Keywords

1 Introduction

Orchids are extremely fascinating plants that surpass all the plant groups in the “Plant kingdom.” It belongs to the Orchidaceae family, which is the second largest as well as the highly advanced family among flowering plants. It encompasses approximately 850 genera and 35 thousand species (Stewart and Griffith 1995; Gutierrez 2010). Orchids are better known for their alluring, enchanting attractive floweret, which are extremely precious globally in floricultural trades. Orchids became the second most top-selling cut flowers as well as potted floricultural products due to their increasing demand in the globe for trading. Their aristocratic, adorable, and wonderful colors, sometimes-intricate forms, have enchanted men and women through the ages. Orchids lend a charming beauty with their extraordinary flower heterogeneity, in terms of size, shape, structure, number, density, color, and fragrance. Besides their adorning values, the orchids are also mentioned specially for their therapeutic medicinal properties as well as economic importance especially in the traditional pharmacopeias extensively since time immemorial (Withner 1959; Kaushik 1983). Earlier in China and Japan orchids were used as herbal medicine for different illnesses nearly 3000–4000 years ago, respectively (Reinikka 1995; Bulpitt 2005; Jalal et al. 2008).

Many species of Vanda, Dendrobium, Habenaria, Malaxis, Cymbidium, Coelogyne, Cypripedium, Anoctochilus, Bletilla, Calanthe, and Cymbidium, etc. are significantly important for having medicinal importance. Medicinal orchid plays an outstanding part in therapeutics with the presence of important phytochemicals such as alkaloids, flavonoids, carotenoids, sterols, saponins, anthocyanins, and polyphenols either in their pseudo bulb, tubers, leaves, stems, flowers, roots, or in the complete plant (Okamoto et al. 1966; Williams 1979; Majumder and Sen 1991; Majumder et al. 1996; Zhao et al. 2003; Yang et al. 2006; Singh and Duggal 2009). Several ailments like arthritis, tumors, fever, malaria, snakebite, scorpion bite, depression, tuberculosis, cervical carcinoma, diabetes, and biliousness, etc. are cured by medicinal orchids (Szlachetko 2001). These orchids were also employed as food and fodder, and local medicine by rural communities for their livelihoods and revenue generation. Moreover, uprooting the whole plant from its habitat for sale to the traders as well as over-exploitation by rural communities causes the extinction of many important orchid species (Kala 2004). Other than that native environment of many orchids is rapidly declining due to hefty desertification, habitat loss, urban sprawl, and usage of land for farming and cultivation. Therefore in medicinal orchids, it leads to a wide gap between booms and busts.

In recent years, in Western countries, the growing use of herbal medicine and its demand is increasing. Ultimately, this type of over-exploitation requisites an intense protection measure. But in situ or ex situ of medicinal orchids conservation in their natural habitat is not sufficient for propagation as their rate is low. Orchid seeds are small, have no endosperm, and require fungal pathogens to germinate; therefore, germination rates in nature are very low (Arditti 1992). It takes a long time to obtain the desired number of orchids through asexual reproduction by rhizomes, bulbs, or rooting branches. Hence, it needs proactive mass distribution and re-establishing them in nature. To meet their growing pressure and to reduce collection pressure on wild species, biotechnological approaches such as the plant tissue culture technique has contributed immensely to plantlets production on large scale and developed different protocols for rapid cloning of desired genotypes using various types of explants. This technique has come up as a key drive in the production of planting quality material for commercially and medicinally important orchids to fulfill the increasing demand and to reduce the collection pressure on wild orchids.

Under the above circumstances, biotechnological approaches enhance the in vitro propagation as well as conservation and mass multiplication of important medicinal orchids has raised high hopes by adopting asymbiotic seed germination, vegetative explants materials, artificial seed technology and secondary metabolites production, in vitro acclimatization of raised plantlets and their establishment in nature, etc. This chapter briefly endows the state-of-the-art information mediated on tissue culture with biotechnological interventions in some medicinal orchids through micropropagation, along with its societal impacts such as ethnomedicinal properties, phytochemistry, biological activities, and economics that being the need of the hour.

2 In Vitro Propagation

To establish a successful propagation of orchids explants type selection is the most crucial factor. Among the various vegetative explants materials, the leaf has been utilized as a potent and potential source of explants for the mass multiplication of orchids. Leaf has the viability for producing a large number of uniform plantlets from a single leaf or leaf segment through direct embryogenesis or organogenesis. Knudson (1922) explored the asymbiotic seed germination in orchids under the aseptic condition, which was the first feasible technique of in vitro propagation that formed the base of modern biotechnology (Knudson 1922). Later on, Rotor (1949) developed a method to culture Phalaenopsis using uni-nodal flower stalk cuttings but all credit goes to George Morel for developing a micropropagation technique for orchids at a large scale (Rotor 1949). Virus-free Cymbidium clones were obtained from in vitro shoot meristem culture (Morel 1960). Later on, Morel (1964) reported that it was possible to produce million of plantlets within a year using a single bud by frequent sub-culturing of protocorm-like bodies (PLBs) that motivated the orchid growers (Morel 1964). The present-day micropropagation in both basic and practical aspects is much more organized than it was in the beginning. Though shoot-tips have remained the most commonly used explants for propagating orchids, the regeneration potential of other explants like axillary buds, stem discs, inflorescence segments, floral stalks, leaves, leaf peels, perennating organs (pseudobulbs, rhizomes, tubers), and roots has also been utilized successfully (Vij et al. 2004; Arditti 2008).

2.1 Seed Germination

To produce firm seeds and flowers, it takes 5–10 years for an orchid plant. Orchid seeds are one of the most distinctive features of the Orchidaceae family. They are tiny, very small, and powdered, and are produced in large quantities, with 1300–4000,000 seeds per capsule (Harley 1951; Arditti 1961). Very fragile, relatively undifferentiated, and without endosperms or cotyledons, seeds are produced from the majority of orchid species (Mitra 1971).

Due to a lack of metabolic machinery and functional endosperm, the natural germination rate of orchid seeds is very poor. Only 0.2–0.3% germinates in natural conditions (Prasad and Mitra 1975). It is well known that the seeds of almost all orchids are entrusted to mycorrhizal fungi for germination in natural conditions. Symbiotic fungi have been extensively exhibited to induce seed germination in both terrestrial and epiphytic orchids for seedling development. But, asymbiotic seed germination has imparted a systematic way for the mass multiplication of orchids (Chen et al. 2022).

2.1.1 Asymbiotic Seed Germination

The ability of orchid seeds to germinate asymbiotically by in vitro means was demonstrated for the first time by Knudson in Cattleya species (Knudson 1922). Asymbiotic in vitro seed germination of orchids occurred by culturing immature ovules often known as either embryo, fruit, or pod (Fig. 1a–d). The germination potential of immature embryos was much better than that of mature ones and varied with their developmental stages. Due to pH, dormancy, and other metabolic factors, very young orchid oocytes cannot germinate and thus cannot form suitable explants (Withner 1953). During in vitro seed germination of orchids, the intermediate protocorm stage is followed by subsequent seedling development (Fig. 1e–f). A protocorm is a chlorophyll-like, hairy, and pear-like bulbous or oblong structure that originates from the apical or lateral suture of the seed coat and provides nutrients like cotyledons during embryonic development and subsequent seedling growth (Lee 1987). Protocorms have been inconsistently assessed as uniform callus structures or distinct shoots (Kanase et al. 1993). The protocorm-like body specified the orchids for the regeneration of multiple plantlets which is a blessing to the world floricultural market (Fig. 1g–j).

Fig. 1
A set of 13 photos illustrates the in vitro micropropagation process of Cymbidium aloifolium. The photos depict the different stages, including seed germination, formation of P L Bs, shoot and root formation, and acclimatization of the plantlets.

In vitro micropropagation of Cymbidium aloifolium, (a) Mother plant, (b) Seed capsule, (c) In vitro seed germination, (d) Swelling of seeds, (e) PLBs formation, (f) Enlargement of PLBs, (g) Shoot formation, (h) Formation of shoot and root, (i) Shoot elongation, (j) Shoot multiplication, (k) Hardening and acclimatization, (l, m) Acclimatized plantlets ready for ecorestoration

Full size image

Asymbiotic seed germination of orchids was exploited for in vitro mass production of orchids with commercial and medicinal importance for conservation and ecorestoration. It was reported by several investigators from time to time.

Half strength of Murashige and Skoog (MS) medium (Murashige and Skoog 1962) were used for seed germination of Bletia purpurea (Dutra et al. 2008), Coelogyne stricta (Parmar and Pant 2016), Cymbidium giganteum (Hossain et al. 2010), Cymbidium goeringii (Gong et al. 2018), and Spathoglottis plicata (Aswathi et al. 2017; Hossain and Dey 2013). Accordingly, Cymbidium aloifolium was germinated in 1.0 mg/L 6-benzylaminopurine (BAP) and 0.5 mg/L α-naphthaleneacetic acid (NAA) supplemented (Paul et al. 2019). However, a modified half-strength MS medium was tested for in vitro germination of Dendrobium ovatum (Shetty et al. 2015).

Six different media compositions for testing were examined for their effectiveness towards the growth of Dactylorhiza hatagirea (Warghat et al. 2014) and Bletia purpurea seeds in BM-1 (Van Waes and Debergh 1986); 1/2 MS, Vacin and Went modified (VW) medium (Vacin and Went 1949); Malmgren modified terrestrial orchid medium (MM) (Malmgren 1996) and Knudson C (KC) medium (Knudson 1946). Dendrobium macrostachyum seeds were accomplished on MS, VW, and KC medium having different accumulation, amalgamation of growth hormones, and other additives. Among them, VW basal medium tested with 0.5 mg/L BAP and 5 mg/L NAA was more acceptable for plantlet formation (Li et al. 2018). Dactylorhiza hatagirea was cultured on Heller and Lindemann (LD) medium (Warghat et al. 2014), MM, VW, MS, and KC media. Both MS and KC medium were examined for asymbiotic seed germination of Eria bambusifolia (Basker and Bai 2010). MS, KC, and KC-modified Morel medium were used for Satyrium nepalense (Mahendran and Bai 2009) seed germination. Seeds from mature capsules of Dendrobium trigonopus were augmented in B5, MS, and 1/2 MS with NAA, BAP, and bark powder for in vitro germination (Pan and Ao 2014). MS + 1.0 mg/L BAP + Phytamax™ were provided for seed germination of Dendrobium aphyllum (Hossain et al. 2013).

