Evaluation of somatic embryo production in three elite cassava farmer preferred Ugandan genotypes (Nasse13, Narocass1, and Nase19) and genetic transformation of Narocass1 using agrobacterium-mediated gene transfer

Abstract

Friable embryogenic callus (FEC) production is a prerequisite for the genetic transformation of cassava (Manihot esculenta Crantz), however, its induction first requires the generation of organised embryogenic structures (OES). The production of OES and FECs in cassava is dependent on the genotype. The objective of this study was to optimise conditions suitable for somatic embryo production in the Ugandan elite cassava genotypes; NASE13, NASE19, and NAROCASS1, and to demonstrate genetic transformation. OES were induced by initiating leaf-lobe explants on Murashige and Skoog (MS) media, supplemented with 24D at varying concentrations of 5, 8, and 11 mg L-1, half-strength MS containing 50 µM picloram, and MS full strength with 50 µM picloram as the control. To produce FEC, resulting OES were established on Gresshoff and Doy (GD) media before culturing on regeneration media (MS 5 µM 1-Naphthaleneacetic acid (NAA)) to determine the regeneration frequencies of each genotype. The generated NAROCASS1 FECs were cocultured with an OD600 of 0.05 of Agrobacteria suspension containing the enhanced green fluorescence protein (eGFP). There was a highly significant difference among the different media compositions in the percentage response to OES formation (p < 0.001). NASE 13 showed the highest regeneration frequency of 38.4%; while NAROCASS1 had the lowest regeneration frequency of 10.5% recorded. The presence of eGFP gene in NAROCASS1 was confirmed by polymerase chain reaction (PCR) analysis of the transformed lines. These findings present opportunities to utilise elite Ugandan cassava genotypes already adopted by farmers to augment them with desirable traits.