Biotechnological approaches for in vitro propagation, conservation and secondary metabolites production in Bulbophyllum, an endangered orchid genus: a review

Abstract

Bulbophyllum represents the largest genus in the family Orchidaceae. The orchid species of this genus are widely used in the traditional medicine systems in different Asian countries such as China, India, Indonesia and Thailand. Studies on the secondary metabolites of Bulbophyllum have revealed the presence of important phytochemicals such as phenols, flavonoids, alkaloids, tannins, triterpenoids, sesquiterpenoids, steroids and glycosides. Some species of Bulbophyllum are reported to be of horticultural importance for their unique flowers. Habitat destruction and unsustainable utilization of different species of Bulbophyllum have led to a decline in the natural populations. The present review provides insights into the phytochemistry and ethnomedicinal uses of different species of Bulbophyllum, and highlights the biotechnological approaches developed for its conservation and sustainable utilization. Overall, the details provided in the present review can potentially be used for genome editing and biotechnological advances to develop plants with improved traits, which will be essential for the judicious utilization of the Bulbophyllum species so as to conserve and save the populations in the wild.

Introduction

Orchidaceae is one of the largest families of angiosperms consisting of nearly 28,000 species with over 736 genera (Kim et al. 2020). Orchids are nature's most extravagant group of flowering plants distributed throughout the world from the tropics to high alpines and display an incredible range of diversity (Debnath and Kumaria 2023). They have both aesthetic and medicinal values, and are also regarded as ecological indicators because of their low tolerance to habitat disturbance (Joshi et al. 2009; Lal and Singh 2020). In recent times, growing orchids has become an international business as it covers around 8% of the world’s floriculture trade which possesses a great economic potential for a country (Chugh et al. 2009). Traditionally, orchids have been widely used for their therapeutic values. Around 300 orchids are used in Chinese Traditional Medicine and their utility can be traced back to 3000 BC (Shengji and Zhiwei 2018). In India, orchids have been an important part of herbal medicines since the Vedic period due to their curative properties (Hossain 2011). However, the natural populations of orchids are shrinking rapidly due to various anthropogenic stresses such as habitat destruction, climate change, illegal trade and indiscriminate collection for medicinal and ornamental purposes. At present, the entire Orchidaceae family is included in the IUCN Red Data List of threatened species and is also enlisted in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) (Gale et al. 2018; Liu et al. 2023; https://www.iucnredlist.org).

The genus Bulbophyllum, belonging to the subfamily Epidendroideae of Orchidaceae and subtribe Bulbophyllinae is one such an example of endangered medicinal orchids. It ranks among the largest genera in angiosperms with around 2200 species having pantropical distribution ranging from Continental Africa, Madagascar, East Indian Islands, Asia, Australia and the tropical Pacific islands to the Neotropics. The Asia-specific region is considered as the centre of origin for Bulbophyllum. Madagascar and New Guinea are the two main centres of diversification with more than 200 endemic species (Tang et al. 2020; Hermans et al. 2021). The representatives of Bulbophyllum are sympodial epiphytic or lithophytic herbs with creeping rhizomes and round and fleshy pseudobulbs. The leaves are entire, thick, leathery, glabrous, either single or multiple growing terminally on the pseudobulb and ranges from oblong to lanceolate shape. The flowers are small and arranged in an unbranched raceme arising from the base of the pseudobulb (Sharifi-Rad et al. 2022). The leaves, roots and pseudobulbs, or in a few cases the whole plant of many species of this genus are reported to be rich sources of bioactive molecules that possesses tremendous therapeutic values (Hossain 2011). Numerous studies on the profiling of the secondary metabolites of Bulbophyllum have revealed the presence of important phytochemicals such as phenols, flavonoids, alkaloids, tannins, triterpenoids, sesquiterpenoids, steroids and glycosides (Soni et al. 2018).

Habitat destruction, however, due to various anthropogenic activities in addition to over-exploitation for ornamental and medicinal purposes have caused tremendous pressure on the natural populations of this species-rich genus leading to the reduced population sizes of the species. According to the IUCN Red Data List (2022), about 18 species of Bulbophyllum have been categorized globally as Critically Endangered (CR), 51 species as Endangered (EN), 39 species as Vulnerable (VU), 4 species as Nearly Threatened (NT) and many as Data Deficient (DD) due to lack of suitable studies. Therefore, conservation of the genus Bulbophyllum along with other genera of the Orchidaceae family is highly critical, and requires special attention to save their germplasm diversity. The invention of in vitro propagation techniques has been considered a boon to ex situ conservation strategies. This technique is accepted as an efficient and potent method not only for the mass-scale propagation and conservation of rare, threatened and endangered plants, but also for the sustainable extraction of phytochemicals (Kumar et al. 2022). The plant tissue culture technology employs the cellular totipotency mechanism of plants to regenerate and mass propagate the plants using different explants like shoot tips, stems, rhizomes, protocorms, nodal buds and seeds under an aseptic and controlled environment (Pant 2013). The in vitro propagation or micropropagation is more advantageous than conventional methods due to rapid multiplication, year-round availability, disease-free production, and scope for plant improvement and upscaled production of plant metabolites (Hussain et al. 2012). Although, Bulbophyllum possess great scope for future pharmaceuticals and noble drug discovery, much of this genus remains unexplored. This review aims to highlight the various conservational approaches available and to identify the potential areas of research which are yet to be explored to avail maximum benefit from this medicinally important genus. Figure 1 shows the various explored and unexplored areas of research in the genus Bulbophyllum.

Fig. 1.

