Table 2.

Some examples of the examples of in vitro culture of protocorm-like bodies for the production of specialized metabolites

Species	Specialized metabolite	Main use	Culture conditions			References
			Nutrient medium	Culture type	Culture conditions (Temperature, light, photoperiod)
Anoectochilus formosanus	Phenolics, flavonoids, and polysaccharides	Antioxidants and immunostimulants	Protocorm induction: MS medium supplemented with 0.2 mg L−1 TDZ + 0.5 mg L−1 NAA + 3% sucrose PLB suspension culture: MS liquid medium containing 3% sucrose. Effect of cytokinin BA (0–7 mg L−1) and KN (0–7 mg L−1); Ammonia/nitrate (NH4 Cl/KNO3), NH4+:NO3− at 0:60, 10:50 20:40, 30:30, 40:30, 50:10 and 60: 0 mM; sucrose, glucose, and fructose (3% each) and sucrose: glucose (1:1), glucose and fructose (1:1), fructose and sucrose (1:1) were tested	PLB culture in shake flasks	Cultures were maintained under a 10 h day and 14 h dark photoperiod and a PPFD of 20 µmol m−2 s−1	Zhi-Wei et al. (2012)
Dendrobium candidum	Phenolics, flavonoids, coumarins, and polysaccharides	Antioxidants and immunostimulants	Protocorm induction: MS medium supplemented with 0.2 mg L−1 NAA + 0.5 mg L−1 BA + 3% sucrose PLB suspension culture in shake flasks: MS liquid medium containing 3% sucrose. PLB suspension culture in bioreactors: PLB suspension cultures in 3 L balloon-type bubble bioreactors containing 2 L MS medium supplemented with 0.5 mg L−1 NAA + 2.5% sucrose + 150 mg L−1 NaH2PO4 + 1% banana homogenate. Effect of inoculum density (10, 30, 50, 70, and 90 g L−1), aeration volumes (0.05, 0.1, 0.2, and 0.3 vvm), and culture methods (raft, immersion, and Ebb and flood) were tested	PLB culture in shake flasks and bioreactors	Cultures were maintained at 25 ± 1 °C and under a 16 h day and 8 h dark photoperiod and a PPFD of 40 µmol m−2 s−1	Cui et al.  (2014a, b, 2015)
Dendrobium candidum	Alkaloids and polysaccharides	Antioxidants and immunostimulants	Protocorm induction: MS medium supplemented with 0.5 mg L−1 NAA + 2.0 mg L−1 BA + 10% coconut water + 3% sucrose PLB suspension culture in bioreactors: PLB suspension cultures in 3 L balloon-type bubble bioreactors containing 2 L MS medium supplemented with 0.5 mg L−1 NAA + 2.0 mg L−1 BA + 10% coconut water + 3% sucrose. Effect of carbon source (30 g L−1 sucrose, glucose, and fructose), salt strength of MS medium (1/4x, 1/2x, 3/4x, and 1x), phosphate concentration (0, 0.6, 1.2, 1.8, 2.4, and 3 mM) and NH4+/NO3− ratio (0/30/, 10/20, 15/15, 20/10. And 30/0 mM using NH4Cl and KNO3 using a constant nitrogen source of 30 mM) were tested	PLB culture in bioreactors	The bioreactors were aerated at 0.1 vvm and kept at 25 ± 1 °C and 16 h photoperiod and a PPFD of 40 µmol m−2 s−1	Yang et al. (2015)
Dendrobium candidum	Alkaloids, phenolics, flavonoids, and polysaccharides	Antioxidants and immunostimulants	PLB suspension culture in shake flasks: 3 g of PLBs were inoculated in 25 mL MS medium taken with 50 mL of Erlenmeyer flasks. The cultures were elicited with 25, 50, 75, 100, and 125 µM MeJA. Another set of cultures was treated with 75 µM MeJA or 75 µM SA or 75 µM MeJA + 75 µM SA or 37.5 µM MeJA + µM SA. PLB suspension culture in bioreactors: 20 g PLB suspension cultures in 3 L balloon-type bubble bioreactors containing 2 L MS medium and treated 75 µM MeJA after 30 days of culture, and treated for 2, 4, 6, 8, or 10 days of elicitation.	PLB culture in shake flasks and bioreactors	Both flask and bioreactor cultures were kept at 25 ± 1 °C and 16 h photoperiod and a PPFD of 40 µmol m−2 s−1. Bioreactors were aerated with 0.1 vvm.	Wang et al. (2016)
Dendrobium Enopi x Dendrobium Pink Lady	Phenolics, and flavonoids	Antioxidants	PLB proliferation in Petri plates: PLBs were cultured with half-strength MS medium 2% sucrose without growth regulators.	PLB culture	Cultures were maintained at 24 ± 2 °C and 16 h photoperiod. Effect of LED lights such as white light (400–700 nm), far-red (730 nm), green (530 nm), blue (440 nm), red (660 nm), and blue-red (440 and 660 nm) tested. In another experiment effect of LED light intensities such as white (4.63, 5.18, 17.0 µmol m−2 s−1), far-red (1.11, 9.12, 20.8 µmol m−2 s−1), green (0.77, 6.15, 16.9 µmol m−2 s−1), blue (0.91, 6.72, 15.7 µmol m−2 s−1), red (1.26, 15.40, 29.30 µmol m−2 s−1) and blue-red (2.01, 20.30 and 44.80 µmol m−2 s−1) were tested	Yeow et al. (2020)
Dendrobium Sabin Blue	Anthocyanin: Petunidin	Food coloring agent	PLB proliferation in Petri plates: PLBs were cultured with half-strength MS medium 2% sucrose + 1 mg L−1 BA. Effect of elicitors such as MeJA (25–200 µM), SA (25–200 µM), and melatonin (0.1–25 µM)	PLB culture	Cultures were maintained at 25 ± 2 °C and 16 h light and 8 h dark photoperiod	Malik et al. (2021)
Dendrobium Sabin Blue	Alkaloid: Dendrobine and Anthocyanin: Petunidin	Dendrobine is used as analgesic and antipyretic. Petunidin is used food coloring agent	PLB proliferation in Petri plates: PLBs were cultured with half-strength MS medium 2% sucrose + 1 mg L−1 BA. Effect of elicitors such as Yeast extract (50–200 mg L−1), chitosan (0.5–10 mg L−1), salicylic acid (5-200 mg L−1), silver nitrate (0.1–20 mg L−1) and glutamine (50–200 mg L−1) were tested	PLB culture	Cultures were maintained at 25 ± 2 °C and 16 h light and 8 h dark photoperiod	Chin et al. (2021)
Dendrobium officinale	Alkaloids, flavonoids, and polysaccharides	Dendrobium alkaloids are useful in Alzheimer’s treatment, and flavonoids and polysaccharides are immunostimulants	PLB regeneration and proliferation: PLBs were regenerated on half MS medium + 0.5 mg L−1 NAA + 1.0 mg L−1 BA + 2.0 mg L−1 2,4-D and 100 g L−1 potato homogenate	PLB culture	Cultures were maintained at 25 ± 2 °C and 16 h light and 8 h dark photoperiod	Wang et al. (2021)

2,4-D: 2,4-dichlorophenoxyacetic acid; BA: N⁶-benzyladenine; LED: Light emitting diodes; MeJA; Methyl jasmonate; MS: Murashige and Skoog (1962) medium; NAA: α-naphthalene acetic acid; PPFD: Photosynthetic photon flux density; PLB: Protocorm like bodies; TDZ: Thidiazuron; vvm: air volume/medium volume/minute