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Item Germinability and Viability of Pollen of Selected Commercial Dendrobium Hybrids(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Kahagalla, T.H.; Herath, H.M.; Attanayake, R.N.; Senanayake, S.P.Dendrobium is one of the highly demanding ornamental orchid genera in Sri Lankan floriculture industry. The assessment of functional quality of the pollinium on the basis of germinability and viability is useful for the development of breeding programs for crop quality improvement and pollen storage protocols in conservation. This research was carried out to determine in vivo and in vitro pollen germinability and pollen viability of eight commercial Dendrobium hybrids (A-H). Percentage of pollen viability was studied by placing crushed pollinia on a cavity slide with 1% 2,3,5-triphenyltetrazolium chloride solution and incubating at dark for 12 hours. Pollinia of Dendrobium flowers were placed on the stigma of the same flower on the second day after opening. After three days, pollinia were stained with lacto phenol cotton blue and percentage of in vivo pollen germination was calculated. In vitro pollen germination of the Dendrobium hybrids was studied in three different media. Pollinia were removed from the anthers on the second day after flower opening and placed in (i) 5%, 10%, 15%, 20% and 25% sucrose solutions and (ii) a semi solid medium containing 10% sucrose, 100 mgL-1 H3BO3, 300 mgL-1 Ca(NO3).2H2O, 200 mgL-1 MgSO4.7H2O, 200 mgL-1 KNO3, 8 gL-1 agar and stigmatic fluid of Dendrobium flower and incubated at room temperature. Pollinia of Dendrobium hybrids A, G and H placed in a (iii) semi solid medium containing 1%, 2%, 3% sucrose, 75 mgL-1 H3BO3 and 0.5%, 1%, 2% agar were incubated at 24 oC, 26 oC, and 28 oC. After 48 hours, percentage of in vitro pollen germination was calculated. Means were compared using one-way ANOVA with Tukey’s pairwise comparison test in IBM SPSS Statistics 22 software. As the percentage values of pollen germination and viability were not normally distributed, the values were transformed to arcsin square root values prior to statistical analysis. There was no significant deference in pollen viability of different hybrids which varied between 94% and 77%. The highest percentage of in vivo pollen germination was found in Dendrobium cultivar D (67%) and there was a significant deference in in vivo pollen germination among the hybrids. Correlation analysis was performed using IBM SPSS Statistics 22 software to determine the relationship between pollen viability and in vivo pollen germination and found that there was no significant relationship (�= 0.237). The percentage of in vitro pollen germination was low and the highest in vitro pollen germination (7.2%) was observed in the semisolid medium containing 1% sucrose, 75 mgL-1 H3BO3, 1% agar and incubated at 26 oC for Dendrobium cultivar H.Item Evaluation of the Survival of Dendrobium crumenatum Swartz. Pollen Stored at Different Temperatures(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Silva, W.E.R.; Attanayake, R.; Ratnayake, S.; Senanayake, S.P.Dendrobiums have a high demand in the Sri Lankan floriculture industry as cut flowers and potted plants. Most Dendrobium cultivars found in the Sri Lankan market are imported from countries like Thailand, Singapore and Malaysia. Development of new cultivars and commercial cultivation of orchids has become a billion-dollar profitable industry in the world. In order to uplift the local orchid industry, cultivars with Sri Lankan identity should be developed. As Sri Lankan Dendrobiums have high floral diversity with economically important characters like fragrance, patterns and shapes, such characters could be introduced to the popular cultivars through breeding programs. Pollinia of these species and cultivars may not be always available throughout the year. Therefore, a suitable pollen storage method that maintains pollen viability and germinability, should be developed. The objective of this study was to develop a pollen preservation technique for Dendrobium crumenatum (E: Pigeon orchid, S: Sudu pareyi mal) by storing at different temperatures in order to use them in breeding programs. Pollen were collected into 0.2 mL tubes from fully opened fresh flowers at the onset of the anthesis around 7.00 – 8.00 am in July 2019. A set of pollen was dried in silica for 24 hours before storage and