A note on in vitro callus induction and shoot development in Paphiopedilum druryi (Bedd.) Stein – a Critically Endangered and Horticulturally Promising Orchid

A note on in vitro callus induction and shoot development in Paphiopedilum druryi (Bedd.) Stein – a Critically Endangered and Horticulturally Promising Orchid | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report A note on in vitro callus induction and shoot development in Paphiopedilum druryi (Bedd.) Stein – a Critically Endangered and Horticulturally Promising Orchid Selvaraju Parthibhan, Raju Ramasubbu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7205302/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract An efficient pilot protocol for callus induction and shoot development was established in a narrow endemic and Critically Endangered terrestrial orchid Paphiopedilum druryi (Bedd.) Stein. 27.78% green compact callus was produced from stem TCLs on half-strength MS medium (½ MS). The callus remained viable for 2 months and then turned to yellow and brown. Axillary shoot explants cultured on ½ MS medium supplemented with 10X (MS) vitamins, 170 mg L − 1 NaH 2 PO 4 , 0.5% PVP, 2.0 mg L − 1 TDZ with 0.1 mg L − 1 1-naphthaleneacetic acid (NAA) favoured shoot development on 50% explants up to 3 months of culture period. Subsequent symbiotic cultures of the shoots with Tulasnella calospora produced new leaf and root tip, which were successfully recovered to asymbiotic culture after sterilization. These pilot protocols can be applied on large scale with modification following wider screening of physical and chemical factors for improved results. Terrestrial orchid Thin cell layers Axillary shoot Tulasnella calospora Figures Figure 1 Figure 2 Figure 3 Introduction The genus Paphiopedilum Pfitz. belongs to the family Orchidaceae (subfamily Cypripedioideae) is commonly known as lady’s slipper orchids [ 5 ]. They are the most popular and high-priced commercial orchids in the floriculture industry for their large, peculiar, attractive and beautiful flowers with a modified shoe-shaped labellum. There are about 80 species of Paphiopedilum habitats in three life forms, namely terrestrial, facultative epiphytic and obligatory epiphytic [ 16 ]. They are widely distributed in India, Southeast Asia, Myanmar, China, Indonesia, New Guinea, the Solomon Islands and the Philippines [ 6 ][ 16 ][ 1 ]. In India, eight species of Paphiopedilum have been recorded from North East India and one species from the Southern Western Ghats. Out of which, Paphiopedilum druryi (Bedd.) Stein is an only narrow endemic and critically endangered terrestrial orchid distributed between 1400-1550m asl in the Agasthyamalai Biosphere Reserves of Tamil Nadu and Kerala, Southern Western Ghats [ 28 ]. This sympodial orchid has a short stem, clasping glossy green lanceolate leaves, and beautiful, solitary, long-lasting yellowish green to golden-yellow spectacular flowers with long petals and exquisite greenish-yellow sepals and a black median band, which highly attracts horticulturists and illegal traders. Besides, the flowers of P. druryi are traditionally used as medicine as an aphrodisiac [ 31 ], and the leaves contain flavonoids, cyanogenic glycosides and tannin [ 19 ]. Due to these horticultural and medicinal properties, the whole plant and flowers of these wild populations have been overexploited by ruthless collection for regional and international trade. Habitat degradation, human disturbance, deforestation, fire and tourism are also responsible for the population reduction and threat to the existing population. As a result, the plant has been recognized under Critically Endangered (CR) category since 1994 by the International Union for Conservation of Nature and Natural Resources (IUCN) and is now under Critically Endangered (A2abcd + 3bcd + 4abcd; B1ab (ii, iii, v) + 2ab (ii, iii, v) C1) (ver 3.1) IUCN, 2025-1, [ 28 ]. As per the Wildlife Protection Act (Schedule VI), except seedlings or tissue-cultured saplings obtained through solid or liquid media in sterile containers are not subject to the provisions of the convention. Although artificially propagated hybrids are exempt from the provisions of the convention for a few species of Paphiopedilum [ 4 ]. As per the latest IUCN (2020-1) records, the extent of occurrence and area of occupancy of this little-known species remains 4 km2, with extreme fluctuations recorded in the natural habitat. The number of mature individuals recorded was 3,459 [ 22 ] and about 250 [ 14 ]. With the projection of a 90% decline expected in the next 100 years, and severe international and national laws have to be enforced against the illegal collection of these plants from the wild. However, immediate attention is required for the conservation of the existing plant stocks to restore and substitute the wild orchid resource. Biotechnological tools such as micropropagation, in vitro flowering, cryopreservation and low temperature preservation can aid in the restoration of any threatened species [ 34 ], especially the species with poor regeneration ability. Correspondingly, effective conservation studies were achieved only through in vitro asymbiotic seed germination in several Paphiopedilum species, such as, Paphiopedilum villosum var. boxallii [ 7 ], P. villosum [ 15 ], P. callosum var. sublaeve [ 35 ], P. exul [ 11 ], P. insigne [ 8 ], P. venustum [ 13 ], etc. Similarly, callus-mediated shoot production was recorded from seeds, protocorms, protocorm-like bodies, node and root explants of Paphiopedilum such as, P. rathschildianum [ 21 ] [ 20 ], P. niveum [ 33 ], P. hangianum [ 37 ], P. callosum and P. delenatii [ 18 ], P. rothschildianum [ 25 ], etc. Being the only orchid reported from India as Critically Endangered (IUCN 2020-1), no effective conservation measures have been undertaken except for in vitro seed germination trials by Jawaharlal Nehru Tropical Botanic Garden and Research Institute (JNTBGRI), Kerala [ 30 ]. Conventional propagation of Paphiopedilum in general, including P. druryi, through axillary buds and natural seed germination, is incompetent, time-consuming and not successful. Moreover, the present exploration and phenological investigation have confirmed that the flowering and fruiting of this little-known endemic orchid were unpredictable, and an alternative source of explants has to be identified for the in vitro multiplication and subsequent conservation. In this background, an in vitro propagation study was carried out to multiply the P. druryi through in vivo explants. Materials and methods Plant material and explant sterilisation Young axillary shoots of P. druryi collected from Kalakkad Mundanthurai Tiger Reserve (KMTR), Tamil Nadu, were used as the source of explant for direct and indirect organogenesis (Figs. 1 & 2 a). The collected specimens were identified by Dr.R. Ramasubbu, Associate Professor & Herbarium (GUD) In-charge, The Gandhigram Rural Institute, Tamil Nadu, India. The specimens were numbered (GUD 113) and deposited in the GUD herbarium. The young leaves excised from the clasping axillary shoots and the remaining leafless stem segments were used as a source for callus induction. The entire axillary shoots after leaf excision, with or without root initials, were used as explants for direct organogenesis. All the explants were soaked in 0.1% polyvinyl pyrrolidone (PVP) immediately after excision for 10 min. Then, the explants were washed in running tap water with few drops of soap solution (Teepol, India) for 10–15 min. Surface sterilisation was screened separately on each explant under aseptic conditions using different sterilants like HgCl 2 , NaOCl, H 2 O 2 and AgNO 3 from 0.1 to 1.0% either alone or in combination with 0.5% streptomycin or