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Rapid Development of Plbs Derived from Immature Seeds and Mass Multiplication of Vanda Tessellata (roxb.) Hook. Ex G. Don - a Threatened Orchid Species in Bangladesh

Bakul Bhattacharjee
Plant Genetic Engineering Lab., Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

S. M. Shahinul Islam*
Plant Genetic Engineering Lab., Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

Sreeramanan Subramaniam
School of Biological Sciences, University Sains Malaysia (USM), Penang, Malaysia

Abstract/ Executive Summary:

A rapid and an efficient protocol for germination of immature seeds of Vanda tessellata, PLBs development and plant regeneration system have been developed. The results indicate that immature capsules performed better in terms of germination (100.0%) and development of PLBs (90.0%). Around 45 - 60 days old PLBs were transferred to half strength of MS supplemented with different concentrations of auxins and cytokinins. The highest weight of PLBs (5.53 g), the best plant regeneration (16.50) and shoot length (3.25 cm) were recorded when the medium was supplemented with BAP (1.0 mg/l) and NAA (1.5 mg/l). The highest number (5.0) and length (3.22 cm) of roots, the maximum number of plantlets (3.75) were obtained in the medium supplemented with 1.5 mg/l BAP and 1.0 mg/l IAA. The maximum numbers of leaves (3.33) were recorded in the medium fortified with 1.5 mg/lBAP + 1.00 mg/lIAA; the highest length of leaves (2.59 cm) was observed in the medium combining with of BAP and IAA (2.0 mg/l). Well shooted and rooted plants were acclimated in pots that contained two types of compost. Here, type-II showed better performance on plant development.

Keywords: Rapid development, Medicinal orchids, Immature seeds, Regeneration

Location:

Start Date:

End Date:

Program Area: Variety and Species

Non-commodity: Performance

Objectives:

The present work was undertaken to study the effects of various media for seed germination, development of protocorm like bodies, their regeneration, mass multiplication of this species.

Methodology:

The immature capsules of Vanda tessellata used as seed source were collected from Rajshahi, Bangladesh. At first, the capsules were washed by running tap water and surface sterilized by detergent. Then capsules were treated with 0.2% (w/v) HgCl₂ solution for 10 min and finally dipped in 70% ethanol for 10 - 12 sec. Then they were washed with sterile distilled water and sterilized capsules were cut longitudinally by a sterile surgical blade. Around 200 mg seeds were cultured per vessel. Various media viz. MS, B5 (Gamborg et al. 1968) and PM (PhytamaxTM, Sigma, USA) were used in this study. The basal media (MS and B5) amended with 3% (w/v) sucrose and the PM was amended with 2% (w/v) sucrose. The pH for all media was adjusted at 5.6 - 5.8. Inoculated vessels were maintained in the culture room under a period of 16 hrs light and 8 hrs dark at 25 ± 2°C. After two weeks of inoculation, a few seeds were taken out and dispersed in one drop of water on a glass slide and examined under a light microscope. Germination percentages were calculated employing the following formula: No. of seeds showing swelling of the embryo × 100/ No. of total seeds. Once the spherules were formed, developmental stages of the protocorms were recorded every week up to 8-10 weeks. Then PLBs were transferred to ½MS supplemented with different concentration of auxins and cytokinins. Furthermore, regenerated plants were transplanted and grown outside the environment for ex vitro establishment; complete plantlets were recorded within 60 - 70 days of culture initiation. The well rooted plants were removed from the culture bottles and hardened successfully in clean plastic pots of 8.0 cm diameter containing different mixtures of compost viz., (i) brick and charcoal pieces (1:1), (ii) brick, charcoal and decaying litter (1 : 1 : 1), (iii) brick, charcoal, decaying litter and sawdust (1 : 1 : 1 : 1), (iv) brick, charcoal, decaying litter and coconut husk (1 : 1 : 1 : 1). All substrates were covered with a layer of 10.0 g sphagnum moss. The plantlets were watered in alternate evening and fed with MS nutrient salt solutions (diluted 10 times) fortnightly for about a month and eventually established under natural condition following the protocols of Das et al. (2007). On the basis of seed germination and its developmental stages, PLBs formation, regeneration, shoot and root developments were recorded. Each value represents an average of 10 replicates. Data were subjected to analysis of variance (ANOVA) and means were separated by DMRT.

Source of Information: Plant Tissue Cult. & Biotech. 25(2): 181-191, 2015 (December)

Article Link (Online Journal): http://www.banglajol.info/index.php/PTCB/article/view/26253/17634

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In conclusion, the above findings showed that MS is the best for seed germination and protocorm formation. Similarly MS supplemented with BAP (1.0 mg/l) and NAA (1.5 mg/l) was found to be the most suitable for PLBs development and plantlet formation. On the other hand, the combined effect of BAP and IAA was the suitable hormonal combination for root development. Thus, the results clearly demonstrated that the above culture condition was the most effective for mass multiplication of Vanda tessellata and this protocol will be useful to commercial growers for mass multiplication.

Knowledge Management: Journal