A system for rapid gene introgression into cassava immature leaves and subsequent recovery of transformed lines

Abstract

Friable embryogenic callus (FEC) currently forms the tissue of choice for gene transfer to cassava tissues. However, this technology has not been sufficiently adopted in most laboratories, partly because of the challenging protocols involved in FEC production. Therefore, the development of transformation systems targeting readily accessible explants could enable the rapid improvement of this essential root tuber. This study developed a simple, efficient, and rapid transformation protocol for three elite cassava cultivars (TMS 60444, 08/354, KME 1) using immature leaf lobes (ILLs). Factors affecting callus and somatic embryo formation, such as auxin concentration and culture photoperiod, were optimized. Cassava transformation was achieved by co-cultivation of ILLs and callus with Agrobacterium strain EHA101 harboring pTF102 plasmid. We report a positive interactive effect between dark treatment and an increasing auxin concentration. Exposure to light was found to negatively affect the formation and viability of SEs by promoting tissue browning. The results further show that cassava transformation success depends on the cultivar, explant type, and age. Using this simple protocol, transgenic plants of TMS 60444, 08/354, and KME 1 with an average transformation frequency of 0.4% (0.5, 0.4, and 0.4%, respectively) were obtained in 6 months. Reverse transcription PCR, Southern blot, and histochemical GUS staining confirmed the stable integration of the gusA gene into the regenerated plantlets. This optimized transformation system for cassava is rapid, efficient, and simple to operate and will facilitate more genetic applications in cassava research.