The explored and unexplored research areas of the genus Bulbophyllum

Phytochemistry of Bulbophyllum

The bioactive compounds present in plants have immense therapeutic potential and serve as the template for the production of synthetic or semi-synthetic drugs. The genus Bulbophyllum is a reservoir for a wide range of phytochemicals such as phenols, flavonoids, alkaloids, tannins, terpenoids, sterols, bibenzyls, phenanthrenes and dihydrodibenzoxepins, which are responsible for a range of bioactivity including antioxidant, anti-inflammatory, cytotoxic, antimicrobial, neuroprotective, etc. (Hossain 2011; Huehne et al. 2020). The ethnomedicinal uses of various plants lead to the discovery of new drugs and small therapeutic molecules (Moraes et al. 2021). Leong et al. (1997) reported new phenanthrenes, 9,10-dihydrophenanthrenes and triterpenoid friedelin from the hexane extract of B. vaginatum. Also, two new dimeric phenanthrenes (reptanthrin and isoreptanthrin) have been isolated from B. reptans (Majumder et al. 1999). Additionally, Wu et al. (2006) had reported three new dihydrodibenzoxepins from the tubers of B. kwangtungense along with three known compounds namely, cumulatin, densiflorol A and plicatol B. These compounds exhibited anti-tumor activities against HeLa and K562 human tumor cell lines. The presence of phenanthrene, phenanthraquinone, biphenanthrenes and dihydrostilbenes classes of compounds such as coelonin, densiflorol B, gigantol, batatasin III, tristin, vanillic acid, bulbophythrin A and bulbophythrin B have been reported in B. odoratissimum (Majumder and Sen 1991; Chen et al. 2007; Xu et al. 2009; Hossain 2011; Tsering et al. 2017). Further, alkaloids, saponin glycosides, tannins, phenols, flavonoids, steroids and reducing sugars are found to be present in the species B. neilgherrense (Kumari et al. 2013). Anticholinesterase (AChE) inhibitors extracted from plants of B. neilgherrense could be potential drugs in the inflammatory treatment of neurodegenerative disorders (Priya and Krishnaveni 2005). Chinsamy et al. (2014) reported high antioxidant activity in the roots and pseudobulbs of B. scaberulum. Moreover, its root extract was found to be more effective in inhibiting anticholinesterase activity as compared to the commercial product galantamine. Fang et al. (2018) identified new phenylpropanoids from the tubers of B. retusiusculum, and reported that the species possesses strong antimicrobial properties. The analgesic and anti-inflammatory activities of B. neilgherrense pseudobulbs using different rat models revealed analgesic activity against radiant heat-induced pain, moderate anti-inflammatory activity against carrageenan-induced acute inflammation and mild or negligible activity against formalin-induced subacute inflammation and pain in rats (Nair et al. 2018). These medicinal properties were attributed to the presence of flavonoids (chrysin and quercetin), glycosides, tannin, phenolic compounds and calcium. Reports have shown that most of the Bulbophyllum species have high antioxidant activities (Soni et al. 2018; Huehne et al. 2020). Extensive investigations are still warranted on the bioactive compounds and their potential bioactivity from unexplored species of Bulbophyllum which will pave the way for the discovery of new and efficient drugs (Sharifi-Rad et al. 2022).

Ethnomedicinal uses of Bulbophyllum spp.

The genus Bulbophyllum has been extensively used in folk medicine in different parts of the world especially Asia and Africa (Bhinija et al. 2021). Various formulations prepared with different plant parts like pseudobulbs, leaves, roots and fruits, as well as whole herbs are used to treat numerous diseases such as fever, tuberculosis, cardiac disorders, rheumatism, fractures and cancers. In traditional Chinese pharmacopoeia, a few species of Bulbophyllum is mentioned, for e.g., B. andersonii, B. veginatum, B. ambrosia and B. griffithii. The decoction from whole plant of B. andersonii is used to treat rheumatism and the soup prepared with chicken meat is consumed for feminine weakness (Cidian 1986; Wu 1994). The tubers of B. kwangtungense are used for treating fever, pulmonary tuberculosis and bleeding (Gutierrez 2010). The juice of roasted fruits or pseudobulbs of B. veginatum is reported to treat ear ache (Hossain 2011). It was reported that Myanmar women use the pseudobulbs of various species of Bulbophyllum to prepare hair tonic and shampoo (Kurzweil and Lwin 2015). The whole plant decoctions of B. ambrosia prepared by boiling dried plant or fresh plant are used in the treatment of hepatitis, cough and heat in the lungs (Teoh 2016). Moreover, the people of Yunnan province in China use the paste of fresh pseudobulbs and roots of B. griffithii externally to treat breast abscesses or infected traumatic injuries, and the paste of boiled pseudobulbs or dried powder for sore throats and bronchitis (Wu 1994; Teoh 2016). In India, the root paste of B. cariniflorum is taken for inducing abortion during the first trimester by the people of Mondanala and Sutanguni districts of Orissa (Dash et al. 2008; Hosseini and Dadkhah 2016). Also, the fresh pulp of pseudobulbs and leaf juice of B. leopardinum is applied on burns (Pant 2013; Ninawe and Swapna 2017), while the pseudobulb extracts of B. lilacinum mixed with water are taken as a drink to treat tiredness, anxiety, inflammations, diabetes, anaemia, tuberculosis and asthma (Hosseini and Dadkhah 2016; Akhter et al. 2017). In Vietnam, the people use B. coweniorum traditionally for treating haemoptysis, pneumonia, sore throat and chronic gastritis (Hoang 2017). In case of B. sterile, the pseudobulbs are chopped, boiled in coconut oil and applied externally as analgesic, anti-inflammatory and skin anti-allergic (Nair et al. 2018). The fine paste of pseudobulbs and leaves of B. neilgherrense is orally administered for leukoderma, weakness, tuberculosis and heart diseases whereas the whole plant of B. odoratissimum is ground into powder and taken either orally to treat cough, toothache, cancer and chronic inflammation or applied externally for fractures (Zhang et al. 2007; Ninawe and Swapna 2017; Sharifi-Rad et al. 2022). In addition to their therapeutic values, the volatile compounds of Bulbophyllum spp. such as B. vinaceum and B. patens are also used in integrated pest management strategies (Jaleel et al. 2018). In addition, formulations prepared from various parts of Bulbophyllum spp. and their uses are illustrated in Table 1.

Table 1.

Ethnomedicinal uses of various Bulbophyllum spp.

