Establishment of an in vitro regeneration system and genetic transformation of the Tunisian 'Maltese half-blood' (Citrus sinensis): an agro-economically important variety.

Abstract

An efficient in vitro regeneration system using epicotyl segments was developed and then used for optimizing genetic transformation of the Tunisian 'Maltese half-blood' (Citrus sinensis) variety using phosphinothricin (PPT) resistance as a selectable marker. The maximum regeneration efficiency was achieved after incubating epicotyl explants (excised in an oblique manner) in MT culture media containing BAP (4mg/l) and IAA (0.3mg/l) hormonal combination in the dark for 3weeks before their transfer to light. Data from the genetic transformation assays indicated that the highest number of regenerated-transformants was reached when the selection phase was conducted in MT culture media containing PPT (0.25mg/l) and Carbenicillin (500mg/l) for 3weeks in the dark followed by 8weeks of light. After that, transformed buds were maintained for eight additional weeks in the same culture media but with reduced PPT concentration (0.125mg/l) before decreasing Carbenicillin dose (250mg/l) at the second half of this last incubation period which allowed both a good shoot proliferation and an optimal rooting efficiency. Based on molecular analyses, the transgenicity of 21.42% of the regenerated vitroplants was confirmed. The developed regeneration and transformation procedures of the elite 'Maltese half-blood' variety can be used for orchard renewal as well as for functional studies and genome editing purposes to develop new cultivars with the desired genetic traits.