Browsing by Author "Senanayake, S. P."

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Alternative gelling agents to develop cost effective medium for in vitro propagation of Dendrobium cv. “Big Jumbo White”
(Faculty of Science, University of Kelaniya Sri Lanka, 2022) Kanishka, W. S.; Lakmali, G. B. T.; Umesha, M. S. P.; Priyadarshan, A. I. S.; Senanayake, S. P.
In vitro propagation is the most common method of Dendrobium propagation. Commonly, agar is used as an effective gelling agent though, it is expensive. However, the developing a cost-effective method is beneficial for small-batch productions and is recommended. The objective of the current study was to evaluate the efficacy of ‘kithul’ flour (starch extracted from the pith of Caryota urens) and xanthan gum as alternative gelling agents to develop an efficient, cost-effective medium for in vitro propagation of Dendrobium. Seeds of Dendrobium cv. “Big Jumbo White” was cultured using full-strength Murashige and Skoog (MS) medium. After 90 days of incubation, 1g of Protocorm Like Bodies (PLBs) and plantlets of Dendrobium cv. “Big Jumbo White” were transferred to twelve different MS media supplemented with BAP (Benzyl Amino Purine) (2.5 mg/L) and NAA (Naphthalene Acetic Acid) (0.5 mg/L) and solidified with either agar (1:0) or with alternative agents; kithul flour (0:1) and xanthan gum (0:1) or a series of combinations of each alternative gelling agent with agar (1:4, 2:3, 3:2 and 4:1) totalling to 12 treatments with six number of replicates per each. The weight of plantlets and PLBs, number of plantlets, number of leaves, number of roots, and length of the roots were recorded as growth parameters in fortnight intervals for a period of 90 days to assess the growth performance. After 90 days of incubation, the MS medium of agar: kithul flour (1:4) of the treatment series with kithul flour and agar: xanthan gum (2:3) of the treatment series of xanthan gum, have shown significantly high performance in the growth and development of PLBs and plantlets. Four growth parameters, number of plantlets: 44, number of leaves per plantlet: 5, number of roots per plantlet: 12 and root length: 1.5 cm, were significantly high in the agar: kithul flour (1:4) medium than in the agar: xanthan gum (2:3) medium. Moreover, compared to the conventional tissue culture media prepared using only agar, the modified medium using agar: kithul flour (1:4) has shown 73% of cost reduction. In conclusion, the use of agar and kithul flour (1:4) as an alternative gelling agent can be recommended as a cost-effective medium for the development of PLBs into plantlets of Dendrobium cv. “Big Jumbo White” on a small scale in vitro culture system.
Antimicrobial potential of different Aporosa spp. (S. Kebella) in Sri Lanka
(Faculty of Science, University of Kelaniya Sri Lanka, 2024) De Silva, A. G. K. S.; Thathsarani, Y. K. D.D.; Senanayake, S. P.
The genus Aporosa belongs to the family Phyllanthaecae and locally referred to as ‘S: Kebella’ is reported to be rich with medicinal properties. Despite sharing the same vernacular name, these plant species exhibit significant variation in morphological traits leading drug adulteration. This suggests establishment of accurate identification methods of these species while assessing their potential medicinal properties. The present study was focused to evaluate the antimicrobial activity of ethanolic extract of the leaves of five Aporosa species found in Sri Lanka and thereby revealing the best plant/s with the inhibition properties of pathogenic microorganisms. Five Crude extracts of authenticated plant specimens were prepared using Dimethyl sulfoxide (DMSO). Antibacterial activity was evaluated against Escherichia coli and Staphylococcus aureus, while antifungal activity was tested against Candida albicans. The well diffusion method was employed across four extract concentrations (0.5, 1, 2, 4 mg/mL) and the results revealed concentration-dependent effects. Microorganisms were quantitatively assessed by the presence or absence of inhibition zones. Clear inhibition zones indicated the compounds showed antimicrobial activity against the tested microorganisms. According to the results all the tested extracts have produced zones of inhibitions against E. coli indicating their antibacterial activity against E. coli. High potential of antibacterial activity against E. coli was shown by Aporosa cardiosperma morphotype 3 at the concentrations of 4.00 mg/ mL and 2.00 mg/mL, with their respective diameter zones of inhibition ranging from 