Population growth, coupled with the need to transition from a petrochemical-based economy towards a more sustainable bio-based one, is predicted to increase the demand for wood and other forest-based products three-fold by 2050 [1]. This increased demand, together with the challenges associated with climate change and the need to increase agricultural production, will put further pressure on the area and quality of natural forests. It has been estimated that planted forests, which comprise only 7% of the global forest area, have the potential to supply two-thirds of global roundwood demand [2] and offer a route to sustainably increase the production of forest products and reduce pressure on natural forests. To meet the increased demand, it will be necessary to further increase the productivity of planted forests.
There is a long history of productivity improvements in commercially planted conifer species through traditional breeding and silviculture [3, 4]. The use of genomic-based breeding technologies, particularly the implementation of genomic selection are also showing promise for implementation into breeding programs [5–7]. Long breeding cycles, large and complex genomes, variable juvenile-mature correlations, emerging pests and diseases, climate, and market changes provide challenges to breeding approaches that have to date led to moderate gains in conifers [6, 8].
Direct manipulation of conifer genomes offers a potentially more rapid route to trait improvement and allows the introduction of novel traits as well as improvement of existing ones. Demonstrated trait modifications in conifers include; insect resistance [9, 10], herbicide tolerance [11, 12], wood pulping efficiency [13, 14], stress tolerance [15] and sterility [16]. These technologies also enable production of rationally designed trees that produce biochemicals and biomass for specific purposes [17], yet, no modified conifers have been commercialized. These modification technologies require the introduction of new genes either via Agrobacterium or biolistic based methods [18]. However, the transformation of conifers is challenging, relying on complex somatic embryogenesis protocols, with many species or genotypes proving recalcitrant to somatic embryogenesis protocols and/or transformation [18]. The lack of efficient transformation systems for elite germplasm intended for large-scale production remains a major challenge for genetically modified varietal forestry [19].
Over the last decade, genome editing, particularly the CRISPR/Cas9 system, has been widely used in plants, both for fundamental research and precision breeding [20–22], with the first genome-edited food introduced into the market in 2019 [23]. Novel traits or traits difficult to achieve by breeding, such as biotic- and abiotic-stress resistance [24–26], and sterility [27] can be generated by knockout-mediated trait improvement. Desirable traits can be fine-tuned by generating a range of alleles through either genome editing or base editing [28–31]. Successful demonstrations of editing have included trees like poplar and eucalyptus [32–35]. As far as we are aware, genome editing is yet to be demonstrated in coniferous trees.
Globally, organisms that have had foreign DNA introduced into their genome are considered to be GMOs and are subject to various levels of regulation. However, genome edited plants where the transgene has been removed by crossing and segregation, are not regulated as GMOs in many countries, including Australia, Argentina, Canada, Japan, and the USA [36, 37]. Yet, the European Union and New Zealand still considers such mutated plants to be GMOs and regulates them accordingly [38].
In conifers, removing transgenes by segregation is challenging due to their long breeding timescales. Genome editing mediated by direct delivery of CRISPR/Cas9 ribonucleoproteins (RNPs), circumvents the introduction of new DNA into the plant genome, and as above would not be regulated as GMOs in many countries [39]. The ability to produce edited plants without the requirement to undergo time consuming breeding to remove transgenes makes the use of RNP-mediated editing particularly attractive for slow-breeding conifers.
Pinus radiata D. Don., a conifer species native to California, is the world’s most extensively planted exotic softwood [40] due to its high productivity and suitability for the construction timber, furniture, pulp and paper industries [41]. It is predominantly planted in Australia, Chile, and Spain and is the dominant species in New Zealand planted forests, where it comprises 90% of the planted production forest area and contributes 1.6% to GDP [42]. We have investigated the use of CRISPR/Cas9 to edit the P. radiata glucuronic acid substitution of the xylan 1 (GUX1) gene [43, 44] and demonstrated genome editing using DNA and RNPs.