Sl. no.	Species name	Parts used	Therapeutic uses	References
1	B. affine Wall. ex Lindl	Whole plant	Used to treat phlegm, bacterial infection, bleeding and as a tonic	Ou et al. (2003), Marasini and Joshi (2012)
2	B. albidum (Wight) Hook.f		Strengthening of a weak uterus for conception	Lalitharani et al. (2011)
3	B. ambrosia (Hance) Schltr	Whole plant	Used to treat hepatitis, coughs and heat in the lungs	Teoh (2016)
4	B. andersonii (Hook.f.) J.J.Sm	Whole plant	Used to treat rheumatism, coughs, feminine weakness, body dampness, retention of food and poor blood flow	Cidian (1986), Wu (1994)
5	B. barbigerum Lindl	Leaves; Whole plant	Used to treat ear pain and side pain	Fonge et al. (2019)
6	B. calyptratum Kraenzl	Leaves; Whole plant	Used to treat skin diseases (measles, poxes abscesses, rashes); wounds and burns	Fonge et al. (2019)
7	B. careyanum (Hook.) Spreng	Pseudobulbs; leaves	Burns, abortion and recovery during childbirth	Pant (2013), Ninawe and Swapne (2017), Tsering et al. (2017)
8	B. cariniflorum Rchb.f	Roots	Induce abortion	Dash et al. 2008), Hosseini and Dadkhah 2016
9	B. coweniorum J.J. Verm. et P.O’Byrne	Whole plant	Used to treat pneumonia, sore throat, chronic gastritis and haemoptysis	Hoang 2017
10	B. cylindraceum Wall. ex Lindl	Whole plant	Used to treat joint pain and numbness	Wu (1994)
11	B. flabellum-veneris (J.Koenig) Aver	Pseudobulb; whole plant	Used to treat oedema and liver dysfunction	Chuakul (2002)
12	B. griffithii (Lindl.) Rchb.f	Roots; Pseudobulbs	Used to treat fractures, abscesses, chronic coughs, bronchitis, sore throats, fractures, infected breasts, abscesses, and all types of sores	Wu (1994), Teoh (2016)
13	B. inconspicuum Maxim	Whole plant	Used to treat fever, coughs, bronchiectasis, tuberculosis, painful and swollen joints, external injuries, skin ulcers, facial acne, menstrual irregularities, toothache	Teoh (2016)
14	B. intertextum Lindl	Whole plant	Body pain	Fonge et al. (2019)
15	B. kaitesse Rechib	Pseudobulb	Used to treat cancer, inflammations and bacterial infection	Kalaiarasan and John (2012)
16	B. kwangtungense Schltr	Tubers; whole plant	Used to treat sore throat, coughs, pulmonary tuberculosis, bleeding, fever, rheumatism, arthritic pain, traumatic injuries and mastitis	Gutierrez (2010)
17	B. laxiflorum (Blume) Lindl	Whole plant	Used to treat phlegm, dry throat, haemoptysis and stomach problems, trauma and fractures, rheumatism, high fever and epilepsy	Cidian (1986), Wu (1994)
18	B. leopardinum (Wall.) Lindl. ex Wall	Whole plant	Burns	Pant 2013), Ninawe and Swapne (2017)
19	B. levinei Schltr	Whole plant	Used to treat swellings and superficial infections	Teoh (2016)
20	B. lilacinum Ridl	Pseudobulb; whole plant	Used to treat tiredness, anxiety, aphrodisiac, inflammation, rheumatism, hypertension, diabetes, anemia, tuberculosis, cough, asthma, jaundice, heavy menstruation, leucorrhea, eye disease and wounds	Hosseini and Dadkhah (2016), Akhter et al. (2017)
21	B. lobbii Lindl	Leaves	Used to treat burns	Chuakul (2002)
22	B. maximum (Lindl.) Rchb.f		Used to treat madness	Hossain (2011)
23	B. modestum Hook.f	Stems	Used to treat ear infection	Kitirattrakarn and Anantachoke (2003)
25	B. mutabile (Blume) Lindl	Leaves	Fever	Samuel et al. (2010)
26	B. neilgherrense Wight	Pseudobulbs; leaves; Whole plant	Used to treat heart diseases, rheumatism, leukoderma, weakness, tuberculosis, chronic inflammation, fractures, scabies and tonic	Sharifi-Rad et al. (2022)
27	B. odoratissimum (Sm.) Lindl. ex Wall	Whole plant	Used for cough, toothache, tuberculosis, chronic inflammation, fracture, Anti-phthisis, rheumatism and cancer	Zhang et al. (2007), Ninawe and Swapna (2017), Prasad et al. (2021), Sharifi-Rad et al. (2022)
28	B. pectenveneris (Gagnep.) Seidenf	Whole plant	Used to treat stasis of blood, poor circulation and joints, muscles and bone pain	Ou et al. (2003)
29	B. pectinatum Finet	Whole plant	Used in the treatment of tuberculous cough, asthma, sprains and fractures	Wu (1994)
30	B. pumilum (Sw.) Lindl	Whole plant	Used to treat epilepsy	Fonge et al. (2019)
31	B. reptans (Lindl.) Lindl. ex Wall	Whole plant	Used to treat sudden hearing loss and indigestion, dry throat, trauma and fractures	Wu (1994)
32	B. retusiusculum Rchb.f	Whole plant	Used as tonic	Chuakul (2002), Teoh (2016)
33	B. rufinum Rchb.f	Whole plant	Used as a tonic and to treat asthma	Chuakul (2002), Teoh (2016)
34	B. sterile (Lam.) Suresh	Pseudobulbs	Analgesic and anti-inflammatory activities; used for heart disease, leukoderma, skin allergy and rheumatism	Nair et al. (2018)
35	B. umbellatum Lindl	Whole plant	Used to enhance congeniality	Pant (2013), Ninawe and Swapna (2017)
36	B. veginatum (Lindl.) Rchb.f	Fruits, Pseudobulbs	Used to treat ear ache	Hossain (2011)