Vacin and Went (1949) medium was alone tested for seed germination of Dendrobium parishii (Vacin and Went 1949; Kaewduangta and Reamkatog 2011). Likely, on VW medium mature seeds of Dendrobium lasianthera were enhanced with the incorporation of different concentrations of peptone of 1, 2, and 3 gm/L (Utami et al. 2017). Mature seeds of Cypripedium macranthos were sown on hyponex-peptone (HP) medium that contained 1 μM NAA and BAP after sterilization (Shimura and Koda 2004). Mature capsules of Ansellia africana were tested on Vasudevan and Van Staden (2010) medium for seed germination in vitro (Vasudevan and Van Staden 2010; Bhattacharyya et al. 2017a). However, in vitro germination of Dendrobium nobile Lindl. (Bhattacharyya et al. 2014), D. thyrsiflorum (Bhattacharyya et al. 2015), D. heterocarpum (Longchar and Deb 2022), Cymbidium iridioides (Pant and Swar 2011), C. kanran (Shimasaki and Uemoto 1990), Cypripedium debile (Hsu and Lee 2021), and C. macranthos (Shimura and Koda 2004) was reported in MS medium of full strength. Cymbidium iridioides young pods were cultured on MS medium containing 1 mg/L of NAA and BAP (Longchar and Deb 2022). Immature seeds of Cymbidium kanran were inoculated on MS medium for shoot multiplication (Shimasaki and Uemoto 1990). Young pods of Cymbidium iridioides were cultured on MS medium having NAA (1 mg/L) + BAP (1 mg/L) for micropropagation (Pant and Swar 2011).

2.2 Micropropagation of Orchids Via Vegetative Explants Materials

In orchids, as a result of out crossing, heterozygous offspring were produced from seeds. Therefore, it is necessary to augment various vegetative parts of mature plants to validate micropropagation protocols in orchids. Georges Morel was the pioneer for culturing Cymbidium shoot tips and attained protocorm-like bodies (PLBs) from contaminated plants to regenerate mosaic virus-free plants (Morel 1960). He introduced the term “protobulb (PLB)” in his work published in the Bulletin of the American Orchid Society (Arditti 2010). At the same time, a number of orchid species have yielded fruitful results, including Lycaste, Cattleya, Ondontoglossum, Dendrobium, Phaius, Miltonia, and Vanda (Arditti and Ernst 1993).

Large-scale propagation of medicinal orchids through in vitro method, different vegetative explants sources such as shoot tip, axillary bud, leaves, nodal segments, and inflorescence were augmented through callus formation or PLB mediation or direct shoot bud formation as described below:

2.2.1 Shoot Tip Culture

To induce efficient clonal propagation of medicinal orchids, shoot tips have been efficiently cultured. It was first implemented in Cymbidium by Morel (Morel 1960). This technique enables the rapid propagation of Vanda coerulea (Seeni and Latha 2000). Response of bud formation is obtained from the shoot tips in vitro and mature plants in a medium having 8.8 μM BAP and 4.1 μM NAA. For forming multiple shoots in Vanda tessellate BAP and NAA combination was found to be more effectual as compared to indole-3-acetic acid (IAA), NAA, and kinetin at single action (Rahman et al. 2009). Shoot primordium of Doritis pulcherrima was cultured for rapid propagation and regeneration of plantlets (Mondal et al. 2013). In VW medium, Dendrobium shoot tip was cultured containing 15% coconut water plus 10 ppm NAA for a rapid proliferation of PLB and plantlet formation as well as the growth of seedlings (Soediono 1983). Sixty days old Dendrobium chrysotoxum shoot tips was inoculated on MS + 0.1 mg/L NAA + 3% sucrose + 0.5 mg/L BAP for proliferation, shoot induction (Gantait et al. 2009).

2.2.2 Nodal/Internodal Culture

Dendrobium fimbriatum segments were conferred for shoot induction, and proliferation in MS + 0.2–0.5 mg/L NAA + 1.0–4.0 mg/L BAP (Huang et al. 2008). But MS medium with NAA and BAP at 17.76 μM recorded maximal regeneration (14.0 ± 0.47) of shoots (Paul et al. 2017). Stem nodes of Dendrobium devonianum cultured at MS + 0.01–0.5 mg/L NAA + 1.0–4.0 mg/L BAP for PLB and shoot induction and proliferation in vitro (Li et al. 2011, 2013a). 0.5–1.0 cm nodal segments excised with axillary buds from 4–5-month-old Dendrobium chrysanthum seedlings grown in vitro, half strength MS + 0.1 mg/L NAA + 6 mg/L BAP + 3% sucrose + 0.65% agar (Mohanty et al. 2013a).

Nodal explants of Malaxis acuminata were cultured on MS + sucrose (3% w/v) + 3 μM NAA + 3 μM BAP and resulted in well-developed plantlets with shoots and root growth (Arenmongla and Deb 2012). Young healthy nodal shoot segments from the newly grown branches of wild Bulbophyllum odoratissimum were taken and cultured on BAP (4.0 mg/L) and IBA (0.5 mg/L) fortified MS medium for producing maximum shoot proliferation (Prasad et al. 2021). Nodal cultures of Ansellia africana were tested in an MS medium supplemented with 5 μM NAA and 10 μM of meta-topolin (mT) for multiple shoot induction (Bhattacharyya et al. 2017a). Pseudo-stem segments of Dendrobium nobile with nodes (0.5–1 cm) was used as explants for induction of PLBs with varied concentration of thidiazuron (TDZ) for culture (Bhattacharyya et al. 2014). Malaxis acuminata internode cultures responded to MS + 0.5 mg/L NAA + 3 mg/L TDZ; MS + 0.5 mg/L NAA + 3 mg/L TDZ + 0.4 mM spermidine (spd); MS + 1.5 mg/L activated carbon (AC) + 4 mg/L IBA was used for shoot induction (Cheruvathur et al. 2010).

2.2.3 Leaf Culture

Leaves and leaf tips of young orchids were cultured in vitro for PLB initiation and shoot proliferation. Wimber (1965) showed the potential of Cymbidium leaves (Wimber 1965). Growth stimulation in the nutrient pool, donor axis location, and physiological age of the mother plant strongly determine the regeneration potential (Trunjaruen and Taratima 2018). Therefore, factors like growth hormones, medium nutrients composition, leaf part, leaf source (in vivo/in vitro), explants preparation, leaf maturity, etc. determine the efficiency of a leaf explants micropropagation protocol (Chugh et al. 2009).

The leaf base of Vandaceous orchids evinced greater proliferative potential than leaf tips (Na and Knodo 1995; Jena et al. 2013; Seeni and Latha 1992; Nayak et al. 1997). Younger leaves perform better than older leaves. Leaves of mature Vanda coerulea did not respond to bud formation or PLB in vitro (Seeni and Latha 2000). Whereas, mature plants of V. spathulata (L.) Spreng the regeneration potential of leaf explants was noticed with 28.5 μM IAA + 66.6 μM BAP medium (Mitra et al. 1976).

2.2.4 Axillary Bud Culture

Axillary bud culture also played a very important role in medicinal orchid micropropagation. Cymbidium elegans’s axillary buds were responsive to PLBs formation (Pant and Pradhan 2010). Axillary bud culture of Dendrobium longicornu was tested in MS medium with 0.8% agar + 3% sucrose + 5 μM NAA and 15 μM BAP (Dohling et al. 2012). In Cypripedium formosanum a quarter concentration of MS medium containing 22.2 or 44.4 mM BAP was sufficient to propagate 6.3 and 7.1 shoots per explant with an average length of 10.6–11.7 mm to produce cultures after 90 days (Lee 2010). Five species of Dendrobium (D. crumenatum, D. fimbriatum, D. moschatum, D. nobile, and D. parishii) induced multiple shoots when axillary buds were cultured in vitro (Sobhana and Rajeevan 1993). Field-grown axillary buds of Lycaste hybrids were grown in half-strength MS basal medium supplemented with 0.5 mg/L BAP and 1.0 mg/L TDZ and 2% (w/v) sucrose (Huang and Chung 2011). Six to seven millimeter long shoot tips of Aranda Deborah hybrids grown in VW medium supplemented with coconut water (20% v/v) produced an average of 2.7 PLB after 45 days (Lakshmanan et al. 1995).

2.2.5 Pseudobulb Culture

The pseudobulb of Coelogyne cristata was cultured with basal medium + BAP (1–10 mg/L) + kinetin (1–10 mg/L) alone and in combination with NAA (1–10 mg/L). In parallel sets of experiments, 0.2% AC was used in the medium for shoot multiplication (Sharma 2021); 6-BAP (2.0 mg/L) + NAA (0.5 mg/L) induced shoot proliferation in C. flaccid (Parmar and Pant 2016). The pseudobulb of Malaxis acuminata was cultured on MS + 1.0 mg/L BAP + 1.0 mg/L NAA + 2.0 g/L AC for PLB formation (Suyal et al. 2020).

2.2.6 Flower Bud Culture

Ascofinetia, Neostylis, and Vascostylis were the first species to culture the young flower buds or inflorescence for medicinal orchid micropropagation (Intuwong and Sagawa 1973). Similarly, Phalaenopsis, Phragmipedium, and Cymbidium were also cultured equivalently (Kim and Kako 1984). The floral buds were exposed to either higher auxin levels or higher cytokinin levels and anti-auxin levels (Zimmer and Pieper 1977; Tanaka and Sakanishi 1978; Reisinger et al. 1976). Younger floral buds or inflorescence were more responsible than the matured ones in terms of shoot or PLB proliferation in Oncidium Gower Ramsey, Phalaenopsis capitola, Dendrobium Miss Hawaii, Ascofinetia (Intuwong and Sagawa 1973; Mitsukuri et al. 2009; Nuraini and Shaib 1992).

2.2.7 Root and Rhizome Segment Culture

The in vitro root culture was so far attempted with success in a few species of medicinal orchids. The capacity of orchid root to induce shoot regeneration was very low as reported earlier (Kerbauy 1984). Thereafter roots of Catasetum, Cyrtopodium, and Rhyncostylis were utilized to regenerate plantlets a very high proliferation rates (Kerbauy 1984; Sanchez 1988; Sood and Vij 1986). Root tips excised from Vanda hybrids and Rhyncostylis were cultured in 1.0 mg/L IAA, 1.0 mg/L BAP and 200 mg/L of casein hydrolysate for a speedy shoot proliferation rate (Chaturvedi and Sharma 1986). Rhizome of Cymbidium goeringii responded to MS + 0.2% (w/v) AC, 3% (w/v) sucrose, 0.2% (v/v) coconut water, and 0.8% (w/v) agar powder (Park et al. 2018). Moreover, auxin, particularly NAA was responsible for stimulating rhizome formation of some medicinal orchids and ultimately new shoots were developed from a rhizome in a cytokinin-enriched medium of C. kanran Makino (Shimasaki and Uemoto 1990), C. forrestii (Paek and Yeung 1991), and Geodorum densiflorum (Roy and Banerjee 2002).

Rhizome tips were also tested for PLB formation and shoot development (Udea and Torikata 1972). In a few cases, cytokinins were inductive for stimulation of shoots from rhizome segments of medicinal orchids such as Cymbidium forrestii (Paek and Yeung 1991) and Geodorum densiflorum (Lam.) Schltr. (Roy and Banerjee 2002; Sheelavantmath et al. 2000). Sometimes BAP was responsible for the reduction of rhizome growth and branching but induced certain rhizome tips gradually into shoots (Paek and Yeung 1991).