Bavistin for 1 to 15 min. The surface-sterilised explants were finally rinsed with sterile distilled water 3 to 5 times and placed in a sterile petri dish containing 0.1% PVP solution to avoid and control browning during explant preparation. After excision of the sterilant-exposed cut ends, 0.5 cm 2 leaf segments and about 1 mm transverse thin cell layers (tTCLs) from stem segments were prepared and inoculated on the callus induction medium. The axillary shoot explants were prepared with shoot bud and secondary axillary buds by excision of all the mature leaves around the shoot bud and leaves covering the dormant secondary axillary buds. All the excisions were made with the help of sterile surgical knives under 0.1% PVP solution in a petri dish. The explants were washed again with PVP solution in another petri dish and then cultured directly onto the shoot induction medium. Callus induction and proliferation Murashige and Skoog medium [ 23 ] containing half-strength macro nutrients, full-strength micro nutrients and vitamins (½ MS) and 2% sucrose was used as a basal medium for callus induction and proliferation. Different plant growth regulators (PGRs) such as,6-benzyladenine (BA), thidiazuron (TDZ)each at 0.1 to 2.0 mg L − 1 , and 2,4-dichlorophenoxyacetic acid (2,4-D)at 0.1 to 10.0mg L − 1 were used either alone or in combinations with NaH 2 PO 4 (170 mg L − 1 ), polyvinyl pyrrolidone (PVP)at 0.5 to 1.0%, coconut water (CW)at 10 to 15%, activated charcoal (AC)(1.0g L − 1 ) and ascorbic acid (AA) (20 mg L − 1 ) for callus induction from leaf and stem TCLs. The callus initials were further subcultured on the same basal medium containing NaH 2 PO 4 (170 mg L − 1 ), PVP (0.5%), AA (20%), CW (15%) and AC (0.5 g L − 1 ) with TDZ 2.0 mg L − 1 and 2,4-D 5.0 mg L − 1 for callus proliferation. Direct organogenesis Half strength MS medium (½ MS) modified with 10X vitamins and Gamborg B5 medium [ 9 ] supplemented with 170 mg L − 1 NaH 2 PO 4 , 0.5% PVP and 1.0 to 2.0 mg L − 1 TDZ or 5.0 to 10.0 mg L − 1 BA either alone or in combination with 0.1 mg L − 1 of 1-naphthaleneacetic acid (NAA) were used for direct shoot development from in vivo axillary shoot explants. Some of the explants that responded to shoot growth on TDZ 2.0 mg L − 1 were subcultured on the same media composition with 0.5 g L − 1 AC and its root fungal symbiont Tulasnella calospora [ 27 ]. Finally, the symbiotic cultures were treated with 0.5% HgCl 2 to recover the plantlets free from the symbiont. Culture condition All the media were solidified with 0.7% (w/v) agar and pH of the media was adjusted to 5.6 ± 0.1using 0.1 M NaOH and 0.1 M HCl before autoclaving at 121°C at 1.06×10 5 Nm − 2 for 20 min. All the cultures were maintained under controlled conditions of 23 ± 2°Cand 16/8 h (light/dark) photoperiod for direct organogenesis, and dark (0/24 h light/dark) for callus induction. Experimental design and data analysis All the experiments were conducted in a completely randomised design. The callus induction treatments contained six explants per concentration and the experiments were repeated thrice, although the direct organogenesis treatment contained three explants per concentration and the experiment was repeated twice. All the cultures were observed at regular intervals of twice a week. The percentage data on culture response were subjected to arcsine transformation before statistical analysis. The data means were compared with analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS) software version 18.0.0. (IBM SPSS, Chicago, IL). Means were differentiated at P ≤ 0.05 level of significance using Duncan's Multiple Range Test (DMRT). All the chemicals were purchased from Himedia, Bengaluru, India. Results and discussion Sterilisation screening with 0.5% HgCl2 treatment for ≥ 45s to 2 min with and without antibiotics and or fungicides was found to be effective for leaf, stem TCL and axillary shoot explants. Maximum response of ˃90% contamination-free explants was obtained with HgCl2 treatments alone(Fig. 2 b). Additionally, 0.1% HgCl2 treatment for 2 min exhibited maximum (˃90%) contamination-free explants in the leaf. Above or below these optimal treatments, the explant becomes brown and turns necrotic or contaminated. Other sterilants resulted in very low responses (10%) or completely failed to produce sterilised explants. This is the first report on a successful sterilisation experiment of in vivo explants (other than pod and seeds) in Paphiopedilum druryi . Previous literature on in vitro studies of Paphiopedilum is highly focused on micropropagation through seeds, seed-derived ( in vitro ) protocorms, callus, stem, root and leaves of seedlings [ 32 ] [ 21 ] [ 2 ] [ 37 ] [ 25 ] [ 17 ]. In the present study, callus induction was experimented with in vivo leaf and stem explants of P. druryi . Both explants on ½ MS medium supplemented with NaH 2 PO 4 (170 mg L − 1 ), PVP (0.5 to 1.0%), coconut water (10 to 15%), activated charcoal (1 g L − 1 ) and ascorbic acid (20 mg L − 1 ) with individual BA or TDZ or 2,4-D treatments (each at 0.1-2.0 mg L − 1 ) failed to respond callus induction until 90 days of culture period (Table 1 , Fig. 2 c). Similar inability of leaves to produce callus has previously reported in Paphiopedilum philippinense hybrids [ 3 ], P . rothschildianum [ 21 ], P. ‘Alma Gavaert’ [ 2 ], P. var. densissimum , P . insigne , P . bellatulum and P . armeniacum [ 17 ] with TDZ alone and 2,4-D combination. In contrast, the treatment with kinetin (4 µM) alone resulted in 75% callus induction from in vitro nodal stem segments of P. rothschildianum [ 25 ]. However, most of the leaf explants treated with the supplements (170 mg L − 1 NaH 2 PO 4 , 0.5% PVP, 20% AA, 15% CW and 0.5% AC) and PGRs (TDZ, 2,4-D and BA) remained green until 4 weeks compared to PGR alone(Fig. 2 f). A similar culture response was reported earlier in P. villosum var. densissimum , P. bellatulum , P. insigne and P. armeniacum on ¼ MS medium with supplementsand similar PGR combinations [ 17 ]. Table 1 Effect of PGRs on ½ MS medium supplemented with NaH 2 PO 4 (170 mg L − 1 ), PVP (0.5%), AA (20%), CW (15%) and AC (0.5 g L − 1 ) on callus induction from leaf and stem thin cell layers of P. druryi , after 90 days of culture. PGRs Conc. (mg L − 1 ) Frequency of response (%) Type of callus Leaf Stem tTCL Leaf Stem tTCL BA 0.1 0.00 0.00 - - 0.5 0.00 0.00 - - 1.0 0.00 0.00 - - 1.5 0.00 0.00 - - 2.0 0.00 0.00 - - TDZ 0.1 0.00 0.00 - - 0.5 0.00 0.00 - - 1.0 0.00 0.00 - - 1.5 0.00 0.00 - - 2.0 0.00 0.00 - - TDZ (1.0) + 2,4-D 0.1 0.00 0.00 - - 0.5 0.00 0.00 - - 1.0 0.00 0.00 - - 3.0 0.00 0.00 - - 5.0 0.00 27.78 - Pale green, compact 7.0 0.00 0.00 - - 10.0 0.00 0.00 - - ‘- ‘ no response. The combinational treatment of TDZ (1.0 mg L − 1 ) and 2,4-D (5.0 mg L − 1 ) on ½ MS medium supplemented with NaH 2 PO 4 (170 mg L − 1 ), Peptone (1 g L − 1 ), CW (10%) and PVP (0.5%) induced callus from 27.78% stem transverse thin cell layers after 4 weeks of culture period (Fig. 2 e; Table 1 ). Similar callus induction response with TDZ (0.1 or 1.0 mg L − 1 ) and 2,4-D (0.5 or 5.0 mg L − 1 ) or Dicamba (5.0 mg L − 1 ) combinations at 1:5 or 2:5 ratio were recorded from root tip explants of Paphiopedilum ‘Alma Gavaert’ (2), Seed, protocorm and secondary protocorms of P. rathschildianum [ 21 ], protocorms of P. hangianum [ 37 ] and PLBs of P. niveum [ 33 ]. In addition, 1:3, 1:10, and 1:100 ratio treatments of TDZ and 2,4-D were also reported to produce callus in P. rathschildianum [ 20 ], P. niveum [ 12 ], and P. callosum and P. delenatii [ 18 ]. The callus initials first appeared as green protuberances near the pericycle region of the stem TCL, the outermost layer of the vascular system (Fig. 2 d). The green protuberances grew