In vitro propagation of Spiraea betulifolia subsp. aemiliana (Rosaceae) and comparative analysis of phenolic compounds of microclones and intact plants

Abstract

In vitro culture of Spiraea betulifolia subsp. aemiliana (C.K. Schneid.) H. Hara from two natural populations—on Sakhalin and Kunashir Islands—was induced and an analysis of phenolic compounds from in vitro microshoots as compared with in vivo plants was performed for the first time. Active adventitious shoot formation for the Sakhalin plants was implemented on the Murashige-Skoog (MS) nutrient medium supplemented with 0.1 μM 6-benzylaminopurine (18.8 ± 6.0 shoots per explant; 90% regeneration frequency) and for Kunashir plants on MS containing 5.0 µM 6-benzylaminopurine and 1.0 µM 1-naphthalene acetic acid (28.8 ± 3.6 and 91%, respectively). Indole-3-butyric acid was found to be effective for the in vitro rooting; the best result was obtained on ½ MS supplemented with 0.1 µM indole-3-butyric acid. By high-performance liquid chromatography, it was determined that the profile of phenolic compounds was more diverse in intact plants than in the plants from in vitro culture: 26 versus 13–21 phenolic substances, respectively. Concentrations of chlorogenic acid, kaempferol, and “phenolic acid 4” were 1.5–2.0-fold higher in the in vitro plants (depending on the cultivation stage) compared to the intact plants, while concentrations of hyperoside, astragalin, and quercetin were higher in the intact plants. Our results indicate the feasibility of using S. betulifolia subsp. aemiliana plants from in vitro culture as a valuable alternative source of phenol carboxylic acids and flavonoids as well as potential usefulness of further studies on the accumulation of secondary phenolic compounds in this subspecies in vitro. For the first time, concentration of phenolic acids and flavonoids in shoot extracts of in vitro plants of Spiraea betulifolia subsp. aemiliana from axillary bud explants was determined and compared with intact-plant extracts.