Modifying fatty acid profiles through a new cytokinin‐based plastid transformation system

Abstract

SUMMARYThe widespread use of herbicides and antibiotics for selection of transgenic plants has not been very successful with regard to commercialization and public acceptance. Hence, alternative selection systems are required. In this study, we describe the use of ipt, the bacterial gene encoding the enzyme isopentenyl transferase from Agrobacterium tumefaciens, as a positive selectable marker for plastid transformation. A comparison between the traditional spectinomycin-based aadA selection system and the ipt selection system demonstrated that selection of transplastomic plants on medium lacking cytokinin was as effective as selection on medium containing spectinomycin. Proof of principle was demonstrated by transformation of the kas-III gene encoding 3-ketoacyl acyl carrier protein synthase III into tobacco plastids. Transplastomic tobacco plants were readily obtained using the ipt selection system, and were phenotypically normal despite overexpression of isopentenyl transferase. Over-expression of KASIII resulted in a significant increase in 16:0 fatty acid levels, and a significant decrease in the levels of 18:0 and 18:1 fatty acids. Our study demonstrates use of a novel positive plastid transformation system that may be used for selection of transplastomic plants without affecting the expression of transgenes within the integrated vector cassette or the resulting activity of the encoded protein. This system has the potential to be applied to monocots, which are typically not amenable to traditional antibiotic-based selection systems, and may be used in combination with a negative selectable marker as part of a two-step selection system to obtain homoplasmic plant lines.

The Engineered Chloroplast Genome Just Got Smarter

“…The aadA gene, first successfully used for Chlamydomonas chloroplast transformation [34] and later in tobacco chloroplasts [35] on spectinomycin selection, is the only marker that has worked reproducibly to regenerate transplastomic events in a number of different plant species. In recent years, several antibiotic free selectable markers including D-amino acid oxidase [36], positive selectable marker isopentenyl transferase (ipt)[37], as well asanthranilate synthase [alpha]-subunit ( ASA2 ) gene [38] have been developed (Figure1B). Removal of antibiotic selectable markers could be achieved using direct repeats or cre-lox recombination approaches [39].…”

A mutant Synechococcus gene encoding glutamate 1-semialdehyde aminotransferase confers gabaculine resistance when expressed in tobacco plastids

“…Recently, the use of Agrobacterium tumefaciens ipt, encoding the enzyme isopentenyl transferase, as a selectable marker for plastid transformation has been demonstrated (Dunne et al 2014). …”

Transplastomic plants for innovations in agriculture. A review

“…The company is a spinout from the University of Stavanger, with the main profile to produce foreign proteins such as kinases and proteins from parasites in tobacco chloroplasts, and to develop plastid transformation technologies in maize and wheat. A core asset in the portfolio of Plastid AS is the utilization of plant hormone-based selection and differentiation (Dunne et al 2014), which addresses a major bottleneck in the generation of transplastomic plants. To our knowledge, the company currently has no field tests in progress.…”

“…Non-antibiotic, native plant genes that offer dominant and portable selectable markers like ASA2 (Barone et al 2009) or confer additional agricultural advantage to the transformed crops such as BADH conferring salt or drought tolerance to transformed crops Kumar et al 2004a) may be good alternatives to antibiotic selection markers, and a few examples have also shown improved and in some cases inducible Lössl et al 2005;Buhot et al 2006;Tungsuchat et al 2006;Verhounig et al 2010) transgene expression in different organs and plastid types (e.g., Valkov et al 2011;Zhang et al 2012;Caroca et al 2013). A recently developed positive selection protocol utilizes the Agrobacterium tumefaciens isopentenyl transferase (ipt) gene involved in early stage of cytokinin biosynthesis and allows cell proliferation and differentiation into shoots without the use of exogenous cytokinin in the selection medium (Dunne et al 2014). However, further basic research and developments towards commercial applications are necessary.…”

“…During the proof-ofconcept study of a new, cytokinin-based selection method for the production of transplastomic tobacco plants, the insertion of kasIII into the plastid genome resulted in altered fatty acid composition of the leaves (Dunne et al 2014). …”