Interactions of Bulbophyllum and pollinators

In nature, flowering plants adopt different mechanisms of pollination which is important for their adaptability and continuity in environment. Depending only on wind and rain for successful pollination is not enough and, therefore, plants have evolved different forms of interactions with animals, particularly flies (Van der Kooi and Ollerton 2020). In this context, understanding orchids’ pollination mechanism will be crucial for planning effective conservational approaches (Phillips et al. 2020). Numerous studies have been already conducted on the nature of interactions between Bulbophyllum species and fruit flies. The temporal variation in the pollinarium sizes in two closely related species B. involutum and B. ipanemense, were reported by Borba and Semir (1999). This variation due to the removal of pollinarium was found to have prevented pollinators from self-pollination and instead facilitated cross pollination, which can lead to greater gene flow. However, the smaller diameter of the stigmatic cavity in B. involutum had reduced the chances of interspecific pollination with B. weddellii and maintained isolation between these sympatric species, which share the same pollinators and have synchronized flowering. Many species of Bulbophyllum were found to produce floral fragrance to attract Euglossine bees or specific species of Bactrocera fruit flies (Tephritidae) as pollinators (Gerlach and Schill 1991; Tan et al. 2002). The species B. cheiri is reported to secrete a floral fragrance consisting of methyl eugenol (ME) which lures fruit fly species (Bactrocera carambolae, B. papayae, and B. umbrosa). The primary components of the floral exudates are reported to be methyl eugenol, 2-ally-4,5-dimethoxyphenol, and its o-methyl ether, euasarone compounds which the males then transform into the female-attracting pheremones, 2-allyl-4,5-dimethoxyphenol and trans-coniferyl alcohol (Nishida et al. 2004). Teixeira et al. (2004) studied on the lip structures of Bulbophyllum species for wind-assisted fly pollination (B. involutum, B. ipanemense and B. weddellii) and non-wind assisted fly pollination (B. epiphytum, B. glutinosum, B. regnellii and B. rothschildianum) to investigate the role of secretory tissues in pollination. It was observed that the lip structures of wind-assisted fly pollination had nectaries while the non-wind assisted fly pollination had osmophores which played role in facilitating pollination in different ways. The species B. vinaceum was reported to produce floral exudates which had phenylpropanoids that could lure tephritid fruit fly males and then subsequently converted to pheromone components. Tan and Nishida (2007) found that zingerone present in the floral synomone of B. baileyi attracted Bactrocera fruit flies during pollination. In another similar study, B. variegatum was found to display a typical sapromyiophilous pollination syndrome as it secreted an unpleasant floral scent that attracted specific fly from the Platystomatidae (Humeau et al. 2011). Nakahira et al. (2018) also reported about the presence of methyl eugenol (ME) and raspberry ketone (RK) which are attractants for the Dacini fruit flies, in the genus Bulbophyllum. The non-nectar producing and non-resupinate solitary flowers of B. pratervisum are reported to emit specific and pleasant floral fragrances to attract Dacini male fruit flies (Tan and Tan 2018). The unique floral characteristics and pollination mechanisms of orchids have led to their intense selective pressures for outcrossing to avoid inbreeding depression. This is an evolutionary adaptive mechanism which needs to be explored in detail so that proper conservation strategies can be taken up for orchids in general and Bulbophyllum in particular (Gamisch et al. 2014; Jiang et al. 2020).

Plants and microbes’ interaction

The microorganisms play a vital role in the plants’ survivality in nature. In orchids too, the mycorrhizal fungi play important roles in the seed germination and subsequent growth which otherwise would take a long time due to absence of endosperm. The research of fungi in orchid roots, especially dominant mycorrhizal fungi is critical for the protection of orchids. Calvert (2017) identified orchid mycorrhizal fungi (OMF) from the roots of B. exiguum, B. bracteatum, B. minutissimum, B. elisae and B. shepherdii based on the internal transcribed spacer (ITS) gene sequences and ascertained their phylogenetic relationships. A diversity of OMF genera were found to be present which included Tulasnella, Serendipita and Ceratobasidium. The species Bulbophyllum exiguum, B. bracteatum and B. elisae were observed to harbour a single Tulasnella sp. which indicated narrow OMF specificity and suggested their common sub-clade within Bulbophyllum. Similarly, Liang et al. (2022) conducted a study on the OMF composition in the roots, rhizomes and rhizosphere soil of B. tianguii from three terrestrial environments using the second-generation sequencing technology. The species annotation, phylogenetic tree and co-occurrence network analysis revealed that symbiotic relationship existed between the fungi Sebacina, Exophiala, Cladosporium and the orchid. Petrolli et al. (2022) highlighted that a few orchid species were found to be opportunistic in their mycorrhizal interactions, as illustrated by B. prismaticum which was found to grow on both tree species and in association with a broad spectrum of OMF. These studies on OMF provide a theoretical basis for the orchids and fungal interactions. As Bulbophyllum is the largest orchid genus, exploring the interactions of various species with their fungal associates may help in devising effective conservation strategies.

Conservation approaches

In the present era, the loss of biodiversity has become a global concern. Various anthropogenic activities such as over-exploitation, deforestation and urbanization are destroying the natural habitats of orchids leading to their diminishing natural populations (Bazzicalupo et al. 2023). Orchids have a complex interdependent relationship with the surrounding ecosystem for their habitat, germination, pollination and survival, and therefore, the conservational methods include both ex situ and in situ approaches which help to preserve their essential ecological processes and ecosystems (Jalal 2012). In addition, orchid conservation strategies should include the use of various biotechnological tools to document orchid diversity, identify the threatened taxa, safeguard, multiply using in vitro propagation and produce secondary metabolites because the conventional methods alone are inadequate due to slow growth, poor germination, susceptibility to pests and nutritional deficiencies (Gantait et al. 2021). The biotechnological methods which are popularly used for the ex situ conservation include molecular marker technology, molecular diagnostics, in vitro technologies and cryopreservation techniques have been applied successfully for different orchid species (Tandon and Kumaria 2005; Bhattacharyya et al. 2018; Gantait and Mitra 2019).

Molecular approaches

An efficient conservation procedure needs accurate identification of plants, its microbiome and proper assessment of the threat level of the germplasm. The use of DNA markers has made the identification and assessment of genetic diversity, population connectedness easier and competent especially for rare and endangered plants thus promoting a holistic approach of plant conservation (Mukherjee and Ramakrishnan 2018). The popular DNA-based markers assisted techniques include SPAR method (single primer amplification reaction) comprising of RAPD (random amplified polymorphic DNA), DAMD (direct amplification of minisatellite DNA) and ISSR (inter simple sequence repeats). Besides, SCoT (start codon targeted) is another dominant marker which is more recently explored for assessing the genetic polymorphism in the species (Bhattacharyya et al. 2018; Rai 2023). Along with these, SSRs (simple sequence repeats), AFLP (amplified fragment length polymorphism) and RFLP (restriction fragment length polymorphisms) are other commonly used markers system (Kumar et al. 2018). These techniques are useful for the assessment of plant genetic diversity and assist plant conservation programmes as the molecular documentations illustrate the ability of a particular marker to detect variation, quantify diversity and provide tools in understanding the trends in evolution (Treccarichi et al. 2023). Studies have shown the utilization of molecular marker techniques to devise effective conservation approaches for many endangered and medicinally important orchid species belonging to a wide range of genera (Li et al. 2014; Qian et al. 2014; Tsai et al. 2015; Tikendra et al. 2019). In case of Bulbophyllum genus, the genetic and morphological variations in B. exaltatum was studied by Ribeiro et al. (2008) which suggested that hybridization or incipient differentiation contributed to the elevated genetic identity observed among the different populations. The cloning and analysis of rDNA ITS sequences from plants in Bulbophyllum provided a foundation for molecular identification and genetic diversity studies (Jiang et al. 2012). The genetic variability among Bulbophyllum spp. has also been studied using morphological and molecular markers, such as SDS-PAGE protein profiles and RAPD analysis by Ramesh et al. (2016). It was observed that the high diversification of vegetative characters was exemplified by adaptation to various habitats. In addition, the anatomical data suggested that not only the geographical conditions and type of habitat but also the nutrient supply of host-tree on which orchids grow, plays a vital role in survivability of the epiphytic orchids. Jaros et al. (2016) studied the spatial patterns of diversity in B. occultum using ALFP. Hence, the molecular marker technology is now an upcoming advanced practice to sample the germplasm systematically (Gantait and Mitra 2019). Rao (2020) has reported that studies on the spatial and temporal chromosomal distribution of Bulbophyllum species are the need of the hour for understanding the evolutionary pattern and phylogenetic relationship of this genus. The chloroplast genomic diversity in Bulbophyllum section Macrocaulia was studied to get insights into species divergence and adaptive evolution (Tang et al. 2021). However, the genus Bulbophyllum is still less explored in this area of research as the literature does not provide enough information.

