Humic Acid and Auxins Induced Metabolic Changes and Differential Gene Expression during Adventitious Root Development in Azalea Microshoots

Auxins and humic acid (HA) were investigated for their roles in adventitious root induction in azalea microshoots in our previous study. To reveal the regulatory mechanisms of auxins and HA in this process, measurements of the levels of reactive oxygen species (ROS), carbohydrates, and phenolic compounds and gene expression during in vitro root development were performed. During the adventitious root induction process, ROS levels in shoots treated with auxins and HA increased compared to untreated shoots, especially during the earliest period after transfer to the media. Media supplemented with NAA experienced increases in H2O2 contents by 480%and 250%, respectively, after 7 and 14 days of culture. The phenolic compound levels were also enhanced in the shoots treated with auxins and HA, reflecting the different rooting-promoting abilities of both auxins and HA. The highest levels of total phenolic [68.6 mg·g−1 fresh weight (FW)], polyphenolic acids (121.72 μg caffic acid/g FW), and total flavonols (162.42 μg quercetin/g FW) were recorded after 21 days for NAA media, but the maximum levels of anthocyanins (49.76 μg cyanindin/g FW) were recorded after 21 days for IBA medium. Soluble carbohydrate, starch, and soluble protein levels were increased in the shoots treated with all treatments; however, the influence of NAA treatments was stronger than that of other treatments for most investigated parameters. The NAA significantly enhanced soluble carbohydrates by 30%, 37%, and 25%, respectively, at 14, 21, and 28 days compared with untreated microshoots. Expression of the POD1 gene increased in the shoots submitted to HA treatment media. Expression levels of auxin response factors (ARFs) increased with IBA- and NAA-treated explants, suggesting that ARFs may have diverse regulatory roles in adventitious root induction in evergreen azalea. Moreover, the profiles of the IAA1, IAA9, IAA14, and IAA27 transcripts were analyzed to reveal their roles in the adventitious rooting of evergreen azalea microshoots. These results indicate that auxins and HA promote adventitious root induction in Rhododendron plants through their impact on ROS, carbohydrate contents, phenolic compound levels, and expression levels of different genes related to root development in evergreen azalea plants.