2.2.8 Thin Cell Layer Culture

Longitudinal or transverse sections of the thin cell layers are isolated from different plant parts such as leaves, floral primordia, stems, or PLBs. The efficiency of normal plant tissue culture and thin cell layer culture techniques is compared very methodically (Rout et al. 2006). In vitro raised seedlings of Dendrobium chrysotoxum, cross-section (2 mm thickness) of stem-nodes is grown in MS medium (semi-solid and liquid) supplemented with BAP 4.44 μM and Kinetin 4.65 μM induced shoot buds (Kaur 2017).

2.2.9 Protoplasts Culture

Different explants of orchids like stem, root, leaf disc, petal, and protocorm were used for the isolation of protoplasts. Chris K. H. Teo (Malaysian scientist) and K. Neumann (German botanist) first introduced the induction and synthesis of orchid protoplasts (Teo and Neumann 1978a, b). Since then studies were carried out in this field for the isolation of orchid protoplasts. However, during the screening of more than 24 orchid species, from bases of juvenile leaves of medicinal orchid Cymbidium aloifolium protoplast culture was achieved (Seeni and Abraham 1986).

2.3 Root Induction

Concentrations of different auxins were incorporated into basal media either singularly or in combination for testing their root-promoting efficiency in medicinal orchids. For root induction of Dendrobium fimbriatum with 100% rooting frequency, MS + 0.5 mg/L NAA or 0.3–1.0 mg/L IBA and a combination of 0.5 mg/L IBA and NAA were used (Huang et al. 2008). IBA, IAA, and phenolic elicitor PG containing MS medium were responsible for root induction of Ansellia africana within 6 weeks interval (Bhattacharyya et al. 2017a). IBA was responsible for root promotion of medicinal orchids viz., 1.0 mg L/1 IBA in Acampe praemorsa (Nayak et al. 1997) and Cymbidium iridioides (Pant and Swar 2011), and 1.5 mg L/1 IBA in Dendrobium densiflorum (Pradhan et al. 2013).

A decline in root number and length was reported with increased concentration of IBA. In Dendrobium nobile, IBA was better than NAA in maximizing root numbers (Asghar et al. 2011). MS + 3% sucrose + 2 g/L AC + 0.2 mg/L IBA was used in Dendrobium chrysotoxum (Gantait et al. 2009). Whereas, in the root formation of Vanilla planifolia and Geodorum densiflorum, NAA exhibited a conducive effect (Sheelavantmath et al. 2000; Tan et al. 2011).

In Dendrobium transparens (Sunitibala and Kishor 2009) and Dendrobium primulinum (Pant and Thapa 2012) supplementation of IAA increased the rate of root proliferation whereas its affectivity was poor during root formation. However, rooting of Vanda spathulata shoots was observed within 3–9 weeks in a medium containing 75 g/L banana pulp and 5.7 μm IAA. In vitro shoots of 2–5 cm in length developed two to five roots easily in pots at 80–90% survival rates instead of hardening (Decruse et al. 2003).

2.4 Photoperiodic Condition

In vitro seed culture and micropropagation of medicinal orchids were influenced by ambiance conditions, like photoperiod (PP) for efficient early culture development.

Cool white light, 16/8-h PP, 1000 lux light intensity, 25 ± 2 ° C, and pH 5.2 have been reported for Dendrobium moschatum (Kanjilal et al. 1999). Fluorescent light, 12/12-h PP, 60 μL mol m−2 s−1, 25 ± 2 °C was provided in D. parishii (Kaewduangta and Reamkatog 2011). D. trigonopus was probably supplemented with 14/12-h PP, 25 ± 2 °C, 50 μL mol m−2 s−1 (Pan and Ao 2014). In D. aphyllum provide 14/12-h PP, 60 μL mol m−2 s−1, cool white fluorescent, 25 ± 2 °C (Hossain et al. 2013). 1000–1500 lux, 12/12-h PP, white fluorescent tube, 25 ± 1 °C extended to D. candidum (Zhao et al. 2008). 50 μL mol m−2 s−1, 12/12-h PP, 25 ± 2 °C was furnished in D. chrysanthum (Mohanty et al. 2013a). In D. chrysotoxum 16/8-h PP, 30 μL mol m−2s−1, white fluorescent tube, 60% RH, 25 ± 2 °C was supplied (Gantait et al. 2009). Originally, 25 ± 2 °C in the dark for 2 weeks, 23 μL mol m−2s−1 25 ± 2 ° C, 16/8-h PP, (callus + PLB) was described in D. crumenatum (Kaewubon et al. 2015). 350–500 lux 16/8-h PP, 25 ± 2 °C was supplied in D. densiflorum (Pradhan et al. 2013). 1500–2000 lux, 12/12-h PP, 25 ± 2 °C and pH 6.0 was suitable for D. devonianum (Li et al. 2011, 2013a). Cool white fluorescent tubes, 12/12-h PP, 40 μL mol m−2 s−1, 25 ± 2 °C were used in D. draconis (Rangsayatorn 2009). 2000 lux, 12/12-h PP, 25 °C and pH 5.4–5.6 was reported in D. fimbriatum (Huang et al. 2008). Cultures of Ansellia africana were maintained in cool white fluorescent tubes in a culture room with a light intensity of 40 μ mol m−2 s−1 at 25 ± 2 °C under a dark and light cycle of 12 h (Bhattacharyya et al. 2017a). D. fimbriatum was cultured under a photoperiod of 14 h with a light intensity of 50 μ mol m−2 s−1 using cool-fluorescent tube lights, at 25 ± 2 °C (Paul et al. 2017).

2.5 Hardening and Acclimatization

Hardening and acclimation of in vitro cultured plantlets are important steps of micropropagation for better survival and successful plant establishment under ex vitro conditions. The percentage of plant loss or damage is higher during the transfer of in vitro growing plants to ex vitro conditions. Regenerates have to adapt to many abnormal conditions such as high irradiance, low humidity, and water hydraulic conductivity of the root and root-stem connections in an ex vitro environment (Fila et al. 1998). Acclimatization of regenerates with gradually reducing humidity will overcome this threat (Bolar et al. 1998).

Well-rooted micropropagated orchid plantlets were ready for acclimatization after attaining sufficient growth in terms of root or shoot length. After removal from flasks, the well-rooted plants were cleaned thoroughly to remove the remnant of artificial media such as sucrose and nutrient agar. Thereafter, clean plantlets were soaked in an effective fungicide solution before shifting them into pots or poly sleeves having a potting mixture. The blending of various potting mixtures plays an important part in the survivability of orchid plantlets raised in vitro. A combination of the potting mixture was pounded of dried coconut husk or coco peat, tiny pieces of tree cortex, peat moss or sphagnum moss, and pieces of broken bricks or charcoals in various ratios. The ideal potting mixture should have water retaining capacity along with draining out of extra water and aeration for proper hardening and acclimatization of plants (Diaz et al. 2010; Kang et al. 2020) (Fig. 1k–m).

Brick pieces and charcoal chunks (1:1) mixture were fruitful for acclimatization of Dendrobium chrysanthum with a topmost cover of moss (Mohanty et al. 2013a). Plantlets of Dendrobium moschatum were shifted for hardening to a blending of charcoal, brick, coal, sand, and soil (1:1:1:1:1) with 48% survivability (Kanjilal et al. 1999). Rooted shoots of Dendrobium macrostachyum were provided with a perlite and peat moss mixture and kept in the green house for acclimatization (Li et al. 2018). In the mixture of coco peat, litter, and clay in the ratio of 2:1:1 with a covering of sphagnum moss Cymbidium aloifolium plantlets were acclimatized with an 85% survival rate (Pradhan et al. 2013). Acclimatization was carried out for hardening plantlets of Dendrobium draconis and shifted to cocopeat and perlite (1:1) composition with 92% achievement (Rangsayatorn 2009). In Coelogyne cristata, the composition of pine bark, brick, moss, and charcoal pieces (1:1:1:1) was used for transplanting (Sharma 2021). In Coelogyne finlaysonianum, brick, charcoal, coco peat and litter (1:1:1:1); brick, charcoal, litter, and saw dust (1:1:1:1); brick, charcoal, and litter (1:1:1); and brick and charcoal (1:1) were utilized for survival (Islam et al. 2015). A mixture of humus and sand (1:1) was tested in Changnienia amoena (Jiang et al. 2011). A composition of brick, charcoal, coconut husk, and sand (1:1:1:1) was provided for acclimatization of Spathoglottis plicata (Grell et al. 1988). In Cymbidium iridioides, plantlets were acclimatized by using cocopeat, peat moss, and brick (Pant and Swar 2011). In the ratio of 1:1:1 substrate of brick, charcoal, shredded bark, and a moss cover were imparted for the survivability of Dendrobium longicornu in a greenhouse (Jaime et al. 2015). Eria bambusifolia was tested on coconut husk, charcoal, brick pieces, broken tiles, and perlite (Basker and Bai 2010). Hardening plantlets of Satyrium nepalense were transferred to a 1:1:1 ratio of a mixture containing vermicompost, sand, and coconut husk in plastic pots (Mahendran and Bai 2009). Rhynchostylis retusa was adapted in small plastic pots containing (2:1) moss and bark (Naing et al. 2010). Cypripedium macranthos was hardened in a plastic bag that contain wet vermiculite and acclimatized in a soil mixture of coarse volcano ash and clay granules (Shimura and Koda 2004). Dactylorhiza hatagirea was survived in a potting mixture consisting of (1:1:1) cocopeat, vermiculite, and perlite (Warghat et al. 2014). Rooted plantlets of Dendrobium lasianthera were planted in a composition of coconut husk and sphagnum moss (3:1) and achieved a 90% survivability rate (Utami et al. 2017). In vitro rooted Ansellia africana plantlets were tested with a mixture of vermiculite, sand, and decaying litter (1:1:1) and found 87% survivability after 60 days (Bhattacharyya et al. 2017a). Dendrobium nobile plantlets were acclimatized with various compositions of mixture viz., (1) charcoal and bricks in the ratio 1:1; (2) in the ratio 1:1 of decaying litter and brick; (3) in the ratio 1:1:1 of brick chips, leaf litter, and charcoal; and (4) brick chips, leaf litter, and charcoal in the ratio 1:1:1 in addition to the topmost coating of moss. Among various compositions brick, charcoal, and decaying litter treatment as well as moss covering received the highest 84.3% survivability (Bhattacharyya et al. 2014). Composition of (a) brick and charcoal (1:1) (b) brick and coco peat in the ratio 1:1 (c) coco peat, brick, charcoal pieces in the ratio 1:1:1; and (d) leaf mold, brick chips, and cocopeat in the ratio 1:1:1 were supplied for transplantation of Bulbophyllum odoratissimum in Green house condition with 90% relative humidity (RH) and 91.66% survival rate. Among the different treatments, brick chips, charcoal, and coco peat (1:1:1) containing the mixture was best for high water retention as well as good aeration capacity (Prasad et al. 2021).