14.70–16.00 mm. Next highest inhibition zone of 13.70 was shown by A. cardiosperma at the concentrations of 1.00 mg/mL, 2.00 mg/mL, 4.00 mg/mL. However, S. aureus has shown response to none of the tested leaf extracts in all concentrations. All the extracts have produced zones of inhibitions against C. albicans indicating their antifungal activity against C. albicans. High potential of antifungal activity against C. albicans was shown by A. cardiosperma morphotype 2 at the concentrations of 2.00 mg/ mL and 4.00 mg/mL, with their respective diameter zones of inhibition ranging from 15.70–16.70 mm. The next highest inhibition zones ranging from 14.0–15.30 mm were shown by both A. cardiosperma morphotype 1 and A. cardiosperma at the concentrations of 1.00 mg/mL, 2.00 mg/mL, 4.00 mg/mL. Comparative analysis with standard antibiotics revealed distinct efficacy profiles. Notably, Aporosa extracts showed considerable inhibitory effects although smaller than antibiotics. The findings of the research concluded the potential of utilizing the tested Aporosa spp. for their antimicrobial properties, underlying their capabilities in combating pathogenic microbes. These findings underscore the therapeutic promise of leaf extracts of Aporosa spp. in antimicrobial applications, warranting further exploration for pharmaceutical development.
Host Plant Preference of Genera Dendrobium and Bulbophyllum (Family: Orchidaceae) in Sri Lanka.
(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Sandamali, P. M. H.; Senanayake, S. P.; Benjamin, S. P.; Rajapakse, S.; Athukorala, N. P.
Orchids are a well-known plant family for their restricted distribution in the wild. Most epiphytic orchids are hosted by specific trees and hence it’s abundance may be influenced by the availability of suitable host trees and epiphytic composition. The distribution of many epiphytic orchids in nature indicates that they display strong species specificity in selecting hosts and noticeably lacking on other trees. Dendrobium and Bulbophyllum are epiphytic genera and mostly have restricted distribution in the natural environments. Furthermore; they display a biased distribution towards host trees. Therefore, the study aims to investigate the host tree preference of the species of Dendrobium and Bulbophyllum with a view to provide important information for the conservation of these species. Wet forests in Matale (Knuckles mountain range), Nuwara Eliya (Hakgala nature reserve, Horton Plains National Park, Pidurutalagala mountain, Kandeela forest reserve), Baddulla (Namunukula mountain), Kegalle (Makandawa forest reserve), Ratnapura (Sinharaja rain forest, Peak wilderness), Kurunegala (Kankaniyammulla forest), Galle (Hiyare rain forest, Kottawa forest, Morningside forest) districts were explored for the distribution of two genera; Dendrobium and Bulbophyllum. Specimens were collected randomly from the selected sites; three plots of 500m× 500m size per each site, and relevant data of host plants were recorded such as tree height, girth at breast height (GBH), tree bark characteristics (bark colour, nature of the moss cover and bark texture) and the abundance of orchid species on the host tree. Further, herbarium specimens of host trees and the orchids were prepared for identification. Presence of orchid species on the identified host trees was counted and, percentage of frequency was calculated. It was revealed that the orchids are mostly associated with hosts with dense moss cover and rough tree barks. Sixty five percent of species of Dendrobium and Bulbophyllum were found on hosts having 51-100 cm GBHs. Further, these orchids have shown preference to plant families; Myrtaceae (26%), Lauraceae (23%) and Phylanthaceae (16%) as their hosts, whereas, with respect to the genus specificity, highest percentage of these orchids have shown preference to genera Syzygium (family Myrtaceae) and Glochidion (family Phylanthaceae) as their hosts. Findings of the present study have revealed that the species diversity and species richness of genera Dendrobium and Bulbophyllum were high in the montane forests located in Nuwara Eliya (Horton Plains Nature Park, Kandeela forest reserve and Pidurutalagala mountain) and Matale (Knuckles mountain range) districts. This might be due to the fact that these forests were characterized by high humidity and high elevation. Chemical composition, distribution of moss cover and fungi associated with tree barks of these preferred host trees, would provide new insight for the conservation of these epiphytic orchid species.