very slowly during subcultures on the same medium composition at 4-week intervals, while the callusing base gradually turned brown. Most of the calli that developed remained green for the first and second subcultures, while some of them had turned yellow within the first subculture period. Likewise, stem thin cell layer explants above 1 mm thickness showed early browning without severe exudation as reported in Paphiopedilum var. densissimum , P. insigne , P. bellatulum , and P. armeniacum [ 17 ]. However, at the end of 90 days of culture period, the callus, including green callus, turned to yellow to brown compact callus and became necrotic (Fig. 2 d, e). Further subculture of the callus on the same medium or callus proliferation medium failed to respond till the next 5 weeks of observation (Table 2 ). Table 2 Effect of TDZ and 2,4-D on ½ MS medium supplemented with NaH 2 PO 4 (170 mg L − 1 ), PVP (0.5%), AA (20%), CW (15%) and AC (0.5 g L − 1 ) on callus proliferation from stem tTCL derived callus of P. druryi , after 90 days of culture. PGRs Conc. (mg L − 1 ) Frequency of response (%) Leaf Stem tTCL TDZ (2.0) + 2,4-D (5.0) 0.1 0.00 0.00 0.5 0.00 0.00 1.0 0.00 0.00 1.5 0.00 0.00 2.0 0.00 0.00 ‘- ‘ no response. Axillary shoots with the secondary axillary buds were cultured on ½ MS medium and Gamborg B5 medium with TDZ 2.0 mg L − 1 , which produced shoot growth and axillary bud development with a 33.33% response with 0.33 average shoots (Fig. 3 a; Table 3 ). Shoots cultured on BA (5.0-10.0mg L − 1 ) and lower TDZ concentration (1.0 mg L − 1 ) turned brown and became necrotic within a week. Although shoots cultured on TDZ (2.0 mg L − 1 ) showed no severe browning except near the cut ends of mature leaves and basal stems. Initial explant preparation and subsequent excision of leaf and stem cut ends under 0.1% PVP solution, following fresh subcultures, facilitated moderate survival of the responded shoot buds up to 90 days. Further shoot buds continued to grow with the existing small leaves and later produced fresh leaves and root initials. Hence, the addition of PVP was found to oxidise the phenolic exudation released by the explants and prevent media browning [ 29 ]. Similarly, NAA 0.1 mg L − 1 combination with TDZ (2.0 mg L − 1 ) resulted in an increased frequency of shoot bud development to 50% with 0.5 shoots per explant on ½ MS medium (Fig. 3 b). The remaining explants were gradually turned brown due to higher phenolic exudation on very young axillary buds. The developing shoots cultured along with the fungal symbiont T. calospora grew well with new leaves and roots up to 8 weeks (Fig. 3 c).The symbiotic growth response was affected due to effective uptake of ammonium present in the medium (½ MS). Functional transporters of ammonium and several amino acids present in the genome of T. calospora facilitate effective nitrogen uptake from ammonium rather than nitrate [ 26 ]. Similar associations of T. calospora have been recorded in several species of Paphiopedilum such as, P. spicerianum , P. micranthum , P. armeniacum, P. dianthum , P.sukhakulii and P. villosum [ 10 ] [ 36 ] [ 24 ].Then the symbiotic plantlets were successfully transformed as fungus-free plantlets on ½ MS medium containing additives and TDZ (2.0mg L-1) after frequent sterilisation with 0.5% HgCl 2 for 1 min (Fig. 3 d). Table 3 Effect of PGRs on ½ MS and Gamborg B5 medium on direct shoot development from axillary shoots of P. druryi , after 60 days of culture. PGRs (mg L − 1 ) ½ MS medium B5 medium Freq. of response (%) Avg. shoot no. per response Freq. of response (%) Avg. shoot no. per response TDZ 1.0 0.00 0.00 ± 0.00 b 0.00 0.00 ± 0.00 a TDZ 2.0 33.33 0.33 ± 0.21 ab 33.33 0.33 ± 0.21 a BA 5.0 0.00 0.00 ± 0.00 b 0.00 0.00 ± 0.00 a BA 10.0 0.00 0.00 ± 0.00 b 0.00 0.00 ± 0.00 a TDZ 2.0 + NAA 0.1 50.00 0.50 ± 0.22 a 33.33 0.33 ± 0.21 a BA 10.0 + NAA 0.1 0.00 0.00 ± 0.00 b 0.00 0.00 ± 0.00 a ‘- ‘ no response. Conclusion This is the first attempt at successful callus induction from in vivo stem transverse thin cell layers of Paphiopedilum druryi (Bedd.) Stein. As per the literature survey, it is very hard to establish in vitro cultures from in vivo explants, due to very slow growth response and yet no literature has reported callus induction from in vivo stem explants in Paphiopedilum in general. Further experiments with extensive screening of medium composition, nitrogen source and additives are necessary to obtain more viable callus and shoots production. Establishment of initial culture stock from minimal explants can be possible only with thin cell layer technology. Hence, the thin cell layer technique would be an appropriate and efficient method for in vitro micropropagation of this critically endangered Paphiopedilum druryi (Bedd.) Stein. Abbreviations AC- activated charcoal TDZ -thidiazuron MS -Murashige and Skoog CW -coconut water PVP - polyvinyl pyrrolidone NaH 2 PO 4 - Monosodium phosphate NAA- 1-naphthaleneacetic acid TCL - thin cell layer HgCl 2- Mercury chloride NaOCl- Sodium hypochlorite H 2 O 2 - Hydrogen peroxide AgNO 3- Silver nitrate tTCLs - transverse thin cell layers AA- ascorbic acid HCl- Hydrochloric acid NaOH- Sodium hydroxide BA- 6-Benzylaminopurine PGRs- Plant growth regulators 2,4-D- 2 , 4 - Dichlorophenoxyacetic acid Declarations Acknowledgements We sincerely acknowledge the financial support from the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India (PDF/2017/000982). We also thank PCCF & Chief Wildlife warden, Chennai (Ref.No. WL(A)/45497/2018, Permit No. 19/2019), and APCCF & Field Director, KMTR, Tirunelveli (C.No. M/6149/2018), Tamil Nadu forest Department for the permission to carry out the research work. Ethics declarations: Ethics approval and consent to participate: The collection permit for the explants from the forest areas was made through PCCF & Chief Wildlife warden, Chennai (Ref.No. WL(A)/45497/2018, Permit No. 19/2019), and APCCF & Field Director, KMTR, Tirunelveli (C.No. M/6149/2018), Tamil Nadu Forest Department, India Clinical Trial: Not applicable Ethics declaration: The study does not contain any human participants or animal handling. Consent to Participate : not applicable. Consent for publication: All the authors consent to the publication of the work. Competing Interest: The authors declare no competing interests. Data Availability: All data will be available based on requests from the readers. Conflict of interest The authors declare no conflicts of interest. Author Contribution Selvaraju Parthibhan: Methodology, data analysis, execution of works, and Preparation of MS. 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Optimization of protocorm-like bodies tissue culture and clonal propagation conditions for endangered lady’s slipper orchid ( Paphiopedilum niveum (Rchb.f.) Stein). Pak J Biotechnol. 2015;12:105–14. Teixeira da Silva JA, Tsavkelova EA, Zeng S, Ng TB, Parthibhan S, Dobra´nszki J, Cardoso JC,RaoMV. Symbiotic in vitro seed propagation of Dendrobium : fungal and bacterial partners and their influence on plant growth and development. Volume 242. Planta; 2015. pp. 1–22. Wattanapan N, Nualsri C, Meesawat U. ) In vitro propagation through transverse thin cell layer (tTCL) culture system of lady’s slipper orchid: Paphiopedilum callosum var. sublaeve . Songklanakarin J Sci Technol. 2018;40:306–13. Yuan L, Yang ZL, LiSY, Hu H, Huang JL. Mycorrhizal specificity, reference, and plasticity of six slipper orchids from South Western China. Mycorrhiza. 2010;20:559–68. Zeng S, Wang J, Wua K, Teixeira da Silva JA, Zhanga J, Duan J. (2013) In vitro propagation of Paphiopedilum hangianum Perner & Gruss. Sci Hortic 151:147–156. Zeng S, Huang W, WuK, Zhang J, Teixeira da Silva JA, Duan J. (2016) In vitro propagation of Paphiopedilum orchids. Crit Rev Biotechnol 36:521–534. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7205302","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":515758680,"identity":"d78ba8b8-5064-428e-89c8-92dda7e30fac","order_by":0,"name":"Selvaraju Parthibhan","email":"","orcid":"","institution":"The Gandhigram Rural Institute (Deemed to be University)","correspondingAuthor":false,"prefix":"","firstName":"Selvaraju","middleName":"","lastName":"Parthibhan","suffix":""},{"id":515758681,"identity":"840c087e-21db-4bd9-9a14-1742a86145f1","order_by":1,"name":"Raju Ramasubbu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYPCCAwkM7A0MzCRq4TlAshaJBCK1mLcffva4ouZOHv/MN4afCypsGPjbuxPwapE5k2ZueObYs2KJ2znG0jPOpDFInDm7Aa8WCYYcNskGtsOJDbdzDKR52w4zGEjkEtDC/wao5d/hxPk3zxj/Jk6LBNCWxrbDiRtu8JgRaYvEM3PDxr5niRvPpJVZ85xJ4yHsF/7kZw8bvt1JnHf88ObbPBU2cvztvfi1AAEblOYwAJE8hJQja2F/QIzqUTAKRsEoGIEAAMchSekIO33HAAAAAElFTkSuQmCC","orcid":"","institution":"The Gandhigram Rural Institute (Deemed to be University)","correspondingAuthor":true,"prefix":"","firstName":"Raju","middleName":"","lastName":"Ramasubbu","suffix":""}],"badges":[],"createdAt":"2025-07-24 11:53:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7205302/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7205302/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91552516,"identity":"1ec85981-ec1f-4bf1-aeea-37c3515359b5","added_by":"auto","created_at":"2025-09-17 16:21:48","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4747458,"visible":true,"origin":"","legend":"\u003cp\u003eNatural habitat and morphology of \u003cem\u003ePaphiopedilum druryi\u003c/em\u003e (Bedd.) Stein.\u003c/p\u003e\n\u003cp\u003ea. Natural population recorded from KMTR, Tamil Nadu \u003cem\u003eb\u003c/em\u003e. habit \u003cem\u003ec\u003c/em\u003e. adult individual with flower initiation\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7205302/v1/bc75cec2a43f78efd4bf5838.jpg"},{"id":91553206,"identity":"2591cfe9-3797-44be-873b-9dc9e3479bd5","added_by":"auto","created_at":"2025-09-17 16:29:48","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8417645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e callus induction from stem thin cell layers and leaf explants of \u003cem\u003ePaphiopedilum druryi\u003c/em\u003e (Bedd.) Stein.\u003c/p\u003e\n\u003cp\u003ea. Habit of \u003cem\u003eP. druryi\u003c/em\u003e; \u003cem\u003eb.\u003c/em\u003eSterilized axillary shoot, the \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;explants source; \u003cem\u003ec\u0026amp; d\u003c/em\u003e. Callus induction and origin from the pericycle region of stem thin cell layers; \u003cem\u003ee\u003c/em\u003e. Callus on proliferation medium showing enlargement and later browning; \u003cem\u003ef\u003c/em\u003e. Leaf explant remained green without callus induction until 60 days of culture. (Bars: \u003cem\u003ea \u003c/em\u003e= 2 cm; \u003cem\u003eb, c\u003c/em\u003e = 1.5 cm; \u003cem\u003ed, e\u003c/em\u003e = 0.2 cm; \u003cem\u003ef\u003c/em\u003e = 8 mm).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7205302/v1/8b35c1e467ade24f48946891.jpg"},{"id":91553539,"identity":"10b45db1-45c5-49f5-8a86-45111b7f1e97","added_by":"auto","created_at":"2025-09-17 16:37:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8088343,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro \u003c/em\u003eshoot development of \u003cem\u003ePaphiopedilum druryi\u003c/em\u003e (Bedd.) Stein.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ea. In vivo\u003c/em\u003e shoot bud induction on TDZ 2.0 mg L\u003csup\u003e-1\u003c/sup\u003e; \u003cem\u003eb\u003c/em\u003e. Shoot bud development on TDZ 2.0 mg L\u003csup\u003e-1\u003c/sup\u003e with NAA 0.1 mg L\u003csup\u003e-1\u003c/sup\u003e; \u003cem\u003ec\u003c/em\u003e. Shoot development with new leaf (NL) and new root (NT) on medium with \u003cem\u003eT. calospora\u003c/em\u003e; \u003cem\u003ed\u003c/em\u003e. Established plantlet from symbiotic culture growing on ½ MS medium containing TDZ. (Bars: a, b, d = 1.5 cm; c = 1.0 cm).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7205302/v1/63d5e77e737ae3adddc9590a.jpg"},{"id":95227804,"identity":"991763c9-d4aa-4145-bd5e-a39caf57698a","added_by":"auto","created_at":"2025-11-05 16:32:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":22047994,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7205302/v1/6152f0da-f21e-43a7-a875-a29cb664dbb4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A note on in vitro callus induction and shoot development in Paphiopedilum druryi (Bedd.) Stein – a Critically Endangered and Horticulturally Promising Orchid","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe genus \u003cem\u003ePaphiopedilum\u003c/em\u003e Pfitz. belongs to the family Orchidaceae (subfamily Cypripedioideae) is commonly known as lady\u0026rsquo;s slipper orchids [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. They are the most popular and high-priced commercial orchids in the floriculture industry for their large, peculiar, attractive and beautiful flowers with a modified shoe-shaped labellum. There are about 80 species of \u003cem\u003ePaphiopedilum\u003c/em\u003e habitats in three life forms, namely terrestrial, facultative epiphytic and obligatory epiphytic [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. They are widely distributed in India, Southeast Asia, Myanmar, China, Indonesia, New Guinea, the Solomon Islands and the Philippines [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e][\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e][\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In India, eight species of \u003cem\u003ePaphiopedilum\u003c/em\u003e have been recorded from North East India and one species from the Southern Western Ghats. Out of which, \u003cem\u003ePaphiopedilum druryi\u003c/em\u003e (Bedd.) Stein is an only narrow endemic and critically endangered terrestrial orchid distributed between 1400-1550m asl in the Agasthyamalai Biosphere Reserves of Tamil Nadu and Kerala, Southern Western Ghats [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis sympodial orchid has a short stem, clasping glossy green lanceolate leaves, and beautiful, solitary, long-lasting yellowish green to golden-yellow spectacular flowers with long petals and exquisite greenish-yellow sepals and a black median band, which highly attracts horticulturists and illegal traders. Besides, the flowers of \u003cem\u003eP. druryi\u003c/em\u003e are traditionally used as medicine as an aphrodisiac [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and the leaves contain flavonoids, cyanogenic glycosides and tannin [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Due to these horticultural and medicinal properties, the whole plant and flowers of these wild populations have been overexploited by ruthless collection for regional and international trade. Habitat degradation, human disturbance, deforestation, fire and tourism are also responsible for the population reduction and threat to the existing population. As a result, the plant has been recognized under Critically Endangered (CR) category since 1994 by the International Union for Conservation of Nature and Natural Resources (IUCN) and is now under Critically Endangered (A2abcd\u0026thinsp;+\u0026thinsp;3bcd\u0026thinsp;+\u0026thinsp;4abcd; B1ab (ii, iii, v)\u0026thinsp;+\u0026thinsp;2ab (ii, iii, v) C1) (ver 3.1) IUCN, 2025-1, [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. As per the Wildlife Protection Act (Schedule VI), except seedlings or tissue-cultured saplings obtained through solid or liquid media in sterile containers are not subject to the provisions of the convention. Although artificially propagated hybrids are exempt