High-frequency plant regeneration and genetic fidelity assessment of regenerants by molecular and biochemical markers in Woodland Sage (Salvia nemorosa L.)

Salvia nemorosa L. is a valuable medicinal and ornamental plant from Lamiaceae. A high-frequency protocol for in vitro regeneration from S. nemorosa leaf explants was developed based on our results. Various plant growth regulators were evaluated for their effects on direct and indirect organogenesis, callogenesis, shoot multiplication, and rooting of explants at different concentrations. The maximum percentage of direct shoot regeneration (97.5%) and the number of shoots per explant (8.6) were obtained on MS medium supplement with 8 mu M TDZ plus 8 mu M NAA. The result revealed that media amended by 16 mu M 2, 4-D with 8 or 16 mu M BA was the best treatment for callus induction. Media containing the combination of 8 mu M TDZ plus 2 mu M NAA provided the highest rate of shoot induction (84%) and the highest number of shoots (5.4) from calli. Regenerated shoots were multiplied efficiently with an average of 7.4 shoots and 32.4 leaves per explant on media containing 12 mu M TDZ. In the present study, TDZ and BA treatments led to the highest proliferation rate; however, adenine sulfate was preferred for proliferation because it did not produce hyperhydric plants. The genetic fidelity of 19 plantlets (one donor plant, nine direct and nine indirect regenerants) was appraised by 10 ISSR markers. The molecular analysis revealed that direct organogenesis produced true-to-type plantlets and could be employed for clonal multiplication of Woodland Sage. Indirect regenerants showed polymorphic bands (12.5-33.33%), confirming the somaclonal variation occurrence during indirect organogenesis. Also, HPLC analysis of regenerants showed that two indirect regenerants accumulated higher rosmarinic acid content than mother plants, while three of them accumulated lower rosmarinic acid content. As part of this study, molecular and biochemical markers have been employed to investigate somaclonal variation in in vitro regeneration of the Salvia genus for the first time, which could be utilized to develop reliable methods for multiplying elite lines and achieving high-yield lines.