the other set was directly stored at -80 °C, -20 °C, -1 °C, 9 °C and 28 °C temperatures. Cryopreservation of pollen at -80 °C and -20 °C were also conducted in modified Murashige and Skoog medium (pH 5.7). Treatments were performed in triplicates. Samples of stored pollen were removed after 1, 3, 7, 14, 30 and 60 days and in vitro germinability and viability were tested in 10% sucrose and 2,3,5-Triphenyltetrazolium chloride (TTC) respectively. To confirm pollen viability and germinability, pollen of D. crumenatum stored at 9 °C were used in controlled cross pollination with Dendrobium ‘Pink Stripe,’ Dendrobium ‘Sonia Red’ and Dendrobium ‘Pink New Splash’. D. crumenatum had 54.97±12.22 % germinability and 76.0±4.58 % viability prior to storage. Pollen stored at subzero temperatures or subjected to cryopreservation lost viability and germinability. Pollen stored at 9 °C without drying, were viable for 14 days and produced pollen tubes (8.63±0.71 %). Although, percent in vitro germination of D. crumenatum pollen reduced to less than 20% after 7 days of storage (19.57±6.55 %) at 9 °C, pollination of stored pollen resulted in fruit set in Dendrobium ‘Pink Stripe,’ and Dendrobium ‘Pink New Splash’. This study found that seasonally flowering D. crumenatum pollen can be stored for short periods at 9 °C while maintaining their viability and germinability and can be used for cross pollination in breeding programs. However, subzero temperatures and cryopreservation may not be appropriate for storage of D. crumenatum pollenItem Interspecific Relationships of Piper Species in Sri Lanka as Revealed by DNA Barcode ITS(Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2016) Jayarathna, N.; Senanayake, S.P.; Rajapakse, S.; Jayasekera, R.; Paranagama, P.A.The genus Piper which is the largest genus in the family Piperaceae is economically important as it comprises with valuable crop species as well as several wild species. Although ten species of genus Piper are recorded in Sri Lanka, their interspecific relationships were not well studied. Therefore, the aim of this study was to determine interspecific relationships of Piper species in Sri Lanka using internal transcribed spacers from nuclear ribosomal DNA )ITS(. Ten varieties of Piper nigrum L., two varieties of each P. betle L.and P. longum L., P. chuvya )Miq.( C. DC., P. siriboa L., P. sylvestre Lam., P. walkeri Miq. and P. zeylanicum Miq. were used for the study. The genomic DNA was extracted from tender leaf samples using CTAB method, PCR amplified using ITS primer pair and subjected to DNA sequencing. The DNA sequence alignment analysis was carried out and a dendrogram was constructed using the multiple sequence alignment programme MUSCLE. According to the dendrogram clear formation of two major clusters can be seen and ten P. nigrum varieties were clustered together however, variety Kuching has shown a separation from the main cluster. P. longum, P. walkeri, P. sylvestre, P. siriboa, P. betle, P. chuvya and P. zeylanicum formed the other cluster in which P. siriboa has shared close similarities with P. betle, while P. sylvestre and P. walkeri displayed close relationships and clustered together with P. longum varieties. Interspecific relationships between wild species and cultivated species )P. nigrum, P. betle and P. longum( indicated the potential of using the wild species in breeding programs for crop improvement. However, further studies to reveal genetic and phenetic relatedness of those species are needed to deepen the knowledge on interspecific relationships of Piper species.Item Qualitative phytochemical analysis and phenetic variation of Piper species in Sri Lanka(Faculty of Graduate Studies, University of Kelaniya, 2015) Jayarathna, S.P.N.C.; Senanayake, S.P.; Jayasekera, L.R.The genus Piper is represented in Sri Lanka by ten species of which three are endemics. Piper nigrum L. (black pepper) and P. betle L. (betel) are the main crops that make the genus commercially important and some species are used in traditional medicine. This study aimed to reveal the interspecific relationships of selected Piper species based on the variation in qualitative phytochemical screening and phenetic approach. Piper nigrum L., P. betle L., P. longum L., P. chuvya (Miq.) C. DC., P. siriboa L., P. sylvestre Lam. and P. zeylanicum Miq. were collected from natural habitats and from the cultivations. The air dried leaves were powdered