Mahbubur Rahman
Silviculture Genetics Division, Bangladesh Forest Research Institute, P.O. Box No-273, Chattogram 4000, Bangladesh
In vitro propagation, Seed germination, Dendrocalamus giganteus, Giant bamboo
Mehergona Forest Range office campus, under Cox’s Bazar North Division of Bangladesh Forest Department and stored at Silviculture Genetics Division (SGD) of Bangladesh Forest Research Institute (BFRI).
Development of Host and Medicinal Plants
Bamboo, In vitro
Plant materials In 2016, seeds of D. giganteus were collected from Mehergona Forest Range office campus, under Cox’s Bazar North Division of Bangladesh Forest Department and stored at Silviculture Genetics Division (SGD) of Bangladesh Forest Research Institute (BFRI). To maintain the seed viability it was stored in plastic bag in refrigerator at 4°C temperature until use. Healthy seeds were selected carefully and used for raising axenic culture through in vitro germination. The experiments were carried out at the tissue culture laboratory and the nursery of Silviculture Genetics Division BFRI, Chattogram, Bangladesh.
Sterilization and in vitro seed germination Collected seeds were surface sterilized to get rid of all microorganisms before inoculation. Accordingly seeds were rinsed under running tap water for 40-60 minutes to remove debris and dehusked. The dehusked seeds were rinsed in distilled water and soaked for 2 hours. Then the imbibed seeds were washed by double distilled water (DDW) with 2-3 drops of Tween-20 for 12 minutes with agitation to physically remove most microorganisms and to remove some debris. The seeds were then treated with 70% ethanol for 1 minute under laminar air flow cabinet. After pretreatment with ethanol, the seeds were rinsed with autoclaved distilled water three times, to lower the toxic effect of ethanol followed by treatment with 0.1% Mercuric Chloride (HgCl2) for 10 minutes with gentle shaking. Finally the seeds were washed with DDW thrice to remove the traces of HgCl2.
Culture media and culture condition The shoot tips collected from the axenic culture were inoculated onto MS medium comprising 3% sucrose as carbon source and 2.8 gm/L gelrite as solidifying agent for initial growth. Various plant growth regulators such as; cytokinins (BAP and Kn) and auxins (IBA and NAA) were used to prepare MS medium for the in vitro seed germination, regeneration of multiple shoots and development of roots from the base of excised shoots. The pH of the medium was adjusted to 5.8 before addition of gelrite and sterilization. The medium was autoclaved at 1.08 kg/cm2 pressure and 1210C for 20 minutes. The cultures were incubated at 25±2°C under cool white and fluorescent light of 2000-2500 lux, maintaining about 60-80% relative humidity and 16/8 hours light and dark period in the growth chamber, respectively. These culture conditions were used in all the experiments mentioned below unless otherwise stated. Observations were made at regular intervals and tabulated.
In vitro seed germination and multiple shoots production MS medium supplemented with or without different concentrations of (0.0, 0.5, 1.0 and 1.5 mg/L) BAP was employed to find out suitable seed germination medium. Germinated shoots were multiplied in MS medium supplemented with different concentrations (0, 1.0, 2.0, 3.0 and 4.0 mg/L) and combinations of BAP and Kn. Number of shoots per explants and their morphology were observed periodically. To optimize the shoot production, effect of sub culturing and the strength of sucrose level in culture medium were evaluated. Rate of multiplication of shoots and their growth were recorded up to 4-8 weeks of culture.
Development of roots at the base of the shoot, hardening and acclimatization of plantlets In vitro elongated shoots (6-7 cm.) with at least 3-4 nodes were taken out from the culture vessel and transferred to half strength MS medium with different concentrations (0, 1.0, 2.0, 3.0 and 4.0 mg/L) of IBA for root induction. When the plantlets developed few leaves and roots on the rooting medium, they were brought to the green house and kept 2-3 days losing the cap of culture bottles. The plantlets were taken out from the culture vessels, washed thoroughly under running tap water to remove the debris of gelling agent with care and transferred to a pot (10 cm x 9 cm) filled with 2:1 garden soil and compost. The potted plantlets were kept inside the green house for adaptation and maintain the humidity and temperature though misting. Within 10 - 15 days the potted plants began to form new leaves and resumed new growth. The tissue culture plants were brought out from the greenhouse successively and kept under full sunlight in the nursery for further growth up to the plantation season.
Statistical analysis All experiments were performed as Completely Randomized Design (CRD). Data were analyzed using statistical analysis system (SAS v9.3) and means were statistically compared using LSD test. The significance level was set up at p < 0.05. Three replications were considered for each treatment and repeated thrice.
Bangladesh Journal of Forest Science Vol. 36 (1), 10-21, 2020
Journal