Efficient transformation and regeneration of transgenic cassava using the neomycin phosphotransferase gene as aminoglycoside resistance marker gene

Abstract

Cassava is one of the most important crops in the tropics. Its industrial use for starch and biofuel production is also increasing its importance for agricultural production in tropical countries. In the last decade cassava biotechnology has emerged as a valuable alternative to the breeding constraints of this highly heterozygous crop for improved trait development of cassava germplasm. Cassava transformation remains difficult and time-consuming because of limitations in selecting transgenic tissues and regeneration of transgenic plantlets. We have recently reported an efficient and robust cassava transformation protocol using the hygromycin phosphotransferase II (hptII) gene as selection marker and the aminoglycoside hygromycin at optimal concentrations to maximize the regeneration of transgenic plantlets. In the present work, we expanded the transformation protocol to the use of the neomycin phosphotransferase II (nptII) gene as selection marker. Several aminoglycosides compatible with the use of nptII were tested and optimal concentrations for cassava transformation were determined. Given its efficiency equivalent to hptII as selection marker with the described protocol, the use of nptII opens new possibilities to engineer transgenic cassava lines with multiple T-DNA insertions and to produce transgenic cassava with a resistance marker gene that is already deregulated in several commercial transgenic crops.

Exploiting the Combination of Natural and Genetically Engineered Resistance to Cassava Mosaic and Cassava Brown Streak Viruses Impacting Cassava Production in Africa

“…The transformation protocol for the TME 7 cassava landrace (locally referred to as Oko-Iyawo) was optimized using the improved transformation protocol [20], [30]. Multiplication of TME 7 friable embryogenic callus (FEC) in the dark combined with extended regeneration steps following Agrobacterium -mediated FEC transformation sufficed to implement a high regeneration frequency of transgenic plantlets with the TME landraces [31].…”

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

“…To determine the percentage of transgenic maturing embryos appearing on the regeneration medium, embryos can be selected and tested using the GUS assay by following the steps as earlier described [18]:…”

“…The instability of the transformation system renders the establishment of cassava transformation technology under less favourable conditions more challenging [14-16]. Recent progress in the optimization of the transformation protocol has substantially increased efficiency and robustness [17,18]. The improved transformation protocol was subsequently established in laboratories located in Africa based on hands-on workshops and training of local scientists [15,16].…”

Modification of Cassava Root Starch Phosphorylation Enhances Starch Functional Properties

“…Hairpin constructs containing an antisense fragment, a loop, and a sense fragment designed for MeSEX4 or MeLSF2 , were subcloned into a modified pCAMBIA1301 vector containing the S. tuberosum patatin B33 promoter. Transgenic cassava lines were obtained via Agrobacterium-mediated friable embryonic callus (FEC) transformation as described in ( Bull et al, 2009 ; Niklaus et al, 2011 ). Transgenic plantlets selected by antibiotic resistance and confirmed for the presence of the transgene by PCR on genomic DNA were transferred to soil.…”