Somatic embryogenesis and shoot organogenesis from the hypocotyl slices and free radical scavenging activity of regenerants of collard greens (Brassica oleracea L. var. acephala)

  • Nevena BanjacEmail author
  • Branka Vinterhalter
  • Dijana Krstić-Milošević
  • Jelena Milojević
  • Ljiljana Tubić
  • Nabil Ghalawenji
  • Snežana Zdravković-Korać
Original Article


Brassica oleracea var. acephala is an important leafy vegetable that has been widely consumed as a high-nutrient, low-calorie food. Because of the plant’s biennial and self-incompatibility nature, biotechnological approaches are alternative way for propagation and breeding improvements. Since tissue culture studies have been focused in other B. oleracea representatives, the aim of the present study was to achieve effective regeneration protocol distinctive for collard greens, and evaluate the total phenolic content and antioxidant activity of regenerants. The effect of 3 cytokinins [thidiazuron (TDZ), 6-benzyladenine (BA) and 6-furfuryladenine (kinetin, KIN)] at increasing concentrations (0, 5, 7.5, 10, 20 or 30 µM) in combination with tenfold lower concentration of 1-naphtaleneacetic acid (NAA) (0, 0.5, 0.75, 1, 2 or 3 µM, respectively) on the regeneration from hypocotyl slices was studied. Histological analysis revealed the two regeneration pathways, somatic embryogenesis and shoot organogenesis, simultaneously occurred in the same explant, regardless of the cytokinin/NAA combinations used. The regeneration frequency of 95.9%, with 7.5 morphogenic structures regenerated per explant, and the healthy appearance of regenerated plants indicated the optimal combination 20 µM TDZ + 2 µM NAA. TDZ at 5 µM provided the high somatic embryo proliferation rate by generation of secondary embryos (7.79) along with the lowest rate of their abnormalities. Embryo-developed plants were successfully acclimatised (above 90%). The plants regenerated and proliferated on TDZ-containing media had higher total phenolic content that correlated with the highest free radical scavenging activity (IC50 = 19.09 µg ml− 1).

Key message

Somatic embryogenesis and shoot organogenesis from hypocotyl slices of the collard greens were established. The combination 20 µM TDZ + 2 µM NAA was optimal for regeneration providing higher total phenolic content.


Adventitious shoots Collard greens Cytokinins Regeneration Somatic embryos Somatic embryos proliferation Total phenolics 



The authors would like to express their gratitude to the Ministry of Education, Science and Technological Development of the Republic of Serbia for financial support through Contract No. 173015.

Author contributions

NB supervised the whole study and wrote the manuscript. BV performed hystological analysis. DKM performed phytochemical analysis. JM and LJT produced and maintained in vitro cultures. NG cultivated plants in greenhouse. SZK helped with experimental design and contributed to the writing and correction of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors have no conflicts of interest to declare.


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© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Institute for Biological Research “Siniša Stanković”University of BelgradeBelgradeSerbia

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