The molecular markers assisted techniques are also commonly employed for genetic fidelity test of the in vitro-raised orchid cultures to screen for any somaclonal variations which may hamper the qualitative traits of the parental orchids (Debnath and Kumaria 2023). In the in vitro propagation approach, several factors such as media composition, the concentration of plant growth regulators and culture duration might induce genetic instability. Therefore, evaluation of genetic stability is necessary for large-scale propagation of orchids to safeguard potential source of secondary metabolites for future drug discovery. Presently, advanced markers such as mitochondrial and chloroplast-based microsatellites and retrotransposon markers are being used. In addition, flow cytometry analysis can ascertain the genetic fidelity of in vitro-raised plants by evaluating the ploidy level and genome size (Mohapatra et al. 2022). The marker-assisted clonal fidelity has been successfully evaluated for a few species of Bulbophyllum such as B. odoratissimum and B. auricomum (Than et al. 2011a, b; Prasad et al. 2021).

Micropropagation techniques for mass propagation of Bulbophyllum spp.

The micropropagation techniques have been a tremendous boost to floriculture and horticultural plants for mass propagation to meet the growing commercial demands. In orchid species too, this has brought great reward in the conservation and utilization of different ornamentally and medicinally important species as growing orchids through the conventional vegetative propagation using rhizomes and offshoots is a very slow process and difficult (Pant 2013). Though orchids produce 2–3 million seeds per capsule but the seeds are non-endospermic in nature and need suitable mycorrhizal association for germination; therefore only 2–5% of seeds germinate in natural conditions (Mitra 1986; Paul et al. 2012). Orchid propagation was revolutionized after the establishment of asymbiotic seed culture technology by Knudson (1922). Other than seeds, different plant parts such as pseudobulb segments, nodal buds, shoot tips, and root tips are more widely used as explants for commercial varieties and hybrids to maintain identical genotypes (Dohling et al. 2007). The in vitro propagation technique is now being extensively used on a commercial scale for mass multiplication of various ornamental orchid genera like Cattleya, Cymbidium, Dendrobium, Epidendrum, Oncidium, Phalaenopsis, Paphiopedilum, Vanda and many more (Chugh et al. 2009; Bhattacharyya et al. 2015; Paul et al. 2017).

Although many rare and endangered species of Bulbophyllum have immense therapeutic potential, but they are comparatively less explored and very few reports are available on their in vitro propagation (Bhadra et al. 2004; Than et al. 2009; Pakum et al. 2016). Plant tissue culture can play an important role in minimizing the pressure on the orchid’s natural populations (Pant 2013). Therefore, it is pertinent to explore various micropropagation techniques to select the best-suited tissue culture approach for different species of orchids. The success of micropropagation techniques depends on various factors such as nature and maturity of the explant, sterilization procedure and in vitro physiochemical conditions. Table 2 illustrates the different micropropagation techniques developed for various species of Bulbophyllum.

Table 2.

Different micropropagation protocols developed for various species of Bulbophyllum

