Browsing by Author "Silva, W.E.R."

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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 pollen
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.
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.