Table 2.
In vitro clonal propagation of Cannabis sativa via indirect organogenesis *.
| Explant | Explant/Decontamination | Steps and Culture Medium | Experimental Outcome | Pros | Cons | References |
|---|---|---|---|---|---|---|
| Seeds | Seeds: Sterilised in 5% Ca (ClO)2 for 6, 8 and 15 min |
Culture initiation MS medium |
Best sterilisation time was achieved after 15 min (5% hypochlorite solution) | None | Hemp seeds were highly contaminated | [68] |
| In vitro young leaves, petioles, internodes and axillary buds |
Callus induction/indirect regeneration MS medium + KN (1–4 mg/L), NAA (0.5–2 mg/L), 2,4-D (2–4 mg/L), DIC (2–3 mg/L) |
Callus was obtained from all explant types. Petiole explants with 2–3 mg/L DIC had the highest frequency of callus formation with 82.7% of explants | Explants derived from plants growing in pots | Low frequency of callus from internodes and axillary buds. Efficiency of plant regeneration is low | [68] | |
| In vitro regenerated plantlets |
Rooting MS medium + IAA (1 mg/L) and NAA (1.0 mg/L) |
69.95% of the plantlets formed roots | None | Further experiments needed to develop an efficient plant regeneration system | [68] | |
| Seeds | Seeds: sterilised in 70% ethanol for 10 s and in 1% NaClO for 20 min |
Culture initiation DARIA medium |
Explants of cotyledons, stems, and roots were excised from plantlets | None | None | [69] |
| In vitro cotyledons, stems, roots |
Callus induction DARIA medium + KN (1 mg/L) + NAA (0.05 mg/L) |
Callus was obtained from all explant types | The highest efficiency of morphogenic callus induction was noticed from cotyledon explants | Callus formed at root explants was incapable of morphogenesis and plant regeneration | [69] | |
| In vitro explants |
Indirect regeneration DARIA medium + BA (0.2 mg/L) + NAA (0.03 mg/L) |
Stem explants showed the highest regeneration rate percentage and cotyledon explants showed the highest efficiency in callus induction | The use of three media, DARIA ind+, DARIA pro +, and DARIA root +, supplemented with PGRs, enabled regeneration of plants with relatively high efficiency | None | [69] | |
| In vitro explants |
Rooting DARIA medium + IAA (2 mg/L) |
Rooted plants were transferred to soil | None | None | [69] | |
| Seeds | Seeds: sterilised with 70% ethanol for 30 s, 2% NaOCl for 20 min and 0.05% HgCl2 for 5 min |
Culture initiation MS medium |
Seeds produced seedlings for obtaining explants | None | None | [70] |
| In vitro cotyledon and epicotyl |
Callus induction MS medium + BA (0.1–3 mg/L), TDZ (0.1–3 mg/L) with or without IBA 0.5 mg/L |
Cotyledon explant showed better response compared to epicotyl explants in terms of the mass and size of the calli produced in various hormonal combination | The first response of explant to callus formation was observed after 11 days. The addition of IBA in various concentrations of BA had positive influence on callus induction | None | [70] | |
| In vitro calli |
Shoot induction MS medium + BA (0.1–3 mg/L), TDZ (0.1–3 mg/L) with or without IBA 0.5 mg/L |
Epicotyl explants showed better regeneration rate compared to cotyledon. Epicotyl explant callus treated with 2 mg/L BA and 0.5 mg/L IBA showed high shoot regeneration rate | None | None | [70] | |
| In vitro regenerated shoots |
Rooting MS medium + NAA (0.1–1 mg/L), IBA (0.1–1 mg/L) |
IBA (0.1 mg/L) showed highest rooting rate | None | Burning was observed in the shoots cultured in media supplemented with NAA hormone | [70] | |
| Young leaves | Young leaves: sterilised using 0.5% NaOCl, 15% (v/v) bleach |
