Agrobacterium‐Mediated Gene Transfer to Cereal Crop Plants: Current Protocols for Barley, Wheat, Triticale, and Maize

Abstract

The development of powerful “omics” technologies has enabled researchers to identify many genes of interest for which comprehensive functional analyses are highly desirable. However, the production of lines which ectopically express recombinant genes, or those in which endogenous genes are knocked down via stable transformation, remains a major bottleneck for the association between genetics and gene function in monocotyledonous crops. Methods of effective DNA transfer into regenerable cells of immature embryos from cereals by means of Agrobacterium tumefaciens have been modified in a stepwise manner. The effect of particular improvement measures has often not been significantly evident, whereas their combined implementation has resulted in meaningful advances. Here, we provide updated protocols for the Agrobacterium-mediated generation of stably transgenic barley, wheat, triticale and maize. Based upon these methods, several hundred independent transgenic lines have been delivered, with efficiencies of inoculated embryos leading to stably transgenic plants reaching 86% in barley, 10% in wheat, 4% in triticale, and 24% in maize.

PROTEIN DISULFIDE ISOMERASE LIKE 5-1is a susceptibility factor to plant viruses

“…Naturel was cloned into the binary vector pIPKb002 (53) to generate ZmUbi_PDIL5-1_ox. The obtained destination plasmid was transformed into Agrobacterium tumefaciens strain AGL-1 (54). Isolation and culture of immature embryos, Agrobacterium-mediated transformation and regeneration of transgenic barley plants were conducted on the resistance parent W757/612 as previously described (54), with minor modifications.…”

“…The obtained destination plasmid was transformed into Agrobacterium tumefaciens strain AGL-1 (54). Isolation and culture of immature embryos, Agrobacterium-mediated transformation and regeneration of transgenic barley plants were conducted on the resistance parent W757/612 as previously described (54), with minor modifications. Prior to inoculation with Agrobacterium immature embryos were cultured on BCIM with dicamba at a final concentration of 5 mg/L for five days at 24°C in the dark.…”

Molecular Farming: Hosting Plants as Green Factories

“…One of the major beneficial outcomes of this technology is genetically modified (GM, transgenic) plants in which one or several genes have been inserted into plant genome from various sources by using different means such as Agrobacterium tumefaciens or particle bombardment [1,2]. Organel genome of certain plant species also become as target to generate transplastomic plants [3,4].…”

Repair of Site-Specific DNA Double-Strand Breaks in Barley Occurs via Diverse Pathways Primarily Involving the Sister Chromatid

“…The resulting cassette pUbi_I-SceI was then transferred into the binary vector p7U (DNA Cloning Service) using SfiI and HindIII restriction sites. Then, all three reporter constructs and the I-SceI construct were stably introduced into barley (Hordeum vulgare cv 'Golden Promise') via Agrobacterium-mediated gene transfer to immature embryos as described previously (Hensel et al, 2009). Plants were regenerated on selective media, and the presence of the transgene was confirmed by PCR.…”