ptxD gene in combination with phosphite serves as a highly effective selection system to generate transgenic cotton (Gossypium hirsutum L.)

Abstract

This report demonstrates the usefulness of ptxD/phosphite as a selection system that not only provides a highly efficient and simple means to generate transgenic cotton plants, but also helps address many of the concerns related to the use of antibiotic and herbicide resistance genes in the production of transgenic crops. Two of the most popular dominant selectable marker systems for plant transformation are based on either antibiotic or herbicide resistance genes. Due to concerns regarding their safety and in order to stack multiple traits in a single plant, there is a need for alternative selectable marker genes. The ptxD gene, derived from Pseudomonas stutzeri WM88, that confers to cells the ability to convert phosphite (Phi) into orthophosphate (Pi) offers an alternative selectable marker gene as demonstrated for tobacco and maize. Here, we show that the ptxD gene in combination with a protocol based on selection medium containing Phi, as the sole source of phosphorus (P), can serve as an effective and efficient system to select for transformed cells and generate transgenic cotton plants. Fluorescence microscopy examination of the cultures under selection and molecular analyses on the regenerated plants demonstrate the efficacy of the system in recovering cotton transformants following Agrobacterium-mediated transformation. Under the ptxD/Phi selection, an average of 3.43 transgenic events per 100 infected explants were recovered as opposed to only 0.41% recovery when bar/phosphinothricin (PPT) selection was used. The event recovery rates for nptII/kanamycin and hpt/hygromycin systems were 2.88 and 2.47%, respectively. Molecular analysis on regenerated events showed a selection efficiency of ~ 97% under the ptxD/Phi system. Thus, ptxD/Phi has proven to be a very efficient, positive selection system for the generation of transgenic cotton plants with equal or higher transformation efficiencies compared to the commonly used, negative selection systems.

New Breeding Techniques for Greenhouse Gas (GHG) Mitigation: Plants May Express Nitrous Oxide Reductase

“…Indeed, the global advance promulgating engineered crops is pillared on today's artificial intelligence-guided plant codon optimization rules offered by both large and small boutique DNA houses. However, there has been success expressing native bacterial sequences in plants, i.e., in the case of cotton expressing the native sequence of the P. stutzeri gene ptxd (PHOSPHONATE DEHYDROGENASE) [127,128]. One can dare to fathom how a universally-functional nosZ expression system could conceivably redirect some aspects of GHG mitigation research.…”

The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas

“…Furthermore, the use of metabolic markers helps address the regulatory and safety concerns associated with the environmental spread of antibiotic resistance genes by horizontal gene transfer (HGT) during commercial cultivation (EFSA GMO Panel 2004;Beacham et al 2017). Various reports have shown that ptxD can serve as an efficient marker for generation of transgenic plants (López-Arredondo and Herrera-Estrella 2013; Nahampun et al 2016;Pandeya et al 2017), yeasts (Kanda et al 2014) and cyanobacteria (Selão et al 2019). However, whilst the very low abundance of Phi in the natural environment means that HGT of the ptxD marker to other microbial species is unlikely to confer any selective advantage, this could still compromise its use as for crop protection.…”

Growth and selection of the cyanobacterium Synechococcus sp. PCC 7002 using alternative nitrogen and phosphorus sources

“…Recently, the use of ecologically rare or xenobiotic sources of macronutrients has been explored as a means to generate selective pressure towards the growth of genetically modified organisms without the use of antibiotics (Kanda et al, 2014;Loera-Quezada et al, 2016;Pandeya et al, 2017;Polyviou et al, 2015;Shaw et al, 2016) as well as to allow genetically modified plants to outcompete weeds, while consuming considerably less phosphorus (Lopez-Arredondo and Herrera-Estrella, 2012). To this end, phosphite dehydrogenase (PtxD), an enzyme converting phosphite, an ecologically rare form of phosphorus, into phosphate, has been introduced into a variety of organisms, and in some cases used as a selectable marker (Kanda et al, 2014;Lopez-Arredondo and Herrera-Estrella, 2012;Nahampun et al, 2016;Pandeya et al, 2017). A synthetic pathway to utilize melamine, a xenobiotic nitrogen-rich compound, has also been devised and introduced into different organisms (Shaw et al, 2016).…”