Effects of anti-browning agents on protocorm-like body induction using thin cell layers of Endangered Lady’s Slipper Orchid: Paphiopedilum callosum var. sublaeve

Main Article Content

นรารัตน์ วัฒนาพันธ์ วุฒิชัย ศรีช่วย ล้อมพงศ์ กลิ่นนาวี อุปถัมภ์ มีสวัสดิ์


Paphiopedilum callosum var. sublaeve, a lady’s slipper orchid native to southern Thailand, is under threat of species extinction and listed on Appendix I of CITES. In vitro seed germination for its micropropagation
provides a beneficial mode to re-establish plants in the wild. The experiments were conducted to study
the influence of anti-browning (ascorbic acid and activated charcoal) on PLB induction using thin cell layers of . callosum var. sublaeve. Aseptic shoots (1-1.5 cm height) from mature seeds of P. callosum var. sublaeve was firstly used for adventitious PLBs, shoots, and roots induction from transversed thin section within a month. he explants were pretreatment by soaking with or without ascorbic acid (0.1 mg/L) and inoculated on MVWmedium supplemented with activated charcoal (2 g/L) or ascorbic acid (0.1 mg/L). Shoots were pretreated ith ascorbic acid and cultured on MVW medium supplemented with activated charcoal gave the lowest percentage of browning (45.83%). The highest percentage of survival rate (59.09 %), PLB induction
(50.89%), shoot induction (29.17%), and the number of PLBs/explant (1.46) were obtained from the explants
that were pretreated without ascorbic acid (0.1 mg/L) and inoculated on MVW medium supplemented
with activated charcoal (2 g/L). Meanwhile, the explants with no ascorbic acid pretreatment cultured on
MVW medium containing 0.1 mg/L ascorbic acid provided the highest percentage of browning (70.83%).


Article Details



Achakzai, A. K. K., Achakzai, P., Masood, A., Kayani, S. A., & Tareen, R. B. (2009). Response of Plant Parts and Age on the Distribution of Secondary Metabolites on Plants Found in Quetta. Pakistan Journal of Botany, 41(5), 2129-2135.

Anjum, S., Zia, M., & Chaudhary, M. F. (2006). Investigations of Different Strategies for High Frequency Regeneration of Dendrobium malones ‘Victory’. African Journal of Biotechnology, 5(19), 1738-1743.

CITES. (2013). Conventional on International Trade in Endangered Species of Wild Fauna and Flora, Appendices I. Geneva, Switzerland. Retrieved March 9, 2018, Retrieved from https://www.cites.org/eng/disc/text.php

Chen, T. Y., Chen, J. T., & Chang, W. C. (2004). Plant Regeneration through Direct Shoot Bud Formation from of Paphiopedilum Orchids. Plant Cell Tissue and Organ Culture, 76, 11-15.

Elmore, H. W., Samples, B., Sharma, S., & Harrison, M. (1990). Influence of Cultural and Physiochemical Factors on
Ascorbate Stability in Plant Tissue Culture Media. Plant Cell Tissue and Organ Culture, 20, 131-135.

Fernando, S. C., Santha, E. S., & Hewarathna, D. J. A. (2010). Activated Coconut Shell Charcoal as a Component
of Tissue Culture Media of Cocos nucifera L. Journal of the National Science Foundation of Sri Lanka, 8, 181-185.

Fridborg, G., Pedersen, M., Landstrom, L., & Ericksson, T. (1978). The Effect of Activated Charcoal on Tissue
Cultures: Adsorption of Metabolites Inhibiting Morphogenesis. Physiologia Plantarum, 43, 104-106.

Gantait, S., & Sinniah, U. R. (2012). Rapid Micropropagation of Monopodial Orchid Hybrid (Aranda Wan Chark Kuan ‘Blue’ x Vanda coerulea Grifft. Ex. Lindl.) through Direct Induction of Protocorm-like Bodies from Leaf Segments. Plant Growth Regulation, 68, 129-140.

Islam, M. O., Islam, Md. S., & Saleh, A. (2015). Effect of Banana on Growth and Development of Protocorm like Bodies in Dendrobium, sp. orchid. The Agriculturists, 13(1), 101-105.

Julkifle, A. L., Poobathy, R., Samain, R. & Subramaniam, S. Histological Analyses of PLBs of Dendrobium Sonia-28 in the Recognition of Cell Competence for Regeneration and Agrobacterium infection. Plant Omics, 5(6), 514-517.

Kariyana, K., & Nisyawati. (2013). Effect of Ascorbic Acid, Activated Carbon and Light Duration on Explant Browning of Banana Cultivar barangan (Musa acuminate L.). International Journal of Research and Reviews in Applied Sciences, 16(1), 118-123.

Ko, W. H., Su, C. C., Chen, C. L., & Chao, C. P. (2009). Control of Lethal Browning of Tissue Culture Plantlets of Cavendish Banana cv. Formosana with Ascorbic acid. Plant Cell Tissue and Organ Culture, 96, 137-141.

Lakshmanan, P., Loh, C., & Goh, C. (1995). An in vitro Method for Rapid Regeneration of a Monopodial Orchid Hybrid Aranda Deborah Using Thin Section Culture. Plant Cell Reports, 14, 510-514.

Lee, Y., Hsu, S., & Yeung, E. C. (2013). Orchid Protocorm-like Bodies are Somatic Embryos. American Journal of Botany, 100(11), 1211-1231.

