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Clonal Propagation of Rhynchostylis Retusa (lin.) Blume Through in Vitro Culture and Their Establishment in the Nursery

Pinaki Sinha
Biological Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR),Dr.Kudrat E Khuda Road, Dhaka

Miskat Ara Akhter Jahan
Biological Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR), Dr. Kudrat E Khuda Road, Dhaka

Abstract/ Executive Summary:

For high frequency regeneration of Rhyncnostylis retusa (Lin.) Blume apical nodal segments were used. Half strength MS + 2% sucrose + 1.5 mg/l BA + 0.5 mg/l NAA + 2 g/l peptone + 10% (v/v) coconut water (CW) + 0.5 g/l activated charcoal (AC) was the best nutrient medium, on which 89% cultures induced 8 microshoots per culture. Subculture of microshoots for further 8 weeks on the same nutrient medium enhanced the number of microshoots up to 95. For further proliferation of microshoots, their development into shoots as well as formation of secondary microshoots from the base of the old ones, the best medium was half strength of MS + 2% sucrose + 2 g/l peptone + 10% (v/v) CW + 0.5 g/l AC + 150 mg/l L‐glutamine. Plantlets with roots were obtained in half strength of MS + 2% sucrose + 2 g/l peptone + 10% (v/v) CW + 0.5 g/l AC + 5.0 g/l banana powder, on which cent per cent shoots rooted within eight weeks. The pH of all the categories of cultures were maintained at 5.6 before adding 2.2 g/l gelrite and autoclaving, and the cultures were incubated at 2000 ‐ 3000 lux for 16/8 hrs light/dark at 24 ± 2ºC. Regeneration of plantlets continued due to repeated subculture of microshoots and regenerants were acclimatized and established in the nursery.

Keywords: Orchid, Rhyncnostylis retusa, In vitro culture, Regeneration

Location: Patuakhali district

Start Date:

End Date:

Program Area: Knowledge Management

Commodity:

Objectives:

Various protocols developed for orchid microcloning show considerable variations with respect to media composition and environmental conditions. Because there is no extensive literature on in vitro clonal propagation of R. retusa, the present study attempts to address this deficiency.

Methodology:

Mature plants were collected from a Rhynchostylis retusa colony naturally growing on the trees in the villages of Patuakhali district, Bangladesh, and used as source materials. Apical nodal segments (NS) were used as explants. Surface sterilization was made following Sinha et al. (2010), and the surface sterilized explants were prepared for inoculation as follows. For direct induction of protocorm like bodies (PLBs)/microshoots, explants were cut into 10 mm long nodal segments with one node in each segment. Explants were cultured initially in KC (Knudson 1946), VW (Vacin and Went 1949), half strength of MS and MS media, each supplemented with different concentrations and combinations of sucrose, auxins, cytokinins, peptone, coconut water, and activated charcoal. The cultures were gelled with 2.2 g/l gelrite (Duchefa, the Netherlands). The pH of the medium was adjusted to 5.6 before autoclaving at a pressure of 1.06 kg cm‐² at 121ºC for 20 min. Medium was poured in 25 × 150 mm culture tubes containing 20 ml of medium or 100 ml conical flasks containing 40 ml of medium. The medium that responded best was used in subsequent experiments. For proliferation of microshoots and their development into shoots, each clump of microshoots induced on explants was subcultured for 8 weeks on the same nutrient medium on which microshoots were induced. The microshoots clump was then dissected vertically into 8 pieces and subcultured on specific medium with specific additives along with different concentrations of casein hydrolysate (50 ‐ 200 mg/l) and L‐glutamine (50 ‐ 200 mg/l). Media were gelled with 2.2 g/l gelrite. In all experiments the explants were cultured in 250 ml conical flasks or jam bottles (86 x 120 mm) containing 50 ml medium. The pH of the media was adjusted to 5.6. All cultures were incubated at 24 ± 1^oC, and under cool white fluorescent light of 2000‐3000 lux for 16 hr per day.

Source of Information: Plant Tissue Cult. & Biotech. 22(1): 1-11, 2012 (June)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In the present study the high frequency proliferation of microshoots was possible due to the synergistic effect of the organic compounds present in CW and L‐glutamine. CW and L‐glutamine in the culture medium positively affected the multiplication rates of somatic embryos just as they did for other plant species. Likewise PLBs positively affected the rates of other orchid species. Encouraging results were also obtained in Cattleya cultured in half strength of MS supplemented with CW and activated charcoal. For rooting of regenerated shoots of Rhynchostylis gigantea chlofenuron (CPPU) was used. In the present study of Rhynchostylis retusa addition of banana powder in the medium was highly effective for root induction in 100% of the cultures. In the present experiment identical shoots were separated from the culture and subcultured for their development into plantlets in obtaining a large number of identical plantlets. In the present study callus induction was not needed rather microshoots were induced directly from the explants cultured in medium containing limited doses of BA and NAA. The protocol for high frequency regeneration of Rhynchostylis retusa as established through this study could be utilized in commercial cultivation for sustainable use and conservation as well

Knowledge Management: Journal