Chrysanthemum vestitum, a closely related hexaploid wild species of cultivated C. × morifolium has the same and typical flat, tubular and mixed floret forms in its natural population as cultivated chrysanthemum and is an ideal material for studying the petal variation in chrysanthemum. The development progress of this ray floret is affected by key differential genes such as auxin and floral organ development, but the lack of stable and efficient regeneration systems for different floret forms has restricted the study of petal type-related genes in C. vestitum. In this paper, the authors used three floret forms of C. vestitum collected from Funiu Mountain in Henan Province to establish regeneration systems using leaves and transverse thin cell layers (tTCLs) as explants. The results showed that the optimal media for callus induction and adventitious bud differentiation were MS+1 mg∙L^-1 NAA+2 mg∙L^-1 6-BA with flat floret leaves as explants, and the callus induction rate was 100% at 20 days after inoculation. The differentiation rate of adventitious buds was 100%. The optimal rooting medium was 1/2MS+0.2 mg∙L^-1 NAA, and the rooting rate of regenerated plants was 100%. The optimal leaf regeneration system for the flat floret strain was also suitable for determining both the tubular and mixed floret strains. The differentiation rates of adventitious buds were 83.46% and 91.67%, respectively, and the rooting rates were 100%. The flowering plants were observed after transplanting, and it was found that the flower types of the regenerated plants with three different floret forms obtained by the leaf regeneration system were stable, which provided a technical method for the subsequent analysis of the morphological variation mechanism of ray florets by using different floret forms of C. vestitum.

This study aims to establish a rapid and efficient method for qualitative and quantitative determination of plant acid hormones by ultra-high performance liquid chromatography/high resolution time-of-flight mass spectrometry (UPLC- TOF-MS). Several derivatization reagents, 2-bromoacetophenone (BP), 2-dimethylaminoethylamine (DMED), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 3-bromoactonyltrimethylammonium bromide (BTA) were selected for derivatization with abscisic acid, gibberellic acid, indoleacetic acid, jasmonic acid and salicylic acid, respectively. The effect of derivatization reagent was evaluated by comparing the response value of the derivative hormones. The plant hormone standards were diluted in gradient and reacted with the derivatization reagent, and the detection limit and quantitation limit of the derivative hormones were determined. Reagent with the best derivatization effect was reacted with the plant crude extract to test its application effect. All the four derivatized reagents could improve the MS response value of acidic hormone, among which the MS sensitivity of hormone derived by DMED is the best. Reaction with DMED was stable and reproducible. The quantitation limits of ABA-DMED, GA₃-DMED, IAA-DMED, JA-DMED and SA-DMED were 0.05, 0.2, 0.1, 0.1 and 0.5 ng∙mL^-1, respectively. Compared with non-derivatized hormones, the quantitative limits of ABA, GA₃, IAA, JA and SA were reduced by 100, 25, 50, 50 and 10 times, respectively. So, DMED derivatization increased the mass spectrometry sensitivity of several hormones by 10-100 times. The method was applied to the determination of endogenous acidic hormones in rice, wheat and broad bean, and the sensitivity was significantly higher than that before derivation. A simple, rapid and reproducible UPLC-TOF-MS method based on derivatization was established, which greatly improved the sensitivity of the determination of acidic hormones in plants.