Improved micropropagation, morphometric traits and photosynthetic pigments content using liquid culture system in Spathoglottis plicata Blume

Shekhawat, Mahipal S., Priyadharshini, S., Manokari, M.

Research Articles | Published: 18 October, 2021

Volume: 35, Issue: 1, March 2022
Print ISSN : 0970-4078.
Online ISSN : 2229-4473.
Pub Email:
Doi: 10.1007/s42535-021-00303-0
First Page: 9
Last Page: 18
Views: 197

Keywords: Acclimatization, Chlorophylls, Garden orchid, In vitro, Protocorms


An effective plant regeneration protocol via a liquid culture system has been ascertained in Spathoglottis plicata Blume (Orchidaceae), an important orchid of the horticulture industry. The seeds were germinated asymbiotically on the agar-gelled Murashige and Skoog’s (MS) medium supplemented with 1.0 mg L− 1 6-benzylaminopurine (BAP) and about 93.0 % seeds were germinated via the formation of protocorms. The highest rate of shoot proliferation (72.3 ± 0.26 shoots with average 8.0 ± 0.19 cm length per protocorm after 4th subculture) was obtained with liquid MS medium containing 1.0 mg L− 1 BAP and 0.5 mg L− 1 α-naphthalene acetic acid (NAA). The liquid culture system also enhanced the qualitative foliar morphometric parameters (7.3 ± 0.17 cm length x 1.2 ± 0.25 cm width). Half strength MS medium fortified with 1.5 mg L− 1 indole-3-butyric acid (IBA) was observed most suitable for rooting of the shoots. The shoots derived through liquid culture responded better (94.0 %) in rooting experiments (13.0 ± 0.22 roots per shoot, 4.0 ± 0.25 cm length), as opposed to 79.0 % in semi-solid medium with 6.5 ± 0.29 roots and 3.3 ± 0.19 cm length. Additionally, the liquid culture system enhanced qualitative and quantitative parameters of the shoot and root systems, and photosynthetic pigments (chlorophyll a, b, and carotenoids) which assisted in acclimatization and survival success of plantlets (100 %). This is the first attempt to use of the liquid medium for in vitro regeneration of S. plicata which could be adopted for large-scale propagation and conservation of this important but vulnerable orchid species.

Acclimatization, Chlorophylls, Garden orchid, In vitro, Protocorms

*Pdf Download Buy Printed Copy

(*Only SPR Members can download pdf file; #Open Access;)


Acevedo-Rodríguez P, Strong MT (2012) Catalogue of the seed plants of the West Indies. Smithsonian Institution, Washington, p 1192

Ackerman JD, Falcon W, Molinari J, Vega C, Espino I, Cuevas AA (2014) Biotic resistance and invasional meltdown: consequences of acquired interspecific interactions for an invasive orchid, Spathoglottis plicata in Puerto Rico. Biol Invasions 16:2435–2447.

Aewsakul N, Maneesorn D, Serivichyaswat P, Taluengjit A, Nontachaiyapoom S (2013) Ex vitro symbiotic seed germination of Spathoglottis plicata Blume on common orchid cultivation substrates. Sci Hortic 160:238–242

Arab MM, Yadollahi A, Shojaeiyan A, Shokri S, Ghojah SM (2014) Effects of nutrient media, different cytokinin types and their concentrations on in vitro multiplication of G × N15 (hybrid of almond × peach) vegetative rootstock. J Genet Eng Biotechnol 12:81–87.

Aremu AO, Bairu MW, Szucova L, Dolezal K, Finnie JF, Van Staden J (2012) Assessment of the role of meta-topolins on in vitro produced phenolics and acclimatization competence of micropropagated ‘Williams’ banana. Acta Physiol Plant 34:2265–2273

Aswathi, Shibu S, Gopinath A, Mohan A (2017) In vitro propagation of Spathoglottis plicata Blume via asymbiotic seed germination. Int J Adv Res 5:431–438

Beltrame E (2006) Hardy orchids: Spathoglottis–inside and out. The Orchid Review, pp 68–71

Bhattacharyya P, Kumaria S, Tandon P (2016) High frequency regeneration protocol for Dendrobium nobile: a model tissue culture approach for propagation of medicinally important orchid species. S Afr J Bot 104:232–243

