Clonal Fidelity of Micro-propagated Phalaenopsis Plantlets Based on Assessment Using Eighteen Ph-Pto SNAP Marker Loci

Erick Raynalta, Juanita Elina, Sudarsono Sudarsono, Dewi Sukma

Abstract


Phalaenopsis amabilis is an Indonesia native orchid species having large, white flowers with yellow labellum coloration. This studies aimed to develop Phal. amabilis micropropagation methods and evaluate the regenerated plantlet fidelity. Media supplemented with Thidiazuron (TDZ) and Polyvinylpyrrolidone (PVP) and medium pH adjustment effects to induce protocorm-like bodies (PLBs) from leaf explants and proliferate secondary PLBs were investigated. Clonal fidelity among regenerated plantlets was evaluated using eighteen SNAP marker loci. The results showed that the ½ MS medium supplemented with 3 mg L-1 TDZ and 0.5 g L-1 PVP was the best for PLB induction while the ½ MS medium supplemented with 0.5 mg L-1 TDZ was the best for PLB proliferation. For PLB induction, the media pH was adjusted into pH=7 for efficient PLB regeneration. Based on the assessment using 18 SNAP marker loci, four variant alleles in three loci (11.8%) out of a total 34 plantlets were detected. The mutation frequency at the evaluated SNAP marker loci was 2.5 x 103 (0.25%). Changes in SNP alleles may not always result in phenotype changes and allele variant occurrences may not affect phenotype fidelity of micro-propagated Phal. amabilis plantlets. Therefore, further studies about the phenotype fidelity among plantlets are necessary.

Keywords


Micro-propagation; Phenotype fidelity; PLBs; Protocorm-like bodies; Thidiazuron

Full Text:

PDF

References


Ahmad, I., Hussain, T., Ashraf, I., Nafees, M., Maryam, Rafay, M., & Iqbal, M. (2013). Lethal effect of secondary metabolites on plant tissue culture. American-Eurasian Journal Of Agricultural & Environmental Sciences, 13(4), 539–547. crossref

Ajijah, N., Hartati, R. S., Rubiyo, Sukma, D., & Sudarsono. (2016). Effective cacao somatic embryo regeneration on kinetin supplemented DKW medium and somaclonal variation assessment using SSRs markers. AGRIVITA Journal of Agricultural Science, 38(1), 80–92. crossref

Antensari, F., Mariani, T. S., & Wicaksono, A. (2014). Micropropagation of Phalaenopsis ‘R11xR10’ through somatic embryogenesis method. Asian Journal of Applied Sciences, 2(2), 145–150. Retrieved from website

Bairu, M. W., Aremu, A. O., & Van Staden, J. (2011). Somaclonal variation in plants: Causes and detection methods. Plant Growth Regulation, 63(2), 147–173. crossref

Balilashaki, K., Naderi, R., Kalantari, S., & Soorni, A. (2014). Micropropagation of Phalaenopsis amabilis cv. Cool “Breeze” with using of flower stalk nodes and leaves of sterile obtained from node cultures. International Journal of Farming and Allied Sciences, 3(7), 823–829. crossref

Chen, C.-C., Bates, R., & Carlson, J. (2015). Effect of environmental and cultural conditions on medium pH and explant growth performance of Douglas-fir (Pseudotsuga menziesii) shoot cultures. F1000Research, 3, 298. crossref

Chen, F.-C., Yu, J.-Y., Chen, P.-Y., & Huang, Y.-W. (2008). Somaclonal variation in orchids and the application of biotechnology. Acta Horticulturae, (766), 315–322. crossref

Chen, G., Chen, D., Wang, T., Xu, C., & Li, L. (2012). Analysis of the proteins related to browning in leaf culture of Phalaenopsis. Scientia Horticulturae, 141, 17–22. crossref

Chen, L.-R., Chen, J.-T., & Chang, W.-C. (2002). Efficient production of protocorm-like bodies and plant regeneration from flower stalk explants of the sympodial orchid Epidendrum radicans. In Vitro Cellular & Developmental Biology - Plant, 38(5), 441–445. crossref

Chugh, S., Guha, S., & Rao, I. U. (2009). Micropropagation of orchids: A review on the potential of different explants. Scientia Horticulturae, 122(4), 507–520. crossref

Crain, B. J., & Tremblay, R. L. (2014). Do richness and rarity hotspots really matter for orchid conservation in light of anticipated habitat loss? Diversity and Distributions, 20(6), 652–662. crossref

Crain, B. J., & Tremblay, R. L. (2017). Hot and bothered: Changes in microclimate alter chlorophyll fluorescence measures and increase stress levels in tropical epiphytic orchids. International Journal of Plant Sciences, 178(7), 503–511. crossref

Doyle, J. J., & Doyle, J. L. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 19, 11–15. Retrieved from website

Elina, J., Sukma, D., Giyanto, & Sudarsono. (2017). Isolasi dan karakterisasi gen Pto asal 20 aksesi anggrek Phalaenopsis [Isolation and characterization of Pto gene from 20 Phalaenopsis orchid genotypes]. Jurnal Agronomi Indonesia, 45(2), 204–211. Retrieved from website