Intracellular peloton colonization and mycorrhizal associations in epiphytic orchid roots
(Faculty of Science, University of Kelaniya Sri Lanka, 2024) Disanayaka, D. M. H. A.; Thathsarani, Y. K. D. D.; Edirisinghe, P.; Senanayake, S. P.
Orchids are highly valued for their esthetic beauty and economic significance, playing a vital role in the global floriculture industry. In their natural habitats, orchids heavily depend on mycorrhizal associations, which influence seed germination, protocorm growth, and nutrition. Despite their importance, understanding these associations remains a challenge. In Sri Lanka, there is limited knowledge exists on the root-associated fungal species interacting with epiphytic orchids and their ecological roles. Exploring the endophytic fungal microbiome in these orchids helps in identifying fungi that form mycorrhizae in epiphytic habitats, paving the way for effective cultivation and developing novel conservation strategies. Intracellular entangled hyphal coils that colonize within the cortex of the roots of epiphytic orchids are known as pelotons. Knowledge of the presence and the distribution of pelotons is crucial for understanding the symbiotic relationships between orchids and mycorrhizal fungi. Therefore, the objective of this study was to investigate the distribution of pelotons in the roots of selected epiphytic orchids. In the present study, sixteen wild-grown and cultivated epiphytic orchid roots belonging to genera, i.e. Dendrobium, Arachnis, and Vanilla were randomly collected from four study sites in tropical lowland, wet and intermediate zones in Sri Lanka. Microscopic imaging of microtome sections was employed to observe the peloton colonization within the randomly sampled orchid roots. Longitudinal and cross-sectional analyses of six orchid root samples revealed sparse colonization of fungal hyphae. Extensive peloton colonization was observed only in the roots of two specimens of Dendrobium sp. and in one specimen of each Arachnis sp. and Vanilla sp. indicating the presence of mycorrhizal fungi in the roots. However, peloton colonization was not found in all the sampled roots of the studied genera. Furthermore, microscopic observations showed that intracellular pelotons were predominantly colonized in the cortex at approximately two centimeters beyond the tip of the roots that are adhered to the substrate. Pelotons were observed as thin hyphal masses ranging from circular and irregular to elongated shapes with hyphae often knotted or spiraled in various dimensions. The study highlighted that wild Dendrobium sp. from Avissawella, Western province, exhibited the highest peloton colonization (5–6 per root cortical cell) with both circular and elongated shapes. In contrast, Arachnis sp. had one irregularly shaped peloton per root cortical cell. Dendrobium sp. from Mahawa, Northwestern province showed both circular and irregular pelotons while Vanilla sp. from Kelaniya, Western province displayed the lowest colonization with irregular pelotons. Irregularshaped pelotons are predominantly found except in Dendrobium sp. from Mahawa, Northwestern province underscoring geographic and genus-specific influences on peloton morphology. These findings enhance the understanding of mycorrhizal associations in orchids and provide valuable information for future research on orchid-mycorrhizal symbioses and their ecological significance.
Isolation of mycorrhizal fungi in selected epiphytic orchids and investigation of the effect of potential host bark extracts on seed germination under in vitro condition.
(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Premachandra, J. A. H. P.; Senanayake, S. P.
Orchids (Family: Orchidaceae) are one of the largest, diverse plant groups in the world with high ornamental value. Orchid seed germination, in nature, is reported to be influenced by the mycorrizal fungi associated in the bark of the host plants. These fungi penetrate the seeds by their hyphae and supply the nutrients for developing embryo resulting the formation of a globular shape structures referred as protocoms. Fungal hyphae infect the cortical cells of the orchids and forming pelotons, that are digested by the orchid cells for their nutrition. This study was focused on isolation of mycorrhizal fungi from six selected epiphytic orchids: Dendrobium aphyllum, Dendrobium sp., Bulbophyllum sp., Eria sp., Coelogyne sp., an unidentified species, and evaluation of in vitro seed germination using host bark extracts inoculated with isolated fungi from orchid roots. Specimens of wild orchids and the host plants were collected from their natural habitats in low-land wet zone and montane zone in Sri Lanka. The mycorrhizal fungi in roots of selected orchid species were isolated and identified as Trichoderma sp. and Rhizoctonia sp. by comparing with the authenticated morphological characters. Preliminary phytochemical screening was performed for each bark extract of host plants using TLC. Presence of secondary metabolites including alkaloids, flavonoids, terpenoids and phenolic were observed in all the bark extracts. According to the spot observations of TLC, although similar type of secondary metabolites was present in the bark extracts of hosts plants, intensity of the secondary metabolites was detected as different. The TLC analysis revealed the presence of high concentration of chemical compounds in methanolic and aqueous extracts while less amounts were in dichloromethane and hexane extracts. In vitro seed germination of the orchid species was assessed using the V/V basis of host bark extracts with the inoculation of isolated fungi from orchid roots. Effect of host bark extracts with mycorrhizal fungi inoculum on seed germination of orchid seeds was analyzed by performing two-way ANOVA using Minitab statistical software. Comparatively high percentage of seeds were germinated in the aqueous extracts (~30%) and hexane extracts (~25%) while very low percentage was in methanolic (0%) and dichloromethane (~0%) extracts of host barks. The findings suggest that secondary metabolites present in the barks of the plants influence the promotion or inhibition of the orchid seed germination whereas fungal inoculation too has a significant effect on orchid seed germination.