from the provisions of the convention for a few species of \u003cem\u003ePaphiopedilum\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As per the latest IUCN (2020-1) records, the extent of occurrence and area of occupancy of this little-known species remains 4 km2, with extreme fluctuations recorded in the natural habitat. The number of mature individuals recorded was 3,459 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and about 250 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. With the projection of a 90% decline expected in the next 100 years, and severe international and national laws have to be enforced against the illegal collection of these plants from the wild. However, immediate attention is required for the conservation of the existing plant stocks to restore and substitute the wild orchid resource. Biotechnological tools such as micropropagation, in vitro flowering, cryopreservation and low temperature preservation can aid in the restoration of any threatened species [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], especially the species with poor regeneration ability. Correspondingly, effective conservation studies were achieved only through \u003cem\u003ein vitro\u003c/em\u003e asymbiotic seed germination in several \u003cem\u003ePaphiopedilum\u003c/em\u003e species, such as, \u003cem\u003ePaphiopedilum villosum\u003c/em\u003e var. \u003cem\u003eboxallii\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], \u003cem\u003eP. villosum\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], \u003cem\u003eP. callosum\u003c/em\u003e var. \u003cem\u003esublaeve\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], \u003cem\u003eP. exul\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], \u003cem\u003eP. insigne\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], \u003cem\u003eP. venustum\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], etc. Similarly, callus-mediated shoot production was recorded from seeds, protocorms, protocorm-like bodies, node and root explants of \u003cem\u003ePaphiopedilum\u003c/em\u003e such as, \u003cem\u003eP. rathschildianum\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], \u003cem\u003eP. niveum\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], \u003cem\u003eP. hangianum\u003c/em\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], \u003cem\u003eP. callosum\u003c/em\u003e and \u003cem\u003eP. delenatii\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], \u003cem\u003eP. rothschildianum\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], etc. Being the only orchid reported from India as Critically Endangered (IUCN 2020-1), no effective conservation measures have been undertaken except for \u003cem\u003ein vitro\u003c/em\u003e seed germination trials by Jawaharlal Nehru Tropical Botanic Garden and Research Institute (JNTBGRI), Kerala [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Conventional propagation of \u003cem\u003ePaphiopedilum\u003c/em\u003e in general, including P. druryi, through axillary buds and natural seed germination, is incompetent, time-consuming and not successful. Moreover, the present exploration and phenological investigation have confirmed that the flowering and fruiting of this little-known endemic orchid were unpredictable, and an alternative source of explants has to be identified for the \u003cem\u003ein vitro\u003c/em\u003e multiplication and subsequent conservation. In this background, an \u003cem\u003ein vitro\u003c/em\u003e propagation study was carried out to multiply the \u003cem\u003eP. druryi\u003c/em\u003e through \u003cem\u003ein vivo\u003c/em\u003e explants.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant material and explant sterilisation\u003c/h2\u003e\u003cp\u003eYoung axillary shoots of \u003cem\u003eP. druryi\u003c/em\u003e collected from Kalakkad Mundanthurai Tiger Reserve (KMTR), Tamil Nadu, were used as the source of explant for direct and indirect organogenesis (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The collected specimens were identified by Dr.R. Ramasubbu, Associate Professor \u0026amp; Herbarium (GUD) In-charge, The Gandhigram Rural Institute, Tamil Nadu, India. The specimens were numbered (GUD 113) and deposited in the GUD herbarium. The young leaves excised from the clasping axillary shoots and the remaining leafless stem segments were used as a source for callus induction. The entire axillary shoots after leaf excision, with or without root initials, were used as explants for direct organogenesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAll the explants were soaked in 0.1% polyvinyl pyrrolidone (PVP) immediately after excision for 10 min. Then, the explants were washed in running tap water with few drops of soap solution (Teepol, India) for 10\u0026ndash;15 min. Surface sterilisation was screened separately on each explant under aseptic conditions using different sterilants like HgCl\u003csub\u003e2\u003c/sub\u003e, NaOCl, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and AgNO\u003csub\u003e3\u003c/sub\u003e from 0.1 to 1.0% either alone or in combination with 0.5% streptomycin or Bavistin for 1 to 15 min. The surface-sterilised explants were finally rinsed with sterile distilled water 3 to 5 times and placed in a sterile petri dish containing 0.1% PVP solution to avoid and control browning during explant preparation. After excision of the sterilant-exposed cut ends, 0.5 cm\u003csup\u003e2\u003c/sup\u003e leaf segments and about 1 mm transverse thin cell layers (tTCLs) from stem segments were prepared and inoculated on the callus induction medium. The axillary shoot explants were prepared with shoot bud and secondary axillary buds by excision of all the mature leaves around the shoot bud and leaves covering the dormant secondary axillary buds. All the excisions were made with the help of sterile surgical knives under 0.1% PVP solution in a petri dish. The explants were washed again with PVP solution in another petri dish and then cultured directly onto the shoot induction medium.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCallus induction and proliferation\u003c/h3\u003e\n\u003cp\u003eMurashige and Skoog medium [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] containing half-strength macro nutrients, full-strength micro nutrients and vitamins (\u0026frac12; MS) and 2% sucrose was used as a basal medium for callus induction and proliferation. Different plant growth regulators (PGRs) such as,6-benzyladenine (BA), thidiazuron (TDZ)each at 0.1 to 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 2,4-dichlorophenoxyacetic acid (2,4-D)at 0.1 to 10.0mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ewere used either alone or in combinations with NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (170 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), polyvinyl pyrrolidone (PVP)at 0.5 to 1.0%, coconut water (CW)at 10 to 15%, activated charcoal (AC)(1.0g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and ascorbic acid (AA) (20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for callus induction from leaf and stem TCLs. The callus initials were further subcultured on the same basal medium containing NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (170 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), PVP (0.5%), AA (20%), CW (15%) and AC (0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with TDZ 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2,4-D 5.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for callus proliferation.