Stable Inheritance of Transgene and Yellow Fluorescent Protein Gene Expression in Progeny of Transgenic Cacao (Theobroma cacao) Plants

Genetic engineering tools have the potential to rapidly and precisely improve the genome of slow-to-breed cacao. We previously developed an efficient protocol for transforming cacao using cotyledonary explants derived from secondary somatic embryos via <i>Agrobacterium tumefaciens</i>. In this study, we demonstrate that our transformation protocol is successful in elite cultivars, INIAPG-038 and Matina 1-6, producing fertile seeds with stable visual marker inheritance regardless of whether the transgenic plants were used as the pollen or ovule donor. Three vectors were used in the transformations, each containing genes for <i>enhanced yellow fluorescent protein</i> (<i>eyfp</i>) and <i>neomycin phosphotransferase</i> II (<i>npt</i>II). Three transgenic INIAPG-038 events and one transgenic Matina 1-6 event were used to evaluate seed fertility and the stability of transgene inheritance in cacao seeds and plants. The T₁ progeny of these four transgenic events were analyzed for YFP expression and transgene presence. YFP expression segregated at a 1:1 ratio in all events when the transgenic plants were crossed with non-transgenic plants, while a 3:1 segregation was observed when transgenic events were crossed with each other. The transgenic plants exhibited a normal phenotype compared to non-transgenic control plants, producing seeds with a 97% germination rate.