Overexpression of GRF-GIF genes enhances plant regeneration in cassava (Manihot esculenta)

Abstract

The GROWTH-REGULATING FACTOR (GRF) and its INTERACTING FACTOR (GIF) have been shown to stimulate regeneration of transgenic plants with studies reporting increased transformation efficiency in multiple species, including wheat, beet, and citrus. The present work evaluated the effects of overexpressing GRF4-GIF1 and GRF5 on the regeneration of transgenic plants in cassava (Manihot esculenta Crantz). Effects of GRF4-GIF1 and GRF5 sequences derived from Vitis vinifera and Arabidopsis thaliana were assessed by cloning expression cassettes under control of strong constitutive promoters. Friable embryogenic callus from cassava varieties 60444 and NASE 13 were transformed with Agrobacterium tumefaciens strains LBA4404 and LBA4404 THY-, and multiple independent transgenic plant lines recovered. Expression of the morphogenic genes did not enhance transformation efficiency, nor efficiency or timing of somatic embryo regeneration or whole plant recovery above the green fluorescent protein (GFP) control. Organogenesis experiments were carried out to observe effects of transgenic expression of these genes on morphogenesis from petiole, leaf-petiole, and stem explants. Results differed between the two genotypes evaluated. Expression of Vitis vinifera GRF4-GIF1 was effective for stimulation of rapid organogenesis and shoot regeneration at 30 to 37% from leaf-petiole explants of cultivar 60444. In contrast, Arabidopsis thaliana GRF5 was superior in stimulating shoot regeneration in cultivar NASE 13 with 40 to 50% of leaf-petiole explants regenerating shoots. In both cultivars, caulogenesis occurred rapidly within 3 to 4 wk culture on medium containing the cytokinin meta-topolin. Effects of overexpression of these morphogenic genes at the whole plant level were accessed by establishing plants in the greenhouse. GRF4-GIF1 overexpression resulted in significantly shorter plants with increased leaf size and total leaf area while AtGRF5 stimulated above average storage root weight.