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

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.

Somaclonal Variation—Advantage or Disadvantage in Micropropagation of the Medicinal Plants

“…Moreover, SSR markers are prone to have "null alleles", i.e., no amplification of the intended PCR product, due to the mutation that can occur in the primer annealing sites, which may lead to errors in scoring. 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].…”

Plant Regeneration via Organogenesis in Jerusalem Artichokes and Comparative Analysis of Endogenous Hormones and Antioxidant Enzymes in Typical and Atypical Shoots

“…Contrarily, when using random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and start codon targeted (SCoT) marker techniques, polymorphisms were detected among in vitro-induced cultures of Jerusalem artichoke by Abdalla et al [14]. Ioannidis et al [47] found that SSR markers could not effectively detect all genetic variability in the micropropagated plants of Cannabis sativa because of its large genome size. Therefore, more molecular markers and more primers should be adopted to obtain more molecular information about regenerated plants in the future.…”

Challenges and potentials of new breeding techniques in Cannabis sativa

“…Other types of markers have also been shown to be useful; autosomal microsatellite markers and markers based on mitochondrial and chloroplast DNA. SSR’s have been used to differentiate between samples ( Soler et al., 2016 ; Dufresnes et al., 2017 ; Houston et al., 2017 ; Soler et al., 2017 ; Houston et al., 2018 ; de Oliveira Pereira Ribeiro et al., 2020 ; Ioannidis et al., 2022b ). It seems that a limited number of markers, from 6 to 13, is enough, not only to sort Cannabis from hop, but to individualize and differentiate between types (drug versus hemp) and to say something about geographic origin ( Houston et al., 2017 ; de Oliveira Pereira Ribeiro et al., 2020 ).…”