Factors influencing transient expression of Agrobacterium-mediated transformation of gus gene in embryogenic callus of date palm

摘要

A system for Agrobacterium-mediated gene transfer to embryogenic callus of date palm was developed. The callus was produced from shoot tip explants; grown on callus induction medium (CIM), which contained basal MS salts, B5 vitamins, 30 g/l sucrose, 10 mg/l 2,4-D, 3 mg/l 2ip, 170 mg/l KH2PO4 and 3 g/l activated charcoal. Microcalli were efficiently mass propagated on MS medium supplemented with 0.4 mg/l NAA and 0.1 mg/l 2ip, which showed superiority in callus fresh weight and growth measurements, compared with three other tested media. Factors influencing transient expression of the beta-glucuronidase (gus) gene were evaluated following infection of date palm embryogenic callus with Agrobacterium tumefaciens. The obtained results indicated that high bacterial density (OD600 1-1.5) and prolonged infection (2 hr) gave the highest percentage of gus-expressing calli. A further improvement of transient gus expression was obtained through desiccation generated by exposing the embryogenic calli to a gentle air-flow at 45 degrees C, followed by rehydration in Agrobacterium suspension, which significantly enhanced transient gus expression. In contrast, sonication pre-treatment of the calli negatively affected the frequency of gus expression. The results presented indicate that Agrobacterium-mediated gene transfer to date palm cells can be achieved, and thus represent a first step towards the establishment of stable genetic transformation of this agro-economically important monocot crop species.