Table 1.
In vitro clonal propagation of Cannabis sativa via direct organogenesis *.
| Explant | Explant/Decontamination | Steps and Culture Medium | Experimental Outcome | Pros | Cons | References |
|---|---|---|---|---|---|---|
| Seeds | Seeds: sterilised in 75% (v/v) EtOH for 1 min, rinsed in 5% (v/v) active NaCl for 15 min |
Culture initiation PGR-free MS medium |
Best explant response (59–70%) and highest number of shoots per explant recorded for shoot tip explants cultured on medium supplemented with TDZ | Did not utilise PGRs with cytokinin activity, which minimised the risk of soma clonal variation | Regeneration was low, 74% of nodal segments and 82% of shoot tips not growing | [61] |
| In vitro shoot tips and nodal segments with one axillary bud without leaves (seedlings) |
Shoot induction MS medium + BAP (0.5–2.0 mg/L), TDZ (0.1–0.5 mg/L), mT (0.1–1.0 mg/L) |
Best regeneration rate obtained from TDZ at 0.2 mg/L. Nodal segments less responsive and growth of only one shoot per explant regardless of the tested PGR | Shorter micropropagation duration time. Does not require elongation step | TDZ use related to phenotypic vitrification, leaf rolling, leaf narrowing and supressed growth of shoots. High BAP and mT concentrations also related to phenotypic changes in regenerated plants | [61] | |
| In vitro plantlets |
Rooting ½ MS medium + IAA (0.25–0.75 mg/L) and or IBA (0.25–0.75 mg/L) |
No significant difference observed in tested auxins in terms of rooting rates | It can be used for germplasm conservation and breeding. Rooting limited to 21 days due to rapid growth of shoots in culture. Plantlets obtained within 66–70 days | Number of plantlets from single explant was low. Protocol thus not suitable for industrial application | [61] | |
| Seeds | Seeds: surface sterilised in 75% (v/v) ethanol for 2 min and 30 s, soaked in NaClO for 25 min |
Culture initiation ½ MS medium |
Hypocotyl was significantly better than cotyledon leaves in terms of shoot organogenic potential | This is the first report of direct in vitro regeneration of plants from hypocotyls | Leaves displayed a poor ability to promote shoot organogenesis | [47] |
| In vitro cotyledons, hypocotyls and true leaves |
Shoot induction Medium + TDZ (0.4–1.0 mg/L), NAA (0.02–0.2 mg/L), BAP (0.5–2.0 mg/L), IBA (0.5 mg/L), 2,4-D (0.1 mg/L), ZEARIB (1.0–2.0 mg/L), BAPRIB (1.0 mg/L), 4-CPPU (1.0 mg/L) |
Medium containing (TDZ 0.4 mg/L + NAA 0.2 mg/L) was the best, achieving the highest shoot induction rate of 22.32% | None | Medium without PGRs and ZEARIB 1 mg/L + NAA 0.02 mg/L were the worst treatments, without any explant showing response in terms of shoot organogenesis | [47] | |
| Seeds | Seeds: surface sterilised by washing under running water with a few drops of detergent, 0.2% mercury chloride for 13 min |
Culture initiation MS medium |
Plantlets were grown from seeds | None | None | [63] |
| In vitro shoot tips |
Shoot induction MS medium + BAP (0.4 mg/L)/TDZ (0.1 mg/L)/mT (0.5 mg/L) + NAA (0.1 mg/L)/IAA (0.1 mg/L)/GA3 (2.3 mg/L) |
In both varieties, the highest stem was observed when cultured on medium supplemented with TDZ and GA3, and the shortest stem recorded on medium supplemented with TDZ and NAA |
None | The presence of NAA strongly influenced callus formation and general shoot architecture. Difficult to tell which extent longer stems are a genotypic trait |
[63] | |
| In vitro plantlets |
Rooting MS medium + IBA (0.5 mg/L) + activated charcoal |
The most vital plantlets of both genotypes with the highest number of roots were observed on medium where phytohormones were not present or on medium supplemented with mT (0.5 mg/L) | Culture media supplemented with mT without any phytohormones produced the best overall appearance of plantlets | None | [63] | |
| Seeds | Seeds: soaked with H2SO4 for 20 s, sterilised in 75% ethanol for 2 min, 3% (v/v) NaClO for 20 min |
Culture initiation MS medium |
Seeds grew up to seedlings and cotyledons were excised as explants to induce in vitro shoots | None | None | [52] |
| Cotyledons excised from seedlings (aseptic seedlings obtained from sterilised seeds) |
Shoot induction MS medium + TDZ (0.1–0.4 mg/L), BA (4–8 mg/L), ZT (0.5–1.5 mg/L) with or without NAA (0.1–0.6 mg/L) |
Cotyledon cultured in medium containing TDZ with or without the addition of NAA were capable of inducing formation of a nodular callus. Induction rate lower when using only TDZ. Peak of 51.7% induction frequency in MS medium + TDZ (0.4 mg/L) + NAA (0.2 mg/L) | Rapid shoot regeneration. No limitation of cultural season due to the use of cotyledons | This regeneration protocol is genotype dependent | [52] | |