Morphometry and pollen germinability of selected commercial Dendrobium cultivars
(2018) Kahagalla, T. H.; Hearth, H. M.; Attanayake, R. N.; Senanayake, S. P.
Dendrobium is one of the highly demanding ornamental plant genus in Sri Lanka. However, taxonomic identification of Dendrobiums are not straightforward and it is often complicated with the introduction of large number of commercially attractive hybrids and cultivars. Therefore, morphometry was used for taxa identification in the present study. Further, plant growers produce hybrids using these modern cultivars. However, recently, it has been found that artificial pollination of modern hybrids is unsuccessful. It was hypothesized that the modern hybrids bear infertile pollens. Eight commercial Dendrobium cultivars (A-H) with different floral morphology were selected for the study . Sixty different floral and vegetative characters were observed and recorded. Morphological characters were subjected to cluster analysis using PAST 3.1 software package. Two Dendrobium cultivars (F and G) were closely related and have separated from the other taxa. Dendrobium cultivar B has shown a clear separation from the other studied taxa. Pollinia of Dendrobium flowers were deposited on the stigma of the same flower. After 72 hr, a small amount of stigmatic fluid was stained with lacto phenol cotton blue, observed under a light microscope and percentage pollen germination was calculated. Pollen viability was tested by placing crushed pollinia on a cavity slide with 1% solution of 2,3,5-triphenyltetrazolium chloride, incubating at dark for 6 hr and observing under a light microscope. The highest percentage of pollen germination was observed in Dendrobium cultivar D (67%) while the lowest germination was observed in cultivar A (24%). The pollen viability of the cultivars varied from 77% to 94%. Successful observations in planta pollen germination concludes that the modern cultivars still have the potential of using in plant breeding programs.
Phylogenetic relationships of selected commercial Dendrobium hybrids in Sri Lanka
(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Kahagalla, T. H.; Herath, H. M.; Attanayake, R. N.; Senanayake, S. P.
Nuclear rDNA-ITS regions and chloroplast matK genes are useful in delineating plant species. In this study, genetic relatedness of eight commercial Dendrobium hybrids (A-H) with a range of attractive flower colours was studied using nuclear rDNA-ITS and chloroplast matK sequences. Genomic DNA was extracted from fresh, young leaves using a modified cetyltrimethylammonium bromide based protocol. rDNA-ITS and matK were amplified using PCR in 25 μl reactions containing 1X PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 1.25 U Taq DNA polymerase, 0.4 μM forward and reverse primers and 1.00 μl of genomic DNA. The optimized thermal cycling conditions were initial denaturation at 95 oC for 5 minutes, 35 (rDNA-ITS) and 40 (matK) cycles of denaturation at 95 oC for 40 seconds, annealing at 55 oC (rDNA-ITS) and 48 oC (matK) for 40 seconds, extension at 72 oC for 40 seconds and final extension at 72 oC for 10 minutes. rDNA-ITS and matK PCR products were subjected to Sanger sequencing. Sequences were manually edited using BioEdit 7.0.5.3. and ContigExpress software. Sequences were aligned to the nucleotide database in the National Center for Biotechnology Information using mega BLAST program. Forty-three related sequences were obtained from GenBank and the sequences were aligned using ClustalW implemented in MEGA 7.0.26 software. Phylogenetic analysis was performed by generating trees of ITS, matK and concatenated sequences of ITS and matK. The phylogenetic relationships were analyzed using Maximum Likelihood analysis with 1000 bootstrap replications. Phalaenopsis aphrodite, Liparis kumokiri and Malaxis spicata were used as outgroups. Combined gene-tree was estimated using RAxML-HPC BlackBox tool in CIPRES Science Gateway platform. Resulting trees were viewed using Figtree v1.4.3. In the combined gene tree, selected hybrids were clustered into two distinct groups. Dendrobium hybrids