Optimizing embryo and shoot tip derived callus production and high frequency plant regeneration in the model grass Brachypodium distachyon (L.) P. Beauv

Abstract

Brachypodium distachyon has been recently proposed as a new model system for monocot functional genomics because of its unique properties such as small size, short generation time, self-fertile, chromosome base number five and small genome size. In this study, we have standardized a protocol to obtain 100% green plant regeneration from embryo and shoot tip explants. The optimum callus induction in terms of percentage of cultures responding and callus growth was observed on Murashige and Skoog (MS) medium supplemented with 4 mg/l 2, 4 dichlorophenoxy acetic acid (2, 4-D) and 0.5 mg/l kinetin (Kn). On this medium, 88% and 100% explants responded from embryo and shoot tip explants, respectively. Comparatively, shoot tip explants produced better response than embryo explants. The callus multiplied and maintained on MS medium supplemented with 2, 4-D (4 mg/l) and Kn (0.5 mg/l) for about 10 months without decline in growth rate. The callus subcultured on MS medium supplemented with thidiazuron (TDZ; 0.5–7 mg/l) or 6-benzyladenine (BA; 0.5–7 mg/l) alone or in combination with naphthalene acetic acid (NAA; 0.5 mg/l) for regeneration. Maximum shoot regeneration was observed on MS medium supplemented with 5 mg/l TDZ and 0.5 mg/l NAA. On this medium, 87% and 96% cultures responded with mean shoot number 10.2 and 16.6 from embryo and shoot derived callus, respectively. The shoots were rooted on MS medium supplemented with either NAA (2–8 mg/l) or indole-3-butyric acid (IBA; 2–8 mg/l). The optimum response (98%) was observed on MS medium supplemented with 4 mg/l IBA with 4.5 roots per shoot. The rooted plantlets were successfully transplanted with 90% success. The acclimatized plants produced flowers and viable seeds.