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Item Effect of growth regulators on in vitro regeneration of Gerbera jamesonii.(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Perera, H.S.M.; Swarnalatha, K.D.S.; Senanayake, S.P.Gerbera jamesonii (Gerbera), belongs to the family Asteraceae, is considered as one of the important cut flowers in the global floriculture industry. The propagation process of Gerberas require improvements to fulfill the commercial demand and in vitro propagation was considered as a suitable solution to overcome this problem. The aim of the present study was to determine the effect of various concentrations of growth regulators on callus formation and shoot regeneration of Gerbera using leaves as explants. Healthy mature leaves were taken from Gerbera grown in the plant house, University of Kelaniya for obtaining suitable explants. Excised explants were washed with teepol (0.1%) solution for 06 mins followed by washings pre-treated with 10% (w/v) captan and sterilized distilled water (SDW). The explants were surface sterilized with Clorox solution (5%) with addition of Tween 20, followed by washing with SDW. Sterilized leaves were cut into 5-10 mm pieces. Callus induction, shoot proliferation and root regeneration were accomplished on Murashige and Skoog (MS) basal medium supplemented with different concentrations (0, 0.5, 1.0, 1.5 and 2.0mg/L) of 6-benzylaminopurine (BAP) and (0, 0.5 and 2mg/L) of naphthaleneacetic acid (NAA). For rooting, MS medium was supplemented with different concentrations of BAP (0, and 2.0 mg/L) and NAA (0, 2.0 mg/L). The highest callus initiation was observed in MS medium with 2 mg/L NAA and 1.0 mg/L BAP (60 days) while highest shoot initiation was obtained in MS medium with 2 mg/L BAP and 0.5mg/L NAA. Number of shoot formation per explant was 80% and shoots were visible after 26 days. When BAP was used as the sole growth regulator in the culture medium without NAA, limited shoot proliferations and translucent short shoots were observed (54%), whereas, NAA alone as the sole growth hormone did not initiate any shoot proliferation. With up to 2 mg/L NAA in the medium, BAP had a negative effect on shoot development, multiplication rate and the height of the shoots. MS medium supplemented with, 2 mg/L NAA, was identified as the best response for rooting initiation and number of roots per explant were 23.2±1.34. The first roots appeared after 1–2 weeks of culturing and a well developed root system was noted in 4–5 weeks. Subsequently, plants were removed from the medium and transferred to coir dust for acclimatization. The findings indicate that above three media compositions could be utilized for callus initiation, shoot proliferation and root formation from leaf explants of Gerbera jamesonii.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.Item Evaluation of anti-inflammatory and antioxidant properties of Genus Piper in Sri Lanka. International Research Symposium on Pure and Applied Sciences,(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Athapattu, R.S.K.; Jayarathna, S.P.N.C.; Paranagama, P.A.; Senanayake, S.P.In Sri Lanka, the genus Piper is represented by ten species including five wild and two cultivated species. Most of the species of the genus Piper are important as medicinal plants and used in various systems of medicine.This study aims to evaluate the bioactivity (anti-inflammatory and antioxidant properties) of the selected five Piper spp. (P. nigrum L., P. longum L., P. betle L., P. chuvya (Miq.) and P. sylvestre). Specimens of leaves and fruits were collected from their natural habitats and cultivations, and were subjected to the extraction of essential oils by steam distillation. Three replicates were done for each concentration and data were analyzed using the MINITAB 17 statistical package. Antioxidant activity was tested using 2,2-diphenyl-1- picrylhydrazyl (DPPH) free radical scavenging assay and ferric reducing antioxidant power assay. The isolated essential oils of Piper species exhibited remarkable DPPH free radical scavenging ability at different concentrations. The results revealed that the essential oil of P.betle exhibited the highest radical scavenging activity (92.8% at 1 mg/mL) and the value is higher than even of the standard, Butylated Hydroxy Toluene (BHT), (74.5% at 1 mg/mL). Further, essential oils of P. chuvya and P. longum have shown 91.8% and 63.1%inhibition percentages at 1 mg/mL. Essential oil isolated from P. betle has showed remarkable ferric reducing antioxidant power (mean absorbance, 1.89at 1 mg/mL) compared to the standard, BHT, (2.01at 1 mg/mL). Further, it was observed that its antioxidant activity increased with the increment of the concentration of essential oil (0.03-1 mg/mL). These results suggested the potential of using essential oils of leaves of P.betle, P. chuvya (1.31) and P. longum (1.28) as sources of natural antioxidants. This study also focused on evaluating anti-inflammatory activity by using Human Red Blood Cell (HRBC) membrane stabilizing method.Higher mean inhibition percentage was observed for the essential oil of P. betle leaves (79.6%at 1 mg/mL) compared to the standard (aspirin) and the activity significantly increased with higher concentrations (76.4% at 0.5 mg/mL and 79.6% at 1 mg/mL). Further, 62 % and 58.3 % of inhibitions percentages were shown by essential oils of P. chuvya and P. longum respectively. According to the findings of this study, bioactivity properties of the leaves and fruits of these Piper species can be used in future