High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum

Agrobacterium-mediated transformation methods have allowed the stable introduction of target genes into the nuclear genomes of recipient plants. Among them, the floral dip approach represents the simplest due to its straightforwardness and high transformation efficiency. In a standard floral dip protocol that most researchers follow, Agrobacterium cells are grown to stationary phase (OD 600≈2.0) in large cultures and resuspended in inoculation medium to OD 600≥0.8. Here, we tested the effects of low Agrobacterium inoculum on transformation rate. Our data revealed that the floral dip method still guarantees a relatively high transformation rate in the Arabidopsis thaliana Col-0 ecotype even with very low Agrobacterium inoculum (OD 600=0.002). Our finding thus simplifies the floral dipping protocol further, which allows transformation with small bacterial culture and enables high-throughput transformation of large numbers of constructs in parallel.

High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum

Agrobacterium-mediated transformation methods have allowed the stable introduction of target genes into the nuclear genomes of recipient plants. Among them, the floral dip approach represents the simplest due to its straightforwardness and high transformation efficiency. In a standard floral dip protocol that most researchers follow, Agrobacterium cells are grown to stationary phase (OD600≈2.0) in large cultures and resuspended in inoculation medium to OD600≥0.8. Here, we tested the effects of low Agrobacterium inoculum on transformation rate. Our data revealed that the floral dip method still guarantees relatively high transformation rate in Arabidopsis thaliana Col-0 ecotype even with very low Agrobacterium inoculum (OD600=0.002). Our finding thus simplifies the floral dipping protocol further, which allows transformation with small bacterial culture and enables high-throughput transformation of large numbers of constructs in parallel.

Agrobacterium-mediated floral-dip transformation of the obligate outcrosser Capsella grandiflora

Plant transformation by floral dip has been essential for research on plant genetics. The plant family Brassicaceae is one of the most well studied plant families and contains both established and emerging genetic model species. Two emerging model species that bear on the evolution of the selfing syndrome are Capsella grandiflora, an obligate outcrosser, and C. rubella, an inbreeder. While the selfing syndrome has been well characterized at the genomic level the genetic mechanisms underlying it remain elusive, in part due to the challenges of establishing mutation lines in C. grandiflora. Here, we describe an efficient method for transforming C. grandiflora by Agrobacterium-mediated floral-dip while simultaneously tracking self-incompatibility loci. With the ability to transform both C. grandiflora and C. rubella, researchers have gained a valuable tool to study the progression to selfing at the genetic level.

“Floral-dip” transformation of Amaranthus caudatus L. and hybrids A. caudatus × A. paniculatus L.

After “floral-dip” transformation of Amaranth plants with Agrobacterium tumefaciens strain GV3101 carrying pCBV19 gene vector that contained bar and gus genes, transgenic seeds were obtained. The functioning of the tran+sferred genes in Amaranthus tissues was confirmed with herbicide selection (PPT herbicide – phospinotricin) and gus gene activity. Positive results were obtained for cultivars “Karmin” and “Kremoviy rannii”. The percentage of GUS positive samples was 1% (for “Karmin”), 2.2% (for “Kremoviy rannii”) from the total initial quantity of plants that was prior to selection with the herbicide. The seeds of six amaranth cultivars were received after treatment with A. tumefaciens by the method “floral dip”. The lowest lethal dose of herbicide PPT was established – 40 mg/l. After spraying with herbicide, resistant plants were obtained for cultivars: “Kremoviy rannii” (21%) and “Karmin” (20%). After conduction of PCR analysis, positive results were obtained for four cultivars. The percentage of bar positive plants was 0.3% (“Helios”); 0.26% (“Sterkch”); 0.06% (“Kremoviy rannii”); 0.3% (“Rushnichok”) from total initial quantity of plants.