3 Ecorestoration

Ecosphere restoration is the “task reconstructing of an ecosystem that has been damaged due to manmade catastrophe” (Libini et al. 2008). The main objective of restoration is to re-establish the environmental system that is disturbed by various factors with respect to its structure and functional properties.

After successful acclimatization, in vitro-raised Vanda coerulea plantlets were transferred to tree trunks of forest segments, for successful ex situ harbor by using the binding medium like moss and coconut husk with 70–80% survivability rate for ecorestoration. Such a study commencing in India for restoring the natural habitat is of great interest from a horticultural and conservation point of view (Seeni and Latha 2000). Similarly, Epidendrum ilense and Bletia urbana were also shifted to the forest ecosystem or typical natural habitat for ecorestoration (Christenson 1989; Rublo et al. 1989). During the lab to land transfer strategy, it was observed that host trees with rough bark were selected and the in vitro-raised orchids were fixed either to the tree trunks with the roots or tree bark for ecorestoration efforts (Decruse et al. 2003; Aggarwal and Zettler 2010; Aggarwal et al. 2012; Gangaprasad et al. 1999; Grell et al. 1988; Kaur et al. 2017). Micropropagated plantlets of Smithsonia maculate showed 48% survival after one year reinforced at Karamana river of Peppara Wildlife Sanctuary, Kerala, India. The pilot trial on restoration through micropropagation was useful for further reintroduction and population enhancement for the practical conservation of this orchid (Decruse and Gangaprasad 2018). In vitro rooted plantlets of Vanda spathulata were observed with a 50–70% survival rate, which were introduced into forest segments at Ponmudi and Palo de in the Southern Western Ghats of India (Decruse et al. 2003).

The reintroduction trials of orchid plantlets should be conducted with well-established in vitro-rooted plantlets during the monsoon period to corroborate the maximum survival rate of the plantlets for ecorestoration or eco-rehabilitation study.

4 Artificial Seed Technology

The concept of artificial or synthetic seed was first coined by Murashige and at present it is well known by some different names such as manufactured seed, synthetic seed, or synseed (Murashige 1977). Artificial seeds were originally defined as “encapsulated single somatic embryos” by Murashige (1978), i.e., a clonal product that can grow into plantlets at in vitro or ex vivo conditions if used as real seeds for sowing, storage, and transport (Murashige 1978). Gray and Purohit (1991) also define somatic embryos with practical usage in commercial plant production (Gray et al. 1991). Therefore, the production of synthetic seeds has previously been restricted to those plants where somatic embryogenesis has been reported. Although somatic embryogenesis is restricted to selective plant species, to overcome this limitation, exploration of a suitable alternative to somatic embryos, i.e., non-embryogenic vegetative propagules like shoot tips, segmental/axillary buds, protocorm-like bodies (PLBs), organs or embryogenic callus is practiced (Ahmad and Anis 2010; Ara et al. 2000; Danso and Ford-Llyod 2003).

However, artificial/synthetic seeds or beads production was reported first time by Kitto and Janick (Kitto and Janick 1985). Since then, several flowering plant species have extensively utilized this technique including orchids. Production of synthetic seeds opens a new vista in plant tissue culture technology by adding many fruitful improvements on a commercial scale. Artificial seeds were utilized for transformation into plantlets under in vitro and in vivo circumstances. It was applied for the multiplication of rare, threatened, and endangered plant species which are hard to propagate by normal propagation process and by natural seeds.

Synthetic seed production in orchids is especially important as they produce minute non-endosperm seeds. Corrie and Tandon (1993) have used protocorms to produce synthetic seeds of Cymbidium giganteum which are transferred to a nutrient medium or sterile sand and soil medium developed healthy seedlings (Corrie and Tandon 1993). Comparable conversion frequencies of 100%, 88%, and 64% were obtained on in vitro, sand, and sand-soil mixture condition, respectively. These observations enable the direct transplantation of aseptically grown protocorms into the soil as well as reduce the cost of growing plantlets in vitro and subsequent acclimatization. As orchids produce tiny and non-endospermic seeds, the production of artificial seeds was beneficial.

Several reports on encapsulation using somatic embryos have been carried out (Ara et al. 2000; Danso and Ford-Llyod 2003; Castillo et al. 1998; Ganapati et al. 1992). For synthetic seed production, meristematic shoot tips or axillary buds were also utilized in orchids along with somatic embryos or PLBs (Ganapati et al. 1992; Bapat et al. 1987; Piccioni and Standardi 1995). Encapsulation of PLBs is well reported in many orchids such as Cymbidium giganteum, Dendrobium wardianum, Dendrobium densiflorum, Phaius tonkervillae, and Spathoglottis plicata (Danso and Ford-Llyod 2003; Saiprasad and Polisetty 2003; Vij et al. 2001).

In Dendrobium orchid, Saiprasad and Polisetty found that fractionated PLB was best suited for encapsulation at leaf primordia stage 13–15 days after culture (Saiprasad and Polisetty 2003). Encapsulation matrices prepared with MS medium (3/4 strength) + 0.44 μMB BAP + 0.54 μM NAA result in 100% conversion of encapsulated PLBs when cultured on MS medium + 0.44 μMB BAP + 0.54 μM NAA (Dendrobium). Sarmah et al. (Sarmah et al. 2010) production of synthetic seeds in an endangered monopod orchid, i.e., Vanda coerulea by leaf-based encapsulating PLBs with 94.9% conversion frequency on immediate inoculation in Ichihashi and Yamashita (IY) medium (Ichihashi and Yamashita 1977). 95% conversion was achieved on encapsulating PLB of Flickingeria nodosa in Burgeff medium (Withner 1955) + 2% sucrose + 2 mg/L Adenine sulfate + 1 mg/L IAA at 4 °C for 3 months (Nagananda et al. 2011). Alginate encapsulation of Aranda × Vanda PLB was also reported (Gantait et al. 2012). Three percent sodium alginate and 75 mM calcium chloride support better encapsulation of individual PLBs (4 mm long). Plant growth regulator (PGR)-free MS medium (1/2 strength) reported 96.4% of conversion. Likely, short-term storage of PLBs of Dendrobium shavin (Bustam et al. 2012); 60-day-old PLBs in Dendrobium nobile (Mohanty et al. 2013b) and Coelogyne breviscapa (Mohanraj et al. 2009); 30-day-old PLBs in Geodorum densiflorum (Datta et al. 1999); PLB of Spathoglottis plicata Blume (Haque and Ghosh 2017); somatic embryos in Dendrobium candidum (Guo et al. 1994) were used for encapsulation with varied binding solution, polymerization time, and conversion percentage. During the sowing of artificial seeds contamination is one of the main barriers to the commercialization of encapsulation technology. However, Chitosan was used as a fungal growth retardant.

5 Genetic Stability

The somaclonal variations are a phenomenon of plant tissue culture that is dependent on medium composition, multiplication, explants type, adventitious shoots formation, culture period, and plant genotype (Côte et al. 2001). Despite several experiences of in vitro regeneration, either genetic uniformity or variability was observed in micropropagated plantlets (Larkin and Scowcroft 1981). Micropropagation provides a feasible substitute to seed propagation as it entitles rapid propagation of elite stock cultivars in a fairly short duration of time. For the raising of quality plant material, the genetic consistency of micropropagated plants is a prerequisite factor. In contrast, genetic instability occurs in the in vitro-regenerated plants (somaclonal variation) due to the use of hyper-optimum potency of growth regulators and continuous sub-culturing. Orchid micropropagation was interrupted with an intervening callus phase, which interfered with the integrity of the regenerated clonal plantlets (Nookaraju and Agrawal 2012); on the other hand, micropropagation via meristem culture was considered as uniform culture (Rani and Raina 2000).

To examine the in vitro protocols, whether propagation was either true-to-type or not clonal fidelity was tested with various Single Primer Amplification Reaction (SPAR)-based methods such as Inter Simple Sequence Repeats (ISSR), Random Amplified Polymorphic DNA (RAPD), and Direct Amplification of Minisatellite DNA (DAMD) markers (Zietkiewicz et al. 1994; Williams et al. 1990; Heath et al. 1993). In addition, a recently invented molecular marker, the Start Codon-Targeted (SCoT) polymorphism (Collard and Mackill 2009) has gained popularity as a powerful tool for the evaluation of clonal fidelity or genetic diversity in regenerated orchid plants (Bhattacharya et al. 2005; Ranade et al. 2009) (Table 1).

Table 1 Genetic stability analysis of some medicinal orchids with various markers
Full size table

Very few studies were endured for testing of clonal fidelity of micropropagated orchids. Among them, the genetic stability of micropropagated Dendrobium plantlets was screened by Random Amplified Polymorphic DNA (RAPD) marker (Ferreira et al. 2006). Likely, in Habenaria edgeworthii (Giri et al. 2012a); Aerides crispa (Srivastava et al. 2018); Anoectochilus elatus (Sherif et al. 2017); Changnienia amoena (Li and Ge 2006); Cymbedium finlaysonianum (Worrachottiyanon and Bunnag 2018); Cymbidium giganteum (Roy 2012); Cymbidium aloifolium (Sharma et al. 2011; Choi et al. 2006); Dendrobium densiflorum (Mohanty and Das 2013); Dendrobium chrysotoxum (Tikendra et al. 2019a); Dendrobium fimbriatum (Tikendra et al. 2021); Dendrobium heterocarpum (Longchar and Deb 2022); Dendrobium moschatum (Tikendra et al. 2019b); Dendrobium nobile (Bhattacharyya et al. 2014); Eulophia dabia (Panwar et al. 2022); Rhynchostylis retusa (Oliya et al. 2021); Spathoglottis plicata (Auvira et al. 2021); Vanda coerulea (Manners et al. 2013) and in Vanilla planifolia (Sreedhar et al. 2007) genetic uniformity was tested by RAPD marker.

