Abstract
Imperfect T-DNA processing is common during Agrobacterium-mediated transformation, which integrates vector backbone sequences into the plant genome. However, regulatory restrictions prevent such transgenic plants from being developed for commercial deployment. The binary vector pCAMBIA2300 was modified by incorporating multiple left border (Mlb®) repeats and was tested in BY2 cells, tobacco, and cassava plants to address this issue. PCR analyses confirmed a twofold increase in the vector backbone free events in the presence of triple left borders in all three systems tested. Vector backbone read-through past the LB was reduced significantly; however, the inclusion of Mlbs® did not effectively address the beyond right border read-through. Also, Mlbs® increased the frequency of single-copy and vector backbone free events (clean events) twice compared to a single LB construct. Here, we briefly narrate the strength and limitations of using Mlb® technology and reporter genes in reducing the vector backbone transfer in transgenic events.
Highlights
Cassava (Manihot esculenta Crantz) is the largest sources of dietary calories in tropical and subtropical regions after rice and maize (Food and Agriculture Organization of the United Nations [FAO], 2008)
The current study reports on the outcome of deploying Pure multiple left border (Mlb R) (Multi-left border) technology in cassava transformation to reduce vector backbone (VBB) integration, plus the use of visual scorable markers GFP and a gene encoding for phytoene synthase (PSY) as tools for early detection and elimination of plants carrying VBB sequences
Independent putative transgenic BY2 cell lines transformed with p602 (GFP in VBB) and p603 (Phytoene Synthase; PSY in VBB) were screened for reporter gene expression 6 weeks post Agrobacterium coculture
Summary
Cassava (Manihot esculenta Crantz) is the largest sources of dietary calories in tropical and subtropical regions after rice and maize (Food and Agriculture Organization of the United Nations [FAO], 2008). Cassava production is under pressure due to drought, weeds, pests, viral and bacterial diseases, and rapid post-harvest physiological deterioration (Patil and Fauquet, 2009; Naziri et al, 2014; Ekeleme et al, 2019; Orek et al, 2020). Genetic barriers such as high heterozygosity, irregular flowering, poor seed set and inbreeding depression acts as major bottlenecks for conventional breeding approaches in cassava (Elegba et al, 2021). Cassava transformation retains challenges such as genotype-dependent transformation methods, low regeneration rates, Vector Backbone Studies in Cassava and changes in gene expression following embryogenesis and in some cases loss of resistance to Cassava Mosaic Disease (CMD) during tissue culture (Zainuddin et al, 2012; Ma et al, 2015; Beyene et al, 2017; Chauhan et al, 2018)
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