Enhanced single copy integration events in corn via particle bombardment using low quantities of DNA

Abstract

Transgene copy number is an important criterion for determining the utility of transgenic events. Single copy integration events are highly desirable when the objective is to produce marker free plants through segregation or when it is necessary to introgress different transgenes into commercial cultivars from different transgenic events. In contrast multi-copy events are advocated by several authors for higher expression of the transgene. Till recently, it was thought that employment of the particle gun for transformation results in the production of a high proportion of multi-copy events often with complex integration pattern when compared to Agrobacterium-mediated transformation. However, it has been demonstrated that usage of cassette DNA for bombardment in place of whole plasmids would result in simple insertion pattern of the transgenes. While investigating the effect of varying the cassette DNA amount on stable transformation, the frequency of occurrence of low copy events was observed to increase when lower doses of cassette DNA was employed for bombardment. Large scale experimentation with rigorous statistical analysis performed to verify the above observations employing Helium gun and the Electric discharge gun for gene delivery confirmed the above observations. Helium gun experiments involving production of more than 1,600 corn events consistently yielded single copy events at higher frequencies at lower cassette DNA load (46% at 2.5 ng/shot) as compared to higher cassette DNA load (29% at 25 ng/shot) across 18 independent experiments. Results were nearly identical with the Electric discharge particle gun device where single copy events were recovered at frequencies of 54% at 2.5 ng cassettes DNA per shot as compared to 18% at 25 ng cassette DNA per shot. The transformation frequency declined from 41 to 34% (Helium gun) and from 48 to 31% (Electric discharge gun) with reduction in cassette DNA quantity from 25 to 2.5 ng per shot. This reduction in the transformation frequency is more than compensated by the savings in time and effort involved in the production and screening of events if the desired outcome is single copy events. These results demonstrate the flexibility of the particle gun method for controlling the frequency of production of either low copy or high copy events by altering the quantity of cassette DNA used for bombardment. The transgene expression levels over generations in relation to its integration need further investigations.

High-Efficiency Stable Transformation of the Model Fern Species Ceratopteris richardii via Microparticle Bombardment

“…The complex insertion of multiple transgene copies is a known factor in biolistic-based transformation techniques (Hansen and Wright, 1999) and can in part be mitigated through bombardment with linearized DNA (Lowe et al, 2009). In all of the T0 plants tested, the presence of linked Hyg R and 35S::GUS cassettes was revealed by shared hybridization to large gDNA fragments.…”

Genetic Engineering of Saccharum

“…Despite its utility, this transformation system has raised environmental safety concerns from the introduction of unwanted vector sequences and antibiotic resistance genes into the plant genome. To address this problem, vector-free minimal transgene cassettes (MGC) have been used to eliminate the use of unnecessary DNA and to produce transgenic plants with low copy number (Lowe et al 2009 ;Zhao et al 2007 ) . In a recent study in sugarcane, an increase of up to 160-fold in the level of transient expression of the EYFP reporter gene has been achieved by using a double terminator MGC (Beyene et al 2011 ).…”

Advancing Crop Transformation in the Era of Genome Editing

“…Biolistic gene transfer can be applied to a wider range of genotypes than Agrobacterium-mediated gene transfer (Altpeter et al, 2005) but can be limited by the inability to regenerate plants after bombardment. Regeneration response and transgene performance following biolistic gene transfer depend on particle type, size, quantity and acceleration, DNA amount and structure during particle coating, tissue type, and pretreatment (Klein et al, 1988;Vain et al, 1993;Kausch et al, 1995;Frame et al, 2000;Fu et al, 2000;Popelka et al, 2003;Sandhu and Altpeter 2008;Lowe et al, 2009;Sivamani et al, 2009;Wu et al, 2015). Therefore, there is a need for the development of alternative nano-or microparticles, target tissues, and particle coating and delivery protocols for biolistic gene transfer.…”