Agrobacterium-mediated transformation, selection and regeneration of Vanilla pompona

Abstract

Vanilla is a high-value tropical orchid cultivated for its aromatic fruit capsules that are used in foods, perfumes, and industrial products. Vanilla planifolia (Jacks ex. Andrews) is the most important commercially grown species, but its production is constrained by poor yield, variable quality, low genetic diversity and limited horticultural advancements. A closely related species, Vanilla pompona Schiede, characteristics which could be useful in breeding improved varieties: large fruit, potent aroma, and resistance to Fusarium oxysporum f. sp. vanilla. Here we describe tissue culture-based regeneration and Agrobacterium-mediated stable transformation systems for V. pompona. Vegetatively propagated tissue was used to test the efficacy of hygromycin and phosphinothricin selection and to assess the efficiency of three Agrobacterium tumefaciens strains (EHA105, AGL1.1, GV3101) in transformation protocols. Results revealed that for V. pompona, kanamycin is not an effective selection marker, whereas hygromycin and phosphinothricin can be used for screening transformants. AGL1.1 provided the highest transformation efficiency (37%) as compared to strains EHA105 (11%) and GV3101 (4%). Additionally, we incorporated the use of firefly luciferase as a visual reporter of transformation and were able to demonstrate that it provides more robust assessment than that of green fluorescent protein. Finally, we report a novel quantitative imaging method to assess the growth responses of V. pompona protocorm-like bodies in response to selection that could be useful to other plant transformation and selection efforts.