Recalcitrance of Cannabis sativa to de novo regeneration; a multi-genotype replication study

PLoS One. 2021 Aug 13;16(8):e0235525. doi: 10.1371/journal.pone.0235525. eCollection 2021.

Abstract

Cannabis sativa is relatively recalcitrant to de novo regeneration, but several studies have reported shoot organogenesis or somatic embryogenesis from non-meristematic tissues. Most report infrequent regeneration rates from these tissues, but a landmark publication from 2010 achieved regeneration from leaf explants with a 96% response rate, producing an average of 12.3 shoots per explant in a single drug-type accession. Despite the importance regeneration plays in plant biotechnology and the renewed interest in this crop the aforementioned protocol has not been used in subsequent papers in the decade since it was published, raising concerns over its reproducibility. Here we attempted to replicate this important Cannabis regeneration study and expand the original scope of the study by testing it across 10 drug-type C. sativa genotypes to assess genotypic variation. In our study, callus was induced in all 10 genotypes but callus growth and appearance substantially differed among cultivars, with the most responsive genotype producing 6-fold more callus than the least responsive. The shoot induction medium failed to induce shoot organogenesis in any of the 10 cultivars tested, instead resulting in necrosis of the calli. The findings of this replication study raise concerns about the replicability of existing methods. However, some details of the protocol could not be replicated due to missing details in the original paper and regulatory issues, which could have impacted the outcome. These results highlight the importance of using multiple genotypes in such studies and providing detailed methods to facilitate replication.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cannabis / genetics*
  • Cannabis / growth & development*
  • Genotype
  • Plant Leaves / genetics
  • Plant Leaves / growth & development
  • Plant Roots / genetics
  • Plant Roots / growth & development
  • Plant Shoots / genetics
  • Plant Shoots / growth & development
  • Regeneration / genetics*
  • Reproducibility of Results

Grants and funding

The authors gratefully acknowledge our industry partner, Hexo Corp. for the use of their plant material. The financial support of the Natural Sciences and Engineering Research Council of Canada (Grant No. RGPIN-2016-06252; awarded to AMPJ) is also gratefully acknowledged. Hexo Corp. (https://www.hexocorp.com/) and the Natural Sciences and Engineering Research Council of Canada (https://www.nserc-crsng.gc.ca/index_eng.asp) were not involved in study design, data collection and analysis, the decision to publish or the preparation of the manuscript.