Sl. no.	Bulbophyllum species	Explant (s)	Sterilization	Basal medium	PGRs and additives	Culture condition	Remarks	References
1	B. affine Lindl	Mature capsule		½ MS; MS; KC; VW	2,4-D; TDZ; CW; PE; BE	Cultures maintained at 25 ± 2 ºC; 200–300 lx light intensity at 16/8 h light/dark photoperiod	Basal medium + organic additives screening (after 12 weeks of inoculation): VW + CW (150 ml/l) + PE (100 g/l) + BE (50 g/l) = Highest shoot number (3.75 shoots/explant) KC + CW (150 ml/l) + PE (100 g/l) + BE (50 g/l) = Highest shoot length (1.48 cm); root length (0.93 cm) and root number (12.57 roots/explant) In vitro shoot development (after 12 weeks of culture): VW + Sucrose (20 g/l) + CW (150 ml/l) = Highest shoot number (2.65 shoots/explant) VW + Sucrose (10 g/l) + CW (150 ml/l) = Highest shoot length (3.21 cm) VW + Sucrose (20 g/l) + CW (100 ml/l) = Highest root number (17.81 roots/explant) VW + Sucrose (20 g/l) + CW (50 ml/l) = Highest root length (2.13 cm) In vitro shoot development in PGRs (after 8 weeks of culture): VW + TDZ (1.6 mg/l) + 2,4-D (0.6 mg/l) = Highest shoot number (5.7 shoots/explant)	Maneerattanarungroj et al. (2010)
2	B. auricomum L. cv. Dawei	Green capsule	Dip in 70% ethanol for 5–10 s; followed by quick flaming	½ MS, MS, ½ KC, KC, ½ VW and VW	Kn; IAA; IBA	3% (w/v) sucrose; 0.8% (w/v) agar; pH at 5.2; autoclaving at 121 °C and 1.05 kg cm−2 for 20 min	Basal medium screening (after 12 weeks of inoculation): Full MS medium = highest seed germination In vitro shoot formation (after 30 days of culture): MS + Kn (1.5 mg/l) = Highest shoot number (33.90 shoots/treatment) In vitro bulb formation (after 60 days of culture): MS + IAA (1.0 mg/l) = Highest bulb number (181 bulbs/treatment) In Vitro root formation (after 30 days of culture): MS + IBA (0.75 mg/l) = Highest root number (3.2 roots/treatment); Highest root length (1.5 cm)	Thet and Aye (2018)
3	B. auricomum L. cv. Dawei	Capsule of artificial self-pollination	25% cocorex solution for 15 min; followed by 70% ethanol for 10 min; rinse with autoclaved distil water for 3–4 times	½ MS, MS, ½ KC, KC, ½ VW and VW	NAA; BAP; CW; BE; PE	3% (w/v) sucrose and 0.8% (w/v) agar; pH at 5.8; autoclaving at 121 °C for 15 min Cultures maintained at 25 ± 2 °C; 16/8 h light/dark condition photoperiod with light intensity of 3000 lx	Basal medium screening (after 12 weeks of inoculation): MS medium (Control) = highest seed germination Callus and PLBs formation from in vitro pseudobulbs as explant after 30 days of culture: MS + CW (150 ml/l) = Highest callus fresh weight (1.75 ± 0.08 g) In vitro plantlet development from calli-derived PLBs after 30 days of culture: MS + CW (150 ml/l) + BAP (2.0 mg/l) + NAA (1.0 mg/l) = maximum multiple shootings (3.37 ± 0.17)	Aung et al. (2022)
4	B. capillipes C.S.P. Parish & Rchb.f	Pseudobulb segments		Modified semi-solid VW	Kn; BAP; TDZ; IAA; IBA; NAA	Cultures maintained at 25 ± 2 ℃; 16/8 h light/dark condition photoperiod	In vitro plantlet development after 12 weeks of culture: VW + BAP (1.0 mg/l) = Highest shoot number With no significant difference to the control VW + IAA (2.0 mg/l) = highest root number and root length	Wongsa et al. (2020)
5	B. dhaninivatii Seidenf	In vitro shoots		Semi-solid VW	CW; PE; BE	2% (w/v) sucrose; 2.0 g/l activated charcoal and 0.75% (w/v) agar; pH 5.2; autoclaving at 121 °C for 15–20 min Cultures maintained at 12/12 h light/dark photoperiod in LED white light with 20 µmol. m−2 s−1 PPF at 25-27 ºC	In vitro plantlet development after 12 weeks of culture: VW + CW (150 ml/l) + PE (50 g/l) + BE (50 g/l) = Highest shoot number (7.0 ± 1.4) VW + CW (100 ml/l) + PE (50 g/l) + BE (50 g/l) = Highest root number (12.5 ± 1.5) and leaf number (2.9 ± 0.3) VW + CW (50 ml/l) + PE (25 g/l) + BE (50 g/l) = Highest shoot length (3.59 cm); root length (1.83 cm); leaf length (2.87 cm) and leaf width (0.58 cm)	Kongbangkerd et al. (2016)
6	B. echinolabium J.J. Sm	Mature capsule	20% (v/v) NaOCl for 10 min; rinse 3–4 times in autoclaved distil water	VW	IAA; NAA; BAP; Kn	Cultures maintained at 25 ± 2 ℃ for 12 h photoperiod	Basal medium screening after 12 weeks of inoculation: 86% seed germination rate in VW medium In vitro plantlet development: VW + IAA (2 ppm) + NAA (3 ppm) = Best rootings VW + BAP (3 ppm) + Kn (0.5 ppm) = Best shootings	Warseno et al. (2013)
7	B. lilacinum Ridl	Capsule	0.2% (w/v) HgCl2 for 10 min; dip in absolute ethanol for 5–7 s; rinse for 3–4 times in autoclaved distil water	MS; PM (Phytamax) and VW	BAP; NAA; IBA; IAA; Kn; 2,4-D and Pic	pH at 5.4 for PM and pH at 5.8 for MS; autoclaving at 121℃ for 20 min at 1.9 kg cm−2 pressure Cultures maintained at 25 ± 2℃ and 200–300 lx at 14/10 h light/dark condition photoperiod	Basal medium screening after 8 weeks of inoculation: PM medium = Highest seed germinated (80%) In vitro plantlet development after 12 weeks of culture: MS + IAA (1.0 mg/l) + BAP (2.0 mg/l) + 3% (w/v) Sucrose = Best seedling elongation ½ MS + 1.5% (w/v) Sucrose = Best rootings In vitro pseudobulb segments cultured for 30 days: MS + BAP (2.0 mg/l) + Pic (0.5 mg/l) = Maximum multiple shoot bud (MSB) formation ½ MS + Sucrose (1.5% w/v) + Agar (0.8% w/v) = Best plantlet elongation and rooting In vitro shoot primordia-like structure (SPS) cultured for 30 days: MS + BAP (2.0 mg/l) + NAA (2.0 mg/l) = Best plantlet elongation and culture	Bhadra et al. (2004)
8	B. nipondhii Seidenf	Capsules (open, self and cross pollinated)	Running tap water for 10 min; submerged in 5% v/v bleach solution and 1% v/v detergent for 15 min; dipped in 70% (v/v) alcohol; followed by quick flaming	¼ MS; ½ MS; MS; modified ½ VW and VW	PE; CW	2% (w/v) sucrose; 0.7% (w/v) agar; 2 g/l activated charcoal and pH at 5.2 for ½ VW and VW 3% (w/v) sucrose; 0.8% (w/v) agar; 2 g/l activated charcoal and pH at 5.7 for ¼ MS; ½ MS and MS Autoclaving at 121 °C, 1.05 kg/cm2 pressures for 15 min. Cultures maintained at 25 ± 2 °C, 40 μmol m−2 s−1 light intensity, and 12/12 h light/dark photoperiod	Basal medium screening after 12 weeks of inoculation: VW medium = Highest seed germination (91 ± 1.8%) and better seedling development Open pollinated seeds resulted maximum germination when compared to cross and self-pollination In-vitro plantlet development from pseudobulb segment of seedlings after 6 months of culture: VW + PE (75 g/l) + CW (100 ml/l) = Highest shoot and root proliferation	Pakum et al. (2016)
9	B. odoratissimum (Sm.) Lindl. ex Wall	Shoot segments	Prewashed with Labdet-05 for 10 min; washed in running tap water for 1 h and then dried; followed by 30% (v/v) NaOCl for 15 min; rinse with sterile distil water for 3–4 times	MS	BA; NAA; IBA and IAA	Medium with 3% (w/v) sucrose and 0.8% (w/v) agar Cultures maintained at 25 ± 2 ºC; 16/8 h light/dark condition photoperiod in white fluorescent light at intensity (2000 lx)	In vitro shoot proliferation after 12 weeks of inoculation: MS + BA (4.0 mg/l) = Highest regeneration frequency (77.77%) MS + BA (4.0 mg/l) + IBA (0.5 mg/l) = Highest shoot number (5.31 ± 0.46 shoots/explant); shoot length (3.04 ± 0.60 cm) In vitro root proliferation after 8 weeks of culture: MS + IBA (0.5 mg/l) = Highest root number (3.08 ± 0.46 roots/explant); root length (2.53 ± 0.50 cm)	Prasad et al. (2021)