Moreover, Inter Simple Sequence Repeats (ISSR) marker was tested in Anoectochilus elatus (Sherif et al. 2017, 2018); Anoectochilus formosanus (Lin et al. 2007; Zhang et al. 2010); Bletilla striata (Wang and Tian 2014); Bulbophyllum odoratissimum (Prasad et al. 2021); Cymbidium aloifolium (Sharma et al. 2011, 2013; Choi et al. 2006); Dendrobium aphyllum (Bhattacharyya et al. 2018); Dendrobium chrysotoxum (Tikendra et al. 2019a); Dendrobium crepidatum (Bhattacharyya et al. 2016a); Dendrobium fimbriatum (Tikendra et al. 2021); and in Dendrobium nobile (Bhattacharyya et al. 2014); Dendrobium thyrsiflorum (Bhattacharyya et al. 2015); Habenaria edgeworthii (Giri et al. 2012a); Platanus acerifolia (Huang et al. 2009); Vanda coerulea (Manners et al. 2013; Gantait and Sinniah 2013); and Vanilla planifolia (Gantait et al. 2009; Sreedhar et al. 2007; Bautista-Aguilar et al. 2021) for studying the effectiveness of in vitro protocol. Simple Sequence Repeats (SSR) marker was tested in Vanilla planifolia (Bautista-Aguilar et al. 2021). Amplified Fragment Length Polymorphism (AFLP) marker was tested in Anoectochilus formosanus (Zhang et al. 2010) and Dendrobium thyrsiflorum (Bhattacharyya et al. 2017b). Inter-Retrotransposon Amplified Polymorphism (IRAP) marker was tested in Bletilla striata (Guo et al. 2018) and Dendrobium aphyllum (Huang et al. 2009). Directed Amplification of Minisatellite-region DNA (DAMD) marker was tested on Cymbidium aloifolium (Sharma et al. 2011) and Dendrobium heterocarpum (Longchar and Deb 2022). Start Codon-Targeted Polymorphism (SCoT) was performed in micropropagated plantlets of Anseilla africana (Vasudevan and Van Staden 2010); Bletilla striata (Guo et al. 2018); Dendrobium crepidatum (Bhattacharyya et al. 2016a); Dendrobium fimbriatum (Tikendra et al. 2021); Dendrobium heterocarpum (Longchar and Deb 2022); Dendrobium nobile (Bhattacharyya et al. 2014, 2016b); Dendrobium thyrsiflorum (Bhattacharyya et al. 2015), and Spathoglottis plicata (Manokari et al. 2022) for homogeneity demonstration.

Genetic variation or polymorphism was analyzed in Bulbophyllum odoratissimum as 3.94% (Prasad et al. 2021); 2.76% in Anoectochilus formosanus (Zhang et al. 2010); 2.53% in Dendrobium chrysotoxum; 2% in Dendrobium moschatum (Tikendra et al. 2019a, b); 2.38% in Anoectochilus elatus (Sherif et al. 2018); and 2.88% in Platanus acerifolia (Huang et al. 2009). The results of the ISSR analysis confirmed the feasibility of the micropropagation protocol of orchids although tiny dissimilarity in genomic constituents was noticed. Such negligible variation may be due to the maintenance of in vitro culture for a longer duration, concentration of growth regulators, and in vitro stress conditions that lead to clonal variations (Tikendra et al. 2019a; Razaq et al. 2013; Devarumath et al. 2002).

6 Ethno-Medicinal Properties

Orchids are the backbone of traditional herbal medicines and have been extensively studied because of their pharmacological importance. From ancient times orchids are being used in traditional systems of medicine like Ayurveda, Siddha, Yunani, Homeopathy, Traditional Chinese Medicine (TCM), etc. Chinese described a Dendrobium species and Bletilla striata in Materia Medica of Shen-Nung (twenty-eighth century B.C.) and in many other Chinese writings orchids symbolize friendship, perfection, numerous progeny, noble, and elegant (Reinikka 1995). In India, there are nearly 1600 species that constitute about 9% of the total flora (Medhi and Chakrabarti 2009). The therapeutic importance of Indian orchids in treating ailments is well documented in the literature (Lawler 1984; Handa 1986) (Table 2).

Table 2 Distribution and therapeutic importance of some medicinal orchids
Full size table

Several orchid species have important ingredients in various traditional medicinal formulations. Whole plants or their parts are used as a paste or in boiled form, single or mixed with other food stuffs as therapeutics in several ailments (Pant 2013; Gopalakrishnan and Seeni 1987).

The roots of Acampe papillosa are used in rheumatism, burning, boils, expectorant, biliousness, asthma, bronchitis, eyes, and blood, and help in curing infections, curing secondary syphilis, uterine diseases, tuberculosis, fever, and throat troubles (Hossain 2009; Zhan et al. 2016; Chopra et al. 1969). The root of Acampe praemorsa is used as a tonic for rheumatism and treats neuralgia, sciatica, syphilis, and uterine disorders. Various parts of this orchid are used for the treatment of cough, stomachache, ear-ache, and eyes diseases, reduce body temperature, antibiotic for wounds, traumatic pain, backache, menstruation pain, burning sensation, asthma, bronchitis, and mild uterine diseases (Pant 2013; Perfume workshop n.d.-a; Leander and Lüning 1967; Shanavaskhan et al. 2012; Devi et al. 2015; Panda and Mandal 2013; Nongdam 2014; Mishra et al. 2008). The paste of leaves of Aerides multiflorum is used for wounds, cuts, earaches, and consumed as a tonic (Perfume workshop n.d.-a; Baral and Kurmi 2006; Basu et al. 1971; Behera et al. 2013; Raja 2017). The leaf of Aerides odorata is applied in cuts, wounds, and tuberculosis, the fruit is used to heal the wound. Leave juice and seeds are used in treating boils in ear, nose, and skin disorders (Pant 2013; Perfume workshop n.d.-a; Leander and Lüning 1967; Baral and Kurmi 2006; Basu et al. 1971; Behera et al. 2013). The whole plant of Anocetochilus elatus is used to relief chest and abdominal pain and treats snake bites (Raja 2017; Sherif et al. 2012; Jiang et al. 2015).

The whole plant of Anocetochilus formosanus is used as an antipyretic, in detoxification, and treats tuberculosis, diabetes, bronchitis, infections in the kidney, bladder, cramps, snake bites, stomach ache, inflammation, hematemesis, nocturnal emission, nephritis, vaginal discharge, hepatitis, hypertension, and convulsions The plant possesses antioxidant, anti-hyperglycemic, hepatoprotective, anticancerous properties, and pharmacological effects, such as antiosteoporosis, antihyperliposis, and antifatigue (Perfume workshop n.d.-a; Aswandi and Kholibrina 2021; Nandkarni 1976). The leaf and stem of Ansellia africana are used for treating madness. Besides it also possesses anti-acetylcholinesterase activity in treating Alzheimer’s disease (Saleh-E-In et al. 2021; Bhattacharyya and Staden 2016). The whole plant of Arundina graminifolia is used for curing rheumatic, trauma, bleeding, and snake bites. To relieve body aches root is used. In cracks scrapped bulbous stem is applied on the foot-heels (Pant 2013; Aswandi and Kholibrina 2021; Kumar 2002; Dakpa 2007).

Bletilla striata is used for tonic, against leucorrhea; leaves are used in treating lung disease; tubers are used for regeneration of muscle and other tissues, in hemorrhage dyspepsia, dysentery, fever, malignant ulcers, gastrointestinal disorders, anthrax, malaria, eye diseases, ringworm, tumors, necrosis, silicosis, traumatic injuries, coughs, chest pain, cures tuberculosis, sores, scaling, chapped skin, blood purification, strengthening, and lungs consolidation, malignant swellings, breast cancer, pustules ulcers, demulcent, and expectorant (Perfume workshop n.d.-a; Kong et al. 2003; Bulpitt et al. 2007). The Bulbophyllum odoratissimum plant is used to cure fractures, pulmonary tuberculosis, hernia pain, infusion, or decoction is used to treat tuberculosis and chronic inflammation (Perfume workshop n.d.-a; Chen et al. 2008; Bhattacharjee 1998). The entire plant of Calanthe discolor is used for improving blood flow, circulation, abscesses, scrofula, rheumatism, bone pain, and traumatic injuries, treating skin ulcers and hemorrhoids (Perfume workshop n.d.-a; Yoshikawa et al. 1998). Changnienia amoena plant cools the blood, acts as anti-heat and antitoxic, cures coughs, blood-streaked sputum, sores, and furuncles (Teoh 2016). The pseudo bulbs of Coelogyne cristata are used in constipation and aphrodisiac (Pant and Raskoti 2013; Subedi et al. 2011; Pamarthi et al. 2019). Coelogyne stricta pseudo bulb paste cures headaches and fever (Pamarthi et al. 2019; Yonzone et al. 2012). Coelogyne flaccida pseudo bulb paste cures headache and fever, juice helps in indigestion (Teoh 2016; Pant and Raskoti 2013; Pamarthi et al. 2019).

The rhizome paste of Cymbidium aloifolium is applied on fractured and dislocated bones. Bulbs are used as demulcent agents (Pamarthi et al. 2019). The root of Cymbidium ensifolium decoction used to treat gonorrhea and flower decoction used in eye sore disorders (Tsering et al. 2017). The leaves of Cymbidium giganteum are applied over wounds (Bulpitt 2005; Fonge et al. 2019; Linthoingambi et al. 2013). The seed of Cymbidium goeringii is used to treat cuts and injuries; entire plant parts are used in curing fractures (Teoh 2016). The leaf juice of Cymbidium iridioides is used to cease blood; its powder as a tonic; pseudo bulbs and roots are consumed in diarrhea (Aggarwal and Zettler 2010; Medhi and Chakrabarti 2009; Arditti et al. 1982; Arditti and Ernst 1984). The whole plant of Cymbidium kanran is used in heart purification, cures cough and asthmatic problems, and its roots are used to cure ascariasis and gastroenteritis. The whole plant of Cymbidium lancifolium is used in the treatment of rheumatism, improves blood flow, and traumatic injuries. The whole plant of Cymbidium sinense is used in purifying the heart, lungs; treat cough and asthma (Perfume workshop n.d.-a). The dried powdered pseudo bulb of Cymbidium longifolium is consumed on an empty stomach and fresh shoot is used for nervous disorders, madness, epilepsy, hysteria, rheumatism, and spasms. Salep used as demulcent (Zhan et al. 2016; Teoh 2016; Yonzone et al. 2013).

The powdered roots of Cypripedium calceolus promote sleep and reduce pain and tea prepared by the roots cures nerves and headaches (Singh and Dey 2005). The whole plant of Cypripedium debile is used for improving blood flow, swellings, pain, and diuretic. Likely, Cypripedium formosanum is used to improve blood flow, menses, expels gas, pain and itching whereas roots along with stems are used in treating malaria, snake bites, traumatic injury, and rheumatism. The roots and leaves of Cypripedium guttatum are used in treating epilepsy (Perfume workshop n.d.-a). The rhizomes, roots, and stems of Cypripedium macranthos are used to treat skin disease, promote dieresis, swelling, and pain and improve the flowing of blood; dried flowers are used to stop blood (Shimura et al. 2007). The rhizome of Cypripedium parviflora helps to treat insomnia, fever, headache, neuralgia, emotional tension, tumors, delirium, convulsions, anxiety, menstruate pain, and child birth (Moerman 1986; Grieve 1998; Kumar et al. 2005). The whole plant of Cypripedium pubescens is used as antispasmodic, diaphoretic, hypnotic, sedative, tonic, diabetes, diarrhea, dysentery, paralysis, and malnutrition, also in cases of nervous irritability, functions of the brain and promotes sleep. The dry powder roots are used as drugs for joint pains and treating stomach worms (Singh and Duggal 2009; Khory 1982).