Culture initiation/callus induction MS medium + IAA (0.09–0.35 mg/L), IBA (0.1–0.41 mg/L), NAA (0.09–0.37 mg/L), 2,4-D (0.11–0.44 mg/L) with 0.22 mg/LTDZ |
Optimum callus growth in 0.09 mg/L NAA + 0.22 mg/L μM TDZ | Rapid protocol for producing plantlets from young leaf tissue | The formation and growth of the callus was affected by the type of PGR and concentration applied | [71] |
| In vitro calli |
Shoot induction MS medium + BAP (0.11–2.25 mg/L), KN (0.12–2.15 mg/L), TDZ (0.11–2.2 mg/L) |
Highest shoot induction and proliferation was observed in 0.11 mg/L TDZ | None | None | [71] | |
| In vitro regenerated shoots |
Rooting ½ MS medium + IAA (0.09–1.75 mg/L), IBA (0.10–2.03 mg/L), NAA (0.09–1.86 mg/L) |
Shoots rooted best in ½ MS medium with 0.51 mg/L IBA. The presence of IBA resulted in significantly higher rooting percentage (80–96%) than IAA or NAA | None | None | [71] | |
| Leaves, flowers, 4-day-old seedlings | Leaves, flowers, and 4-day-old seedlings: washing with detergent, 70% EtOH for 3 min, sterilised distilled water for 10 min, 2% NaClO soak for 20 min |
Culture initiation/callus induction MS medium + mesoinositol (100 mg/L), thiamine diHCl (10 mg/L), pyridoxine HCl (1 mg/L), nicotinic acid (1 mg/L), 2,4-D (1 mg/L), sucrose (30 g/L) and agar (10 g/L) |
Flowers gave more callus while the leaves had less callus production | Callus was easily induced in standard medium | Cannabinoids were not produced in Cannabis cell cultures | [73] |
| In vitro calli |
Suspension cultures Liquid MS medium after 2 weeks: one part was maintained in the MS medium while the other was maintained in B5 medium (B5 components, 2,4-D 2.0 mg/L, IAA 0.5 mg/L, NAA 0.5 mg/L, K 0.2 mg/L and sucrose 30 g/L) |
Shoots from seedlings produced more callus than the stems and no callus was formed on the roots | None | None | [73,76] | |
| Immature embryo hypocotyls, true leaves, cotyledons and hypocotyls |
Immature embryo hypocotyls, true leaves, cotyledons and hypocotyls: sterilised using 2% (v/v) NaClO for 25 min followed by 75% (v/v) EtOH for 5 min |
Culture initiation: MS+ nicotinic acid (1 mg/L) + pyridoxine-HCl (1 mg/L) + thiamine-HCl (10 mg/L) + myo-inositol (0.1 g/L) + 3% sucrose + phytagel (2.5 g/L) + 2,4-D (1 mg/L) + KIN (0.25 mg/L) + casein (100 mg/L) Hydrolysate regeneration: 1/2 strength MS + 1.5% sucrose + phytagel (3.5 g/L) + TDZ (0.5 mg/L) + 6-BA (0.3 mg/L) + NAA (0.2 mg/L) + IAA (0.2 mg/L) Rooting: 1/2 strength MS + NAA (0.2 mg/L) + IBA (0.5 mg/L) + ZeaRIB (0.01 mg/L) |
Over 20% of the immature embryo hypocotyls developed embryogenic calli within 5 days. Hypocotyls collected 15 days after anthesis produced more calli than those collected earlier or later |
None | Genotype dependence of Cannabis | [83] |
| Leaf | Leaf material from in vitro shoots: no sterilisation mentioned |
Culture initiation/callus induction MS + TDZ (1.0 μM) Shoot induction MS + TDZ (0.5 μM) |
Callus was effectively induced in all 10 genotypes, yet the subsequent transfer of calli to shoot induction medium failed to initiate shoot organogenesis in any of the tested genotypes. Regeneration of Cannabis from somatic tissues is highly genotype specific |
None | This method is not suitable for inducing de novo regeneration across different genotypes | [45] |
BA/BAP—6-benzylaminopurine, EtOH—ethanol, HCl—hydrochloric acid, H2SO4—sulphuric acid, IAA—indole-3-acetic acid, IBA—indole-3-butyric acid, KIN—kinetin, MS—Murashige and Skoog, mT—meta-Topolin, NAA—1-naphthaleneacetic acid, NaCl—sodium chloride, NaOCl—sodium hypochlorite, PGR—plant growth regulator, TDZ—thidiazuron, ZEA—zeatin, and 2,4-D—2,4-dichlorophenoxyacetic acid. * The list in this table may not be completely exhaustive.