High-efficiency Propagation of Mature ‘Washington Navel’ Orange and Juvenile ‘Carrizo’ Citrange Using Axillary Shoot Proliferation

Abstract

In vitro axillary shoot proliferation can be used to increase availability of citrus (Citrus) types in high demand, while limiting somaclonal variation. However, established protocols could be improved to increase efficiency. Therefore, this study investigated some factors [plant growth regulators (PGRs), basal media, and successive subculturing] which affect the in vitro axillary shoot proliferation of mature ‘Washington Navel’ orange (Citrus sinensis) and juvenile ‘Carrizo’ citrange (C. sinensis × Poncirus trifoliata). In ‘Washington Navel’ orange, maximum axillary shoot induction (66.9% explants producing axillary shoots with a mean of 2.45 shoots per explant) was obtained in Driver and Kuniyuki walnut (DKW) medium supplemented with 0.1 mg·L−1 6-benzylaminopurine (BA), 0.05 mg·L−1 naphthalene acetic acid (NAA) along with 1 mg·L−1 6-furfurylaminopurine [kinetin (kin)], whereas in ‘Carrizo’ citrange, axillary shoot production was greatest (82.6% and 87.5% of explants producing axillary shoots with a mean of 4.3 and 4.1 shoots per explant) at 1.0 or 2.0 mg·L−1 BA in DKW medium. The initial nodal propagules (with basal tissue remaining from removed shoots) were repeatedly subcultured for six times every 4 weeks onto DKW medium with the same levels of PGRs used for initial culturing. Woody plant medium (WPM), Murashige and Skoog medium (MS), and DKW were also compared for rooting at quarter to full strength for salt components, all amended with 2.0 mg·L−1 indolebutyric acid (IBA) and 0.5 mg·L−1 NAA. MS at full strength provided the highest rooting in ‘Carrizo’ citrange (93%) and longest root length (58 mm), whereas half-strength MS provided the highest rooting in ‘Washington Navel’ orange (60% to 61%) and the longest roots (26 mm). Addition of 1 μm spermidine to the rooting medium enhanced root length only for ‘Washington Navel’ orange on full-strength MS, but accelerated rooting for both cultivars on all media. The plantlets were successfully transferred to greenhouse conditions, exhibiting normal development, with high uniformity, and no evidence of somaclonal variation.