International Research Symposium on Pure and Applied Sciences (IRSPAS)
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Item 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 backgroundItem 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.Item Comparative study on the effect of different substrate compositions on performance of tissue cultured Dendrobium bigibbum during the prehardening process(Research Symposium on Pure and Applied Sciences, 2018 Faculty of Science, University of Kelaniya, Sri Lanka, 2018) Perera, H.S.M.; Rodrigo, W.U.M.; Perera, U.I.D.; Swarnathilaka, D.B.R.Development of planting material using tissue culture is an established and widely used mass propagation method in the floriculture industry. However, considerable percentage of tissue culture raised plants may not survive during acclimatization process, due to inability to compete with soil microbes and adverse environmental conditions. The present study aimed to establish a successful pre-acclimatization procedure for in vitro produced Dendrobium bigibbum plants. Plants of 2.5 cm average height, with at least 3 leaves and 2-3 roots were selected for pre-acclimatization practices. Plants were planted in vessels with different compositions of substrates denoted as A and B series (A series-Sand:Compost:Charcoal Powder; A1-1:2:0.5, A2-2:2:0.5, A3-1:1:0.5, A4-1:2:1 and B series-Sand:Compost: Charcoal Chips (4-5 mm size); B1-1:2:0.5, B2-2:2:0.5, B3-1:1:0.5, B4-1:2:1) with Murashige and Skoog (MS) basal medium supplemented with benzylaminopurine (BAP) (0.1 mg/L) and naphthaleneacetic acid (NAA) (2 mg/L). Plants were grown under laboratory conditions at 22 ± 1 0C, and 16/8 hr photoperiod for three months before the acclimatization process. As the control, plants were grown in MS basal medium without any substrates. Oneway ANOVA was used to determine whether there is a significant difference between tested samples using Minitab 16. According to the results obtained, there was no significant difference (p>0.05) in the shoot lengths of “A series” compared to the control. However, “B series” has shown a significant difference in the shoot lengths compared to the control (p<0.05). The highest shoot length of 3.87 ± 0.25 was observed in B1 medium. When fresh weights were compared, in “A series”, only A1 showed a higher significant difference (1.30 ± 0.08) with the control while “B series” performed better compared to the control. Dry weight in both A and B series have shown a significant difference compared to the control. Plants grown in B1 medium has the highest number of roots (16.67 ± 1.53) compared to the other treatments. After acclimatization, the highest survival rate of plants were observed in “B series” (90%) whereas “A series” and control showed survival rates of 60% and 30% respectively. Hence, B series is suitable for shoot multiplication, root generation as well as pre-hardening to survive in vivo.Item 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.Item Molecular identification of selected Dendrobium cultivars.(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Silva, W.E.R.; Attanayake, R.; Senanayake, S.P.The family Orchidaceae includes more than 25,000 species, and the genus Dendrobium consists of over 1,450 species around the world. Today many unidentified Orchid cultivars are available in the market and growers use different vernacular names. Authentication of parental materials is important for conservation and selecting cultivars as parental materials in breeding experiments. However, Dendrobiums are well known for their difficulty in identification due to vegetative similarity among different species and morphological dissimilarity among same species. Since DNA barcoding has been proposed to be one of the most promising tools for accurate identification of taxa, this project was initiated with the objectives of identifying selected commercial Dendrobium cultivars and to determine their phylogenetic relatedness. Twelve commercial Dendrobium cultivars were selected based on the flower morphology. Genomic DNA was extracted from young leaves using a modified Cetyltrimethylammonium bromide (CTAB) method. PCR amplification of DNA was performed using universal ITS 1 and ITS 4 primers. PCR products were sequenced at Genetech Pvt. Ltd., Sri Lanka. Sequences were manually edited using BioEdit software version 7.1.9. Out of 12 samples, 9 samples produced non-specific amplification and only 3 samples produced good quality sequences of nearly 700 bp length. BLAST analysis was performed and sequences were deposited in the GenBank (MF535341, MF535342, and MF535343). Sequences of the current study with other 26 sequences from the GenBank were used in maximum likelihood analysis implemented in Mega 6.0 software with 1000 bootstrap replications. Liparis kumokiri (AY907087) was used as the out group for the analysis. Dendrobium cultivar Triple Fantacy (MF535341) resulted 99% similarity to Dendrobium bigibbum var. bigibbum and Dendrobium bigibbum var. superbum (KP142215 and KP142213) in the BLAST analysis. Unidentified Dendrobium cultivar (MF535343) was 94% similar to Dendrobium bigibbum var. bigibbum (KP142215) and Dendrobium bigibbum var. superbum (KP142214). In addition, both Triple Fantacy and unidentified Dendrobium cultivar, were clustered together with Dendrobium bigibbum var. bigibbum and Dendrobium bigibbum var. superbum. Therefore, Dendrobium cultivars, both Triple Fantacy and unidentified Dendrobium cultivar were identified up to species level as Dendrobium bigibbum. Dendrobium cv. Thailand Tommy (MF535342) resulted 99% similarity to Dendrobium nindii (AY239985) and clustered with Dendrobium nindii with 99% bootstrap support. Thus, the identity of Dendrobium cv. Thailand Tommy was confirmed to be Dendrobium nindii. In summary, DNA barcoding with ITS sequence was successfully used in resolving species identity of selected commercial Dendrobium cultivars in Sri Lanka.