Comprehensive Phytohormone Profiling of Kohlrabi during In Vitro Growth and Regeneration: The Interplay with Cytokinin and Sucrose

Abstract

The establishment of an efficient protocol for in vitro growth and regeneration of kohlrabi (Brassica oleracea var. gongylodes) allowed us to closely examine the phytohormone profiles of kohlrabi seedlings at four growth stages (T1–T4), additionally including the effects of cytokinins (CKs)—trans-zeatin (transZ) and thidiazuron (TDZ)—and high sucrose concentrations (6% and 9%). Resulting phytohormone profiles showed complex time-course patterns. At the T2 stage of control kohlrabi plantlets (with two emerged true leaves), levels of endogenous CK free bases and gibberellin GA20 increased, while increases in jasmonic acid (JA), JA-isoleucine (JA-Ile), indole-3-acetic acid (IAA) and indole-3-acetamide (IAM) peaked later, at T3. At the same time, the content of most of the analyzed IAA metabolites decreased. Supplementing growth media with CK induced de novo formation of shoots, while both CK and sucrose treatments caused important changes in most of the phytohormone groups at each developmental stage, compared to control. Principal component analysis (PCA) showed that sucrose treatment, especially at 9%, had a stronger effect on the content of endogenous hormones than CK treatments. Correlation analysis showed that the dynamic balance between the levels of certain bioactive phytohormone forms and some of their metabolites could be lost or reversed at particular growth stages and under certain CK or sucrose treatments, with correlation values changing between strongly positive and strongly negative. Our results indicate that the kohlrabi phytohormonome is a highly dynamic system that changes greatly along the developmental time scale and also during de novo shoot formation, depending on exogenous factors such as the presence of growth regulators and different sucrose concentrations in the growth media, and that it interacts intensively with these factors to facilitate certain responses.

Phytohormone profiling in an evolutionary framework

“…Additionally, samples of blank medium (containing no biological material and kept in the growth chamber alongside the growing culture) were taken to account for environmental contaminants. (Created with BioRender.com) with IAA in green algae (Jiraskova et al, 2009;Stirk et al, 2013 andZizkova et al, 2017) and land plants alike (Pollmann et al, 2002;Novak et al, 2012;Vankova et al, 2017;Kaneko et al, 2020;Zlobin et al, 2020;Cosic et al, 2022;Fuchs et al, 2022;Prerostova et al, 2021). oxIAA has been studied mainly as an auxin catabolite in angiosperms where it is likely not produced directly from IAA (Hayashi et al, 2021); the upstream metabolites are not found in algae (including this study) and the full complement of known enzymes leading to oxIAA production seems to be angiosperm-specific (Bowman et al, 2021).…”

Accumulation of Toxic Arsenic by Cherry Radish Tuber (Raphanus sativus var. sativus Pers.) and Its Physiological, Metabolic and Anatomical Stress Responses

“…The increase in IAM content in the As100 treatment resulted in a reduced conversion of IAM to IAA. The disturbed dynamic balance between IAA and IAM in cherry radish tubers could be caused by a disturbance in the activity of enzymes responsible for the conversion of Trp to IAA via IAM [ 21 ].…”

In Vitro Growth and Regeneration of Brassica oleracea var. gongylodes: A Decade of Research

“…In the last decade, our team succeeded for the first time in establishing and optimizing protocols for efficient regeneration of kohlrabi in vitro, using both somatic embryogenesis and de novo shoot organogenesis [59,60] (Figure 1). Since then, this system has been used productively as a model system for studying plant morphogenesis in vitro [61][62][63][64]. , where the highest regeneration frequency was obtained on a nutrient medium without plant growth regulators (PGRs).…”

“…A comprehensive phytohormone analysis of VP kohlrabi seedlings during growth and shoot regeneration in vitro was performed for the first time by our group [60,63,64]. Depending on the type of investigated phytohormone and corresponding metabolite, individual changes were observed on a time scale (four developmental stages T1-T4, defined arbitrarily in [63]), showing specific hormone profiles (Figure 2A).…”

“…Growth on PGR-free medium from the T1 stage represented by kohlrabi seedlings with two cotyledons to plantlets with two young and developing true leaves at the T2 stage resulted in a significant increase of the precursor of bioactive GAs, gibberellin GA 20 . Another significant change at T2 involved a decrease in the content of one of the most abundant indole-3-acetic acid (IAA) conjugates, IAA aspartate (IAA-Asp) [64]. In contrast, the content of zeatin nucleobases, comprising transZ, cisZ, and dihydrozeatin (DHZ), increased sharply, reaching the maximum value at T2.…”