Genome-Scale Sequence Disruption Following Biolistic Transformation in Rice and Maize

Abstract

Biolistic transformation delivers nucleic acids into plant cells by bombarding the cells with microprojectiles, which are micron-scale, typically gold particles. Despite the wide use of this technique, little is known about its effect on the cell's genome. We biolistically transformed linear 48-kb phage lambda and two different circular plasmids into rice (Oryza sativa) and maize (Zea mays) and analyzed the results by whole genome sequencing and optical mapping. Although some transgenic events showed simple insertions, others showed extreme genome damage in the form of chromosome truncations, large deletions, partial trisomy, and evidence of chromothripsis and breakage-fusion bridge cycling. Several transgenic events contained megabase-scale arrays of introduced DNA mixed with genomic fragments assembled by nonhomologous or microhomology-mediated joining. Damaged regions of the genome, assayed by the presence of small fragments displaced elsewhere, were often repaired without a trace, presumably by homology-dependent repair (HDR). The results suggest a model whereby successful biolistic transformation relies on a combination of end joining to insert foreign DNA and HDR to repair collateral damage caused by the microprojectiles. The differing levels of genome damage observed among transgenic events may reflect the stage of the cell cycle and the availability of templates for HDR.

High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice

“…It is known that biolistic-mediated DNA delivery can lead to insertion of plasmid DNA, often fragmented, into random genome locations in plants 34,35 . However, this unintended DNA insertion is underreported in the literature and when reported, the detailed data were often not supplied in the publications.…”

An embryo lethal transgenic line manifests global expression changes and elevated protein/oil ratios in heterozygous soybean plants

“…Thus we believe that the effects observed were due to the construct inserting in a gene and interfering with its normal function. A recent study on the effects of biolistic transformation on the broader genome found numerous examples of chromosomal damage, deletions, broken transgenes, and other unintended effects [19]. Table shows the number of plants in each group).…”

Genetic Transformation Methods for Crop Improvement- A Brief Review

“…Tree diagram shows the phylogenetic relationship between Agrobacterium and other non-Agrobacterium species belong to the common order Rhizobiales (Slater et al, 2009). many crops like tobacco,soybean, rice, maize (Liu et al, 2019), wheat (Lazzeri, 1999), cotton (Raj asekaran, 2013),mango (Andres Cruz-Hernandez)etc., the DNA coating over the carrier is very important and many protocols have been developed while the generally used methods were Ca2+ protocol and PEG /MgCl2 protocol. the DNA and adhesive chemical mixture in micro-vial are constantly vortexed to prepare microcarrier (Rajasekaran, 2013).…”

“…The gene gun is fixed inside the aseptic condition generally laminar air flow chamber to facilitate transformation without contamination. The material to be transferred is bombarded to a maximum of one time because more times of bombardment will causetissue damage, chromosome truncations, large deletions, partial trisomy and breakage-fusion bridge cycling, etc., (Liu et al, 2019) which ultimately may cause mortality. The plants that show low response to tissue culture find difficult to use gene gun for the transformation of a gene.…”