Plant growth regulator interactions in physiological processes for controlling plant regeneration and in vitro...

The endogenous auxin and cytokinin contents of in vitro regenerated Tulbaghia simmleri maintained on applied plant growth regulators in Murashige and Skoog (MS) medium were investigated using UHPLC-MS analysis. The highest number of shoots (27.6 per leaf) were produced in MS medium supplemented with 2.5 μM thidiazuron. A higher number of these shoots were rooted with 10 μM 6-(2-hydroxy-3-methylbenzylamino) purine (PI-55, cytokinin antagonist). Production of somatic embryos (SEs: 16.4-4.6, globular to cotyledonary stages) improved significantly with liquid MS medium containing 2.5 μM picloram, 2.5 μM phloroglucinol (PG) and 1.5 μM gibberellic acid or 1.5 μM PI-55 and 1.0 μM trans-zeatin. SEs (torpedo and cotyledonary stages) germinated (100%) in plant growth regulator free MS medium. The plantlets were acclimatized and all survived in the greenhouse. Higher levels of endogenous auxin, 2-oxindole-3-acetic acid (oxIAA, 371.52 pmol/g DW) and indole-3-acetylaspartate (IAAsp, 141.56 pmol/g DW) were detected in shoots from PG treatments. The roots of garden-grown mother plants possessed the highest level of indole-3-acetic acid (IAA, 630.54 pmol/g DW) and oxIAA (515.26 pmol/g DW). Cytokinins [CKs: trans-zeatin-O-glucoside (tZOG), cis-zeatin (cZ) and N⁶-isopentenyladenosine-5'-monophosphate (iPRMP)] levels were relatively high in shoots and roots of plantlets in vitro. However, PI-55 treatments influenced the development of plantlets promoting a higher biosynthesis level of iPRMP (418.06 pmol/g DW in root) and cZRMP (904.61 pmol/g DW in roots and 1427.83 pmol/g DW in shoots). The presented protocols offer organogenesis and somatic organogenesis systems for rapid plant regeneration of T. simmleri. In addition, the importance of exogenous and endogenous hormonal effects on in vitro plant growth and development as well as endogenous hormone metabolism signalling and transport related to the physiological processes of CK metabolism and transport are illustrated for in vitro development of T. simmleri.