\u003c/p\u003e\n\u003ch3\u003eDirect organogenesis\u003c/h3\u003e\n\u003cp\u003eHalf strength MS medium (\u0026frac12; MS) modified with 10X vitamins and Gamborg B5 medium [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] supplemented with 170 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eNaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.5% PVP and 1.0 to 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eTDZ or 5.0 to 10.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BA either alone or in combination with 0.1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of 1-naphthaleneacetic acid (NAA) were used for direct shoot development from \u003cem\u003ein vivo\u003c/em\u003e axillary shoot explants. Some of the explants that responded to shoot growth on TDZ 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were subcultured on the same media composition with 0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e AC and its root fungal symbiont \u003cem\u003eTulasnella calospora\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Finally, the symbiotic cultures were treated with 0.5% HgCl\u003csub\u003e2\u003c/sub\u003e to recover the plantlets free from the symbiont.\u003c/p\u003e\n\u003ch3\u003eCulture condition\u003c/h3\u003e\n\u003cp\u003eAll the media were solidified with 0.7% (w/v) agar and pH of the media was adjusted to 5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1using 0.1 M NaOH and 0.1 M HCl before autoclaving at 121\u0026deg;C at 1.06\u0026times;10\u003csup\u003e5\u003c/sup\u003e Nm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for 20 min. All the cultures were maintained under controlled conditions of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;Cand 16/8 h (light/dark) photoperiod for direct organogenesis, and dark (0/24 h light/dark) for callus induction.\u003c/p\u003e\n\u003ch3\u003eExperimental design and data analysis\u003c/h3\u003e\n\u003cp\u003eAll the experiments were conducted in a completely randomised design. The callus induction treatments contained six explants per concentration and the experiments were repeated thrice, although the direct organogenesis treatment contained three explants per concentration and the experiment was repeated twice. All the cultures were observed at regular intervals of twice a week. The percentage data on culture response were subjected to arcsine transformation before statistical analysis. The data means were compared with analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS) software version 18.0.0. (IBM SPSS, Chicago, IL). Means were differentiated at P\u0026thinsp;\u0026le;\u0026thinsp;0.05 level of significance using Duncan's Multiple Range Test (DMRT). All the chemicals were purchased from Himedia, Bengaluru, India.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eSterilisation screening with 0.5% HgCl2 treatment for \u0026ge;\u0026thinsp;45s to 2 min with and without antibiotics and or fungicides was found to be effective for leaf, stem TCL and axillary shoot explants. Maximum response of ˃90% contamination-free explants was obtained with HgCl2 treatments alone(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Additionally, 0.1% HgCl2 treatment for 2 min exhibited maximum (˃90%) contamination-free explants in the leaf. Above or below these optimal treatments, the explant becomes brown and turns necrotic or contaminated. Other sterilants resulted in very low responses (10%) or completely failed to produce sterilised explants. This is the first report on a successful sterilisation experiment of \u003cem\u003ein vivo\u003c/em\u003e explants (other than pod and seeds) in \u003cem\u003ePaphiopedilum druryi\u003c/em\u003e. Previous literature on in vitro studies of Paphiopedilum is highly focused on micropropagation through seeds, seed-derived (\u003cem\u003ein vitro\u003c/em\u003e) protocorms, callus, stem, root and leaves of seedlings [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the present study, callus induction was experimented with \u003cem\u003ein vivo\u003c/em\u003e leaf and stem explants of \u003cem\u003eP. druryi\u003c/em\u003e. Both explants on \u0026frac12; MS medium supplemented with NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (170 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), PVP (0.5 to 1.0%), coconut water (10 to 15%), activated charcoal (1 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and ascorbic acid (20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with individual BA or TDZ or 2,4-D treatments (each at 0.1-2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) failed to respond callus induction until 90 days of culture period (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Similar inability of leaves to produce callus has previously reported in \u003cem\u003ePaphiopedilum philippinense\u003c/em\u003e hybrids [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], \u003cem\u003eP\u003c/em\u003e. \u003cem\u003erothschildianum\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], \u003cem\u003eP.\u003c/em\u003e \u0026lsquo;Alma Gavaert\u0026rsquo; [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], \u003cem\u003eP.\u003c/em\u003e var. \u003cem\u003edensissimum\u003c/em\u003e, \u003cem\u003eP\u003c/em\u003e. \u003cem\u003einsigne\u003c/em\u003e, \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ebellatulum\u003c/em\u003e and \u003cem\u003eP\u003c/em\u003e. \u003cem\u003earmeniacum\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] with TDZ alone and 2,4-D combination. In contrast, the treatment with kinetin (4 \u0026micro;M) alone resulted in 75% callus induction from \u003cem\u003ein vitro\u003c/em\u003e nodal stem segments of \u003cem\u003eP. rothschildianum\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, most of the leaf explants treated with the supplements (170 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eNaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.5% PVP, 20% AA, 15% CW and 0.5% AC) and PGRs (TDZ, 2,4-D and BA) remained green until 4 weeks compared to PGR alone(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). A similar culture response was reported earlier in \u003cem\u003eP. villosum\u003c/em\u003e var. \u003cem\u003edensissimum\u003c/em\u003e, \u003cem\u003eP. bellatulum\u003c/em\u003e, \u003cem\u003eP. insigne\u003c/em\u003e and \u003cem\u003eP. armeniacum\u003c/em\u003e on \u0026frac14; MS medium with supplementsand similar PGR combinations [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of PGRs on \u0026frac12; MS medium supplemented with NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (170 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), PVP (0.5%), AA (20%), CW (15%) and AC (0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) on callus induction from leaf and stem thin cell layers of \u003cem\u003eP. druryi\u003c/em\u003e, after 90 days of culture.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePGRs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eConc.\u003c/p\u003e\u003cp\u003e(mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eFrequency of response (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eType of callus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLeaf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStem tTCL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLeaf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eStem tTCL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eBA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eTDZ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e\u003cp\u003eTDZ (1.0)\u0026thinsp;+\u0026thinsp;2,4-D\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePale green, compact\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u0026lsquo;- \u0026lsquo; no response.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe combinational treatment of TDZ (1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and 2,4-D (5.