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In Vitro Embryo Morphogenesis and Micropropagation of Dendrobium Aggregatum Roxb.

Mohammad Musharof Hossain
Plant Tissue Culture and Biotechnology Laboratory, Department of Botany, University of Chittagong, Chittagong

Abstract/ Executive Summary:

In vitro embryo morphogenesis and micropropagation of Dendrobium aggregatum Roxb. were described. The gradual developmental stages of embryos to seedlings were traced out. Within two weeks of culture the cells of undifferented embryos underwent repeated aniclinal and periclinal division producing a compact, green parenchymatous cell mass called spherule that emerged out by rupturing the testa. The spherules subsequently differentiated into greenish protocorms were considered as typical seed germination. Germination occurred on both (MS and Phytamax (PM) medium but MS medium proved to be more efficient. The primary protocorms underwent profuse proliferation through production of secondary (2º) protocorms when transferred to different plant growth regulators (PGRs) supplemented MS; the medium fortified with 2.0 mg/l BAP and 1.0 mg/l NAA proved to be most effective for induction of 2º protocorms and seedling development. Multiple shoot buds (MSBs) were induced in pseudobulb segments of the in vitro grown seedlings when cultured on different PGRs supplemented media; and the maximum number of MSBs were obtained MS + 2.0 mg/l BAP + 0.5 mg/l picloram. The MSBs underwent elongation and then they rooted when they were transferred to half strength of MS + 0.5 mg/l IAA. The well rooted plantlets were finally transferred to outside natural environment with 80% survival.

Keywords: Embryo morphogenesis, Seed germination, Organogenesis, Dendrobium aggregatum

Location:

Start Date:

End Date:

Program Area: Resource Development and Management

Commodity:

Objectives:

The protocol reported here suggests that same has a potential of providing mass supply of planting material to meet the demands of orchid growers as well as for conservation. A special attention was also paid to investigate the mode of morphogenesis of embryo during formation of protocorm and seedling development.

Methodology:

Plants of Dendrobium aggregatum Roxb. were collected from their natural habitat of Teknaf forest of Cox’s Bazar district of Bangladesh and grown in the Orchidarium of the Botanical Garden of Chittagong University. Several flowers were hand pollinated on the second and third day of anthesis. The pollinated flowers were bagged with a thin transparent polythene bag for one week and then removed Several capsules were collected after three ‐ four months of pollination and the seeds were used for in vitro germination. MS and PM (Phytamax; Sigma Chemical Co. USA) media were used for in vitro studies. pH of the media was adjusted to 5.4 for PM and 5.8 for MS prior to autoclaving at 121ºC for 20 min. at 15 psi. The capsules were cleaned with 20% Teepol, a commercial detergent (Qualigens Fine Chemicals, Mumbai, India) and washed thoroughly under running tap water. These were then surface sterilized by submerging the material in 0.2% (w/v) HgCl₂ solution for 10 min with occasional agitation followed by a dip in 70% ethanol for 1 min and rinsed two ‐ three times with sterile distilled water and then cut longitudinally with a sterile surgical blade keeping the material on a piece of sterile aluminum slab inside a laminar airflow cabinet. The powdery seeds were taken out and were inoculated on the surface of agar gelled nutrient medium in culture vessels (test tubes 2.5 × 15cm and conical flasks 250 cc) for germination. The culture vessels with inoculated seeds were incubated in a growth room where a cycle of 14/10 hrs light and dark regime was maintained at 60 μmol/m²/s provided by cool white fluorescent lamps (Philips Truelight 36w/86 6500° K B7, Philips India), and 60% RH at 25 ± 2°C. After two weeks of inoculation, some of the seeds were taken out and dispersed in one drop of water on a glass slide and observed under a light microscope. Per cent germination was calculated The experiments were designed following Complete Randomize Design (CRD). Five replicates were taken per treatment for seed culture, whereas for micropropagation and rooting 10 replicates were taken. The effects of different media on germination of seeds, induction of shoot buds and roots in the in vitro experiments were tested applying Duncan’s multiple range test (p > 0.5) in one way ANOVA. The statistical analysis were performed using the programme package Statistica ver. 7 (Statsoft, Tulsa, USA). The experiments were repeated thrice.

Source of Information: Plant Tissue Cult. & Biotech. 23(2): 241-249, 2013 (December)

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Achievement/Findings:

The well‐developed plantlets were transferred from culture room to the outside environment through successive phases of acclimation. Eighty per cent of the in vitro grown seedlings survived and continued to grow in pots in the greenhouse. They were finally established in Orchidarium of the Botanical Garden of Chittagong University. The protocol developed in the present investigation for in vitro propagation of D. aggregatum offers a good opportunity to commercial orchid growers for large‐scale propagation as well as for ex situ conservation of this orchid species. This protocol could be extended to other economically valuable, rare and endangered orchids for mass propagation and conservation

Knowledge Management: Journal