The first crop plant genetically engineered to release an insect pheromone for defence

Abstract

Insect pheromones offer potential for managing pests of crop plants. Volatility and instability are problems for deployment in agriculture but could be solved by expressing genes for the biosynthesis of pheromones in the crop plants. This has now been achieved by genetically engineering a hexaploid variety of wheat to release (E)-β-farnesene (Eβf), the alarm pheromone for many pest aphids, using a synthetic gene based on a sequence from peppermint with a plastid targeting amino acid sequence, with or without a gene for biosynthesis of the precursor farnesyl diphosphate. Pure Eβf was produced in stably transformed wheat lines with no other detectable phenotype but requiring targeting of the gene produced to the plastid. In laboratory behavioural assays, three species of cereal aphids were repelled and foraging was increased for a parasitic natural enemy. Although these studies show considerable potential for aphid control, field trials employing the single and double constructs showed no reduction in aphids or increase in parasitism. Insect numbers were low and climatic conditions erratic suggesting the need for further trials or a closer imitation, in the plant, of alarm pheromone release.

Figure 1. Gas chromatography (GC) analysis of volatiles released from transformed wheat plants 71 days after sowing:

(upper trace) B2812 R9P1 possessing the EβfS gene alone, (lower trace) B28203 R6P1 possessing EβfS and FPPS , both with plastidial targeting sequence,. 1 = myrcene, 2 = (E)- β-farnesene (Eβf). Note different scales on y-axis.

Figure 2. Time spent (mean ± s.e.) by cereal aphids Sitobion avenae (Sa), Rhopalosiphum padi (Rp) and Metopolophium dirhodum (Md) in different regions of a 4-arm olfactometer (n = 10).

Aphids were exposed to four discrete odour streams, one treated with a headspace sample of volatiles collected from wheat seedlings, the other three treated with solvent (redistilled diethyl ether) control. Treatments that are significantly different (P < 0.05) are marked with an asterisk. (a) Aphids were not repelled by volatiles from untransformed Cadenza wheat seedlings. (b,c) All three aphid species spent significantly less time in the olfactometer region containing volatiles from the transformed wheat lines B2812 R9P1 and B2803 R6P1.

Figure 3. Alarm response of cereal aphid colonies (n = 17–19) to 1 μl drops of either samples of volatiles released by, B2812 R9P1 and B2803 R6P1 wheat plants containing 19 and 62 ng respectively, pure synthesised (E)-β-farnesene (1000 ng) or a solvent control:

percentage of aphids that responded and/or moved away from the area after 1 minute (mean + /− s.e.). *indicates significant difference (P < 0.05) in response between treatment and (E)-β-farnesene.

Figure 4. Time spent by foraging Aphidius ervi parasitoids on wheat seedlings (n = 15).

Individual gravid females were released at the centre of the plant and time spent (mean ± s.e.) performing different behaviours was recorded until they left the plant. Compared to the untransformed wheat, total time spent was significantly longer on the transformed wheat lines B2812 R9P1 and B2803 R6P1 (P = 0.002).

Figure 5. Aphid infestation levels in field plot trials.

Ninety-nine plants or tillers were inspected in each plot each week. There were 4 plots of each of the transformed wheat lines B2812 R9P1 and B2803 R6P1 and eight plots of untransformed Cadenza wheat. No significant differences in infestation levels were detected.

Figure 6. Scheme showing predominantly Eβf from the double construct and the production of lower levels of myrcene from geranyl diphosphate (GPP) when the immediate precursor (FPP) is insufficiently available in the single construct (see Table S2).