using a grinder and subjected to successive extraction using petroleum ether, chloroform, ethyl acetate and methanol. All the extracts were subjected to qualitative phytochemical tests. Anatomical studies were carried out by observing epidermal peels and transverse sections of the fresh leaves and photomicrographs were prepared. The phytochemical screening confirmed the presence of tannins, flavonoids, terpenoids, glycosides, alkaloids and phenolic compounds in all the species examined. Various types of trichomes, glands and secretory cells were present in the leaves which indicates the richness of secondary metabolites. Fourteen morphological and anatomical characters of leaves were subjected to cluster analysis using PAST software to infer interspecific relationships. According to the phenogram (Single linkage, Euclidean distance), P. longum, P. zeylanicum and P. sylvestre were clustered to one group showing more similarities while P. nigrum, P. chuvya, P. siriboa and P. betle were separated from the rest. Presence of non-glandular trichomes, symmetry of the leaf base and leaf length were the most discriminating characters.Item Phenetic variation and phenolic composition of leaves of Flueggea leucopyrus willd. (vern: Katupila) collected from different climatic zones in Sri Lanka(University of Kelaniya, 2013) Priyadarshan, A.I.S.; Senanayake, S.P.; Jayetilleke, M.P.; Rajapakse, R.G.S.C.Flueggea is a genus of thorny shrubs belonging to the family Euphorbiaceae, and represented by two species in Sri Lanka. Currently, Flueggea leucopyrus is becoming increasingly popular as an ingredient in several traditional therapeutic systems in Sri Lanka, as well as in other countries. According to a questionnaire survey, carried out using a population sample of hundred, the distribution of F. leucopyrus was found to be common in the dry zone, compared to the wet zone of Sri Lanka. Most of the users are of the opinion that the best ingredients for traditional therapeutic systems can be obtained from plants grown in the dry zone. Therefore, the aim of this study is to interpret the phenetic variation and determine the phenolic composition in leaf extracts and of F. leucopyrus in different climatic zones in Sri Lanka. Floral and vegetative characters of F. leucopyrus in fifteen localities in districts of Colombo, Kurunagala, Puttalam and Hambanthota were observed and cluster analysis was done. Leaf material collected from fifteen different localities were acid hydrolysed and extracted into ethyl acetate and ether. Ethyl acetate extracts were used to isolate flavonoid aglycones using cellulose Thin Layer Chromatography (TLC) plates in solvent 50% acetic acid. Ether extracts were used to isolate phenolic compounds using 2-Dimensional paper chromatography in solvent systems, butanol: acetic acid: water; 6:7:3 and sodium formate: formic acid: water; 10:1:200. Then colors of the spots were observed under UV (366nm) and Rf values were calculated. Then cluster analysis was carried out to determine the distribution pattern of flavoniod aglycones and phenolic compounds. Slight phenetic variation was observed among the different population samples. Intra specific variation can be seen in the distribution of phenolics and flavonoid aglycones in leaves of F. leucopyrus with respect to their habitats in different climatic zones. The variation observed in the specimens of different localities in the dry zone is higher than the wet zone samples. To determine the variation in the efficacy of leaves of F. leucopyrus in preparation of therapeutic systems, further elucidation of phytochemicals is needed.Item Development of suitable propagation systems for Tephrosia spp. and Flueggea leucopyrus in Sri Lanka(University of Kelaniya, 2013) Priyadarshan, A.I.S.; Senanayake, S.P.; Jayetilleke, M.P.; Rajapakse, R.G.S.C.Different species of both genera Tephrosia and Flueggea are commonly used as ingredients in traditional therapeutic preparations. Due to the increasing popularity of these plants as ingredients in traditional therapeutic systems, production of planting materials for commercial cultivation is a timely requirement. This research was carried out to develop suitable propagation systems for T. purpurea, T. villosa, T. noctiflora, T. pumila and F. leucopyrus. Though Tephrosia spp. seeds have shown higher viability, low germination percentage was observed within a week on wet filter paper as well