A, B, C, E and F were clustered with Dendrobium bigibbum var bigibbum and Dendrobium phalaenopsis (72% bootstrap). Hybrids G, H and D were clustered with Dendrobium nindii and Dendrobium taurinum (79% bootstrap). In matK gene tree, all the selected hybrids were clustered together with Dendrobium kingianum (90% bootstrap). In rDNA-ITS gene tree, hybrids A, B, C, E and F were clustered with Dendrobium bigibbum var bigibbum and Dendrobium phalaenopsis while hybrids D, G and H were clustered with Dendrobium taurinum and Dendrobium nindii (81% bootstrap). Therefore, though high variation in floral morphology is observed among the selected imported commercial hybrids, they were represented from a narrow genetic background. This is an indicative of genetic bottleneck most likely due to selective breeding and it is important to incorporate more diverse varieties in future breeding programs to maintain a diverse genetic background
Sea moss as an alternative gelling agent to develop a cost-effective in vitro culture medium for the propagation of Phalaenopsis cv. Pink lip
(Faculty of Science, University of Kelaniya Sri Lanka, 2023) Wishwakulathil, D. T.; Priyadarshan, A. I. S.; Senanayake, S. P.; Lakmali, G. B. T.
Genus Phalaenopsis is an ornamental orchid with the greatest commercial importance in the world floriculture industry. The propagation of Phalaenopsis is difficult by conventional breeding due to delayed flowering and uneven characteristics of flowers. Therefore, conventional Phalaenopsis cultivation is ineffective for large-scale production. At present, the tissue culture technique is extensively used for the mass propagation of Phalaenopsis. Tissue-cultured plants are more expensive than traditionally propagated plants due to the high cost of the chemicals used for the preparation of tissue culture media. Agar is widely used as a gelling agent and the most expensive ingredient in the preparation of tissue culture media. Developing a cost-effective invitro culture media using low-cost components is one strategy to reduce the production costs of tissue-cultured plants. The use of alternative gelling agents to replace the agar can highly contribute to reducing the cost of in-vitro culture media in tissue culture than other components. The main component of sea moss is carrageenan, a gelatinous substance used to thicken or as a solidifying agent. The ability of carrageenan-based hydrogels to produce thermos-reversible gels and viscous solutions makes them a desirable option for extensive use as a gelling agent. The objective of the present research was to assess the performance of sea moss as an alternative gelling agent to determine the effectiveness for in vitro propagation of Protocrom-like bodies (PLBs) of Phalaenopsis cv. Pink lip. Growth performance of PLBs of Phalaenopsis cv. Pink lip was used to assess the effect of sea moss as an alternative gelling agent. PLBs (0.020g) were transferred to ½ MS medium containing agar as a gelling agent, and ½ MS medium containing sea moss as a gelling agent. Cultures were maintained for four months and the growth performance of PLBs was evaluated, with fresh weight as a parameter at 30 days intervals. There was no significant difference observed in the mean fresh weight of PLBs throughout the fourmonth period, incubation in ½ MS medium containing agar, and medium containing sea moss as an alternative gelling agent. Moreover, there was no significant difference between the contamination percentages of the agar-containing medium and sea moss-containing medium. According to the cost calculation, the cost reduction resulting in the medium with sea moss as an alternative gelling agent was 79.81% compared to the conventional agar as the gelling agent. In conclusion, the application of sea moss as a gelling agent in tissue culture media can be utilized to achieve the optimum benefits for in vitro propagation of PLBs of Phalaenopsis cv. Pink lip. Based on the findings, sea moss can be recommended as a cost-effective alternative gelling agent for the propagation of Phalaenopsis cv. Pink lip using protocorm-like bodies.