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

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 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 varieties and demonstrate the reliability of this in vitro propagation system.