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) on \u0026frac12; MS medium supplemented with NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (170 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), Peptone (1 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CW (10%) and PVP (0.5%) induced callus from 27.78% stem transverse thin cell layers after 4 weeks of culture period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Similar callus induction response with TDZ (0.1 or 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and 2,4-D (0.5 or 5.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) or Dicamba (5.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) combinations at 1:5 or 2:5 ratio were recorded from root tip explants of \u003cem\u003ePaphiopedilum\u003c/em\u003e \u0026lsquo;Alma Gavaert\u0026rsquo; (2), Seed, protocorm and secondary protocorms of \u003cem\u003eP. rathschildianum\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], protocorms of \u003cem\u003eP. hangianum\u003c/em\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and PLBs of \u003cem\u003eP. niveum\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In addition, 1:3, 1:10, and 1:100 ratio treatments of TDZ and 2,4-D were also reported to produce callus in \u003cem\u003eP. rathschildianum\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], \u003cem\u003eP. niveum\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and \u003cem\u003eP. callosum\u003c/em\u003e and \u003cem\u003eP. delenatii\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe callus initials first appeared as green protuberances near the pericycle region of the stem TCL, the outermost layer of the vascular system (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The green protuberances grew very slowly during subcultures on the same medium composition at 4-week intervals, while the callusing base gradually turned brown. Most of the calli that developed remained green for the first and second subcultures, while some of them had turned yellow within the first subculture period. Likewise, stem thin cell layer explants above 1 mm thickness showed early browning without severe exudation as reported in \u003cem\u003ePaphiopedilum\u003c/em\u003e var. \u003cem\u003edensissimum\u003c/em\u003e, \u003cem\u003eP. insigne\u003c/em\u003e, \u003cem\u003eP. bellatulum\u003c/em\u003e, and \u003cem\u003eP. armeniacum\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, at the end of 90 days of culture period, the callus, including green callus, turned to yellow to brown compact callus and became necrotic (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e). Further subculture of the callus on the same medium or callus proliferation medium failed to respond till the next 5 weeks of observation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of TDZ and 2,4-D on \u0026frac12; MS medium supplemented with NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (170 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), PVP (0.5%), AA (20%), CW (15%) and AC (0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) on callus proliferation from stem tTCL derived callus of \u003cem\u003eP. druryi\u003c/em\u003e, after 90 days of culture.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePGRs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eConc.\u003c/p\u003e\u003cp\u003e(mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eFrequency of response (%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLeaf\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStem tTCL\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eTDZ (2.0)\u0026thinsp;+\u0026thinsp;2,4-D (5.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u0026lsquo;- \u0026lsquo; no response.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAxillary shoots with the secondary axillary buds were cultured on \u0026frac12; MS medium and Gamborg B5 medium with TDZ 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which produced shoot growth and axillary bud development with a 33.33% response with 0.33 average shoots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Shoots cultured on BA (5.0-10.0mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and lower TDZ concentration (1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) turned brown and became necrotic within a week. Although shoots cultured on TDZ (2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) showed no severe browning except near the cut ends of mature leaves and basal stems. Initial explant preparation and subsequent excision of leaf and stem cut ends under 0.1% PVP solution, following fresh subcultures, facilitated moderate survival of the responded shoot buds up to 90 days. Further shoot buds continued to grow with the existing small leaves and later produced fresh leaves and root initials. Hence, the addition of PVP was found to oxidise the phenolic exudation released by the explants and prevent media browning [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, NAA 0.1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e combination with TDZ (2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) resulted in an increased frequency of shoot bud development to 50% with 0.5 shoots per explant on \u0026frac12; MS medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The remaining explants were gradually turned brown due to higher phenolic exudation on very young axillary buds. The developing shoots cultured along with the fungal symbiont \u003cem\u003eT. calospora\u003c/em\u003e grew well with new leaves and roots up to 8 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).The symbiotic growth response was affected due to effective uptake of ammonium present in the medium (\u0026frac12; MS). Functional transporters of ammonium and several amino acids present in the genome of \u003cem\u003eT. calospora\u003c/em\u003e facilitate effective nitrogen uptake from ammonium rather than nitrate [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Similar associations of \u003cem\u003eT. calospora\u003c/em\u003e have been recorded in several species of \u003cem\u003ePaphiopedilum\u003c/em\u003e such as, \u003cem\u003eP. spicerianum\u003c/em\u003e, \u003cem\u003eP. micranthum\u003c/em\u003e, \u003cem\u003eP. armeniacum, P. dianthum\u003c/em\u003e, \u003cem\u003eP.sukhakulii\u003c/em\u003e and \u003cem\u003eP. villosum\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].Then the symbiotic plantlets were successfully transformed as fungus-free plantlets on \u0026frac12; MS medium containing additives and TDZ (2.0mg L-1) after frequent sterilisation with 0.5% HgCl\u003csub\u003e2\u003c/sub\u003e for 1 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of PGRs on \u0026frac12; MS and Gamborg B5 medium on direct shoot development from axillary shoots of \u003cem\u003eP. druryi\u003c/em\u003e, after 60 days of culture.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePGRs (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e\u0026frac12; MS medium\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eB5 medium\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFreq.\u0026nbsp;of\u003c/p\u003e\u003cp\u003eresponse (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAvg. shoot no. per response\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFreq.\u0026nbsp;of\u003c/p\u003e\u003cp\u003eresponse (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAvg.\u003c/p\u003e\u003cp\u003eshoot no. per response\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTDZ 1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTDZ 2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e33.