Abstract
Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicide-resistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3'-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation.
Highlights
Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicideresistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation
To discriminate between nuclear and chloroplast transgenic lines, an accD-N-Rv primer was designed that would anneal to the endogenous chloroplast genome and enable a 2.3-kb PCR product to be generated when paired with an internal acetolactate synthase (ALS) primer, ALS-1597-Fd (Fig. 2A)
The size of the PCR product amplified in lanes 7 and 8 was 1.2 kb, not 2.3 kb and 0.8 kb, indicating that the mutated ALS (mALS) region was not integrated into the chloroplast genome
Summary
Chloroplast transformation vectors, pLD200-mALS possessing the aadA and mALS (transit peptide truncated). PCR was performed with primer sets comprising rbcL-Fd (annealing to the homologous region of rbcL) and ALS-433-Rv to generate 0.83-kb products in lanes 1 to 4, 6, and 10 (Fig. 2C, bottom; results of P197S), and ALS-1597-Fd and accD-Rv (annealing to the homologous region of accD) to generate 1.2-kb products in lanes 1 to 4, 6 to 8, and 10 (Fig. 2C, middle; results of P197S), confirming the presence of transgenes in candidate lines. Transplastomic lines were identified by PCR with the primer set accD-N-Rv and ALS-1597-Fd, with 2.3-kb products visible in lanes 1 to 4, 6, and 10 (Fig. 2C, top; results of P197S), confirming site-specific integration of the transgenes into the tobacco chloroplast genome. Subsequent studies used the transplastomic plants of lane 2 for A122V, lane 4 for G121A, lane 6 for P197S, and lane 8 for W574L/S653I
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