Chen JC, Chen CT (2008) Correlation analysis between indices of tree leaf spectral reflectance and chlorophyll content. Int Arch Photogram Remote Sens Spat Inf Sci XXXVII. Part B7 Beijing, pp 231–238

Chen Y, Goodale UM, Fan X-L, Gao J-Y (2015) Asymbiotic seed germination and in vitro seedling development of Paphiopedilum spicerianum: an orchid with an extremely small population in China. Glob Ecol Conserv 3:367–378

Dagar HS, Dagar JC (2003) Plants used in ethnomedicine by the Nicobarese of Islands in Bay of Bengal, India. In: Singh V, Jain AP (eds) Ethnobotany and medicinal Plants of India and Nepal. Scientific Publishers (India), Jodhpur, pp 773–784

Daud N, Taha RM, Noor NNM, Alimon H (2011) Potential of alternative gelling agents in media for the in vitro micro-propagation of Celosia sp. Int J Bot 7:183–188.

De LC, Pathak P, Rao AN, Rajeevan PK (2014) Commercial orchids. De Gruyter Open, Warsaw, pp 13–19

Debnath SC (2008) Developing a scale-up system for the in vitro multiplication of thidiazuron-induced strawberry shoots using a bioreactor. Canad J Plant Sci 88:737–746.

Environment Protection and Biodiversity Conservation (EPBC) Act (1999) Spathoglottis plicata conservation advice. Pp 1–3 (accessed 19 August 2020)

Haque MS, Ghosh B (2017) Regeneration of cytologically stable plants through dedifferentiation, redifferentiation and artificial seeds in Spathoglottis plicata Blume. (Orchidaceae). Hort Plant J 3:199–208

Hossain MM, Dey R (2013) Multiple regeneration pathways in Spathoglottis plicata Blume: a study in vitro. S Afr J Bot 85:56–62

Jones DL (2006) The Complete Guide to Orchids of Australia. French Forest, NSW, Reed New Holland

Kaur S (2017) In vitro regeneration of shoots from nodal explants of Dendrobium chrysotoxum Lindl. J Hortic Res 25:27–34

Kunakhonnuruk B, Inthima P, Kongbangkerd A (2019) In vitro Propagation of Rheophytic Orchid, Epipactis flava Seidenf. - a comparison of semi-solid, continuous immersion and temporary immersion systems. Biol 8:72.

Kuria P, Demo P, Nyende AB, Kahangi EM (2008) Cassava starch as an alternative cheap gelling agent for the in vitro micro-propagation of potato (Solanum tuberosum L.). Afr J Biotechnol 7:301–307

Malik M, Warchol M, Kwasniewska E, Pawlowska B (2017) Biochemical and morphometric analysis of emopenRosa tomentosaemclose and emopenRosa rubiginosaemclose during application of liquid culture systems for emopenin vitroemclose shoot production. J Hortic Sci Biotechnol 92:606–613.

Manokari M, Latha R, Priyadharshini S, Shekhawat MS (2021a) Short-term cold storage of encapsulated somatic embryos and retrieval of plantlets in grey orchid (emopenVanda tessellataemclose (Roxb.) Hook. ex G.Don). Plant Cell Tiss Organ Cult 144:171–183.

Manokari M, Priyadharshini S, Jogam P, Dey A, Shekhawat MS (2021b) Meta-topolin and liquid medium mediated enhanced micropropagation via ex vitro rooting in Vanilla planifolia Jacks. ex Andrews. Plant Cell Tiss Organ Cult 146:69–82.

Manokari M, Priyadharshini S, Shekhawat MS (2021c) Direct somatic embryogenesis using leaf explants and short term storage of synseeds in Spathoglottis plicata Blume. Plant Cell Tiss Organ Cult 145:321–331.

Mbiyu M, Muthoni J, Kabira J, Muchira C, Pwaipwai P, Ngaruiya J, Onditi J, Otieno S (2012) Comparing liquid and solid media on the growth of plantlets from three Kenyan potato cultivars. Am J Exp Agricult 2:81–89

Minea M, Piluek C, Menakanit A, Tantiwiwat S (2004) A study in seed germination and seedling development of Spathoglottis orchid. J Nat Sci 38:147–156

Mollik AH, Hossan S, Islam T, Jahan R, Rahmatullah M (2009) Medicinal plants used against rheumatoid arthritis by traditional medicinal practitioners of Bangladesh. Planta Med.

Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:495–497

Nature Conservation Regulation (2006) www. (accessed 22 August 2020)

Oliveira BC, Oliveira MEBS, Cardoso JC (2019) Feasibility of the new method for orchid in vitro rooting using liquid and chemical sterilized culture medium under different sucrose concentration. Ornam Hortic 25:263–269

Pornchuti W, Thammasiri K, Chuenboonngarm N, Panvisavas N (2017) Micropropagation of Spathoglottis eburnea Gagnep, a Thai orchid species through shoot tips. Proc. I International Symposium on Tropical and Subtropical Ornamentals, Acta Hortic 1167: 87–93.

Rasmussen HN, Dixon KW, Jersakova J, Tesitelova T (2015) Germination and seedling establishment in orchids: a complex of requirement. Ann Bot 116:391–402.

Reddy J (2016) Nutrient media used for micropropagation of orchids: a research review. World J Pharma Res 5:1719–1732

Sebastinraj J, Muhirkuzhali S (2004) Asymbiotic seed germination and micropropagation of emopenSpathoglottis plicataemclose Blume. Int J Adv Pharm Biol Chem 3:495–501

Shukla MR, Piunno K, Saxena PK, Jones AMP (2020) Improved in vitro rooting in liquid culture using a two piece scaffold system. Eng Life Sci 20:126–132

Sinha P, Hakim LM, Alam FM (2010) In vitro mass clonal propagation of Spathoglottis plicata Blume. Plant Tissue Cult Biotechnol 19:151–160

Sivritepe N, Erturk U, Yerlikaya C, Turkan I, Bor M, Ozdemir F (2008) Response of the cherry rootstock to water stress induced in vitro. Biol Plant 52:573–576

Sopalun K, Thammasiri K, Ishikawa K (2010) Micropropagation of the Thai orchid Grammatophyllum speciosum blume. Plant Cell Tissue Organ Cult 101:143–150

Stella RY, Priya MT, Begam MFK, Manimekalai V (2015) In vitro seed germination, somatic embryogenesis and protocorm based micropropagation of a terrestrial ornamental orchid--Spathoglottis plicata Blume. Eur J Biotechnol Biosci 3:20–23

Suthar RK, Habibi N, Purohit SD (2011) Influence of agar concentration and liquid medium on in vitro propagation of Boswellia serrata Roxb. Indian J Biotechnol 10:224–227

Thompson DI, Edwards TJ, van Staden J (2006) Evaluating asymbiotic seed culture methods and establishing Disa (Orchidaceae) germinability in vitro: relationships, requirements and first-time reports. Plant Growth Regul 49:269–284

Vylíčilová H, Bryksová M, Matušková V, Doležal K, Plíhalová L, Strnad M (2020) Naturally occurring and artificial N9-cytokinin conjugates: from synthesis to biological activity and back. Biomol 10:832.

Wagner WI, Herbst DR, Sohmer SH (1999) Manual of the Flowering Plants of Hawaii, revised edition. University of Hawaii Press, Honolulu

Wang FH, Wang GX, Li XY, Huang JL, Zheng JK (2008) Heredity, physiology and mapping of a chlorophyll content gene of rice (Oryza sativa L.). J Plant Physiol 165:324–330.

Wu R-Y, Chuang K-C, Hsieh T-F, Chang Y-S (2018) Effect of capsule maturity and desiccation time on viability of Taiwan native orchid, Bletilla formosana seeds (Orchidaceae) after cryopreservation. Taiwania 63:345–350

Zahid NA, Jaafar HZE, Hakiman M (2021) Micropropagation of Ginger (Zingiber officinale Roscoe) ‘Bentong’ and evaluation of its secondary metabolites and antioxidant activities compared with the conventionally propagated plant. Plants 10:630.



Authors MSS and PS are grateful to the National Medicinal Plants Board, Ministry of AYUSH, Government of India for providing financial support to their laboratory (grant number NMPB/IFD/GIA/NR/PL/2018-19/187).

Author Information

Shekhawat, Mahipal S.
Biotechnology Unit, Kanchi Mamunivar Government Institute for Postgraduate Studies and Research, Puducherry, India
Priyadharshini, S.
Biotechnology Unit, Kanchi Mamunivar Government Institute for Postgraduate Studies and Research, Puducherry, India

Manokari, M.
Siddha Clinical Research Unit, Central Council for Research in Siddha (M/o AYUSH), Palayamkottai, India