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e33.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBA 5.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBA 10.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTDZ 2.0\u0026thinsp;+\u0026thinsp;NAA 0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e50.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e33.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBA 10.0\u0026thinsp;+\u0026thinsp;NAA 0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u0026lsquo;- \u0026lsquo; no response.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis is the first attempt at successful callus induction from \u003cem\u003ein vivo\u003c/em\u003e stem transverse thin cell layers of \u003cem\u003ePaphiopedilum druryi\u003c/em\u003e (Bedd.) Stein. As per the literature survey, it is very hard to establish \u003cem\u003ein vitro\u003c/em\u003e cultures from \u003cem\u003ein vivo\u003c/em\u003e explants, due to very slow growth response and yet no literature has reported callus induction from \u003cem\u003ein vivo\u003c/em\u003e stem explants in \u003cem\u003ePaphiopedilum\u003c/em\u003e in general. Further experiments with extensive screening of medium composition, nitrogen source and additives are necessary to obtain more viable callus and shoots production. Establishment of initial culture stock from minimal explants can be possible only with thin cell layer technology. Hence, the thin cell layer technique would be an appropriate and efficient method for \u003cem\u003ein vitro\u003c/em\u003e micropropagation of this critically endangered \u003cem\u003ePaphiopedilum druryi\u003c/em\u003e (Bedd.) Stein.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAC- activated charcoal\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTDZ -thidiazuron \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMS -Murashige and Skoog \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCW -coconut water\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePVP\u003cstrong\u003e\u0026nbsp;-\u003c/strong\u003e polyvinyl pyrrolidone\u003c/p\u003e\n\u003cp\u003eNaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e - Monosodium phosphate\u003c/p\u003e\n\u003cp\u003eNAA-\u003csup\u003e\u0026nbsp;\u003c/sup\u003e1-naphthaleneacetic acid \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTCL - thin cell layer \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHgCl\u003csub\u003e2-\u0026nbsp;\u003c/sub\u003eMercury chloride\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNaOCl- Sodium hypochlorite\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 -\u0026nbsp;\u003c/sub\u003eHydrogen peroxide\u003c/p\u003e\n\u003cp\u003eAgNO\u003csub\u003e3-\u003c/sub\u003e Silver nitrate\u003c/p\u003e\n\u003cp\u003etTCLs - transverse thin cell layers\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAA- ascorbic acid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHCl- Hydrochloric acid\u003c/p\u003e\n\u003cp\u003eNaOH-\u0026nbsp;Sodium hydroxide\u003c/p\u003e\n\u003cp\u003eBA- 6-Benzylaminopurine\u003c/p\u003e\n\u003cp\u003ePGRs- Plant growth regulators\u003c/p\u003e\n\u003cp\u003e2,4-D- \u0026nbsp;\u003cem\u003e2\u003c/em\u003e,\u003cem\u003e4\u003c/em\u003e-\u003cem\u003eDichlorophenoxyacetic acid\u003c/em\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely acknowledge the financial support from the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India (PDF/2017/000982). We also thank PCCF \u0026amp; Chief Wildlife warden, Chennai (Ref.No. WL(A)/45497/2018, Permit No. 19/2019), and APCCF \u0026amp; Field Director, KMTR, Tirunelveli (C.No. M/6149/2018), Tamil Nadu forest Department for the permission to carry out the research work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collection permit for the explants from the forest areas\u0026nbsp;was made through \u0026nbsp;PCCF \u0026amp; Chief Wildlife warden, Chennai (Ref.No. WL(A)/45497/2018, Permit No. 19/2019), and APCCF \u0026amp; Field Director, KMTR, Tirunelveli (C.No. M/6149/2018), Tamil Nadu Forest Department, India\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration:\u0026nbsp;\u003c/strong\u003eThe study does not contain any human participants or animal handling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e : not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eAll the authors consent to the publication of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eAll data will be available based on requests from the readers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSelvaraju Parthibhan: Methodology, data analysis, execution of works, and Preparation of MS. Raju Raamsubbu: Planning of Research Problem, research Lead; Correction and review of Manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlrich P, Higgins W. Illustrated dictionary of orchid genera. Florida: Selby botanical garden, South palm avenue, Sarasota; 2008.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen JT. \u003cem\u003eIn vitro\u003c/em\u003e organogenesis of a slipper orchid, \u003cem\u003ePaphiopedilum\u003c/em\u003e \u0026lsquo;Alma Gavaert\u0026rsquo;. Not Sci Biol. 2018;10:607\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen TY, Chen JT, Chang WC. Plant regeneration through direct shoot bud formation from leaf cultures of \u003cem\u003ePaphiopedilum\u003c/em\u003e orchids. Plant Cell Tissue Organ Cult. 2004;76:11\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCITES. (2011) CITES annual report. 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Crit Rev Biotechnol 36:521\u0026ndash;534.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Terrestrial orchid, Thin cell layers, Axillary shoot, Tulasnella calospora","lastPublishedDoi":"10.21203/rs.3.rs-7205302/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7205302/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAn efficient pilot protocol for callus induction and shoot development was established in a narrow endemic and Critically Endangered terrestrial orchid \u003cem\u003ePaphiopedilum druryi\u003c/em\u003e (Bedd.) Stein. 27.78% green compact callus was produced from stem TCLs on half-strength MS medium (\u0026frac12; MS). The callus remained viable for 2 months and then turned to yellow and brown. Axillary shoot explants cultured on \u0026frac12; MS medium supplemented with 10X (MS) vitamins, 170 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.5% PVP, 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eTDZ with 0.1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e1-naphthaleneacetic acid (NAA) favoured shoot development on 50% explants up to 3 months of culture period. Subsequent symbiotic cultures of the shoots with \u003cem\u003eTulasnella calospora\u003c/em\u003e produced new leaf and root tip, which were successfully recovered to asymbiotic culture after sterilization. These pilot protocols can be applied on large scale with modification following wider screening of physical and chemical factors for improved results.\u003c/p\u003e","manuscriptTitle":"A note on in vitro callus induction and shoot development in Paphiopedilum druryi (Bedd.) Stein – a Critically Endangered and Horticulturally Promising Orchid","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 16:21:43","doi":"10.21203/rs.3.rs-7205302/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f73dc0cd-0f1f-484b-9639-7019da575a1d","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-05T14:54:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 16:21:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7205302","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7205302","identity":"rs-7205302","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}