perspectives for developing novel pharmaceuticals.Item Evaluation of nutrient and fiber content of selected invasive plants in Sri Lanka with a potential for preparing compost(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Hewavitharana, N.; Kannangara, B.T.S.D.P.; Jayasekera, L.R.; Senanayake, S.P.Based on availability, the amount of above ground bio-mass and the degree of use as compost in traditional farming systems Tithonia diversifolia, Chromolaena odorata, Mikania scandens, Lantana camara, Wedelia trilobata, Mimosa pigra and Panicum maximum were selected for nutrient analysis, with an attempt to investigate their potential to be utilized in preparation of cost effective, organic potting medium for cultivation of selected vegetables and ornamental plants. According to the preliminary surveys, Anuradhapura, Madawachchiya, Kandy, Gampaha, Kalutara and Kurunegala areas were selected for sample collection. Leaves and immature stem parts of the selected plants were analyzed by two samples per one location with three replicates. The total N, P and K contents were determined by Kjeldahl, vanadomolybdate and flame photometric methods respectively. Atomic absorption spectrophotometry was used for quantitative analysis of Mg, Ca, Cu Zn, and Fe. Crude fiber content was determined gravimetrically after chemical digestion and solubilization of the materials. Results obtained were statistically analyzed using SPSS statistics-22 software. The nutrient contents of T. diversifolia; N (3.28%), P (0.37%), K (2.50%), Mg (0.83%), Ca (4.92%), Cu (10 mg/kg), Zn (517 mg/kg) Fe (296 mg/kg) and M. scandens; N (3.44%), P (0.35%), K (3.30%) Mg (1.33%), Ca (3.39%), Cu (34 mg/kg), Zn (671 mg/kg), Fe (393 mg/kg) were significantly higher than that of other selected weeds. However, they showed significantly lower crude fiber contents 4.85% and 3.50% respectively. Although the nutrient content in P. maximum was significantly low N (1.27%), P (0.24%), K (1.44%) Mg (0.5%), Ca (1.55%), Cu (9 mg/kg), (Zn 181 mg/kg), Fe (267 mg/kg) and its crude fiber content (20.42%) was significantly higher than that of others. It can be concluded, T. diversifolia and M. scandens as plant materials which have higher amounts of nutrients. P.maximum which has higher content of crude fiber also can be incorporated in preparing compost in order to improve the physical properties of the potting medium.Item Optimization of a genomic DNA extraction technique for genetic diversity studies of selected orchid cvs. with ornamental values(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Farook, F.; Attanayake, R.N.; Senanayake, S.P.Orchidaceae is one of the largest and most diverse families of flowering plants and a major export ornamental crop. Cultivar development is the key for the success of ornamental flower industry and therefore, it is vital to identify the genetic diversity of the cultivars. In such an attempt, the first step is to optimize basic molecular biological techniques involved in genetic diversity analysis. However, DNA extraction from orchids is challenging compared to the other plant species since orchids are rich in polysaccharides and secondary metabolites, which can act as inhibitors in downstream applications. Most of the standardized protocols require liquid nitrogen freezing step, which is not an affordable practice in the laboratories of developing countries. Therefore, optimization of a low cost protocol to obtain pure DNA is necessary. The objective of the current study was to optimize extraction of genomic DNA for molecular marker based genetic diversity studies of selected orchid cultivars with ornamental value. Leaf pieces and pestle and mortar were stored at -80°C for at least three days. DNA extraction was done from frozen orchid leaves (50-100 mg young leaves) using Promega Wizard genomic DNA purification kit (Promega Inc. USA), classical CTAB protocol and modified CTAB protocol. Except modified CTAB protocol, none of the other methods produced high quality DNA as determined by spectrophotometry and agarose gel electrophoresis. However, the method was successful only for the Dendrobium cultivars but not for the Phalaenopsis leaves tested. Successful amplification of orchid rDNA-ITS region confirmed that the quality of extracted DNA is suitable for other PCR based molecular marker studies such as Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeats (SSRs). The modified method was reliable and reproducible.Item Study of pollen germination of Dendrobium cultivars(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Silva, W.E.R.; Kahagalla, T.H.; Senanayake, S.P.; Attanayake, R.Orchidaceae family includes more than 25,000 species, among which Dendrobium genus is one of the most numerous with over 1,200 species in the world. Orchid pollen is packaged as pollinia in which the pollen grains are bound together by viscin threads in masses for effective pollination. Palynological studies of Dendrobium are more important as orchid pollen cannot be dispersed by wind and carried to the stigma for effective fertilization but is mostly done by insects and human beings (hand pollination). Thus, this study was aimed at comparing the efficiency of in vitro pollen germination and natural pollen germination. In vitro pollen germination of commercial Dendrobium variety ‘Thailand tommy’and ‘Burania jet green’ was evaluated using three liquid media containing sucrose (0, 5%, 10%, 15%, 20%) with 0.01% boric acid medium, BK medium containing sucrose, H3B03 (100 mg/L), Ca(N03)2 (300 mg/L), MgSO4 (200 mg/L), and