Control of pollen-mediated gene flow in transgenic trees

Abstract

Pollen elimination provides an effective containment method to reduce direct gene flow from transgenic trees to their wild relatives. Until now, only limited success has been achieved in controlling pollen production in trees. A pine (Pinus radiata) male cone-specific promoter, PrMC2, was used to drive modified barnase coding sequences (barnaseH102E, barnaseK27A, and barnaseE73G) in order to determine their effectiveness in pollen ablation. The expression cassette PrMC2-barnaseH102E was found to efficiently ablate pollen in tobacco (Nicotiana tabacum), pine, and Eucalyptus (spp.). Large-scale and multiple-year field tests demonstrated that complete prevention of pollen production was achieved in greater than 95% of independently transformed lines of pine and Eucalyptus (spp.) that contained the PrMC2-barnaseH102E expression cassette. A complete pollen control phenotype was achieved in transgenic lines and expressed stably over multiple years, multiple test locations, and when the PrMC2-barnaseH102E cassette was flanked by different genes. The PrMC2-barnaseH102E transgenic pine and Eucalyptus (spp.) trees grew similarly to control trees in all observed attributes except the pollenless phenotype. The ability to achieve the complete control of pollen production in field-grown trees is likely the result of a unique combination of three factors: the male cone/anther specificity of the PrMC2 promoter, the reduced RNase activity of barnaseH102E, and unique features associated with a polyploid tapetum. The field performance of the PrMC2-barnaseH102E in representative angiosperm and gymnosperm trees indicates that this gene can be used to mitigate pollen-mediated gene flow associated with large-scale deployment of transgenic trees.

Figure 1.

GUS staining of a young floral bud of tobacco carrying PrMC2_pro-I-GUS. The unopened floral bud was cut longitudinally in the middle using a blade, with the surface of the two anthers being partially cut, and stained for GUS activity overnight at 37°C. The photograph was taken after briefly destaining in ethanol to remove chlorophyll. The bright spots in the photograph are reflections in the ethanol left in the specimen during photographing. The GUS staining was found to be present only in the anthers.

Figure 2.

Visual and microscopic observations of tobacco flowers carrying PrMC2_pro-I-GUS or PrMC2_pro-I-barnaseH102E. A, Images of a single flower of GUS tobacco (left panel) and barnaseH102E tobacco (right panel) taken using a conventional camera. B, Images of a partially opened anther of GUS tobacco (left panel) and barnaseH102E tobacco (right panel) taken using a dissecting microscope with 9-fold magnification. The anthers were cut longitudinally with a blade. C, Images of sampled internal contents from a GUS anther (left panel) and a barnaseH102E anther (right panel) taken using a compound microscope with 200-fold magnification.

Figure 3.

Visual observation of bagged male cone clusters at 15 months after grafting. The GUS-labeled bag contains a PxL pine male cone cluster carrying PrMC2_pro-I-GUS, while the barnaseH102E bag contains a PxL male cone cluster carrying PrMC2_pro-I-barnaseH102E. The clear bag was made of cellulose membrane, which allows air exchange between inside and outside of the bag. The male cone clusters and vegetative shoots were able to grow and develop normally inside the bags without the possibility of pollen release into the environment. The aluminum rings inside the bags were used to anchor and support the bags on the branches of grafts. The open end of the bag was toward the bottom, and it was sealed with a sponge and a cable tie. Massive amounts of yellow-colored pollen grains were readily visible inside the GUS bag, while no pollen grains were visually observed inside the barnaseH102E bag.

Figure 4.

Visual and microscopic observations of bagged male cone clusters from PxL pine grafts carrying either PrMC2_pro-I-GUS or PrMC2_pro-I-barnaseH102E. A, The bagged male cone clusters were taken out of the bags, and the needles were trimmed to expose individual male cones. The GUS male cones were releasing pollen, while the barnaseH102E male cones contained no pollen. B, One GUS male cone and one barnaseH102E male cone in A were cut longitudinally in the middle using a blade and observed using a dissecting microscope. C, The pollen extraction procedure was performed with GUS or barnaseH102E male cones, and the resulting samples were observed using a compound microscope. A normal pine pollen grain has two wing-like appendages as seen in C from a GUS male cone.

Figure 5.

Microscopic observation of E. occidentalis single flowers. The barnaseH102E flower carries the construct pARB598 (Table I), which contains the pollen ablation cassette PrMC2_pro-I-barnaseH102E and an antisense 4CL fragment driven by the loblolly pine 4CL promoter. The images were taken using a dissecting microscope with 9-fold magnification.

Figure 6.

Microscopic observation of internal tissues sampled from different stages of male cones of transgenic PxL pine grafts carrying PrMC2_pro-I-GUS or PrMC2_pro-I-barnaseH102E. A, Image of tetrads from a GUS male cone at late stage 2.0 or early stage 3.0. The male cones were sampled on February 27, 2006, which is 1 week earlier than in B to D. B, Image of immature pollen grains of a GUS male cone at stage 3.3. C and D, Abnormal pollen development in barnaseH102E male cones at stage 3.3, with C showing enlarged tetrads with disorganized nuclear DNA and D showing degenerated tetrads. The magnification is 200-fold for all images.

Figure 7.

Microscopic observation of internal tissues sampled from 7- and 8-mm untransformed (A) and barnaseH102E (B) floral buds of E. occidentalis carrying PrMC2_pro-II-barnaseH102E. The size of the floral buds was classified by the length of the operculum. The floral buds were cut longitudinally in the middle, and internal tissues were sampled and observed using a compound microscope. The magnification was 468-fold for A and B.