Phenotypic Expression and Stability in a Large-Scale Field Study of Genetically Engineered Poplars Containing Sexual Containment Transgenes

Abstract

Genetic engineering (GE) has the potential to help meet demand for forest products and ecological services. However, high research and development costs, market restrictions, and regulatory obstacles to performing field tests have severely limited the extent and duration of field research. There is a notable paucity of field studies of flowering GE trees due to the time frame required and regulatory constraints. Here we summarize our findings from field testing over 3,300 GE poplar trees and 948 transformation events in a single, 3.6 hectare field trial for seven growing seasons; this trial appears to be the largest field-based scientific study of GE forest trees in the world. The goal was to assess a diversity of approaches for obtaining bisexual sterility by modifying RNA expression or protein function of floral regulatory genes, including LEAFY, AGAMOUS, APETALA1, SHORT VEGETATIVE PHASE, and FLOWERING LOCUS T. Two female and one male clone were transformed with up to 23 different genetic constructs designed to obtain sterile flowers or delay onset of flowering. To prevent gene flow by pollen and facilitate regulatory approval, the test genotypes chosen were incompatible with native poplars in the area. We monitored tree survival, growth, floral onset, floral abundance, pollen production, seed formation and seed viability. Tree survival was above 95%, and variation in site conditions generally had a larger impact on vegetative performance and onset of flowering than did genetic constructs. Floral traits, when modified, were stable over three to five flowering seasons, and we successfully identified RNAi or overexpression constructs that either postponed floral onset or led to sterile flowers. There was an absence of detectable somaclonal variation; no trees were identified that showed vegetative or floral modifications that did not appear to be related to the transgene added. Surveys for seedling and sucker establishment both within and around the plantation identified small numbers of vegetative shoots (root sprouts) but no seedlings, indicative of a lack of establishment of trees via seeds in the area. Overall, this long term study showed that GE containment traits can be obtained which are effective, stable, and not associated with vegetative abnormalities or somaclonal variation.

Transgenic Reduction of Cytokinin Levels in Roots Inhibits Root-Sprouting in Populus

“…Transgenic technology has been effectively used to produce poplar trees with a variety of useful traits, including herbicide resistance, biotic and abiotic tolerance, improved growth rate, higher nutrient use efficiency, and improved processing and end-use characteristics (Yang et al, 2009;Chang et al, 2018). Although many transgenic field trials have been carried out in the United States, China, and elsewhere, no transgenic trees have been commercially adopted to our knowledge (Li et al, 2016;Klocko et al, 2018).…”

“…For most tree species, gene flow primarily occurs through seed and pollen (DiFazio et al, 2012). A number of molecular confinement strategies have been reported that may block seed and pollen-mediated transgene flow in tree species (Klocko et al, 2018). We have also recently demonstrated that an AGA-MOUS intron-driven expression of a cytotoxin gene can be used to produce flowerless tobacco (Nicotiana benthamiana), and this technique may also be effective in reducing pollen-and seed-mediated transgene flow in poplar (Li et al, 2016).…”

Strategies for Engineering Reproductive Sterility in Plantation Forests

“…These include genes that regulate the transition to flowering, floral organ identity as well as pollen and ovule development. Although advances in sequencing have enabled the identification of flowering gene homologs in diverse angiosperm trees, there are few cases where in planta functions have been characterized in trees ( Brunner et al, 2017 ; Klocko et al, 2018 ). This is due to the long non-flowering period that can last years to decades and that for most species, genetic transformation is a formidable hurdle.…”

“…Targeting such genes for manipulation can thus result in undesired vegetative effects, such as delayed bud flush, in addition to predicted effects on flowering or no effect on flowering ( Hoenicka et al, 2012 ). However, promising results have also been achieved, such as the delayed flowering without growth reduction demonstrated by overexpressing the poplar ortholog of the floral repressor SHORT VEGETATIVE PHASE (SVP) ( Klocko et al, 2018 ). Manipulation of floral organ identity genes might be less likely to have vegetative effects as these genes may show stronger conservation of reproductive-only function.…”

Vaccination and Biopharming Technology

“…In Pakistan GMO food is being consumed but the labeling law is absent, whereas, equity, consistency, and fairness is the basic human right [7,8] maize, canola, cotton, and soybean are genetically engineered crops being used in different countries [9]. Other plants which are genetically engineered include poplar plant, gaining lots of interest [10]. Most recent development in biotechnology and bioengineering allows researchers to develop plant-based vaccines, plants are genetically manipulated [11] containing antigens to generate active immunity are called edible or green vaccines [12].…”