Development of long-term and reliable in vitro plant regeneration systems for elite malting barley varieties: Optimizing media formulation and explant selection

Abstract

The response to in vitro tissue culture of five important Mexican malting barley cultivars namely ‘Armida’, ‘Esmeralda’, ‘Adabella’, ‘Esperanza’ and ‘Alina’, was evaluated. Callus induction and plant regeneration were evaluated in shoot apices and immature barley embryos harvested eight, 12, 16, 20 and 24 days after pollination. These explants were cultured on 22 media formulations that included Murashige and Skoog (MS) and Chu (N6) media amended with two carbon sources and different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D), 3,6-dichloro-2-methoxybenzoic acid (Dicamba), 6-benzyladenine (BA), casein hydrolyzate and L-proline. No regenerable callus lines could be recovered from the experiments using shoot apices as the starting material. Conversely, immature embryos harvested at 12 to 16 days after pollination (1.0 to 1.5 mm long) and cultured on formulations containing the basal MS medium supplemented with 2 mg L^-1 of 2,4-D or Dicamba resulted in the production of nodular and friable embryogenic calli with regenerative capacity in all barley cultivars. A medium containing MS, Dicamba and maltose generated the highest percentage of embryogenic calli formation (2.1 in ‘Adabella’ vs. 23.4 in ‘Alina’ cultivar) with the maximal regenerative potential (7.2 in ‘Armida’ vs. 9.1 recovered plants per gram FW callus in ‘Esperanza’ cultivar). Using this medium, ‘Esmeralda’ embryogenic callus lines have been maintained for more than five years without loss of regenerability or the capacity for producing normal plants.

Key words: Barley, somatic embryogenesis, plant regeneration, immature embryos.