International Postgraduate Research Conference (IPRC)
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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.