Abstract
Micropropagation is a crucial technique for the large-scale production of genetically uniform and pathogen-free Cannabis sativa . However, genotype-dependent responses and low multiplication rates often limit its efficiency. This study investigated the effects of basal media (Driver and Kuniyuki Walnut (DKW) and Murashige and Skoog (MS), plant growth regulators (thidiazuron (TDZ), indole butyric acid IBA, and gibberellic acid (GA₃)), and the mitogenic factor phytosulfokine-alpha (PSK-α) on the propagation and rooting efficiency of five medical cannabis genotypes. Simultaneous ex vitro rooting and acclimatization were also investigated as a streamlined approach for adapting in vitro plantlets grown under greenhouse conditions. Shoot multiplication was significantly influenced by the interaction between genotype and culture medium. While the number of shoots did not differ significantly between DKW and MS media, DKW promoted a greater shoot length, more nodes per culture, and a higher biomass. TDZ addition increased the shoot multiplication rate but reduced shoot length and increased callus formation. A decline in shoot length and number of nodes was observed across subcultures, particularly on DKW-based media and less on MS-based media. GA₃ effectively promoted shoot elongation and facilitated subsequent rooting and acclimatization. Suboptimal in vitro rooting led to the development of a novel ex vitro rooting protocol that combined rooting and acclimatization and achieved high success rates within 3 weeks, significantly reducing the overall micropropagation time. PSK-α had no significant effect on shoot multiplication or elongation. Furthermore, epigenetic analysis indicated progressive DNA hypomethylation in micropropagated shoots compared to greenhouse-grown plants, which increased with prolonged in vitro culture, possibly affecting gene expression and development. These results provide valuable insights into the processes of cannabis micropropagation and emphasize the need for further research on in vitro -induced epigenetic changes, including high-performance liquid chromatography based global DNA methylation analysis, bisulfite sequencing of differentially methylated genes and their regulatory regions, and the functional consequences of methylation changes on gene expression and plant morphological and physiological traits. • Micropropagation efficiency in cannabis varies by genotype and culture medium. • TDZ enhances shoot proliferation but reduces shoot length and increases basal callus formation. • Ex vitro rooting significantly improves cannabis rooting and acclimatization success. • In vitro culture indicates progressive DNA hypomethylation in cannabis shoots. • MSAP-Seq is a reliable method for analyzing DNA methylation in cannabis.
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Claims 13
Claim · model-written summary
Shoot multiplication was significantly influenced by the interaction between genotype and culture medium
- Taxon
- Cannabis sativa
Cannabis sativae0 - Material
- genotypee1
- Treatment
- culture mediume1
- Outcome
- Shoot multiplicatione1
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pathogen-free Cannabis sativa”
e1“Shoot multiplication was significantly influenced by the interaction between genotype and culture medium”
Claim · model-written summary
DKW and MS media did not differ significantly in shoot number
- Taxon
- Cannabis sativaCannabis sativae0
- Treatment
- DKWe1
- Outcome
- number of shootse1
- Direction
- did not differ significantlye1
- Comparator
- MS mediae1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pathogen-free Cannabis sativa”
e1“While the number of shoots did not differ significantly between DKW and MS media”
Claim · model-written summary
DKW promoted shoot length, nodes per culture and biomass compared with MS media
- Taxon
- Cannabis sativaCannabis sativae0
- Treatment
- DKWe1
- Outcome
- shoot lengthe1
- Direction
- promotede1
- Qualifier
- more nodes per culture, and a higher biomasse1
- Comparator
- MS mediae1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pathogen-free Cannabis sativa”
e1“While the number of shoots did not differ significantly between DKW and MS media, DKW promoted a greater shoot length, more nodes per culture, and a higher biomass”
Claim · model-written summary
TDZ addition increased the shoot multiplication rate
- Taxon
- Cannabis sativaCannabis sativae0
- Treatment
- TDZ additione1
- Outcome
- shoot multiplication ratee1
- Direction
- increasede1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pathogen-free Cannabis sativa”
