The Influence of Cytokinin on the Multiplication Efficiency and Genetic Stability of Scutellaria baicalensis Regenerants in In Vitro Culture Conditions

Abstract

The efficiency and method of regeneration in in vitro culture conditions depend primarily on the plant growth regulators (PGRs) used. Even growth regulators belonging to one group may have different effects, stimulating the process of direct or indirect organogenesis, thus possibly disturbing the genetic stability among regenerants. The main aim of this study was to identify the genetic stability of Scutellaria baicalensis regenerates obtained by in vitro culture method using start codon targeted (ScoT) markers. S. baicalensis nodal explants were regenerated on MS medium supplemented with kinetin (KIN) at concentrations of 0.25, 0.5, 1.0, and 2.0 mg × dm−3 or benzylaminopurine (BAP)—0.25, 0.5, 1.0, and 2.0 mg × dm−3. The effects of the number of propagated shoots, length, number of nodes, and fresh mass of regenerants were assessed. Moreover, the genetic stability of the regenerants was analyzed using start codon targeted (SCoT) markers. Direct shoot organogenesis was observed on an MS medium containing kinetin, while indirect shoot induction occurred on an MS medium supplemented with BAP. The highest average number of shoots (3.6) was achieved for the MS + KIN medium at a concentration of 0.25 and 5.8 for the MS + BAP 1.0 medium. The average length and average number of nodes were the highest on the MS + BAP 0.25 medium (50.0 and 6.0, respectively), while the lowest values of these features were observed on the MS + KIN 2.0 medium (40.3 and 4.9, respectively). A total of 111 amplified bands were exhibited by SCoT primers. Three of the analyzed primers revealed four unique genotype-specific markers. The average percentage of polymorphism obtained was 36.7%. The analysis of genetic similarity revealed a high level of genetic similarity between the donor plant and regenerants obtained on MS “0” (medium without the addition of phytohormones). A slightly lower value of genetic similarity was observed for regenerants obtained by direct organogenesis (MS + KIN medium at all concentrations). Indirect shoot organogenesis observed on the MS + BAP medium (all concentrations) resulted in the highest differentiation, both in relation to the donor plant and MS “0” regenerants. The results of our work indicate that, in the case of S. baicalensis, the maintenance of genetic stability depends primarily on the presence of the cytokinin type in the medium.