In vitro shoot organogenesis and comparative analysis of endogenous phytohormones in kohlrabi (Brassica oleracea var. gongylodes): effects of genotype, explant type and applied cytokinins

Abstract

Kohlrabi (Brassica oleracea var. gongylodes) cultivars Vienna Purple (VP) and Vienna White (VW) were tested for their ability of de novo organogenesis in vitro. Root, cotyledon, hypocotyl explants and intact seedlings were cultivated on Murashige and Skoog (MS) media supplemented with different cytokinins: benzyladenine (BA), thidiazuron (TDZ), trans- or cis-zeatin. All tested cytokinins, including cis-zeatin, induced shoot regeneration from hypocotyl explants and intact seedlings, with seedlings being most successful for regeneration efficiency and viability of regenerated shoots in both cultivars. The highest frequency of shoot regeneration was achieved on MS with BA (60 \%) or TDZ (50 \%) for VP; and with BA (50 \%), TDZ (47.5 \%) or transZ (37.5 \%) for VW. Measurements of the endogenous cytokinin and indole-3-acetic acid (IAA) contents in both hypocotyl explants and seedlings with regenerated shoots (HRSs and SRSs) suggested that the observed differences in organogenic response between these two types of explants were related to their cytokinin and IAA contents. HRSs generally exhibited elevated amounts of total cytokinins, while SRSs displayed a higher IAA/bioactive cytokinins ratio. Shoots regenerated from seedlings were further successfully multiplicated on a medium supplemented with BA (0.5 mg L-1). The rooting potential of multiplicated shoots was tested on media supplemented with 2 or 4 mg L-1 indole-3-butyric acid (IBA), with the higher concentration of IBA leading to more efficient rooting. Rooted plantlets were successfully planted into soil and flow cytometric analysis did not reveal ploidy variations, indicating that the described protocol is fast and efficient for kohlrabi regeneration.