In vitro Protocol Development for the production of Virus-Free Plant Materials for selected Elite Citrus cultivars (Rutaceae)

Abstract

Citrus is one of the most widely grown fruits of the angiosperm subfamily Orantoidia in the family Rutaceae, which plays a key role in the world’s economy and commercially grown in the tropical and sub-tropical regions of more than 140 countries. The production of citrus in Ethiopia adversely affected by biotic and abiotic stress. Several diseases, especially graft transmissible diseases such as Citrus tristeza virus (CTV), Citrus exocortis viroid (CEVd), Citrus psorosis virus (CPsV), and huanglongbing (greening), etc., rigorously affect citrus plant growth, fruit quality, and yield. Under such conditions, advanced tissue culture techniques provide the best possible alternative for producing pathogen free planting material from elite citrus cultivars. The aim of this study was to develop an efficient tissue culture protocol that enables producing virus-free elite three citrus cultivars such as Mexican lime (Citrus aurantifolia), Valencia Rhode and Hamelin (Citrus sinensis). For this experiment, style and stigma explants, aseptically cultured on MS (Murashige and Skoog) medium. Explants (unopened flowers) were collected and sterilized using (1.5% and 2%) of NaOCl for (10, 15 and 20 minutes) of time. The MS culture media supplemented with different concentrations and combination of BAP, BAP+2,4-D, and BAP+NAA were used for callus induction and regeneration. The MS medium without PGRs was used as a control. Study results showed that, sterilizing with sodium hypochlorite (NaOCl) at a concentration of 1.5 % for 20 minutes found to be the optimum concentration and exposure of time (100%) for sterilizing of explant. The highest calli initiation (100%) responses were observed on MS medium supplemented with 6-benzylaminopurine (BAP) at 4.0 mg/L followed by MS medium supplemented with the combination of 6-benzylaminopurine (BAP) at 2.0 mg/L and 1- naphthaleneacetic acid (NAA) at 2.0 mg/L (83.33%) in this experiment. Maximum shoot regeneration (66.66%) responses were obtained using MS medium supplemented with 6- benzylaminopurine (BAP) at 4.0 mg/L. MS medium without plant growth regulators showed the best rooting (83.33%) response. Finally, 66.66% of regenerated plantlets on MS medium were in vitro raised by GA3 at 5 mg/l. Among the studied cultivars, Mexican lime showed relatively higher in terms of regenerate ability, indicating that this cultivar could be targeted for callus induction and regeneration to produce virus-free plant. Therefore, the present study had successfully regenerated from style and stigma tissue culture.