Persistence of Unselected Transgenic DNA during a Plastid Transformation and Segregation Approach to Herbicide Resistance

Abstract

The use of a nonlethal selection scheme, most often using the aadA gene that confers resistance to spectinomycin and streptomycin, has been considered critical for recovery of plastid transformation events. In this study, the plastid-lethal markers, glyphosate or phosphinothricin herbicides, were used to develop a selection scheme for plastids that circumvents the need for integration of an antibiotic resistance marker. The effect of selective agents on tobacco (Nicotiana tabacum) mesophyll chloroplasts was first examined by transmission electron microscopy. We found that at concentrations typically used for selection of nuclear transformants, herbicides caused rapid disintegration of plastid membranes, whereas antibiotics had no apparent effect. To overcome this apparent herbicide lethality to plastids, a "transformation segregation" scheme was developed that used two independent transformation vectors for a cotransformation approach and two different selective agents in a phased selection scheme. One transformation vector carried an antibiotic resistance (aadA) marker used for early nonlethal selection, and the other transformation vector carried the herbicide (CP4 or bar) resistance marker for use in a subsequent lethal selection phase. Because the two markers were carried on separate plasmids and were targeted to different locations on the plastid genome, we reasoned that segregation of the two markers in some transplastomic lines could occur. We report here a plastid cotransformation frequency of 50% to 64%, with a high frequency (20%) of these giving rise to transformation segregants containing exclusively the initially nonselected herbicide resistance marker. Our studies indicate a high degree of persistence of unselected transforming DNA, providing useful insights into plastid chromosome dynamics.

Application of Mutated Acetolactate Synthase Genes for Herbicide Resistance to Crop Improvement

“…We have tried to adapt mALSs for use as selectable markers in chloroplast transformation but have not succeeded to date. As with epsps and bar (Cao et al, 1992, Ye et al, 2003 …”

“…The 'secondary selective markers', bar and epsps, are also derived from bacteria and confer resistance to herbicides such as phosphinothricin ) and glyphosate (Ye et al, 2003), respectively. To date, introduction of mALSs to the chloroplast genome has not been attempted perhaps because ALS was thought to be unsuitable as a selectable marker, as in the case of bar or epspe (Cao et al, 1992, Ye et al, 2003. We therefore attempted to utilize mALSs in chloroplast engineering strategies.…”

Agrobacterium-mediated Genetic Transformation in Cucumber (Cucumis sativus L.)

“…Leaf disc assay and colour viability test for the plants transformed with Agrobacterium strain EHA 105 under the selection of PPT revealed more positive results compared to the wild type control plants [44] . In tobacco primary mode of action for glyphosate and PPT herbicides on non-transformed shoots leads to lethal effect [45] . Transgenic lines arising after the herbicide selection phase were subjected to leaf disc assay showed non lethal effect.…”

Transplastomic plants for innovations in agriculture. A review

“…Herbicides can also be used as selective agents (Table 1); therefore, much effort has been devoted to develop transplastomic plants with resistance to different herbicides such as glyphosate (Daniell et al 1998;Ye et al 2001Ye et al , 2003Chin et al 2003;Roudsari et al 2009), phosphinothricin/ glufosinate ammonium (Iamtham and Day 2000;Lutz et al 2001;Kang et al 2003;Ye et al 2003), sulcotrione (Falk et al 2005), isoxaflutole/diketonitrile (Dufourmantel et al 2007), chlorophenylthio-triethylamine (CPTA) (Wurbs et al 2007), pyrimidinylcarboxylate, imidazolinone and sulfonylurea (Shimizu et al 2008), and paraquat (methyl-viologen) (Le Poage et al 2011;Chen et al 2014). Herbicide resistance is achieved (1) by the insertion of a bacterial marker gene (such as bar) encoding an enzyme that inactivates the herbicide (phosphinothricin/glufosinate ammonium-Iamthan and Day 2000; Lutz et al 2001Lutz et al , 2006, (2) by overexpression of the genes of plastidial metabolic enzymes that are the targets of herbicides (e.g., EPSPS: glyphosate- Daniell et al 1998; hppd: sulcotrione and the isoxaflutole derivative, diketonitrile -Falk et al 2005 andDufourmantel et al 2007, respectively), (3) by expression of the genes of mutant, herbicide-resistant plant enzymes (CP4: glyphosate- Ye et al 2001Ye et al , 2003Roudsari et al 2009; mALS: pyrimidinylcarboxylate, imidazolinone, and sulfonylurea- Shimizu et al 2008), or (4) by expression of enzyme genes involved in antioxidant defense, minimizing this way the metabolic impact of the herbicides via the generation of ROS (DHAR, GST, gor: paraquat/methyl-viologen-Le MnSOD: paraquat/methyl-viologen-Poage et al 2011). For instance, glyphosate is a competitive inhibitor of one enzyme of the plastid aromatic amino acid biosynthesis pathway, namely 5-enolpyruvylshikimate-3-phosphate (EPSPS).…”