Physiological and Biochemical Responses of Merwilla plumbea Cultured In Vitro with Different Cytokinins After 1 Year of Growth Under Ex Vitro Conditions

Since their discovery in the 1950s, cytokinins (CKs) have become integral, almost indispensable, components of plant growth media in in vitro propagation. However, despite the extensive use of exogenous CKs in plant tissue culture systems, there is a paucity of knowledge on the longevity of their physiological effect post in vitro. The objective of this study was to evaluate the long-term physiological effect of CKs (including a novel meta-topolin derivative) applied during in vitro propagation of Merwilla plumbea after 1 year of ex vitro growth. Ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was used for the quantification of endogenous auxins and phenolic compounds in the 1-year-old M. plumbea plants. Compared to the control, exogenous CKs had a significant carry-over effect on ex vitro growth of M. plumbea plants after 1 year growing under ex vitro conditions. M. plumbea plants treated with the novel CK 6-(3-hydroxybenzylamino)-9-(tetrahydropyran-2-yl)purine (mTTHP, 1.0 µM) had significantly higher leaf area per plant, total leaf fresh weight and bulb diameter. Varying concentrations of free indole-3-acetic acid (IAA), oxindole-3-acetic acid (OxIAA) and indole-3-acetyl-l-aspartic acid (IAA-Asp) were detected in leaves of plants originally treated with exogenous CKs, whereas these compounds were absent in leaves of plants without exogenous CKs. Likewise, a CK-type and concentration-dependent response was observed for hydroxybenzoic acids, hydroxycinnamic acids, flavonoids and antioxidant activity of the extracts. The current study showed the influence of type and concentration of exogenous CKs used in vitro on production of phytochemical compounds during ex vitro growth. Taken together, the study provides significant insights on the residual effect of exogenous CKs on the regulation of auxin homeostasis and phenolic acid and/or flavonoid metabolism in 1-year-old M. plumbea plants growing under ex vitro conditions.