Fattmah, & Sukma, D. (2011). Development of sequence-based microsatellite marker for Phalaenopsis orchid. HAYATI Journal of Biosciences, 18(2), 71–76. crossref

Fay, M. F. (2018). Orchid conservation: how can we meet the challenges in the twenty-first century ? Botanical Studies, 59, 16. crossref

Fehér, A. (2015). Somatic embryogenesis - stress-induced remodeling of plant cell fate. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1849(4), 385–402. crossref

Feng, J. H., & Chen, J. T. (2014). A novel in vitro protocol for inducing direct somatic embryogenesis in Phalaenopsis aphrodite without taking explants. The Scientific World Journal, 2014(263642), 1–7. crossref

George, E. F., Hall, M. A., & De Klerk, G.-J. (Eds.). (2008). Plant propagation by tissue culture. Vol 1. The Background (3rd ed.). Netherland: Springer. crossref

Gow, W. P., Chen, J. T., & Chang, W. C. (2008). Influence of growth regulators on direct embryo formation from leaf explants of Phalaenopsis orchids. Acta Physiologiae Plantarum, 30, 507. crossref

Gow, W. P., Chen, J. T., & Chang, W. C. (2010). Enhancement of direct somatic embryogenesis and plantlet growth from leaf explants of Phalaenopsis by adjusting culture period and explant length. Acta Physiologiae Plantarum, 32(4), 621–627. crossref

Gulles, A. A., Bartolome, V. I., Morantte, R. I. Z. A., Nora, L. A., Relente, C. E. N., Talay, D. T., … Ye, G. (2014). Randomization and analysis of data using STAR [Statistical Tool for Agricultural Research]. Philippine Journal of Crop Science, 39(supplement1), 137. Retrieved from website

Handoyo, F. (2010). Orchids of Indonesia (Vol. 1). Indonesia: Indonesian Orchid Society. Retrieved from website

Hill, K., & Schaller, G. E. (2013). Enhancing plant regeneration in tissue culture: A molecular approach through manipulation of cytokinin sensitivity. Plant Signaling and Behavior, 8(10), e25709. crossref

Hofmann, N., Nelson, R. L., & Korban, S. S. (2004). Influence of media components and pH on somatic embryo induction in three genotypes of soybean. Plant Cell, Tissue and Organ Culture, 77(2), 157–163. crossref

Huang, Y. W., Tsai, Y. J., Cheng, T. C., Chen, J. J., & Chen, F. C. (2014). Physical wounding and ethylene stimulated embryogenic stem cell proliferation and plantlet regeneration in protocorm-like bodies of Phalaenopsis orchids. Genetics and Molecular Research, 13(4), 9543–9557. crossref

Ikedo, T. (n.d.). Phalaenopsis species: Ecology, morphology and cultivation. Retrieved from website

Khoddamzadeh, A. A., Sinniah, U. R., Kadir, M. A., Kadzimin, S. B., Mahmood, M., & Sreeramanan, S. (2010). Detection of somaclonal variation by random amplified polymorphic DNA analysis during micropropagation of Phalaenopsis bellina (Rchb.f.) Christenson. African Journal of Biotechnology, 9(40), 6632–6639. Retrieved from website

Khoddamzadeh, A. A., Sinniah, U. R., Kadir, M. A., Kadzimin, S. B., Mahmood, M., & Sreeramanan, S. (2011). In vitro induction and proliferation of protocorm-like bodies (PLBs) from leaf segments of Phalaenopsis bellina (Rchb.f.) Christenson. Plant Growth Regulation, 65, 381. crossref

Knudson, L. (1946). A new nutrient solution for germination of orchid seeds. American Orchid Society Bulletin, 15, 214-217.

Larekeng, S. H., Maskromo, I., Purwito, A., Mattjik, N. A., & Sudarsono, S. (2015). Penyebaran polen berdasarkan analisis SSR membuktikan penyerbukan kelapa dalam Kalianda normal ke kopyor [Pollen dispersal based on SSR analysis proves Kalianda to kopyor coconut pollinations]. Buletin Palma, 16(1), 77–92. crossref

Lee, Y. I., Hsu, S. T., & Yeung, E. C. (2013). Orchid protocorm-like bodies are somatic embryos. American Journal of Botany, 100(11), 2121-2131. crossref

Mahendran, G., & Bai, V. N. (2016). An efficient in vitro propagation, antioxidant and antimicrobial activities of Aphyllorchis montana Rchb.f. Plant Biosystems, 150(5), 1087–1095. crossref

Maskromo, I., Larekeng, S. H., Novarianto, H., & Sudarsono, S. (2016). Xenia negatively affecting kopyor nut yield in Kalianda Tall kopyor and Pati Dwarf kopyor coconuts. Emirates Journal of Food and Agriculture, 28(9), 644–652. crossref

Merritt, D. J., Hay, F. R., Swarts, N. D., Sommerville, K. D., & Dixon, K. W. (2014). Ex situ conservation and cryopreservation of orchid germplasm. International Journal of Plant Sciences, 175(1), 46–58. crossref