The tubers of Dactylorhiza hatagirea are used as food and tonic and help in healing wound and fever and control burns and bleeding (Arditti 1992, 1967, 1968; Aggarwal and Zettler 2010; Arditti et al. 1982; Arditti and Ernst 1984). The leaves and pseudo bulb paste of Dendrobium amoenum are applied on skin diseases, burnt skin, and dislocated bones (Venkateswarlu et al. 2002). The leaf paste of Dendrobium aphyllum is applied on deformed abnormal head of a new born baby in order to form a normal shape (Pant 2013). The leaves of Dendrobium candidum are used to treat diabetes (Wu et al. 2004). The stem of Dendrobium chrysanthum is used as a tonic, enhances the immune system, and reduces fever. Leaves are used as antipyretic and mild skin diseases, which benefit the eyes (Bulpitt 2005; Jalal et al. 2008, 2010; Li et al. 2016). The whole plant of Dendrobium chrysotoxum possesses antitumorous and anticancerous properties, stem and flower extract is used as tonic and leaf extract as antipyretic (Bulpitt et al. 2007; Sood et al. 2006; Joshi et al. 2009). The pseudo bulb paste of Dendrobium crepidatum is used in fractured and dislocated bones. Stems are used as a tonic, in arthritis and rheumatism (Joshi et al. 2009; Reddy et al. 2001; Joshi and Joshi 2001). The leaves of Dendrobium crumenatum are used to cure boils and pimples (Joshi and Joshi 2001). The pseudo bulb pulps of Dendrobium densiflorum are used to cure boils, pimples, and various skin eruptions, leaf paste is applied upon fractures bones, sprains, and inflammations (Arditti 1992; Arditti et al. 1982; Arditti and Ernst 1984). The dried stems of Dendrobium devonianum is used as an enhancer for the immune system (Cakova et al. 2017). The stem of Dendrobium draconis are used in antipyretic and hematinic (Perfume workshop n.d.-b). The whole plant of Dendrobium fimbriatum is used during upset of the liver and severe anxiety; leaves are used in bone fracture and as a tonic, the pseudo bulbs are used in fever (Aggarwal and Zettler 2010; Arditti et al. 1982). The pseudo bulb paste of Dendrobium heterocarpum is used in treating fractured and bone dislocate (Arditti and Ernst 1984). The root, stem, and leaf of Dendrobium lasianthera act as anticancer (Utami et al. 2017).

The whole plant juice of Dendrobium longicornu is added to lukewarm water to bath for fever; roots are boiled to feed the livestock, to remove cough; stem juice is used to treat fever (Perfume workshop n.d.-b). The tender shoot tip juice of Dendrobium macrostachyum is used for earaches (Zhan et al. 2016). The pseudo bulb paste of Dendrobium moschatum is used for dislocated and fractured bone (Reddy et al. 2001). The pseudo bulb extracts of Dendrobium nobile are used in treating burns, and eye infections; the plant is used to cure pulmonary tuberculosis, fever, general debility, flatulence, dyspepsia, reduce salivation, parched, thirsty mouth, night sweats, antiphlogistic, and tonic. Seeds are used to heal wounds; stems to cure fever and tongue dryness; stems are used in longevity, aphrodisiac, stomachic, and analgesic (Aggarwal and Zettler 2010; Arditti et al. 1982; Arditti 1967). Whole plant juice of Dendrobium ovatum cures stomach aches, excites bile, and is a laxative for the intestines, curing constipation (Kirtikar and Basu 1981; Caius 1986). The dried stem of Dendrobium primulinum acts as an enhancer for the immune system (Pant and Thapa 2012). The pseudo bulb paste of Dendrobium transparens is used in treating fractures and dislocated bones (Arditti and Ernst 1984). The stem of Dendrobium trigonopus is used to cure fever and anemia (Perfume workshop n.d.-b). Doritis pulcherrima leaf is used to treat ear infections (Perfume workshop n.d.-c).

The whole plant of Eria bambusifolia is used in treating hyper acidity and various stomach aches (Zhan et al. 2016). The tubers of Eulophia dabia tubers are used as a tonic and aphrodisiac help to cure stomach aches, and stimulate blood flow, also used for consumption mixed with milk, sugar, and flavored species (Panwar et al. 2022). The tuber of Eulophia epidendraea is applied upon boils; controls pain in breast feeding mother; cures tumor and diarrhea; acts as an appetizer, anthelmintic, aphrodisiac, stomachic, worm infestation, stimulate appetite and purifies blood during heart troubles (Narkhede et al. 2016). The whole plant of Eulophia nuda is used in stomachache and snake bites; the stems are used to stop bleeding and trauma pain; a thick paste of tuber is applied on the stomach to kill intestinal worms, cures rheumatoid arthritis, bronchitis, scrofulous glands, tumors, purifies blood, used as a tonic, acts as anti-aphrodisiac, demulcent, and anthelmintic. The leaf is used as a vermifuge (Hada et al. 2020). The tuber of Gastrodia elata is used to cure stroke, tetanus, migraine, headaches, backache, skin boils, ulcers, and pain in the lower extremities; for generalized dermatitis dizziness, sleepiness, insomnia, high blood pressure, blood circulation, rheumatism, numbness, and paralysis (Chen et al. 2014). The root paste of Geodorum densiflorum is applied on insect bites and wounds; the root paste by mixing with ghee and honey to correct menstrual disorders and the poultice made from pseudo bulbs is used as a disinfectant (Sheelavantmath et al. 2000). The stem of Gymnadenia conopsea helps the kidney, treats cough, lactation failure, sexual dysfunction, traumatic injuries, thrombosis, and chronic hepatitis (Gustafsson 2000).

The leaves and roots of Habenaria edgeworthii act as cooling and spermopiotic; the pseudo bulb of Habenaria pectinata is used during diathesis bleeding, burning sensation, fever, and phthisis (Singh and Duggal 2009). The root of Herminium lanceum is beneficial for the lungs and kidneys, strengthens muscles, bones, stops bleeding, and treats tuberculosis (Perfume workshop n.d.-d). The whole plant of Liparis odorata is soaked in wine for external use; tubers are used during stomach disorders (Perfume workshop n.d.-e). The pseudo bulb of Malaxis acuminata is used as a tonic, aphrodisiac, styptic, antidysentery, and febrifuge (Pushpa et al. 2011). The stem and leaves of Papilionanthe teres are used for improving blood flow and reducing swellings. The whole plant of Pholidota articulata is used to remove gas and reduce swelling, treat coughs, headaches, dizziness, ulcers, sores, traumatic injuries, uterine, and menses problems. The roots and pseudo bulb paste of Pholidota pallida are used to cure fever and induce sleep and juice to remove abdomen pain. The whole plant of Platanthera chlorantha is used to strengthen the kidneys and lungs, hernia, and sexual dysfunction (Perfume workshop n.d.-c).

The leaves and roots paste of Rhynchostylis retusa are used in rheumatism, leaf juice is used in constipation, gastritis, acidity, and as emollient; root juice is used to heal cuts and wounds; root is used to treat menstrual pain and arthritis; dry flower is used as emetic (Basu et al. 1971; Dakpa 2007; Bulpitt et al. 2007; Bhattacharjee 1998; Dash et al. 2008). Tubers of Satyrium nepalense are used to treat diarrhea, dysentery, and malaria, consumed as an aphrodisiac, and used as a children’s growth supplement. Juice is used in cuts and wounds (Gutierrez 2010; Baral and Kurmi 2006; Basu et al. 1971; Behera et al. 2013; Bulpitt et al. 2007). The pseudo bulb of Spathoglottis plicata is used in rheumatic swelling; the hot fomentation is pressed on to draw out pus from the infected part, helps in proper blood flow and reduces pain (Friesen and Friesen 2012). The whole plant of Thunia alba is used in treating cough, pneumonia, bronchitis, bone break treatment, and injury (Mathew 2013).

The flower juice of Vanda coerulea is used in treating glaucoma, cataract, and blindness. The root of Vanda roxburghii is used to treat fever, dyspepsia, bronchitis, cough, piles, snake bites, rheumatism, allied disorders, and nervous system disease (Uprety et al. 2010). The dried flower powdered juice of Vanda spathulata are used to treat asthma, depression, enhance memory, antioxidant activity, and alleviate chronic disease, and degenerative ailments such as cancer, autoimmune disorders, hypertension, delay in aging process, and atherosclerosis (Jeline et al. 2021). The leaf of Vanda tessellata is used in inflammation, rheumatism, dysentery, bronchitis, dyspepsia, and fever (Chowdhury et al. 2014). The leaf, root, and flower powdered extract of Vanda testacea is used in nervous disorders, piles, inflammations, rheumatism, bronchitis, and anti-cancerous drugs (Kaur and Bhutani 2009). The fruit of Vanilla planifolia is used to treat intestinal gas and fever, increase sexual desire, used as flavoring syrup and perfume fragrance (Rxlist n.d.).

The phytochemicals such as alkaloids, flavonoids, and glycosides made the orchids therapeutically important (Hossain 2011); they are, however, mainly used as nutraceuticals because the active principles responsible for their medicinal properties are yet to be identified with further accuracy.

7 Phytochemistry

Gas Chromatography and Mass Spectrometry (GC/MS) analyzed the essential oil and the oleoresins for various medicinal orchids. In our present study, we accessed and summarized the phytochemicals of 45 orchid species (Table 3).

