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Cited by 31Plant tissue culture and regenerationGenetic diversity and population structureGenetic Mapping and Diversity in Plants and Animals

Genetic Evaluation of In Vitro Micropropagated and Regenerated Plants of Cannabis sativa L. Using SSR Molecular Markers

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Ioannidis, Kostas, Tomprou, Ioanna, Mitsis, Vangelis, Koropouli, Polyxeni·2022·Plants
DOI 10.3390/plants11192569PMC PMC9573407
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Abstract

Simple sequence repeat (SSR) markers were used to evaluate the genetic stability of the acclimatized micropropagated and regenerated plants of a high cannabidiol (H-CBD) and a high cannabigerol (H-CBG) variety of Cannabis sativa L. Shoot regeneration and proliferation were achieved by culturing calli in Murashige and Skoog basal medium (MS) supplemented with several concentrations of 6-benzyladenine (BA) or thidiazuron (TDZ). Calli derived mostly from stem explants, rather than leaves, cultured on MS supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) or combination of kinetin (KIN) with 1-Naphthaleneacetic acid (NAA) or 2,4-D. Rooting of the regenerated plantlets accomplished on half-strength MS medium supplemented with indole-3-butyric acid (IBA). Previous studies performed have developed an efficient in vitro micropropagation protocol for mass production. Both in vitro methodologies can be employed in genetic breeding via molecular techniques. The genetic stability of micropropagated and regenerated plants was accomplished using twelve SSR primer pairs that produced reproducible and clear bands, ranging from 90 to 330 bp in size, and resulted in amplification of one or two alleles, corresponding to homozygous or heterozygous individuals. The SSR amplification products were monomorphic across all the micropropagated and regenerated plants and comparable to mother plants. The monomorphic banding pattern confirmed the genetic homogeneity of the in vitro cultured acclimatized and mother plants as no somaclonal variation was detected in clones for these specific SSRs. Our results evidently suggest that the developed culture protocols for in vitro multiplication is appropriate and applicable for clonal mass propagation of the C. sativa varieties and demonstrate the reliability of this in vitro propagation system.

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  • In Cannabis sativa callus culture, no variation was observed.

    “No variation observed.”

    From Somaclonal Variation and Clonal Fidelity in Commercial Micropropagation: Challenges and Perspectives (2025) · cites this paper as [128]

    The Results (section 2.2; page unavailable) report no somaclonal variation by 12 SSR markers between regenerated plants and their donor plants. This supports the claim if “variation” means variation detected by those markers in callus-derived plants, but the paper does not establish that no variation occurred in the callus cultures themselves. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • SSR markers are considered genetically stable because, when used, they did not reveal any polymorphism, or only a very low level of it, between the original plants and the regenerants.

    “SSR markers are considered genetically stable because, when used, they did not reveal any polymorphism, or only a very low level of it, between the original plants and the regenerants [81,82,83].”

    From Somaclonal Variation—Advantage or Disadvantage in Micropropagation of the Medicinal Plants (2023) · cites this paper as [83]

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References 108 · Cited by 31

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Authors
Ioannidis, K., Tomprou, I., Mitsis, V., & Koropouli, P.
Affiliations
  • Hellenic Agricultural Organization - DIMITRA (GR)
  • Ekati Alchemy Lab SL, 08180 Moià, Spain
  • Ministry of Education and Religious Affairs (GR)
Corresponding author
Ioannidis, Kostas
ORCID
Venue
Plants
Publisher
MDPI AG
Published
2022-09-29
Volume
11
Issue
19
Pages
2569
ISSN
2223-7747 (ISSN-L)
DOI
10.3390/plants11192569
PMC
PMC9573407
OpenAlex
W4298145888
Cited by
31
References
108
Citations by year
2026: 8 · 2025: 9 · 2024: 8 · 2023: 5 · 2022: 1
FWCI
1.88
Type
journal article
Language
en
Open access
Licence
Locations
  • Plants (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Ekati Alchemy Lab SL
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant tissue culture and regeneration
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic diversity and population structure
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic Mapping and Diversity in Plants and Animals
Keywords
Cannabis sativa, micropropagation, somaclonal variation, genetic stability, shoot regeneration, callus culture, 6-benzyladenine, thidiazuron, 2,4-D, indole-3-butyric acid rooting, cannabidiol, cannabigerol, microsatellite markers, acclimatization

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