Bamboo Research
IHBT has standardized macro and Micropropagation techniques of bamboo. Large scale aseptic cultures of D. hamiltonii, D. asper, D.giganteous, Bambusa bamboos, and B. nudans have been raised.
Initiation of aseptic cultures and shoot multiplication
The explants (single node segments) from HP-1986-2 were surface sterilized (Sood et al., 1994) and inoculated vertically on half-strengthMurashige and Skoog (1962) medium containing 3% (w/v) sucrose and 0.8%agar (w/v) for bud sprouting and screening for any contamination. The sprouted buds (Figure 1A) were then transferred either to half-strength agar or static liquid MS medium supplemented with BA (0.55.0mg l
−1). Elongated shoots were excised and small segments (45 mm) were used for direct organogenesis, multiple shoot formation, rooting, callusing and somatic embryogenesis. The shoots from sprouted buds cultivated in 1/2MS medium were transferred to a medium enriched with BA (2.5 mg l−1). Shoot proliferation began in test tubes, but further sub-culturing was in 500-ml jam jars (Kasablanka, Mumbai) containing 15 ml liquid medium. The cultures were incubated at 25±2◦C with a 12-h photoperiod (20 μmol m−2 s−1). Rooting Various auxins (IAA, IBA, NAA), growth inhibitors (choline chloride, TIBA, B9 and maleic hydrazide), activated charcoal, phenols (catechol, phloroglucinol, coumarin) at different concentrations and combinations, were used for root induction in the excised shoots produced in vitro. Details of hormonal combinations conducive for root induction are listed below:(a) Liquid cultures 1/2MS+IBA (0.5 or 1.0 mg l −1) + sucrose (3%) 1/2MS+NAA (0.5 mg l −1) + sucrose (3%)
(b) Agar gelled media MS + coumarin (9 mg l −1) with or without IBA (0.5 mg l −1) or IAA/NAA (0.1mg l −1) each. MS+ choline chloride (3, 9 mg l −1) + IBA (0.5, 1.0 mg l −1) or IAA/NAA (0.1mg l −1) MS+2,4-D (1.0 mg l −1) + phloroglucinol (0.1 mM) 1/2MS + activated charcoal (0.3%) 1/2MS + IBA (0.5, 1.0 mg l −1)1/2MS + NAA (0.5 mg l −1) MS with NH4 NO3 concentration reduced to half + IBA (1.0mg l −1)
The shoots for rooting were obtained from the excised nodal segments proliferated in BA supplemented (2.5 mg l−1) MS liquid medium (half strength) in bunches of four to five shoots in 500-ml jam jars. All cultures were incubated at 25±2◦C with 12-h photoperiod (20 μmol m−2 s−1). After 3 weeks of root initiation, subculturing to the same medium was done in order to produce healthy rooted plants. Callus formation and somatic embryogenesis For callus induction, small 45 mm long basal segments of the newly sprouted buds from the selected nodal explants were cultured on MS medium supplemented with BA and 2,4-D (1 mg l−1 each). GA3 (0.5 mg l−1) was incorporated into the same medium for induction of embryoids. Hardening and acclimatization of rooted plants The rooted plants grown on agar-gelled MS medium were transferred after 34 weeks to a low sucrose (0.51.0%, w/v) containing half-strength MS medium for 2 weeks and then to same medium without any hormones. After 2 weeks these were thoroughly washed and transferred to Hikko trays (M/s Wimco, India) containing a mixture of garden soil:river bed sand:farm yard manure (1:1:1). Plants grown in liquid medium were directly transferred to the soil mix. These were covered for 810 days and then kept under foggers (DAN, Israel) in polytunnels and later transferred to shade house (Figure 2B). Six-month-old established plants were transferred to pits 2Χ2Χ2 ft in the field at a plant to plant and row to row distance of 6 m. Growth data was recorded from the fields and compared with that of plantlets raised from nodal cuttings and grown under identical conditions. The data was collected from 25 different plants and standard error and mean values were calculated.
The comparative growth performance was evaluated after 6 months in polysleeves from the secondary branches on the main culms through conventional means in the horizontal furrows (68"). These were transferred to fields (Figure 2D) in rows 6 m apart during March 1993. The observations pertaining to shoot number (total culms produced), height of culms, thickness of culms at third internode from the base, were made in October for 6 years on yearly basis (Table 2).
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As the bamboos are cross pollinated and flowering cycles are long drawn, a lot of heterogeneity is observed in the seedling populations which may be important for bio-diversity conservation point of view but highly unsuitable for economic plantations. Therefore, prior selection of the seedlings in the field grown plants of Dendrocalamus hamiltonii was ensured in the Institute before attempting mass propagation both through nodal explants in vivo as well as through tissue culture. The selections were based on the growth performance of the seedlings in the field conditions and the propagation from mature plants of known physiological age ensured better performance in the field.
Protocols have been standardized for somatic embryos from mature explants of D. hamiltonii. Various morphogenetics events leading to somatic embryo formation were studied in detail.
Genetic diversity assessment of bamboo using RAPD, AFLP and STMS are underway (DBT funded project).
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DNA fingerprinting was carried out for development of molecular markers for identification and assessment of genetic diversity of Ochlandra travancorica and Leucaena leucocephala (NMITLI project)
Efforts are underway to standardize protocols for producing transgenic bamboo (DBT funded project) using osmotin and nptII genes.
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