| In vitro shoots |
Rooting ½ MS Medium with IBA (0.2–2 mg/L) |
IBA (0.5–2 mg/L) had 80% root induction | None | None | [52] | |
| Seeds | Seeds: washed for 20 min with 0.1% antiseptic APSA80 liquid detergent, sterilised in 75% (v/v) ethanol for 30 s and 0.1% mercuric chloride for 10–15 min |
Culture initiation ½ MS medium with 10 g/L sucrose and 5.5 g/L agar |
Shoot tips were harvested from 20-day-old sterile plantlets | None | None | [51] |
| Shoot tips harvested from 20-day-old sterile plantlets (aseptic seedlings obtained from sterilised seeds) |
Regeneration MS medium + BA (1–5 mg/L), KT (1–5 mg/L), TDZ (1–5 mg/L) with or without NAA (1–5 mg/L) |
TDZ (0.2 mg/L) provided the best bud induction, producing an average of 3.22 buds. 0.1 mg/L NAA was optimal concentration for auxiliary bud induction | CKs stimulated shoot formation and stem enlargement in each explant | Type of CK affected plantlet morphology | [51] | |
| In vitro plantlets |
Rooting MS medium + IBA (0.1–0.5 mg/L) + NAA (0.05–0.25 mg/L), IAA (0.05–0.25 mg/L) |
85% rooting response in IBA (0.1 mg/L) and NAA (0.05 mg/L) explants | None | None | [51] | |
| Axillary buds | Axillary buds: surface disinfected by maintaining them under stirred tap water for 1 h; 30 min immersion in 15% (v/v) bleach, stirred solution |
Culture initiation and shoot induction MS medium with or without vitamins/Formula βH/Formula βA + 0.48 mg/L mT or 0.37 mg/L NAA + 0.41 mg/L IBA with or without MS basal salts, Formula βH basal salts, Formula βA basal salts, with or without MS vitamins |
100% survival of axillary buds was observed for all cultivars at least under one studied media. Most of the varieties survived and reacted better without the addition of MS vitamins. Use of PGRs was variety dependent: some cultivars responded better to the addition of mT instead to NAA+IBA | This study confirmed that the success of in vitro introduction of C. sativa is cultivar dependent | Different cultivars of the same species have a completely different response to the same medium | [60] |
| Nodal segments with axillary buds | Nodal segments containing young axillary buds: sterilised in 2% NaOCl, 0.1% (v/v) Tween 20 for 5 min |
Culture initiation MS medium + activated charcoal |
None | None | None | [65] |
| In vitro explants |
Shoot induction MS medium + 0.1 mg/L NAA + 0.4 mg/L kinetin |
None | None | None | [65] | |
| In vitro plantlets |
Rooting MS medium + 0.1 mg/L NAA + 0.4 mg/L kinetin + 1.0 mg/L IBA |
None | None | None | [65] | |
| Disinfected axillary buds |
Oryzalin treatments Shoot induction medium + 17.32, 34.62, 51.95 mg/L oryzalin or MS medium + 6.93, 13.85, 20.78 mg/L oryzalin |
62.5% to 87.5% survival rate for explants treated with 6.92 mg/L oryzalin | The treatment of axillary buds with oryzalin is an effective method for chromosome doubling | Poor survival rate of explants treated with high oryzalin concentrations with 0% of explants surviving the 51.95 mg/L | [65] | |
| Shoot tips | In vitro shoot tip cuttings |
Maintenance of stock plants in ventilated glass jars ¼ Rockwool block placed onto glass preservation jars (3 shoot tip cuttings for each block) |
The self-built preservation jars were more suited for the culture of Cannabis as they provided more head space | The stock cultures could be maintained for at least 6 months. Excellent-quality plantlets | Wilting plants (blocks too dry/humidity too low). Deterioration of plants due to the blocks being too wet | [56] |
| In vitro shoot tip cuttings |
Maintenance of stock plants using RITA® system Nutrient solution (20 mL), Canna Aqua Vega Fertiliser. RITA container with 3 rockwool blocks each (2 shoot tip cuttings in each block), nutrient solution (75 mL), jars connected via tubing to a 1 bar pressure pump |
The RITA® system was more practicable in terms of handling because of the wide opening | Relies on industry-based fertiliser, rockwool blocks and forced ventilation. No requirement of growth regulators. No sugar or vitamins required | Stunted plants or yellow leaves (nutrient deficiency) | [56] | |
| In vitro shoot tips |
Rooting Glass vessel, 2 rockwool blocks, nutrient solution (20 mL) |
97.5% of in vitro shoot tip cuttings were rooted and acclimatised within 3 weeks inside the growth chamber | None | None | [56] | |