Table 3 Screening of phytochemicals in some medicinal orchids
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Major phytochemicals reported in Ansellia africana namely n-Hexanal, Mesityl oxide, 4-Heptenoic acid, 3,3-dimethyl-6-oxo-methyl ester, Pentadecanoic acid, Succinic acid, 3,7-dimethyloct-6-en-1-yl pentyl ester, Linoleic acid, Linolenic acid, l-Ascorbyl 2,6-Dipalmitate, Toluene, Ethylbenzene, Mesitylene, Erythro-1-Phenylpropane-1,2-diol, Styrene, Hyacinthin, 2-Ethylbutyric acid, 3-methylbenzylester which possess cytotoxic effect against cancerous cell line (Saleh-E-In et al. 2021). Gramniphenol, a potent marker reported in Arundina graminifolia showed anti-tobacco mosaic virus activity (Gao et al. 2012). Phytochemicals of B. striata showed major biological activity in aiding hemostasis, cytotoxicity, antimicrobial, anti-inflammation, anti-oxidation, immunomodulation, anti-fibrosis, antiaging, and anti-allergy (He et al. 2017). Densiflorol B, the most active compound reported from Bulbophyllum odoratissimum exhibit cytotoxic activity against the five tested cell lines (Chen et al. 2008). Major stilbenoids, flaccidin, oxo flaccidin and isoflaccidin were reported in Agrostophyllum callosum, Coelogyne flaccida (Majumder and Maiti 1988, 1989, 1991; Majumder et al. 1995). 5-hydroxy-3-methoxy-flavone-7-O-[β-d-apiosyl-(1 → 6)]-β-d-glucoside, an alpha-glucosidase inhibitor reported from Dendrobium devonianum (Sun et al. 2014). Sesquiterpene such as alloaromadendrene, emmotin, and picrotoxane from Dendrobium nobile possesses immunomodulatory potential (Ye et al. 2002). Dendroparishiol a marker reported from Dendrobium parishii exhibited antioxidant and anti-inflammatory activity against RAW264.7 cells (Kongkatitham et al. 2018). 9, 10-dihydrophenanthrene, a novel marker reported from Eria bambusifolia showed anticancer activity against the human cell line (Rui et al. 2016). Major aromatic phytochemicals were reported in Platanthera chlorantha namely β-Ocimene, Lilac aldehyde, β-Elemene, α-Bergamotene, Cedrene, Germacrene D, Pentadecane, b-Bisabolene, b-Sesquiphellandrene, 1,2,3-Trimethoxy-5-(2-propenyl) benzene, Tetradecanal, Benzophenone, Galaxolide, Docosane, Tetradecyl benzoate (D'Auria et al. 2020). Quercetin-3-O-α-l-(2″-E-p-coumaroyl-3″-Z-p-coumaroyl)-rhamnopyranoside (E, Z-3′-hydroxyplatanoside and quercetin-3-O-α-l-(2″-Z-p-coumaroyl-3″-E-p-coumaroyl)-rhamnopyranoside (Z, E-3′-hydroxyplatanoside) markers reported from Platanus acerifolia. The leaves exhibit antimicrobial activity against Staphylococcus aureus (Wu et al. 2022). Phytochemicals reported in genus Vanda possess major pharmacological activities, markers such as stigmasterol, γ-sitosterol, β-sitosterol, β-sitosterol-D-glucoside, tetracosylferulate possess anti-aging, antimicrobial, anti-inflammatory, antioxidant, neuroprotective, membrane stabilizing, and hepato-protective activities (Khan et al. 2019).

7.1 Secondary Metabolites

A wide range of secondary metabolites is present in Orchids, of which only a very slight portion was analyzed. Normally several phytochemicals viz., alkaloids, saponins, flavonoids, anthocyanins, carotenoids, polyphenols, sterols, etc. were produced and integrated into in vitro culture of orchids (Mulabagal and Tsay 2004; Yesil-Celiktas et al. 2007; Shinde et al. 2010). Among them, polyphenols were responsible for their crucial role in curing many degenerative and age-linked ailments (Brewer 2011; Procházková et al. 2011). Likely, other bioactive compounds like flavonoids, tannins, and alkaloids were bestowed for the medication of several chronic diseases (Lu et al. 2004; Zhang et al. 2005; Harris and Brannan 2009).

7.1.1 Bioactive Compounds

Various plant parts (leaf, root, and pseudobulb) of orchids possess a group of important phenolic acids such as gentisic acid, gallic acid, salicylic acid, protocatechuic acid, syringic acid, caffeic acid, sinapic acid, ferulic acid as well as flavonoids viz., catechin, apigenin, myricetin, naringin, rutin, quercetin, kaempferol, and alkaloids viz., chysine, drobine, dendronine, grandifolin, crepidine, and vanilin in higher concentration. In in vitro raised plants, bioactive compounds were more dominant than in wild plants of medicinal orchids (Fig. 2).

Fig. 2
A set of 10 chemical structures of Fimbriol A, Rhamnetin, Coelonin, Cephalinone C, Gigantol, Pholidotol A, Shanciol B, Phaindole, Tryptanthrin, and Vanillin.

Chemical structure of bioactive molecules of medicinal orchids (Drawn in Chemdraw 8.0)

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The majority of bioactive compounds viz., ayapin, n-octastylferulate, crepidatin, confusarin, physcion, scopolin, rhein, fimbriatone, and β-sitosterol were reported in Dendrobium fimbriatum which were important for pharmacological point of view (Paul et al. 2017; Bi et al. 2003; Shailajan et al. 2015). However, studies on the phytochemical analysis of medicinal orchids raised in vitro are very few (Bhattacharyya et al. 2014, 2015, 2018, 2016a,b; Bhattacharyya and Staden 2016; Giri et al. 2012b; Bose et al. 2017). A bioactive compound such as bisbenzyl erianin was isolated from the callus culture of Dendrobium chrysotoxum which was the potential as an antioxidant, antitumor, and antiangiogenic agent (Zhan et al. 2016). The presence of polyphenols was reported in Habenaria edgeworthii culture (Giri et al. 2012a). Different biochemical constituents like total phenolic, flavonoid, alkaloids, and tannins contents were analyzed and comparisons were reported between the various parts of mother plants and micropropagated plants of Dendrobium nobile (Bhattacharyya et al. 2014). Compounds with higher concentrations are reported in micropropagated plants of Herminium lanceum (Singh and Babbar 2016) and Habenaria edgeworthii (Giri et al. 2012a) than in wild plants. The phytochemical evaluation of various parts of the mother plant and in vitro propagated plants of Bulbophyllum odoratissimum was performed by using HPLC (Prasad et al. 2021). Extracts of Dendrobium crepidatum contained bioactive compounds like tetracosane, hexadecanoic acid, triacontane, phenol derivatives, and tetradecanoic acids are responsible for antioxidant and cytotoxic activities (Paudel et al. 2019).

7.1.2 Biological Activity

7.1.2.1 Antioxidant Activity

Bioactive components exhibited vigorous antioxidant properties in divergent in vitro methods which showed high scavenging potentiality to various Reactive Oxygen Species (ROS) viz. hydroxyl radical, peroxynitrite, superoxide anion, and hypochlorous acid (Halliwell 2008). Unlike synthetic antioxidants, vigorous studies were conducted on antioxidants present in natural fruits, vegetables and medicinal plants, which are considered less toxic due to their effective free radical scavenging activity.

1,1- diphenyl-2-picrylhydrazyl (DPPH) and Ferric Reducing Antioxidant Power (FRAP) assay were used for the analysis of the antioxidant activity of the plant extracts of mother and micropropagated Dendrobium nobile plants (Cao et al. 2021). Both the assays describe the antioxidant response of Dendrobium nobile determining the high antioxidant potential in samples of leaf due to its high content of polyphenols, alkaloids, and flavonoids. Among the different solvents and plant parts of the tested species, the DPPH activity of the methanolic leaf extraction was the highest (89.8 ± 2.9%), but the activity of radical scavenging of the chloroform leaf extraction was the lowest (28 ± 2.9%) of the micropropagated plant. D. nobile plantlets grown through tissue culture reported higher levels of free radical scavenging activity than mother plants (Bhattacharyya et al. 2014). Total phenol content (TPC), radical scavenging activity DPPH and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), Total Flavonoid Content (TFC) as well as total reducing power ability is being reported from all plant material extracts of mother plants and in vitro-cultured plants of Bulbophyllum odoratissimum (Prasad et al. 2021). DPPH radical scavenging activity was studied in some of the following orchid species viz. Acampe papillosa, Aerides odorata, Bulbophyllum lilacinum, Arundina graminifolia, Cymbidium aloifolium, Dendrobium aphyllum, Papilionanthe teres, Luisia zeylanica, Dendrobium tortile, Rhynchostylis retusa (Rahman and Huda 2021); Rhynchostele rossii (Gutiérrez-Sánchez et al. 2020); Dendrobium candidum (Wang et al. 2016); Dendrobium chrysanthum (Aswandi and Kholibrina 2021); Dendrobium draconis (Sritularak et al. 2011); Pholidota articulata (Singh et al. 2016a); Papilionanthe teres (Mazumder et al. 2010); Geodorum densiflorum (Keerthiga and Anand 2014). DPPH assay measures the total phenolic, alkaloid and flavonoid content by using Folin-Ciocalteu, spectrophotometry and modified acid-alkalimetry methods in Dendrobium crumenatum (Topriyani 2013). DPPH radical, column chromatography Diaion HP-20 or reverse-phase silica gel column chromatography was studied in Gymnadenia conopsea (Shang et al. 2017). A DPPH radical, spectrophotometric method, Liquid Chromatography Mass Spectrometry (LC-MS) was studied in Paphiopedilum villosum (Khamchatra et al. 2016). DPPH and ABTS assay were studied in Cymbidium kanran (Axiotis et al. 2022); Dactylorhiza hatagirea (Kumari et al. 2022); Dendrobium moschatum (Robustelli della Cuna et al. 2018); Geodorum densiflorum (Keerthiga and Anand 2014); Gastrodia elata (Song et al. 2016). DPPH, ABTS radical scavenging assays and reducing capacity assays have been studied in Dendrobium aphyllum (Liu et al. 2017) and Dendrobium macrostachyum (Sukumaran and Yadav 2016). DPPH, ABTS, and metal chelating in Malaxis acuminata (Bose et al. 2017) and in Dendrobium nobile hydroxyl radicals scavenging assay was also studied (Luo et al. 2010). MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay in Dendrobium aphyllum (Liu et al. 2018) and DPPH assay in Dendrobium densiflorum (Pant et al. 2022), and in Dendrobium crepidatum by using GC–MS (Gas Chromatography and Mass Spectrometry) was used to identify the compounds (Paudel et al. 2019). DPPH, ORAC, and deoxyribose assays in Dendrobium parishii (Raja 2017); DPPH scavenging activity, reducing power and chelating activity against iron ions (Fe2+) in Dendrobium candidum (Ng et al. 2012). DPPH and FRAP assay were studied in Dendrobium devonianum (Wang et al. 2018) and Dendrobium fimbriatum (Paul and Kumaria 2020). Deoxyribose assays, non-site-specific scavenging assays, or antioxidants and iron ions, also known as site-specific scavenging assays have been studied in Dendrobium chrysotoxum (Zhao et al. 2007) (Table 4).

Table 4 Testing of antioxidant activity of some medicinal orchids
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7.1.2.2 Antimicrobial Activity

Five different multidrug resistance (MDR) bacterial clinical isolates were used for testing the antibacterial activity of the epiphytic orchid Pleione maculata which includes Escherichia coli (2461), Enterococcus sp. (2449), Staphylococcus aureus (2413), Serratia sp. (2442), and Acinetobacter sp. (2457) along with antimycobacterial activity with Mycobacterium tuberculosis strain (H37Rv) (Bhatnagar and Ghosal 2018). Likely methanolic extracts of tubers of Satyrium nepalense were studied against both Gram-negative and -positive food pathogenic bacteria namely Staphylococcus mutans, Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumonia and 6 mg/100 μL concentration was responsible for the minimal effect against all the tested microorganisms (Mishra and Saklani 2012).

