In vitro callogenesis essays to induce somatic embryogenesis of the Tunisian chickpea

Abstract

The present study was the first report on the somatic embryogenesis of the Tunisian chickpea (Cicer arietinum L.) particularly supposed to be recalcitrant and difficult to manipulate in vitro. An efficient protocol has been developed for inducing indirect somatic embryogenesis derived from immature zygotic embryo axis, young leaflet and hypocotylar explants. Callogenesis was achieved on full-strength Murashige and Skoog's (1962) (MS) basal medium supplemented with two auxin/cytokinin combinations; 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylamonopurine (KIN) or 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) to establish the phytohormones requirement to promote the best induction of friable calli. For somatic embryos induction, embryogenic calli developed from zygotic embryo axes, leaflet and hypocotylar explants were cultured on MS full strength and MS/2, half strength, free of phytohormones media. This study found that all explants exhibited high frequency of callus induction. For immature zygotic embryo axes and young leaflet explants, media containing the combination of 2,4-D and KIN were best effective for inducing callogenesis and friable calli. The most important embryogenic callus percentages were obtained on the culture medium containing the highest concentrations of 2,4-D (2 mg L-1) and KIN (0.5 mg L-1). However, media containing the combination NAA and BAP were best effective for inducing embryogenic calli on hypocotylar explants. A maximal rate of embryogenic calli was reached on medium containing 3 mg L-1 NAA and 1 mg L-1 BAP. The highest number of somatic embryos was obtained with embryogenic calli derived from embryo axis explants and cultured on MS half strength free phytohormones medium. About 56% of somatic embryos converted successfully into fertile plantlets.

Keywords

• callogenesis
• chickpea
• explants
• phytohormones
• somatic embryo