e1“TDZ addition increased the shoot multiplication rate but reduced shoot length and increased callus formation”
Claim · model-written summary
TDZ addition reduced shoot length
- Taxon
- Cannabis sativaCannabis sativae0
- Treatment
- TDZ additione1
- Outcome
- shoot lengthe1
- Direction
- reducede1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pathogen-free Cannabis sativa”
e1“TDZ addition increased the shoot multiplication rate but reduced shoot length and increased callus formation”
Claim · model-written summary
TDZ addition increased callus formation
- Taxon
- Cannabis sativaCannabis sativae0
- Treatment
- TDZ additione1
- Outcome
- callus formatione1
- Direction
- increasede1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pathogen-free Cannabis sativa”
e1“TDZ addition increased the shoot multiplication rate but reduced shoot length and increased callus formation”
Claim · model-written summary
Shoot length and number of nodes declined across subcultures, more on DKW-based media than on MS-based media
- Taxon
- Cannabis sativaCannabis sativae0
- Treatment
- subculturese1
- Outcome
- shoot length and number of nodese1
- Direction
- declinee1
- Qualifier
- particularly on DKW-based media and less on MS-based mediae1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pathogen-free Cannabis sativa”
e1“A decline in shoot length and number of nodes was observed across subcultures, particularly on DKW-based media and less on MS-based media”
Claim · model-written summary
GA₃ promoted shoot elongation
- Taxon
- Cannabis sativaCannabis sativae0
- Treatment
- GA₃e1
- Outcome
- shoot elongatione1
- Direction
- promotede1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pathogen-free Cannabis sativa”
e1“GA₃ effectively promoted shoot elongation and facilitated subsequent rooting and acclimatization”
Claim · model-written summary
GA₃ facilitated rooting and acclimatization
- Taxon
- Cannabis sativaCannabis sativae0
- Treatment
- GA₃e1
- Outcome
- rooting and acclimatizatione1
- Direction
- facilitatede1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pathogen-free Cannabis sativa”
e1“GA₃ effectively promoted shoot elongation and facilitated subsequent rooting and acclimatization”
Claim · model-written summary
The ex vitro rooting protocol achieved high rooting and acclimatization success within 3 weeks
- Taxon
- Cannabis sativaCannabis sativae0
- Treatment
- ex vitro rooting protocole1
- Outcome
- success ratese1
- Direction
- achievede1
- Qualifier
- within 3 weekse1
- Setting
- ex vitroe1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pathogen-free Cannabis sativa”
e1“Suboptimal in vitro rooting led to the development of a novel ex vitro rooting protocol that combined rooting and acclimatization and achieved high success rates within 3 weeks, significantly reducing the overall micropropagation time”
Claim · model-written summary
The ex vitro rooting protocol significantly reduced the overall micropropagation time
- Taxon
- Cannabis sativaCannabis sativae0
- Treatment
- ex vitro rooting protocole1
- Outcome
- overall micropropagation timee1
- Direction
- reducinge1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pathogen-free Cannabis sativa”
e1“Suboptimal in vitro rooting led to the development of a novel ex vitro rooting protocol that combined rooting and acclimatization and achieved high success rates within 3 weeks, significantly reducing the overall micropropagation time”
Claim · model-written summary
PSK-α had no significant effect on shoot multiplication or elongation
- Taxon
- Cannabis sativaCannabis sativae0
- Treatment
- PSK-αe1
- Outcome
- shoot multiplication or elongatione1
- Direction
- no significant effecte1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pathogen-free Cannabis sativa”
e1“PSK-α had no significant effect on shoot multiplication or elongation”
Claim · model-written summary
DNA hypomethylation in micropropagated shoots increased with prolonged in vitro culture, relative to greenhouse-grown plants
- Taxon
- Cannabis sativaCannabis sativae0
- Treatment
- prolonged in vitro culturee1
- Outcome
- DNA hypomethylatione1
- Direction
- increasede1
- Qualifier
- progressivee1
- Comparator
- greenhouse-grown plantse1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“pathogen-free Cannabis sativa”
e1“epigenetic analysis indicated progressive DNA hypomethylation in micropropagated shoots compared to greenhouse-grown plants, which increased with prolonged in vitro culture, possibly affecting gene expression and development”
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