Choice of explant

Choice of explant is very crucial to develop efficient micropropagation techniques and it varies depending on the aim for the establishment of in vitro culture. In the genus Bulbophyllum, capsules are widely used to establish the cultures, followed by shoot segments and pseudobulbs (Maneerattanarungroj et al. 2010; Warseno et al. 2013; Aung et al. 2022). The age, maturity and type of pollination to form the capsule affect the success rate of in vitro seed germination. Pakum et al. (2016) studied the seed germination in B. nipondhii and found that the rate of germination was highest in open-pollinated seeds as compared to the cross-pollinated and self-pollinated seeds wherein the germination rate was very low due to inbreeding depression leading to decreased seed viability and survival. In our preliminary studies on B. griffithii, we have found that micropropagation can be successfully initiated with hand-pollinated seeds resulting in plantlets with multiple shooting (Fig. 2a–e). The orchid propagation through capsules is desirable for conservation purposes as the embryo gives rise to heterogenous plantlets which can be re-introduced in nature (Lee 2011). Successful stories for conservation of rare and endangered orchids through asymbiotic in vitro seed germination have been developed for other genera (Bhattacharyya et al. 2015; Diengdoh et al. 2017; Singh and Kumaria 2019; Debnath and Kumaria 2023). Pseudobulb segments have been used to raise in vitro cultures of B. capillipes and B. nipondhii (Pakum et al. 2016; Wongsa et al. 2020). Also, recently genetically identical cultures have been established from the nodal buds of young shoots of B. odoratissimum through direct organogenesis (Prasad et al. 2021). However, many of the medicinally important Bulbophyllum spp. are still devoid of effective in vitro propagation techniques.

Fig. 2.

In vitro seed germination of Bulbophyllum griffithii. a Plant with flower. b Plant growing in the wild with capsule. c Protocorm formation after 30 days of inoculation (Bar = 1 cm). d Stages of protocorm development under 10X magnification (Bar = 1 cm). e 4-month-old plantlet with multiple shooting (Bar = 1 cm)

Sterilization procedure

The survivability of the explant in the culture is highly dependent on the rate of microbial contamination. In the case of orchids, the contamination rate is dependent on the nature of the explant used as different sterilization procedures are effective on the explant types. Ethanol (70%) treatment followed by quick flaming is the most common procedure to sterilize the capsules of Bulbophyllum. Mercuric chloride (0.2%) and sodium hypochlorite (20%) have been reported to be used to surface sterilize the capsules of B. lilacinum and B. echinolabium, respectively, for 10 min each (Bhadra et al. 2004; Warseno et al. 2013). The treatment of the capsules of B. nipondhii and B. auricomum with bleach prior to ethanol treatment has been found to be beneficial (Pakum et al. 2016; Aung et al. 2022). As the seeds are protected inside the capsule and exposed less to the microbes, extensive sterilization is not required. Prasad et al. (2021) observed that the surface sterilized nodal buds of B. odoratissimum with Labdet-05 detergent under running tap water for 10 min followed by 30% sodium hypochlorite treatment for 15 min and thorough washing with sterile distilled water for 4–5 times was effective.

Nutrient medium

The most commonly used medium for Bulbophyllum micropropagation has been reported to be VW medium (Vacin and Went 1949), however, reports on the use of other media also exist. The effectiveness of VW medium has been reported in several Bulbophyllum species such as B. nipondhii, B. dhaninivatii, B. capillipes and B. echinolabium (Warseno et al. 2013; Pakum et al. 2016; Kongbangkerd et al. 2016; Wongsa et al. 2020). On the other hand, Bhadra et al. (2004) found PM (Phytamax) medium to be most suitable for the seed germination of B. lilacinum and ½ MS medium with 1.5% sucrose effective in better rooting while MS medium (Murashige and Skoog 1962) with 3% sucrose effective for maximum shoot elongation. In case of B. affine, maximum shoot proliferation was observed in VW basal medium but root growth was better on KC (Knudson 1946) basal medium (Maneerattanarungroj et al. 2010). Thet and Aye (2018), studied the efficacy of different media such as ½ MS, MS, ½ KC, KC, ½ VW and VW, and found that MS medium was most suitable for shoot multiplication in case of B. auricomum. The nutrition preference in orchids varies from species to species and hence the suitability of the culture medium also varies. The VW medium is a minimalist medium with low mineral salts concentration, inorganic nitrogen, vitamins and sucrose while on the other hand MS medium is a nutrient-rich medium (Dutra et al. 2008). Thus, the minerals, vitamins and carbohydrates requirement differ widely among the species of Bulbophyllum. In fact, most of the rare and endangered species of Bulbophyllum taken into account for this review have been reported to propagate best in VW medium, and therefore, it may be concluded that the natural habitats of those species are less nutrient dense.

Organic supplements

Among various organic additives, coconut water (CW), potato extract (PE) and banana homogenate (BH) have been commonly used to propagate the species of Bulbophyllum. Organic additives when added in small amounts in the culture medium can cause better growth and morphogenesis (George et al. 2008). The medium supplemented with PE (75 g/l) and CW (100 ml/l) combination was reported to be the most effective medium to proliferate plantlets of B. nipondhii from the pseudobulbs (Pakum et al. 2016). Kongbangkerd et al. (2016) observed that the addition of CW (100 g/l), PE (50 g/l) and BH (50 g/l) in the nutrient medium was beneficial for the shoot regeneration and plantlet development in B. dhaninivatii. The highest amount of callus formation was observed in B. auricomum when the medium was supplemented with 150 ml/l of CW (Aung et al. 2022).

