Rapid Development of in Vitro Germination, Micropropagation and Antimicrobial Activities of Indigenous Orchid Species in Bangladesh

Abstract

In vitro micropropagation, hybridization and cryopreservation techniques are essential tools for orchid development. Considering the importance of rapid and mass propagation and medicinal value of indigenous orchids in Bangladesh this research programme has been under taken with a major view to develop a reproducible micropropagation technique, to estimate antimicrobial activity and to analyze of bioactive compounds of indigenous orchids. For this research five different orchid species were selected. It was observed that immature capsule showed better performance for germination (100%) and development of PLBs (90%). The highest number (90%) and weight of PLBs (5.53g), best plant regeneration (16.50) and shoot length (3.25 cm) were recorded when the medium supplemented with BAP (1.0 mgl-1) and NAA (1.5 mgl-1). On the other hand, somatic embryos were induced from leaf and shoot that cultured on MS medium supplemented with BAP, NAA and kinetin. Among them BAP (0.5 mgl-1) and kinetin (1.0 mgl-1) showed better performance on somatic embryogenesis. Germination and regeneration efficiency of somatic embryos were recorded as highest 60.83% in ½MS medium supplemented with 1.0 mgl-1 BAP. Maximum number of multiple shoots (10.8 ± 0.22) was recorded in MS medium supplemented with 1.5 mgl-1 BAP and 1.0 mgl-1 NAA. So far as we know there is no report regarding the plant regeneration through somatic embryogenesis from leaf and stem explants of V. tessellata. In case of multiple shoot induction and subsequent development, twenty different plant growth regulators (PGRs) were tested. It was revealed that the combination of 1.0 mgl-1 NAA and 1.0 mgl-1 BAP was proved to be the best combination for multiple shoot development and elongation. Another experiment were conducted using different PGRs were applied for in vitro development of protocorms, regeneration and mass multiplication derived from immature seeds of Rhynchostylis retusa (L.) Blume. The maximum percentage of seed germination (72.60%) was recorded after 7-8 weeks of culture initiation. The highest numbers of secondary PLBs (16.0) were obtained from each of primary protocorms in MS medium supplemented with 1.0 mgl-1 BAP and 1.0 mgl-1 NAA. On the other hand, the somatic embryo induction was highest in leaf (75%) and root (50.2%) when 1.0 mgl-1 BAP + 1.5 mgl-1 kinetin was used. Another investigation for in vitro germination and enhancing PLBs development were carried out in three indigenous orchids. Under this study three basal media were tested and among them PM was found to be the best because of the highest rate of seed germination (98.20%), PLBs development and plant formation. The MS with 1.0 mgl-1 BAP and 0.1 mgl-1 picloram was the most efficient medium for seedling growth of A. premorsa and A. khasianum. But in the case of P. cornorerris the best seedling growth was observed using medium supplemented with 0.5 mgl-1 BAP and 0.1 mgl-1 NAA. In case of antimicrobial activity and photochemical screening the whole plant extracts of Vanda tessellata and Rhynchostylis retusa were studied. The various solvent extracts such as chloroform, methanol, ethanol and hexane of this orchid were considered for in vitro antimicrobial activity against five clinical pathogenic bacteria and three fungi by disc diffusion method. A preliminary phytochemical analysis was performed for the detection of alkaloid, terpenoids, flavonoids, phenols, tannins, steroids and glycosides etc. The antibacterial activity against all bacteria with the zone of inhibition ranging from 5 ‐ 15 mm were observed and found that highest inhibition zone (14 - 15 mm) with the concentration of 10.0 mgl-1 of chloroform extract. Chloroform extract showed significant antifungal activity against Penicillium sp., Rhizopus sp. and Aspergillus niger with the highest zone (16‐17 mm) of inhibition. In R. retusa, the antibacterial activity against all bacteria with the zone of inhibition ranging from 5 ‐ 12 mm were observed and found that highest inhibition zone (10 - 12 mm) with the concentration of 10.0 mgl-1 of chloroform extract. Chloroform extract also showed significant antifungal activity against Penicillium, Rhizopus and Aspergillus niger with the highest zone (12‐15 mm) of inhibition. Another attempt of this study was taken to enhance anther culture response of Vanda tessellata by applying physical and chemical pre-treatment factors including with different media. It was observed that 3 days cold pretreatment at 4°C and ½MS medium slightly modified by adding CuSO4.5H2O (2.2 mgl-1) with BAP (10 mgl-1), NAA (1.5 mgl-1), peptone (2.0 mgl-1) and 3% sucrose as carbon sources was the best medium for callus induction. From the experimental findings it might be concluded that the cold pretreatment, different media composition and carbon sources and their interaction clearly influence the anther culture ability of V. tessellata and this protocol will be helpful to commercial growers for mass multiplication of orchids.