Application of fluorescent and UV-Vis detection methods to profile antimicrobial activity of cassava tissues for an efficient Agrobacterium-mediated transformation.

Abstract

The majority of tissue culture and transformation studies in cassava (Manihot esculenta Crantz) focus on the modification of conditions in order to establish a better protocol. Although this is a standard approach for making progress in genetic transformation technology for a target plant variety, serious difficulty still remains due to the limited applicability and adaptability of the given protocol. In the present study, we aim to develop a new concept that focuses on the development of simple but adaptable genetic transformation technology in cassava. In order to establish an efficient transformation protocol, two local edible cassava varieties, R-type, with a broad leaf with reddish petiole, and S-type, with a thin leaf with shiny greenish petiole, were obtained from Okinawa, Japan. Three detection methods, i.e., fluorescence microscopy, thin-layer chromatography (TLC) with detection under an ultraviolet (UV) illumination (254 nm) and light emitting diode (LED) illuminations (365 nm and 500 nm) without any staining, and a spectrum scanning (250-700 nm) by a microplate reader system were employed to identify a series of unique features of the petioles and leaves. Antimicrobial activity of methanol extracts from the tissues was also assayed. We succeeded in the transient expression of the GUS gene using cassava leaves and also established stable introduction of the GUS gene into three organogenic cassava calli by adapting Agrobacterium-mediated transformation. With all the findings, we have identified a flexible tool to create a better match between explants and Agrobacterium strains.