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.

Assessing the extent and the environmental drivers of Eucalyptus globulus wildling establishment in Portugal: results from a countrywide survey

“…Apparently, the only way of eliminating the risk of spread both outside and inside forest stands, would be by producing and planting sterile trees, which appears to be feasible, namely in Eucalyptus spp. (Zhang et al 2012). Indeed, Zhang et al (2012) showed that it is possible to prevent pollen formation in different transgenic plants, including eucalypts, without detrimental effects on vegetative growth.…”

A tapetal ablation transgene induces stable male sterility and slows field growth in Populus

“…Surprisingly, this occurred in spite of coexpression of the barnase inhibitor barstar (Wei et al 2006). In contrast, Zhang et al (2012) did not observe any growth impairment in their field-grown, male-sterile pines and eucalypts, possibly because of the reduced toxicity of the barnase variant employed. They did not, however, present any data or statistical analysis in support of this observation.…”

“…Similarly, Zhang et al (2012) reported complete male sterility over 4 years in pine and over 2 years in eucalypts. Tobacco and silver birch transformed with the BpMADS1::BARNASE and the BpFULL1::BARNASE constructs were highly sterile for three and two consecutive years, respectively (Lännenpää et al 2005).…”

“…These results suggest that barnase toxicity can vary widely depending on species and growth environment. It also supports the need for steps to reduce barnase toxicity due to misexpression, which could include the use of spacers (Jagannath et al 2001), separation of subunits among plants following crossing (Burgess et al 2002;Bihao et al 2012), or attenuated versions of the protein (Zhang et al 2012).…”

“…The employed promoters were derived from the birch BpMADS1 gene (homologous to the Arabidopsis gene SEPALLATA3, previously known as AGL19) and the birch BpFRUITFULL-LIKE1 gene. Zhang et al (2012) showed that a male conedominant promoter from Monterey pine (Pinus radiata), PrMC2, fused to an attenuated version of barnase gave complete and stable male sterility in multi-year field trials of a pine hybrid (Pinus rigida×Pinus taeda) and a eucalypt hybrid (Eucalyptus grandis×Eucalyptus urophylla).…”