Recalcitrance ofCannabis sativatode novoregeneration; a multi-genotype replication study

Abstract

Full title (250 characters max):Recalcitrance of Cannabis sativa to de novo regeneration; a multi-genotype replication study Short title (100 characters max):Recalcitrance of Cannabis sativa to de novo regeneration methods Abstract 2Cannabis sativa is relatively recalcitrant to regeneration from somatic tissues, but several 3 reports have been published demonstrating a response. Most reports show low levels of 4 regeneration from somatic tissues, but a landmark publication by Lata et al. in 2010 reported 5 regeneration from leaf explants with a 96% response rate, producing an average of 12.3 shoots 6 per explant in a single, high-THC genotype. Despite the importance regeneration plays in plant 7 biotechnology this protocol has not been used in subsequent papers in the decade since it was 8 published, raising the concern that it is not reproducible. Many researchers are looking to build 9 research programmes in this growing field, and it is important that the reproducibility and 10 robustness of single-genotype C. sativa regeneration protocols undergo multi-lab validations to 11 ensure they are reproducible across the species. Replication studies in this burgeoning field will 12 help research groups avoid lost time and resources which arise from pursuing protocols that are 13 not reproducible. Here we test the replicability of this protocol across 10 drug-type C. sativa 14 genotypes. This protocol successfully induced callus in all 10 genotypes. Callus size and 15 appearance substantially differed among cultivars, with the most responsive genotype producing 16 6-fold more callus than the least responsive genotype. However, the most successful shoot 17 induction medium developed in the 2010 paper failed to induce regeneration in any of the 18 cultivars tested, resulting in the eventual necrosis of the calli. Based on this replication study, it 19 is evident that the existing regeneration protocol is not robust and could not be replicated in any 20 of the 10 genotypes tested. 4 22 2 Introduction 23 Plant tissue culture provides the foundation for advanced biotechnological techniques 24 such as protoplast systems, microspore culture for double haploid production, transgenics, 25 genome editing, and other important tools which have yet to be explored in Cannabis sativa L. 26 Due to prohibition, early micropropagation studies of Cannabis were limited in number and 27 scope. As a result, the majority of past research relied on single drug-type genotypes or on less 28 regulated industrial hemp (Cannabis sativa with <0. 0.03% w/w THC in the flowering heads) [1] 29 cultivars as a proxy for drug type Cannabis. These early studies found that both drug-type 30 Cannabis (high THC and/or high CBD) and industrial hemp can be maintained in vitro [2-4] and 31 that in vitro grown C. sativa display comparable chemical and physical profiles to greenhouse-32 grown counterparts [2], with no significant effect on the cannabinoid contents of high-THC 33 genotypes [4,5]. Regulations surrounding the production and consumption of Cannabis h...

The Past, Present and Future of Cannabis sativa Tissue Culture

“…While Page et al [77] demonstrated that DKW was more suitable for Stage 2 micropropagation of Cannabis, their findings likely represent an underestimate of the benefits as the study was conducted for only one subculture of Stage 2 plants, while the issues of culture decline generally increase over multiple subcultures. Using DKW based media, several cultivars of Cannabis have now been maintained for multiple years with no obvious signs of decline [89]. Despite the improvement over MS based media, some signs of nutrient deficiency were still observed, and further improvements are likely possible through optimization of basal media.…”

“…Earlier reviews on the state of C. sativa micropropagation and regeneration largely failed to underscore the many challenges in the existing body of research. These challenges include incomplete and ambiguously reported results [28,67,74,81,82,84,86], recalcitrance to regeneration [28,68,81,82,86,88]; genotype and tissue-specific responses to regeneration [68,81,82,87,88]; and a lack of reproducibility of successful protocols in the literature [77,89].…”

“…Reproducibility of existing methods remains a challenge in both regeneration systems [89] and in SM systems [80]. As previously discussed and with most contemporary US-based studies presenting data from a single cultivar [28,78,85,86], it is likely that this lack of genotypic diversity is contributing to the struggle with protocol reproducibility in Cannabis tissue culture.…”

“…Until recently, this decade-old protocol had not been replicated in any publication by an independent research group or used for subsequent biotechnological development by the original authors. A 2020 replication study testing this protocol across 10 SSR-characterized Cannabis cultivars found that callus was formed but no regeneration was observed [89]. The contradictory findings between these two studies raise concerns about the applicability of existing tissue cultures methods across genotypes and/or their reliability.…”

“…Strong cultivar-specific responses to treatments have been noted in Cannabis tissue culture and this likely contributes to the lack of reproducibility in both shoot proliferation and regeneration-based systems [55,74,77,79,87,89]. As Cannabis research becomes more accessible, the implications of drawing sweeping conclusions based on single-cultivar studies have come under scrutiny [12,89] and evidence has called into question the genotype-independent responses implicit in many of these single-cultivar studies [77]. It will be the goal of future studies to establish complete and detailed methodologies applied across a broad genotypic sample to overcome the recalcitrance in the species or at least to identify amenable cultivars to overcome the challenges currently faced in the tissue culture of Cannabis.…”