as in different six potting media. Six potting media were prepared using five raw materials namely coir dust, sand, top soil, cow dung and compost with different compositions to evaluate the seed germination and growth performance of Tephrosia species. Water impermeability of seed coats was identified as the reason for the seed dormancy in Tephroisa spp. and soaking in Con. H2SO4: H2O; 3:1 for 25 mins and rubbing on sandpaper for 2 mins were determined as suitable seed dormancy breaking methods. Variation was observed in the growth performance of Tephrosia spp. in six different potting media. Soil medium was determined as a more suitable medium compared to the hydroponic solution for all four Tephrosia species. Due to less seed production in F. leucopyrus, stem cutting propagation methods in three different potting media (Coir dust, Sand, Coir dust: Sand 1:1) were applied to evaluate the efficiency in propagation. Semi hardwood and hardwood cuttings of F. leucopyrus were well established within a period of a week in sand potting medium. These findings can be used to establish suitable propagation systems for genera Tephrosia and F. leucopyrus for commercial cultivation.Item Phenetic analysis and phytochemical screening of Albizia lebbeck and its substitute plants in Sri Lanka(University of Kelaniya, 2013) Jayasiri, A.P.A.; Senanayake, S.P.; Paranagama, P.; Amarasinghe, A.P.G.Albizia lebbeck (vern: Suriya mara) is a tree belonging to the family, Fabaceae. It is native to tropical Southern Asia, and found widely in India. It has been used in traditional therapeutic systems of Ayurveda, Sidhdha and Unani, for preparation of drugs for many diseases. Due to the limited distribution of A. lebbeck in Sri Lanka, substitute plants are used in the drug manufacturing industry. A questionnaire survey was carried out on a sample population of hundred Ayurvedic physicians, traditional Ayurvedic practitioners, drug suppliers, drug manufacturers and the general public, in order to collect traditional knowledge in the medicinal uses of these plants. The survey revealed that A.odoratissima, Adenathera pavonina and Samanea saman are commonly used substitute plants for A. lebbeck in traditional medicinal systems. To evaluate the phenetic diversity, a morphometric study and cluster analysis were carried out using floral and vegetative characters of A. lebbeck and its substitute plants. Variations in the bark, inflorescences, floral colour and type, texture and colour of pods are found as the important diagnostic characters of these species. Cluster analysis clearly indicated the morphological variation in population samples of all four plant species. Stem bark of the four species were subjected to sequential solvent extraction using hexane, chloroform, methanol and water. Weights of each crude sample were obtained after evaporation of the solvent. Highest yield was obtained from the methanolic extracts which revealed the presence of polar compounds in the species. The extracts were subjected to the preliminary phytochemical screening for carbohydrates, proteins, amino acids, glycosides, tannins, phenolics, alkaloids and saponins, Phytochemical analysis has confirmed the presence of glycosides, flavonoids, tannin, phenolics and phytosteroids in methanolic extracts of the four species. The results of the present study reveal the presence of saponin, tannins and phenolic compounds in the water extracts of the four plants. The distribution of classes of phytochemicals in the four plants was similar to each other except alkaloids as it was found only in Adenathera pavonina and Samanea saman. Therefore, this study has provided supportive evidence for the possibility of the presence of similar medicinal properties in A. lebbeck, A.odoratissima, Adenathera pavonina and Samanena saman. These findings can be considered as valuable facts in the recommendation of the use of these three plants as substitutes of A. lebbeck in medicinal preparations. Further investigation on the similarity in bioactivity of the four plants is needed to confirm this recommendation.Item Taxonomic and Phytochemical Study on Albizia lebbeck and Substitute Plants used in Ayurvedic Drug Preparations in Sri Lanka(University of Kelaniya, 2012) Jayasiri, A.P.A.; Senanayake, S.P.; Paranagama, P.; Amarasinghe, A.P.G.Nowadays consumption of herbal medicines is widespread and has increased dramatically. The main supply of herbal