Containment of herbicide resistance through genetic engineering of the chloroplast genome

Abstract

Glyphosate is a potent herbicide. It works by competitive inhibition of the enzyme 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS), which catalyzes an essential step in the aromatic amino acid biosynthetic pathway. We report the genetic engineering of herbicide resistance by stable integration of the petunia EPSPS gene into the tobacco chloroplast genome using the tobacco or universal vector. Southern blot analysis confirms stable integration of the EPSPS gene into all of the chloroplast genomes (5000-10,000 copies per cell) of transgenic plants. Seeds obtained after the first self-cross of transgenic plants germinated and grew normally in the presence of the selectable marker, whereas the control seedlings were bleached. While control plants were extremely sensitive to glyphosate, transgenic plants survived sprays of high concentrations of glyphosate. Chloroplast transformation provides containment of foreign genes because plastid transgenes are not transmitted by pollen. The escape of foreign genes via pollen is a serious environmental concern in nuclear transgenic plants because of the high rates of gene flow from crops to wild weedy relatives.

Figure 1

Growth curves of E. coli XL1 blue strain containing the plasmids (A) pZS-RD-EPSPS in the presence of 10 mM glyphosate (–Δ–) or 40 mM glyphosate (–○–) and pZS-197 (control) in the presence of 10 mM glyphosate (–◊–) or 40 mM glyphosate (–□–) or (B) pSBL-RD-EPSPS in the presence of 10 mM glyphosate (–Δ–) or 40 mM glyphosate (–○–) and pSBL-ctv2 (control) in the presence of 10 mM glyphosate (–◊–) or 40 mM glyphosate (–□–) in M9 minimal medium.

Figure 2

PCR analysis of the pZS-RD-EPSPS and pSBL-RD-EPSPS transgenic lines. (A) Lanes 2–7: primers for rbcL and aadA; lanes 10–15: external primers for rbcL and aadA. Lanes 1 and 9: unbombarded tissue; lane 8:1 kb ladder. (B) Lanes 1–4: internal primers for 16S rRNA and aadA. Lanes 7–10: external primers for 16S rRNA and aadA. Lanes 5 and 11: unbombarded tissue; lane 6:1 kb ladder.

Figure 3

Southern blot analysis of transgenic plants. (A) EcoRI digested total DNA probed with the EPSPS gene. (B) EcoRI digested total DNA probed with the chloroplast border fragment comprising rbcL-orf512. (A and B) Lane 1: untransformed tobacco plant; lanes 2–4: total DNA isolated from three transgenic lines (15A, 15-2, 15-5). (C) Structure of the chloroplast genome with the site of foreign gene integration represented by a shaded dotted line.

Figure 4

Analysis of maternal inheritance of spectinomycin resistance in the seed progeny of chloroplast transgenic plants. The control and transgenic seeds were germinated in MSO medium containing spectinomycin (500 µg/ml). (A) Control seedlings (B) Transgenic seedlings.

Figure 5

Herbicide resistance in the progeny of chloroplast transgenic plants. Eighteen-week-old plants sprayed with 5 mM glyphosate. (A) Transgenic plants. (B) Control plants.