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Micropropagation Through Seeds and Shoot Segments in Dendrobium Crepidatum Lindl

P. Das Gupta
Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh

M. Al-Forkan
Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh

S. K. Bhadra
Department of Botany, University of Chittagong, Chittagong-4331,Bangladesh

Abstract/ Executive Summary:

Seeds of Dendrobium crepidatum were aseptically grown on four different 0.8% (w/v) agar solidified basal media, namely PM, MVW, MS and MK. Seeds germinated on all these media but they took less time in the former two than in the latter. In vitro germinated seedlings underwent rapid elongation in hormone supplemented liquid and 0.8 % (w/v) agar solidified media. In terms of the increase in shoot system, liquid MS + 2 % (w/v) sucrose + 1.0 mg/l NAA + 3.0 mg/l BAP + 1.5 mg/l Kn was the best followed by liquid MVW + 2 % (w/v) sucrose + 1.0 mg/l NAA + 3.0 mg/l BAP + 1.5 mg/l Kn and the others. The seedlings developed poor root system on the elongation media. Thus in order to induce strong and stout root system, these were transferred onto 0.8% (w/v) agar solidified (i) half strength MS + 1.5 % (w/v) sucrose and (ii) MS + 3 % (w/v) sucrose + 0.5 mg/l IAA. In terms of both number of roots developed and increase in root system, half strength MS was better. For further multiplication, the shoot segments from the in vitro germinated seedlings were cultured on 0.8% (w/v) agar solidified (i) MS + 3 % (w/v) sucrose + 0.01 mg/l IAA + 2.0 mg/l BAP and (ii) MS + 3 % (w/v) sucrose + 0.1 mg/l IAA + 1.0 mg/l BAP media. Multiple shoot buds developed from the nodal zone of the shoot explants in both the media and the former proved better than the latter. The multiple shoot buds underwent elongation on the same media but did not produce root system. The shoot buds of 2 - 3 cm long were individually cultured on two different 0.8 %(w/v) agar solidified media, namely (i) half strength MS + 1.5 % (w/v) sucrose and (ii) MS + 3 % (w/v) sucrose + 0.5 mg/1 lAA. Some of these in vitro grown seedlings were successfully transferred outside where they continued their growth.

Keywords: Dendrobium crepidatum, Micropropagation, Media effect

Location: Department of Botany, University of Chittagong, Chittagong

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Orchid

Objectives:

To developing an efficient and reliable in vitro cultural technique for raising seedlings from the seeds of D. crepidatum and their further multiplication through organ culture.

Methodology:

Four to five months old seed capsules of D. crepidatum were used as the source of seeds for in vitro culture. The capsules were sterilized by submerging them in 0.2 % (w/v) HgCl₂ solution for 10 min when the sterilant was occasionally agitated and then a dip for 10 -12 see in absolute alcohol. Thereafter, these were washed with sterile distilled water. The sterilized pods were then opened and the seeds were taken out with the help of a pair of forcep and inoculated on to the four types of 0.8 % (w/v) agar solidified media, namely MS, MVW, MK and PM taken in cultural vessels (conical flask/test tube). These operations were done in a laminar flow cabinet. The mouth of the cultural vessels was closed tightly with cotton plugs and finally sealed with aluminium foil. Before inoculation of the seeds, the cultural vessels containing medium were autoclaved at 121°C for 20 min at 15 psi. The cultural vessels with inoculated seeds were kept in the culture room where 14 h of continuous light and 10 h of continuous dark period with 25 ± 2°C were maintained. After 15 days of germination, the tiny seedlings were aseptically taken out of the cultural vessels and subcultured at a lower density on 0.8 % (w/v) agar solidified seed germinating media. Further subculturing was done at an interval of 15 days. In order to induce rapid elongation, the seedlings were transferred on to different hormone supplemented 0.8 % (w/v) agar solidified/liquid MS, PM and MVW media. The elongated seedlings were transferred to two types of 0.8 % (w/v) agar solidified rooting media viz., (i) half strength MS + 1.5 % (w/v) sucrose and (ii) MS + 3 % (w/v) sucrose + 0.5 mg/l IAA for induction of strong and stout root system. For further multiplication, the seedlings of 5 - 7 Cm long were cut into two-three segments each having a node. The shoot explants were then aseptically cultured on five types of media and the cultural vessels were maintained in the cultural room under the same conditions as used for seed germination. Subculturing was done at a regular interval of 20 - 25 days and within two - three subcultures new shoot buds came out from the nodal zone of the shoot explants. When the shoot buds attained a height of 2 - 3 Cm, they were individually grown in the rooting media. Two kinds of rooting media viz., (i) 0.8 % ( w/v) agar solidified half sterength MS and (ii) 0.8 % (w/v) agar solidified MS fortified with 0.5 % mg/l lAA.

Source of Information: Plant Tissue Culture, Vol: 8, (1), pp: 1-10, (June) 1998

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Some of the mature in vitro grown seedlings were finally taken outside the culture room and established in wooden orchid pots. The transfer of in vitro produced seedlings from the culture room to outside was different requiring gradual acclimatization of the seedlings with the outside environment. The results, as a whole, supported that the technique of in vitro germination of D. crepidatum and their further multiplication with the in vitro induction of shoot buds is possible and the techniques developed can reliably be used in commercial and research laboratories for both micropropagation and conservation purposes.

Knowledge Management: Journal