Plant growth regulators

Plant growth regulators (PGRs) especially auxins and cytokinins play important role in plant organogenesis and morphogenesis in tissue culture (Hill and Schaller 2013). Cytokinin controls leaf expansion, and promotes the activity of axillary meristems, hence shoot proliferation in culture is highly dependent on the amount and kind of cytokinin supplemented in the medium (Gaspar et al. 1996). In most of the studies reported on Bulbophyllum, BA (6-benzyladenine) has been used either singly (2–4 mg/l) or in combination with other plant growth regulators for shoot proliferation (Bhadra et al. 2004; Warseno et al. 2013; Prasad et al. 2021; Aung et al. 2022). In B. lilacinum, best shoot elongation was found in the medium supplemented with 2 mg/l of BAP (6-Benzylaminopurine) and 1 mg/l of IAA (indole-3-acetic acid) (Bhadra et al. 2004) whereas medium supplemented with 1.5 mg/l of kinetin was optimum for shoot multiplication in B. auricomum (Thet and Aye 2018). In case of B. odoratissimum, the basal medium in combination with 4 mg/l BA and 0.5 mg/l IBA (indole butyric acid) resulted in highest shoot length and shoot number after 12 weeks of explant culture (Prasad et al. 2021). Thidiazuron (TDZ), a cytokinin analog, in combination with 2,4-dichlorophenoxyacetic acid (2,4-D) in the ratio of 1:2 mg/l was reported to be optimum for multiple shoot induction in B. affine (Maneerattanarungroj et al. 2010). However, Aung et al. (2022) observed that a combination of 2 mg/l BA, 150 ml/l CW and 1 mg/l NAA (naphthalene acetic acid) were optimum for multiple shoot induction in B. auricomum.

Successful rooting is necessary and a prerequisite for any in vitro culture for their establishment in nature. Nature and concentration of auxins play an important role in the in vitro rooting along with other factors such as light intensity, composition of the medium and plant genotype, etc. (Fogaça and Fett-Neto 2005). Auxins in the form of IBA, IAA and NAA have been reported to be beneficial for root development in various species of Bulbophyllum. In B. auricomum, the medium supplemented with 0.75 mg/l of IBA resulted in the best rooting while the medium fortified with 1 mg/l of IAA was reported to result in the highest number of bulb formations (Thet and Aye 2018). The basal medium in combination with 2 mg/l of IAA was found to be optimum for root formation and elongation in case of B. capillipes (Wongsa et al. 2020) whereas in B. odoratissimum, the medium supplemented with 0.5 mg/l of IBA was reported to be optimum for root proliferation (Prasad et al. 2021).

Acclimatization procedure

The acclimatization procedure is very important for the structural and physiological adaptations of in vitro plants for their survival in nature. The acclimatization and final transfer of the in vitro plants into green house or natural fields have been a bottleneck for many orchid species. The difficulty faced by most orchids during the acclimatization period is excessive desiccation and infection by bacteria or fungi. This may be due to lower relative humidity, higher light level and septic condition of the outside environment that are stressful to micropropagated plants compared to in vitro conditions (Hazarika 2003). Hence, potting mixture with water holding capacity, good aeration and proper drainage is crucial for the survival of the in vitro plants during acclimatization. Sphagnum mosses are widely used for hardening and growing of orchids as they provide moisture, nutrition and aeration to the orchid roots (Kaveriamma et al. 2019). Although successful acclimatization protocols are developed in recent times for few orchid species, limited reports exist on the acclimatization procedure of micropropagated species of Bulbophyllum. The acclimatization of B. affine was found to be effective with a survival percentage of 93% when the in vitro raised plantlets were hardened either using vermiculite or dry fern for 12 weeks (Maneerattanarungroj et al. 2010). Similar use of vermiculite was reported to be useful during the initial acclimatization of other orchid species (Paul et al. 2017). The in vitro plantlets of B. echinolabium were dipped in vitamin B1 solution and fungicide Benlate (2 g/l) solution prior to potting and sprayed with NPK Growmore® 30:10:10 fertilizer at a dose of 2 g/l 1–2 times a week with frequent watering to acclimatize to the natural conditions (Warseno et al. 2013). The survival rate of the cultures of B. capillipes was reported to be 100% when potted with Sphagnum moss and coconut husk (Wongsa et al. 2020). The in vitro plantlets of B. odoratissimum were hardened with a 91.66% success rate when potted in the substratum containing small brick chips: charcoal pieces: coco peat in the ratio of 1:1:1 (Prasad et al. 2021). The use of similar compost consisting of brick and charcoal pieces along with a top layer of moss was reported to be effective for the successful acclimatization of other orchids (Bhattacharyya et al. 2015; Debnath and Kumaria 2023).

Germplasm storage

Germplasm conservation (or storage) is important to maintain the genetic stock of important plants for future research prospective. It can be useful in developing new plant varieties or improve the existing ones for food, ornamentals, forestry, industrial and medicinal purposes (Das et al. 2021). The short-to-medium term storage of plant germplasm has been successfully accomplished by reducing the temperature at which the cultures are grown. However, this technique of storage has certain demerits of slow-growth response, difficulty in management of large in vitro collections and somaclonal variations (Tandon and Kumaria 2005). In the recent past, synthetic seed technology has emerged as an effective germplasm storage approach for different orchid species (Gantait et al. 2012, 2015; Mohanty et al. 2013; Bhattacharyya et al. 2018; Kundu and Gantait 2018; Gantait and Mitra 2019). The choice of explant source, encapsulating agent and matrix can determine the success of synthetic seed technology in medicinal plants. This method of germplasm storage can be further improved by the stable ‘long-term storage’ also known as ‘cryopreservation’ in ultra-low temperature (Liquid Nitrogen, – 196 °C) (Gantait et al. 2017). Although, many species of Bulbophyllum are reported to possess tremendous medicinal properties and require special attention for their germplasm conservation, no studies have been conducted so far on its germplasm conservation. Therefore, effective cryopreservation approaches need to be explored for the genus Bulbophyllum as it is a potential area of research with great scope for its long-term conservation.

Conclusion

Biotechnological tools serve as a boon to the conservational biology as well as horticultural practices. Molecular marker-assisted study helps in species authentication and identification for crop improvement and devising efficient breeding programmes for conservation of rare, endangered and medicinal orchids. The micropropagation protocols can serve as alternative for the sustainable, commercially viable, genetically stable and affordable sources of medicinal biomass that can be useful for the pharmaceutical industries. However, internal factors of orchids such as the type, nature and age of explants, and their physiological characteristics are genotype dependent which greatly vary among species and in turn influence the nature of micropropagated regenerants. This review highlights the pharmacological importance, and various biotechnological approaches available till date for different species of Bulbophyllum. Hence, extensive and comprehensive scientific studies are still warranted to explore the medicinal and industrial aspects as well as to devise efficient conservation strategies for safeguarding the diverse germplasm of genus Bulbophyllum.

Authors contributions

MT proposed the original idea of the work. Data curation, processing, and manuscript drafting were done by MT and SD. Conceptualization, supervision, review, and editing were done by SK. All the authors have read and agreed to the final version of the manuscript.

Data availability

All the data and materials can be accessed in the manuscript itself.

Declarations

Conflict of interest

The authors do not have any conflict of interest.