Exogenous cytokinins affect genes involved in kohlrabi auxin transport and shoot apical meristem development

Abstract

In most plant species, typical protocols for de novo shoot organogenesis (DNSO) under controlled in vitro conditions include a series of two growth media; the first containing auxin for callus induction, and the second containing cytokinin (CK) which induces shoot regeneration. However, the research conducted on kohlrabi (Brassica oleracea var. gongylodes) has shown that intact seedlings are capable of forming calli and regenerating shoots on media containing only CK, with no requirement for exogenously applied auxins. Analyses of endogenous phytohormonal status during kohlrabi DNSO showed that the presence of CKs in growth media contributed to elevated endogenous auxin (i.e. IAA) levels and IAA/bioactive CKs ratio in kohlrabi shoots. Crosstalk between cytokinins and auxins is an important factor in de novo shoot organogenesis. Thus, the present study was designed to examine whether successful DNSO, occurring in intact kohlrabi seedlings through the influence of cytokinin containing media, was accompanied by distinct alterations in the expression levels of genes implicated in auxin transport, cytokinin homeostasis regulation and de novo shoot apical meristem formation. The activity of PIN3, PIN4, ARR5, RGD3 and CUC1 genes was determined by quantitative PCR analysis. In the most cases, the expression of analysed genes had a characteristic two phase response with two declines in expression. These decreases in respect to expression in non-treated controls corresponded to time points in which formation of calli (for the first decline) and de novo shoots (for the second decline) were observed. The expression of genes demonstrated patterns that clearly correlated with CK-induced morphological manifestations of DNSO. Additional study is needed to further clarify molecular mechanisms underlying kohlrabi in vitro regeneration.