Robust transformation procedure for the production of transgenic farmer-preferred cassava landraces

Abstract

Recent progress in cassava transformation has allowed the robust production of transgenic cassava even under suboptimal plant tissue culture conditions. The transformation protocol has so far been used mostly for the cassava model cultivar 60444 because of its good regeneration capacity of embryogenic tissues. However, for deployment and adoption of transgenic cassava in the field it is important to develop robust transformation methods for farmer- and industry-preferred landraces and cultivars. Because dynamics of multiplication and regeneration of embryogenic tissues differ between cassava genotypes, it was necessary to adapt the efficient cv. 60444 transformation protocol to genotypes that are more recalcitrant to transformation. Here we demonstrate that an improved cassava transformation protocol for cv. 60444 could be successfully modified for production of transgenic farmer-preferred cassava landraces. The modified transformation method reports on procedures for optimization and is likely transferable to other cassava genotypes reportedly recalcitrant to transformation provided production of high quality FEC. Because the three farmer-preferred cassava landraces selected in this study have been identified as resistant or tolerant to cassava mosaic disease (CMD), the adapted protocol will be essential to mobilize improved traits into cassava genotypes suitable for regions where CMD limits production.

Evaluation of red fluorescent protein (DsRed) as alternative visual marker of genetic transformation in cassava (Manihot esculenta Crantz)

“…Hygromycin selective pressure was used to recover the stacked DsRed/GFP transgenic tissues and plants. In our hands selection and recovery of transgenic events with nptII proved more effective than hygromycin and differed from that reported by Zainuddin et al (2012).…”

Tissue- and Organ-Specific Promoters for Expression of Heterologous Genes in Transgenic Cassava (Manihot Esculenta Crantz) Plants

“…Transgenic plants (three) expressing the glgC gene had up to 70% higher AGPase activity than control plants when assayed under conditions optimal for plant and not bacterial AGPase activity. Recently, Zainuddin et al [32] employed Agrobacterium tumefaciens LBA4404 harboring CAMBIA1301 plasmid which contains the hpt II gene for resistance to hygromycin and the UidA reporter gene driven by the constitutive CaMV35S promoter to generate transgenic cassava cultivar TMS60444 plants. Early this year, Oyelakin et al [11] constructed and tested a T-DNA vector with pCsVMV-GUS and CaMV 35S-NPTII cassettes transcribing in opposite direction in cassava transgenic plants.…”

Gene expression analysis provides insight into the physiology of the important staple food crop cassava

“…Despite the importance of these structures, their basic physiology remains largely unknown, especially the molecular genetic basis of storage root development. Similarly, in cassava, the favored target tissue for transgene integration and genome editing is a friable embryogenic callus (FEC) (Taylor et al , 2001; Bull et al , 2009; Taylor et al , 2012; Zainuddin et al , 2012; Nyaboga et al , 2013). Little is known concerning gene expression in this tissue, or its relatedness to the somatic organized embryogenic structures (OES) from which it originates (Gresshoff & Doy, 1974; Taylor et al , 2012; Chauhan et al , 2015).…”