Induction of Early Flowering in Cassava (Manihot Esculenta) and Nicotiana Benthamiana by Overexpression of Cassava Flowering Locus T (Meft1)

Abstract

Flowering is a prerequisite in conventional plant breeding. In cassava, delayed and non-synchronous flowering makes conventional breeding difficult. This study aimed at accelerating flowering by overexpressing the endogenous cassava (Manihot esculenta) Flowering Locus T variant 1 (MeFT1) in cassava and Nicotiana benthamiana. The MeFT1 gene was overexpressed through 1) agroinfiltration into N. benthamiana and four cassava cultivars using the Cassava Mosaic Virus (CMV)-based vectors, and 2) constitutive expression under the 35S promoter in the same cassava cultivars and N. benthamiana to serve as positive controls. Molecular characterization of the regenerated and agro-infiltrated plants was conducted using polymerase chain reaction (PCR), Southern blot hybridization, and reverse-transcription PCR (RT-PCR). Flowering characteristics, including flowering time and the number of flowers, were evaluated for the infiltrated N. benthamiana plants. Southern blot analysis confirmed successful viral delivery in both N. benthamiana and cassava and replication at the point of infiltration (POI) in N. benthamiana. Viral systemic movement was only observed in N. benthamiana and not in cassava. The agro-infiltrated N. benthamiana plants flowered earlier and had more branches and flowers than their controls. For the positive controls, the regenerated N. benthamiana plants tested positive for the transformed gene and flowered early at two weeks post-transfer to rooting media. Recalcitrance to transformation was also observed in the four cassava cultivars used in the study. Combined, the findings from this study report the use of the East African Cassava Mosaic Virus (EACMV) virus vector in gene delivery into N. benthamiana for functional trait characterization. Since flowering was not observed in the infiltrated and regenerated cassava plants despite the successful delivery of the vector, further studies on how to improve the vector structure and infiltration procedures are necessary to achieve a successful flowering induction in cassava.