Ethanolic and hexane extracts of Coelogyne cristata and Coelogyne fimbriata, leaves and pseudobulbs were explored against human pathogens like Gram-positive Bacillus cereus (ATCC 14579), Staphylococcus aureus (ATCC 12600), and Gram-negative Escherichia coli (ATCC 10798), Yersinia enterocolitica (ATCC 9610), and Klebsiella pneumonia (ATCC BAA-3079) bacteria. Only 70% of ethanolic leaf extracts inhibited the growth of the investigated human pathogens (Pyakurel and Gurung 2008; Subedi 2002; Wati et al. 2021; Subedi et al. 2013). Methanolic and water extract of Peristylus densus showed better antimicrobial activity against bacterial and fungal strains with an inhibition zone of 8–10 mm when tested against S. typhi, P. aeruginosa, S. aureus, E.coli, and Aspergillus niger (Jagtap 2015). Methanolic and ethanolic extract of Malaxis acuminata revealed strong antimicrobial activity against P. aeruginosa and S. aureus strain in Minimum Inhibitory Concentration (MIC) assay and Butanol extract showed a strong inhibition zone of 32 mm compared to control 28 mm against Candida albicans (Suyal et al. 2020). Ethyl acetate extract showed significant antimicrobial activity against bacterial strains K. pneumoniae, S. enteric and E. coli with an inhibition zone of 14–18 mm in Pholidota articulata (Singh et al. 2016b). Whereas ethanolic extract of the species showed antimicrobial activity against microbial strains S. aureus, Vibrio cholerae, B. subtilis, E. coli, and K. pneumoniae with inhibition zone ranges from 9 to 12 mm. No activity was observed in V. cholerae (Marasini and Joshi 2012). Ethanolic extract of Pholidota imbricata showed effectiveness against S. aureus, V. cholerae, B. subtilis, E. coli, and K. pneumonia microbial strains with inhibition zone ranges from 8 to 14 mm (Marasini and Joshi 2012). Rhyzopus stolonifer, Candida albicans, and Mucor sp. were tested with the different orchid species. No activity against fungal organisms reported in Coelogyne stricta (leaf), Coelogyne stricta (Pseudobulb), and Dendrobium amoenum. Whereas Pholidota imbricata and P. articulata extracts showed fine activity. Dendrobium nobile, Eria spicata, Rynchostylis retusa, Bulbophyllum affine, and Vanda cristata showed very weak to moderate activity against selected fungal pathogens (Marasini and Joshi 2012).

7.1.2.3 Cytotoxic Activity

The cytotoxic activity of crude extracts from Dendrobium longiflorum plants was determined by the Mean Transit Time (MTT) assay (Mosmann 1983; Sargent and Taylor 1989). This study tested tumor cells of the human brain (U251) and cervical cancer cells (HeLa). The cytotoxicity results of D. longicornu acetonic extract showed a significant cell growth inhibitory effect on the U251 cell line which may be due to high levels of flavonoids, while ethanolic extract had no significant cytotoxic activity on U251 cells. Similarly, the higher flavonoid levels in the ethanolic extract of D. longicornu showed significant results on the cytotoxic activity of the HeLa cell line. The cytotoxic activity of flavonoids has been described by previous researchers (Patel and Patel 2011; Awah et al. 2012; Jeune et al. 2005).

Methanolic extract of the whole plant of Pleione maculata was tested for cell cytotoxicity and found to be within permissible limit, i.e., 7% at MIC assay. This supports scientific evidence in favor of folk medicinal utilization of Pleione maculata for various ailment treatments (Bhatnagar and Ghosal 2018). However, no cytotoxic effect was observed at an extract dosage of 50–100 μg/mL in the methanolic extract of Pholidota articulata, whereas 200–400 μg/mL of the extract showed better activity in HeLa cells (IC50 673.04) compared to U251 cells (IC50 3170.55). The control showed a better cytotoxic effect (Joshi et al. 2020). Similarly in Papilionanthe uniflora no cytotoxic effect was observed at a methanolic extract dosage of 50–100 μg/mL, whereas 200–400 μg/mL of the extract showed better activity in HeLa cells (IC50 781.85) compared to U251 cells (IC50 2585.88) and control showed better cytotoxic effect (Joshi et al. 2020).

The cytotoxic activity of Dendrobium crepidatum was determined against HeLa (Human Cervical Cancer) and U251 (Human Glioblastoma) cell lines. The extract contains bioactive compounds like tetracosane, tetradecanoic acid, triacontane, phenol derivatives, and hexadecanoic acid which cause cytotoxic activity. The percentage of growth inhibition of HeLa cells for extraction of hexane (DCH) at 100 g/mL and chloroform extract (DCC) at 800 g/mL was found to vary from 19.84 to 4.31% and 81.49–0.43%, respectively. Whereas higher growth inhibition percentage was recorded in DCC at 800 μg/mL and in the extraction of acetone (DCA) at 400 μg/mL (74.35–0.59%) of HeLa cell, which was significantly different compared to other extracts. Likewise, ethanol extracts (DCE) at 100 μg/mL and methanol extracts (DCM) at 200 μg/mL showed significantly higher growth inhibition percentages of HeLa cells (Paudel et al. 2019).

8 Economics

Orchids are popular due to their attractive and long-lasting flowers, with unique shapes and forms. This is a flowering plant consisting of diverse genera and species. Nowadays using the micropropagation technique it has become easy to multiply some of the rare medicinal orchids. Flowers have bagged a significant position in present-day contemporary society. Therefore, a potential pressure for flowers was created especially in terms of the orchid flower as they have a plethora of flower forms and colors. As Orchid reproduction is in a very germinal stage in India, different medicinal orchid varieties can be reproduced by adopting a well-planned Orchid augmentation strategy for the cut-flower trade.

Orchids were the first horticultural crop mass multiplied successfully through the micropropagation technique and the commercial aspects of this group were being increasing day by day for their medicinal importance. Commercial Tissue Culture laboratories around the globe have aided the orchid’s mass multiplication and helped the orchid industry revolutionize in the form of cut flower business in several countries. The Indian flower market is expected to grow to INR 661 billion by 2026. North East India, along with Sikkim, Arunachal Pradesh, and Himachal Pradesh, is the orchid-rich state in the country. In southern India, Kerala and Tamil Nadu have high humidity, low temperatures, abundant rainfall, and a pleasant climate suitable for commercial orchid cultivation. The orchid industry in India is in its infancy in terms of in vitro micropropagation or commercial cultivation. This is due to inappropriate or unsuitable planting material for large-scale cultivation, a deficit of technology for commercial mass propagation techniques, a lack of post-harvest commercial techniques for the cut-flower market for international trade, export policies, inappropriate commercial planting methods, etc. However, in India, it can be possible to grow commercially viable orchid varieties such as Cattleya, Cymbidium, Dendrobium, Oncidiums, Phalaenopsis, Paphiopedilum, and Vandaceous for cut flower production. Presently, the inward demand for orchid cut-flower is mainly refilled through imports from outside India. However, with the installation of in vitro propagation technology, cost-effective greenhouses, and post-harvest and storage technology, the orchid cut-flower industry can commence in other parts of India also.

According to the National Horticultural Database released by the National Horticultural Administration, in 2020–2021, the flower planting area in India is 322,000 hectares, producing 2152 thousand tones of scattered flowers and 828,000 tones of cut flowers. Growing orchids is more than just a pleasure these days. This is an international trade that accounts for about 8% of the world’s horticultural business and has the potential to change a country’s economic outlook.

According to Biotech Consortium India Limited (Biotechnology Division) and Agri-Business of Small Farmers’ Consortium, Indian Tissue Culture Market Research, 2005, Dendrobium sp. as cut flowers and Vanilla as spice are the most important plants in India which are suitable for micropropagation. Growing orchids in India, different agro-climatic regions, low labor costs, and accelerating high-end customer markets create a successful impact on society (Singh et al. 2008). But, the orchid cut flower business is consistently retarded by the unruly condition in airports; large numbers of infected and deserted cut flowers; moreover chemically processed flowers are rejected in Indian cities for violation of bio-safety norms (De 2008).

Presently, worth millions of dollars industry of orchid cut flower are flourishing in different countries such as Malaysia, Australia, Thailand, and Singapore among the top ten cut flowers of the world, the cut flower grasps sixth position and 3% Cymbidium orchid alone contributes in this list (De and Debnath 2011).

9 Conclusion

Biotechnological interventions and plant tissue culture techniques are accelerating the large-scale reproduction of the delicate and rare medicinal orchid for its potential uses as therapeutics. Since orchids are exotic breeders, they propagate by seed to produce hybrid plants. Therefore, protocols that allow regeneration from different vegetative parts of the plant are needed to achieve suitable types of micropropagation of medicinal orchids, which have shown amazing developments in germplasm conservation in recent years. Hardening and acclimatization of in vitro-propagated orchids have maintained in different ratios of the organic medium before ex vitro survivability. In recent years, as a research tool addition to being used, plant tissue culture techniques have also been of great industrial significance in the plant propagation field, plant improvement, and secondary metabolites production.

Furthermore, testing of clonal fidelity of micropropagated medicinal orchids by using markers like RAPD, ISSR, and SCOT can be adequately utilized in the sustainable implementation of plant genetic resources by identifying and eliminating the difficulties of somaclonal variations. However, from various parts of the in vitro-raised medicinal orchid many compounds have been isolated which are a good source of bioactive molecules as well as phytochemicals. Antioxidant activities and ethnomedicinal properties have been offering better possibilities for the occurrence of value-added products, for the treatment of diseases with herbal medicines to boost health benefits.

Similar to micropropagation technology, synthetic seed technology has attracted much attention in recent years due to its broader application of germplasm conservation in natural habitats. Although little progress has been made in proving the feasibility of synseeds, their successful implementation in the conservation of orchid ornamental/medicinal genetic resources is achievable.

Emphasis on eco-rehabilitation study provides a new gateway for ex situ conservation of in vitro-raised medicinal orchids in their natural habitats. The host tree and orchid species symbiosis still maintains a proper balance for further reintroduction and population enhancement for the practical conservation of important orchids. Orchids have both flower value and medicinal value and are more demanding in the international market. Endemic and rare orchids have a plethora of flower shapes and colors that require scientific attention for their use in the cut flower industry.

Comprehensive research is still necessary to extensively study the different orchid species for various ailments. However, due to limited understanding and knowledge about the therapeutic values of these locally available plants, the use of orchids in the traditional healing process is restricted. For commercial scale, very less effort has been made for medicinal orchid cultivation due to its small size population and restriction in distribution. Different precious orchid species that have reached either the threatened or extinct category can survive with biotechnological interventions and human support for their mass propagation. Therefore, to meet the current need for medicinal orchids and to reduce the pressure on its natural population, plant tissue culture can be an acceptable alternative for its sustainable utilization which is the need of the hour.