material for ayurvedic drug preparations is from the wild. It causes inherent problems: misidentification, phenotypic variability, extract variability and adulteration. The pharmacognostic evaluation is the preliminary step in the standardization of crude drugs which provides valuable information in morphology and physical characteristics, and the purity and quality of the plant drugs. Albizia lebbeck (Sinhala- Mara, Sanskrit-Mahari, Hindi- Shiris ) is a South Asian medicinal plant widely cultivated and naturalized in tropical and subtropical regions. In Sri Lanka many substitute plants are used due to the restricted distribution which has caused ambiguity in utilizing accurate plant material. The present study focuses on exploring the use of A. lebbeck and its substitutes in ayurvedic drug preparations with reference to their morphological and pharmacognostic similarity. A questionnaire survey was carried out, using a randomly selected sample population of 100, to determine the use of substitute plants and it revealed that three plants in the subfamily Mimosodeae, Albizia odoratissimma (Sinhala-Suriya mara), Adenanthera pavonina (Sinhala-Madatiya) and Samanea saman (Sinhala-Pare mara) are used widely in Sri Lanka, whereas A. odoratissima is being predominantly used in drug preparations. Specimens of the above plants were collected from the natural habitats and indentified using the authenticated specimens at the National Herbarium. Methanolic bark extracts of A. odoratissimma and A. pavonina were subjected to a preliminary phyotochemical screening to detect the different secondary metabolites, such as carbohydrates, proteins, amino acids, glycosides, and alkaloids. Further, phytochemical screening was carried out using solvents; ethyl acetate, methanol and water. Thin Layer Chromatography was performed on each extract, for the qualitative and quantitative analysis. High yield was obtained from methanolic extracts that indicated the presence of polar compounds. Chromatographic properties have showed the variation of chemical profiles in these two bark extracts. These compounds will be characterized by fractional analysis, and their distributional patterns in these plant species will be compared to evaluate the effectiveness as substitutes to A. lebbeck.Item Taxonomic study on Albizzia lebbek and its substitute plants used in Ayurvedic preparations in Sri Lanka(University of Kelaniya, 2011) Senanayake, S.P.; Jayasiri, A.P.A.Consumption of herbal medicines is widespread and increasingly used nowadays. The main supply of herbal materials for ayurvedic preparations is from the wild, which causes inherent problems; for instance, misidentification, phenotypic variability, extract variability and contamination. Albizzia lebbek (Sin- suriyamara, San- mahari, Hin- siris) is a South Asian medicinal plant, widely cultivated and naturalized in tropical and subtropical regions. It is extensively used in Ayurvedic preparations in India whereas many substitute plants are used in Sri Lanka due to the restricted distribution. This has caused ambiguity in utilizing accurate plant material in Sri Lanka. The present study focused to explore the use of Albizzia lebbek and its substitutes in Ayurvedic preparations with reference to their morphometric similarity and geographical distribution. A survey carried out to determine the use of substitute plants through a sample survey questionnaire revealed that three plants in the subfamily Mimosoideae; Albizzia odoratissima (Sin- huriya mara), Adenanthera pavonina (Sin- madatiya) and Samanea saman (Sin- pare mara) were common and widespread, Albizzia odoratissima being predominantly used. The randomly selected sample population of one hundred included traditional Ayurvedic practitioners, Ayurvedic physicians, drug dealers/ suppliers, drug manufacturers and the general public. Further, Albizzia lebbek was recorded only in a few locations in Sri Lanka and use of this plant in Ayurvedic preparations is not reported. A comparative morphometric analysis of these plants was done based on seedling, leaf, bark, floral, pod and seed characters and multi-access key was constructed using DELTA (Description Language for Taxonomy) package which could serve as a pictorial guide for accurate identification.Item Chemotaxonomy and medicinal properties of Sida spp: flavonoids(University of Kelaniya, 2000) Perera, N.I.S.; Senanayake, S.P.; Jayasekara, L.R.