KNO3 (100 mg/ L) and sucrose solutions. Pollen were collected from newly opened flowers of two Dendrobium cultivars, sown on different media and then incubated at room temperature for 24h and examined under a light microscope (10 x 40). Natural pollen germination on stigmatic fluid of Dendrobium cultivars was evaluated using hand pollinated Dendrobium flowers. The pollinia of Dendrobium cultivars were deposited on stigma of newly opened flowers and after 72 hours the small amount of stigmatic fluid was stained with cotton blue in lacto phenol and observed under a light microscope (10 x 40). Pollen tube elongation equal or greater than the diameter of the pollen was considered as germinated pollen. Highest percentage of mean pollen germination was recorded in 0.01% boric acid containing 20% sucrose medium for both ‘Thailand tommy’ (16%) and ‘Burania jet green’ (12.50%) while the lowest mean germination was seen in distilled water medium in ‘Thailand tommy’ (2%) and ‘Burania Jet Green’ (1.50%) in the in vitro pollen germination process. In natural pollen germination process, the percentage of mean pollen germination on stigmatic fluid of Dendrobium cultivars was 26.6% and the pollen tube was elongated 5 times than the pollen diameter.Item Phenetic Diversity and Preliminary Phytochemical Screening of Tephrosia Pers. Spp. (Family Fabaceae) in Sri Lanka(Ceylon Journal of Science (Biological Sciences), 2016) Priyadarshan, A.I.S.; Senanayake, S.P.; Jayatilleke, M.P.; Rajapakse, S.Tephrosia is a genus of medicinally important perennial shrubs or herbs distributed in warm temperate and tropical regions, and belongs to Family Fabaceae. The genus is represented in Sri Lanka with 10 species. The objective of the present study was to interpret interspecific relationships of Tephrosia species in Sri Lanka using phenetic and phytochemical variations. Specimens of six different Tephrosia species including T. purpurea, T. villosa, T. noctiflora, T. tinctoria, T. maxima and T. pumila were collected from their natural habitats in different climatic zones in Sri Lanka. Sixteen floral and vegetative characters of Tephrosia spp. were observed and cluster analysis was performed to interpret phenetic relationships. Herbarium specimens were prepared and voucher specimens were submitted to the National Herbarium, Peradeniya. Phenogram indicated the presence of interspecific relationship with respect to the morphological characters. Air dried leaves were subjected to sequential solvent extractions using solvents with different polarities such as hexane, chloroform and methanol. These extracts were subjected to preliminary phytochemical screening tests to detect the occurrence of carbohydrates, protein and amino acids, glycosides, alkaloids, saponins, flavonoids, tannins, phenolics, and phytosteroids. Phytochemical screening of the different Tephrosia spp. indicated a close relationship among the species and further isolations and purifications are needed to elucidate the chemical constituents to assess the efficacy.Item Phenetic analysis and phytochemical screening of medicinally important Albizia spp. in Sri Lanka(Ceylon Journal of Science (Biological Sciences), 2016) Jayasiri, A.P.A.; Senanayake, S.P.; Paranagama, P.; Amarasinghe, A.P.G.Albizia Durazz. is a genus of 150 species in the tropics and subtropics of the world and belongs to the sub family Mimosaceae in the Family Fabaceae. Of the six species recorded in Sri Lanka, A. lebbeck is used as a shady tree while A. odoratissima is grown mainly for their timber value, However, A. odoratissima and A. lebbeck are found to be used in ayurvedic medicine however, the medicinal properties of these species are not fully understood. A questionnaire survey was carried out using a hundred sample population to identify their medicinal usage. Floral and vegetative characters of the above two Albizia spp. were observed and phenetic relationships were identified. Air dried stem barks of A. odoratissima and A. lebbeck were subjected to sequential solvent extraction using hexane, chloroform, methanol and water, and the crude weight of the yield were obtained. The results revealed that ayurvedic physicians and traditional ayurvedic medical practitioners use A. odoratissima in medicinal preparations whereas the medicinal use of A. lebbeck is not reported. Further, it was revealed that Samanea saman and Adenanthera pavonina are commonly used as substitutes for A. lebbeck. Knowledge of phenetic variation of the two Albizia spp. can be used for accurate identification which prevents adulteration. Highest yield was obtained from the methanolic extracts. These extracts were subjected to preliminary phytochemical screening to assess the occurrence of different phytochemicals. Results have shown the presence of glylcosides, tannins, phenolics, phytosteroids and flavonoids in methanolic extracts A. odoratissima, and A. lebbeck. The present study suggests that further studies should be conducted on the identification of active compounds in these two plant species for their pharmacognostic properties in order to understand their mode of remedial action for ailments.Item Evaluation of phenetic diversity of selected orchid cultivars with ornamental value(The Institute of Biology, Sri Lanka, 2016) Farook, F.; Attanayake, R.N.; Senanayake, S.P.Item Assessment of epiphytic lichen diversity in pine plantations and in adjacent secondary forest in Peacock Hill, Pussellawa, Sri Lanka(2015) de Silva, C.M.S.M.; Senanayake, S.P.
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