Genetically engineered rice endogenous 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) transgene alters phenology and fitness of crop-wild hybrid offspring

Abstract

Genetically engineered (GE) rice endogenous epsps (5-enolpyruvoylshikimate-3-phosphate synthase) gene overexpressing EPSPS can increase glyphosate herbicide-resistance of cultivated rice. This type of epsps transgene can enhance the fecundity of rice crop-weed hybrid offspring in the absence of glyphosate, stimulating great concerns over undesired environmental impacts of transgene flow to populations of wild relatives. Here, we report the substantial alteration of phenology and fitness traits in F₁-F₃ crop-wild hybrid descendants derived from crosses between an epsps GE rice line and two endangered wild rice (Oryza rufipogon) populations, based on the common-garden field experiments. Under the glyphosate-free condition, transgenic hybrid lineages showed significantly earlier tillering and flowering, as well as increased fecundity and overwintering survival/regeneration abilities. In addition, a negative correlation was observed between the contents of endogenous EPSPS of wild, weedy, and cultivated rice parents and fitness differences caused by the incorporation of the epsps transgene. Namely, a lower level of endogenous EPSPS in the transgene-recipient populations displayed a more pronounced enhancement in fitness. The altered phenology and enhanced fitness of crop-wild hybrid offspring by the epsps transgene may cause unwanted environmental consequences when this type of glyphosate-resistance transgene introgressed into wild rice populations through gene flow.

Figure 1

Number of tillers per plant (a: WR1-F2; b: WR2-F2); flowering time (c: WR1-F2; d: WR2-F2); and seed set ratios (e: WR1-F2; f: WR2-F2) of F₂ transgenic (solid squares, dark grey columns) and non-transgenic (empty triangles, white columns) hybrid lineages in pure planting. Solid triangles, empty circles, and solid diamonds (in c and d) indicate days at which 1%, 30%, and 50% plants being flowered, respectively. The comparisons were made between transgenic and non-transgenic F₂ hybrid lineages in pure-planting based on independent t-test (N = 6). Bars represent standard error. * or ** indicates significances at the levels of P < 0.05 or P < 0.01, respectively.

Figure 2

Number of tillers (a, b), panicles (c, d), and filled seeds (e, f) per plant in F₁ transgenic (dark grey columns) and non-transgenic (white columns) crop-wild hybrids derived from WR1 (left panel) and WR2 (right panel), in mixed-planting plots (30 cm, 40 cm, 50 cm plant spacing) with different densities. The comparisons were made between transgenic and non-transgenic F₁ hybrids in mix planting based on paired t-test (N = 6). Bars represent standard error. * or ** indicates significances at the levels of P < 0.05 or P < 0.01, respectively.

Figure 3

Number of panicles (a, b) and filled seeds (c, d) per plant of F₂ transgenic (dark grey columns) and non-transgenic (white columns) crop-wild hybrid lineages in pure-planting (a, c) and mix-planting plots (b, d). The comparisons were made between transgenic and non-transgenic F₂ hybrid lineages in mix planting (30 cm) based on paired t-test (N = 6). Bars represent standard error. * or ** indicates significances at the levels of P < 0.05 or P < 0.01, respectively.

Figure 4

Germination ratios of buried-seeds (a: WR1-F3, b: WR2-F3) and ratios of tiller regeneration (c: WR1-F1 and WR1-F2, d: WR2-F1 and WR2-F2) in transgenic (dark grey columns) and non-transgenic (white columns) crop-wild hybrid lineages. The comparisons were made between transgenic and non-transgenic hybrid lineages based on independent t-test (N = 3 for seed germination ratio; N = 6 for tiller regeneration ratio). Bars represent standard error. * or ** indicates significances at the levels of P < 0.05 or P < 0.01, respectively.

Figure 5

Correlation between the ratios of increased panicles (a, b, and c) or seeds (d, e, and f) and the content of endogenous EPSPS proteins in different transgene recipients (parents) at 60 (a and d), 100 (b and e), and 160 (c and f) days after seed germination. Solid diamonds: wild rice (WR1); empty diamonds: wild rice (WR2); empty circles: weedy rice (WRD1); solid circles: weedy rice (WRD2), and solid triangles: cultivated rice (Minghui-86).

Figure 6

Schematic illustration of the pedigrees to produce F₁-F₃ crop-wild hybrid lineages. T: transgenic; + +, + −, and − −: transgene homozygous, transgene heterozygous, and non-transgenic, respectively. F₁ and F₂ hybrid lineages were used to test differences in fitness; the F₃ hybrid lineages were used to test differences in seed germination after being buried in soils.