Engineered Cleistogamy in Camelina sativa for bioconfinement

Abstract

Camelina sativa is a self-pollinating and facultative outcrossing oilseed crop. Genetic engineering has been used to improve camelina yield potential for altered fatty acid composition, modified protein profiles, improved seed and oil yield, and enhanced drought resistance. The deployment of transgenic camelina in the field posits high risks related to the introgression of transgenes into non-transgenic camelina and wild relatives. Thus, effective bioconfinement strategies need to be developed to prevent pollen-mediated gene flow (PMGF) from transgenic camelina. In the present study, we overexpressed the cleistogamy (i.e. floral petal non-openness)-inducing PpJAZ1 gene from peach in transgenic camelina. Transgenic camelina overexpressing PpJAZ1 showed three levels of cleistogamy, affected pollen germination rates after anthesis but not during anthesis, and caused a minor silicle abortion only on the main branches. We also conducted field trials to examine the effects of the overexpressed PpJAZ1 on PMGF in the field, and found that the overexpressed PpJAZ1 dramatically inhibited PMGF from transgenic camelina to non-transgenic camelina under the field conditions. Thus, the engineered cleistogamy using the overexpressed PpJAZ1 is a highly effective bioconfinement strategy to limit PMGF from transgenic camelina, and could be used for bioconfinement in other dicot species.

Figure 1

Illustration of the effects of the fully opened (A) or cleistogamous (non-opening) (B) flowers on PMGF from transgenic camelina to non-transgenic camelina or wild relatives. Red cross, inhibited PMGF. Bar = 1 mm.

Figure 2

Representative images of the three levels of cleistogamy in the single-copied homozygous transgenic camelina lines overexpressing the PpJAZ1 gene from peach. Day 0, flower bud stage (prior to flowering). Day 1, anthesis stage. Day 2, post-anthesis stage. Day 3, fruit formation stage. Red arrows, different flower developmental stages of the same flower of each line. Bar = 1 mm.

Figure 3

The relative expression levels of the transgene PpJAZ1 in the leaves of transgenic camelina lines overexpressing PpJAZ1 measured by qPCR. The camelina Actin gene was used as the reference gene. qPCR was conducted as described in Zhao et al. [77] and data analysis was conducted using the 2^−ΔΔCt method. The mean values of three independent replicates ± standard errors (vertical bars) are displayed.

Figure 4

The effect of the overexpressed PpJAZ1 gene on the silicle development on the main branches (A), silicle number per plant (B), and seed number per plant (C) of the transgenic camelina lines at Days 1 ~ 3. WT, non-transgenic. Red arrows, the aborted silicles on the main branches. Bar = 1 cm.

Figure 5

The effect of the overexpressed PpJAZ1 gene on pollen germination rates of the transgenic camelina lines at Days 1 ~ 3. (A) Representative images of pollen germination. (B) Pollen germination rate. WT, non-transgenic. Bar = 50 μm.

Figure 6

The effect of the engineered cleistogamy in the best transgenic camelina line on PMGF under the field conditions. One field site per transgene was used for the transgenic camelina line overexpressing the PpJAZ1 or GUSPlus reporter gene. In each field site, the best transgenic camelina line was planted for each transgene in the center of 5 × 5 m as the pollen source, while the non-transgenic camelina plants were planted as the pollen recipients in the four directions with distances of 0.5, 1, 2, 5, 10, 15, and 20 m for each direction. Seeds were harvested from all of the non-transgenic camelina plants at each distance in each direction on each field site, and used for germination on solid media containing the proper antibiotics for selection, followed by PCR confirmation of the presence of the transgene in the antibiotics-resistant seedlings.