Linington, I. M. (1991). In vitro Propagation of Dipterocarpus alatus and Dipterocarpus intricatus. Plant Cell Tissue and Organ Culture, 27, 81-88.

Madhasudhanan, K., & Rahiman, B. A. (2000). The Effect of Activated Charcoal Supplemented Media to Browning of in vitro Cultures of Piper species. Biologia Plantarum, 43(2), 297-299.

Mayer, J. L. S., Stancato, G. C. & Appezzato-Da-Gloria, B. (2010). Direct Regeneration of Protocorm-like Bodies (PLBs) from Leaf Apices of Oncidium flexuosum Sims (Orchidaceae). Plant Cell Tissue and Organ Culture. DOI 10.1007/s11240-010-9782-9.

Meilasari, D. & Iriawati. 2016. Regeneration of Plantlets Through PLB (Protocorm-Like Body) Formation in Phalaenopsis ‘Join Angle X Sogo Musadian. Journal of Mathematical and Fundamental Sciences, 48(3), 204-212.

Mulgund, G. S., Nataraja, K., Malabadi, R. B., & Kumar, S. V. (2011). TDZ induced in vitro Propagation of an Epiphytic Orchid Xenikophyton smeeanum (Reichb.f.). Research in Plant Biology, 1(4), 7-15.

Ndakidemi, C. F., Mneney, E., & Ndakidemi, P. A. (2014). Effects of Ascorbic Acid in Controlling Lethal Browning in in vitro Culture of Brahylaena huillensis Using Nodal Segments. American Journal of Plant Sciences, 5, 187-191.

Ng, C., Saleh, N. M., & Zaman, F. Q. (2010). In vitro Multiplication of the Rare and Endangered Slipper Orchid, Paphiopedilum rothschildianum (Orchidaceae). African Journal of Biotechnology, 9(14), 2062-2068.

Ng, C., & Saleh, N. M. (2011). In vitro Propagation of Paphiopedilum Orchid through Formation of Protocorm-like Bodies. Plant Cell Tissue and Organ Culture, 105, 193-202.

Nhut, D. T., Don, N. T., Vu, N. H., Thien, N. Q., Thuy, D. T. T., Duy, N., & Teixeira da Silva, J. A. (2006). Floriculture, Ornamental and Plant Biotechnology. Volume II. Global Science Books Publishers, UK.

Ozyigit, I., Kahraman, M. V., & Ercan, O. (2007). Relation Between Explant Age, Total Phenols and Regeneration
Response in Tissue Cultured Cotton (Gossypium hirsutum L.). African Journal of Biotechnology, 6(1), 3-8.

Park, S. Y., Murthy, H. N., & Paek, K. Y. (2003). Protocorm-like Body Induction and Subsequent Plant Regeneration from Root Tip Cultures of Doritaenopsis. Plant Science, 164, 919-923.

Prasongsom, S., Thammasiri, K., & Chuenboonngarm, N. (2016). Efficient Adventitious Shoot Regeneration from Shoot Tip Culture of Rhynchostylis gigantea Lindl. (amethyst-purple), a Rare Thai Orchid Species. Walailak Journal, 13(9), 757-767.

Ruzin, S. (1999). Plant Microtechnique and Microscopy. New York: Oxford University Press

Thomas, T. D. (2008). The Role of Activated Charcoal in Plant Tissue Culture. Biotechnology Advances, 26, 618-631.

Titov, S., Bhowmik, S. K., Mandal, A., Alam, Md. S., & Uddin, S. N. (2006). Control of Phenolic Compound Secretion and Effect of Growth Regulators for Organ Formation from Musa spp. cv. Kanthali Bud Explants. American Journal of Biochemistry and Biotechnology, 2(3), 97-104..

Tokuhara, K., & Mii, M. (2001). Induction of Embryogenic Callus and Cell Suspension Culture from Shoot Tips Excised from Flower Stalk Buds of Phalaenopsis (Orchidaceae). In Vitro Cell Developmental Biology-Plant, 37, 457-461.

Toth, K. , Haapala, T., & Hohtola, A. (1994). Alleviation of Browning in Oak Explants by Chemical Pretreatments.
Biologia Plantarum, 36(4), 511-517.

Vacin, E. F., & Went, F. W. (1949). Some pH Change in Nutrient Solutions. Botanical Gazette, 110, 605-613.

Vyas, S., Guha, S., Kapoor, P., & Rao, I. U. (2010). Micropropagation of Cymbidium Sleeping Nymph Through Protocorm-like Bodies Production by Thin Cell Layer Culture. Scientia Horticulturae, 123, 551-557.

Zeng, S., Wu, K., Teixeira da Silva, J. A. Zhang, J., Xia, N., & Duan, J. (2012). Asymbiotic Seed Germination Seedling Development and Reintroduction of Paphiopedilum wardii Sumerh., an Endangered Terrestrial Orchid. Scientia Horticulturae, 138, 198-209.

Zhao, P., Wang, W., Feng, F.S., Wu, F., Yang, Z. Q., & Wang, W. J. (2007). High-Frequency Shoot Regeneration
Transvers Thin Cell Layer Culture in Dandrobium Candidum Wall Ex Lindl. Plant Cell Tissue and Organ Culture, 90, 131-139.