Strategies to improve low copy transgenic events in Agrobacterium-mediated transformation of maize

Abstract

Transgenic plants containing low copy transgene insertion free of vector backbone are highly desired for many biotechnological applications. We have investigated two different strategies for increasing the percentage of low copy events in Agrobacterium-mediated transformation experiments in maize. One of the strategies is to use a binary vector with two separate T-DNAs, one T-DNA containing an intact E.coli manA gene encoding phosphomannose isomerase (PMI) as selectable marker gene cassette and another T-DNA containing an RNAi cassette of PMI sequences. By using this strategy, low copy transgenic events containing the transgenes were increased from 43 to 60 % in maize. An alternate strategy is using selectable marker gene cassettes containing regulatory or coding sequences derived from essential plant genes such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or MADS box transcription factor. In this paper we demonstrate that higher percentage of low copy transgenic events can be obtained in Agrobacterium-mediated maize transformation experiments using both strategies. We propose that the above two strategies can be used independently or in combination to increase transgenic events that contain low copy transgene insertion in Agrobacterium-mediated transformation experiments.

Measurement of Transgene Copy Number in Plants Using Droplet Digital PCR

“…. DNA delivery techniques can be optimized to obtain a higher frequency of transgenic lines with single or low-copy insertions; however, the exact number of transgenes transferred to the plant genome cannot be controlled or limited, and transgenic lines must be individually assessed to determine transgene copy number (Sivamani et al, 2015). Since expression levels, and consequently phenotypes, have long been acknowledged to be influenced by copy number, accurate determination is essential (Hobbs et al, 1993).…”