| Shoots | Shoots from immature and mature inflorescences: surface sterilised in ethanol for 1 min, followed by 10% v/v bleach for 10 min, washed in sterile water for 50 s |
Culture initiation MS medium + TDZ (0–2.2 mg/L) |
TDZ was shown to be among the most effective PGRs for shoot proliferation and de novo regeneration | First known report of shoot regeneration from floral tissues | None | [48] |
| In vitro explants with regenerating shoots |
Shoot regeneration/rooting MS medium + KIN (0.40 mg/L) + NAA (0.10 mg/L) + activated charcoal |
Regeneration was occurring from existing meristematic tissue, but this was not specifically determined | First report of shoot regeneration or plant propagation at reproductive phase | Further work needed to refine the protocol | [48] | |
| Nodal segments with axillary buds | Nodal segments containing axiliary buds: disinfected with 0.5% NaOCl for 20 min |
Shoot induction MS medium + TDZ (0.01–1.10 mg/L) + 500 mg/L activated charcoal |
In TDZ, of the different concentrations tested, the highest average number of shoots was obtained in MS + 0.5 µM TDZ | One step protocol for promoting shoot formation and root induction in the same medium | None | [54] |
| In vitro explants with regenerating shoots |
Shoot formation/Rooting ½ MS medium + IBA (0.01–1.01 mg/L), mT (0.01–1.21 mg/L) |
100% of explants exposed to with 0.48 mg/L mT produced shoots. Shoot number and shoot length was higher when using mT compared to TDZ. The best concentration for rooting was 0.05 mg/L mT | High shoot proliferation rate. Proof of the safety of mT for large-scale production. 96% of regenerated shoots were able to develop roots | mT concentrations higher than 0.97 mg/L were inhibitory to rooting | [54] | |
| Nodal segments with axillary buds | Nodal segments containing auxiliary buds: sterilised using 0.5% NaOCl for 20 min |
Shoot induction MS medium + BA (0.01–2.03 mg/L), KN (0.01–1.94 mg/L), TDZ (0.01–1.98 mg/L) with or without GA3 (2.42 mg/L) |
TDZ was the most effective PGR for shoot proliferation. 100% culture response when using TDZ (0.11 mg/L), with an average of 13 shoots per explant |
Regeneration of many plants in a short period of time. GA3 can act as a replacement for auxins in shoot induction | TDZ concentrations higher than 1.1 mg/L supressed shoot formation | [50] |
| In vitro shoots |
Rooting MS medium + IAA (0.44–0.88 mg/L), IBA (0.51–1.02 mg/L), NAA (0.47–0.93 mg/L) with or without 500 mg/L activated charcoal |
94% response of cultures in IBA (0.51 mg/L) with an average of 4.8 roots per explant | Addition of activated charcoal was effective in root induction | Profuse callus formation was observed when using IAA and IBA | [50] | |
| Nodal segments with axillary buds | Apical nodal segments containing axillary bud: sterilised using 0.5% NaOCl for 20 min |
Shoot initiation MS medium + BA, KN, TDZ (concentrations not mentioned) |
Quality and quantity of shoot regenerants in cultures were best with 0.11 mg/L TDZ | None | None | [64] |
| Apical nodal segments containing axillary bud: sterilised using 0.5% NaOCl for 20 min |
Rooting ½ MS medium + activated charcoal + IAA + IBA + NAA (concentrations not mentioned) |
Highest percentage of rooting achieved in ½ MS with 500 mg/dm3 activated charcoal supplemented with 0.51 mg/L IBA | None | None | [64] | |
| Nodal segments with axillary buds | Nodal segments containing axillary buds: sterilised using 1.67% (C(O)NCl)₂ + Tween 20 for 8 min |
Shoot initiation: MS + TDZ (0.011– 1.76 mg/L), mT (0.012–1.93 mg/L), BAP (1–5 mg/L), IAA (0.1 mg/L) |
MS medium + 0.1 mg/L TDZ resulted in the highest regeneration of shoots. Tissue culture responsiveness was genotype dependent |
None | Results demonstrated the recalcitrance of Cannabis in tissue culture and its poor multiplication rate | [66] |
| Apical shoot tip | Apical shoot tip+ node |
Shoot initiation: DKW medium without PGRs |
The highest number of harvested shoot tips was found in the 46 µmol/m2/s in non-vented vessels |
Unlike traditional micropropagation, this method re-uses the same rooted basal stem section of the initial explant over several apical tip removal cycles, resulting in a higher number of shoot tips | None | [67] |
Abbreviations: BA/BAP—6-benzylaminopurine, 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, RITA—temporary immersion system for tissue culture, TDZ—thidiazuron, ZEA—zeatin, and 4-CPPU—forchlorfenuron. * The list in this table may not be completely exhaustive.