Miyao, A., Nakagome, M., Ohnuma, T., Yamagata, H., Kanamori, H., Katayose, Y., … Hirochika, H. (2012). Molecular spectrum of somaclonal variation in regenerated rice revealed by whole-genome sequencing. Plant and Cell Physiology, 53(1), 256–264. crossref

Mose, W., Indrianto, A., Purwantoro, A., & Semiarti, E. (2017). The influence of thidiazuron on direct somatic embryo formation from various types of explant in Phalaenopsis amabilis (L.) blume orchid. HAYATI Journal of Biosciences, 24(4), 201–205. crossref

Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15(3), 473–497. crossref

Niknejad, A., Kadir, M. A., & Kadzimin, S. B. (2011). In vitro plant regeneration from protocorms-like bodies (PLBs) and callus of Phalaenopsis gigantea (Epidendroideae: Orchidaceae). African Journal of Biotechnology, 10(56), 11808–11816. Retrieved from website

Oktavia, F., Kuswanhadi, K., Dinarty, D., Widodo, W., & Sudarsono, S. (2017). Genetic diversity and population structure of IRRDB 1981 and Wickham rubber germplasm based on EST-SSR. AGRIVITA Journal of Agricultural Science, 39(3), 239–251. crossref

Ossowski, S., Schneeberger, K., Lucas-Lledó, J. I., Warthmann, N., Clark, R. M., Shaw, R. G., … Lynch, M. (2010). The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana. Science, 327(5961), 92–94. crossref

Pesik, A., Efendi, D., Novarianto, H., Dinarti, D., & Sudarsono, S. (2017). Development of SNAP markers based on nucleotide variability of WRKY genes in coconut and their validation using multiplex PCR. Biodiversitas, 18(2), 465–475. crossref

Pornpienpakdee, P., Singhasurasak, R., Chaiyasap, P., Pichyangkura, R., Bunjongrat, R., Chadchawan, S., & Limpanavech, P. (2010). Improving the micropropagation efficiency of hybrid Dendrobium orchids with chitosan. Scientia Horticulturae, 124(4), 490–499. crossref

Ru, Z., Lai, Y., Xu, C., & Li, L. (2013). Polyphenol Oxidase (PPO) in early stage of browning of Phalaenopsis leaf explants. Journal of Agricultural Science, 5(9), 57–64. crossref

Samarfard, S., Kadir, M. A., Kadzimin, S. B., Ravanfar, S., & Saud, H. M. (2013). Genetic stability of in vitro multiplied Phalaenopsis gigantea protocorm-like bodies as affected by chitosan. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 41(1), 177–183. crossref

Samarfard, S., Kadir, M. A., Kadzimin, S. B., Saud, H. M., Ravanfar, S. A., & Danaee, M. (2014). In vitro propagation and detection of somaclonal variation in Phalaenopsis gigantea as affected by chitosan and thidiazuron combinations. HortScience, 49(1), 82–88. Retrieved from website

Santarem, E. R., Pelissier, B., & Finer, J. J. (1997). Effect of explant orientation, pH, solidifying agent and wounding on initiation of soybean somatic embryos. In Vitro Cellular and Developmental Biology - Plant, 33(1), 13–19. crossref

Shi, X., Yang, L., Yan, G., & Du, G. (2017). Medium pH between 5.5 and 7.5 has minimal effects on tissue culture of apple. HortScience, 52(3), 475-478. Retrieved from website

Shimelis, D., Bantte, K., & Feyissa, T. (2015). Effects of polyvinyl pyrrolidone and activated charcoal to control effect of phenolic oxidation on in vitro culture establishment stage of micropropagation of sugarcane (Saccharum officinarum L.). Advances in Crop Science and Technology, 3(4), 10–13. crossref

Soetopo, L., & Purnamaningsih, S. L. (2012). In vitro propagation of Dendrobium and Phalaenopsis through tissue culture for conservation. AGRIVITA Journal of Agricultural Science, 34(2), 115–126. crossref

Sudarsono, S., Haristianita, M.D., Handini, A.S. & Sukma, D. (2017). Molecular marker development based on diversity of genes associated with pigment biosynthetic pathways to support breeding for novel colors in Phalaenopsis. Acta Horticulturae, 1167, 305-312. crossref

Sukma, D., Elina, J., Giyanto, & Sudarsono, S. (2017). Disease resistance breeding of Phalaenopsis spp. for tropical environment and molecular marker development for plant selection. Acta Horticulturae, 1167, 237–244. crossref

Sun, W. Y., Zhao, W. Y., Wang, Y. Y., Pei, C. C., & Yang, W. C. (2011). Natural variation of Pto and Fen genes and marker-assisted selection for resistance to bacterial speck in tomato. Agricultural Sciences in China, 10(6), 827–837. crossref

Sutanto, A., Sukma, D., Hermanto, C., & Sudarsono. (2014). Isolation and characterization of Resistance Gene Analogue (RGA) from Fusarium resistant banana cultivars. Emirates Journal of Food and Agriculture, 26(6), 508–518. crossref




DOI: http://doi.org/10.17503/agrivita.v40i3.1493

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.