Fate of forest tree biotechnology facing climate change

Woody plants have been cultured in vitro since the 1930s. After that time much progress has been made in the culture of tissues, organs, cells, and protoplasts in tree species. Tree biotechnology has been making strides in clonal propagation by organogenesis and somatic embryogenesis. These regeneration studies have paved the way for gene transfer in forest trees. Transgenics from a number of forest tree species carrying a variety of recombinant genes that code for herbicide tolerance, pest resistance, lignin modification, increased woody bio-mass, and flowering control have been produced by Agrobacterium-mediated and biolistic methods, and some of them are undergoing confined field trials. Although relatively stable transgenic clones have been produced by genetic transformation in trees using organogenesis or somatic embryogenesis, there were also unintended unstable genetic events. In order to overcome the problems of randomness of transgene integration and instability reported in Agrobacterium-mediated or biolistically transformed plants, site-specific transgene insertion strategies involving clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) platform offer prospects for precise genome editing in plants. Nevertheless, it is important to monitor phenotypic and genetic stability of clonal material, not just under greenhouse conditions, but also under natural field conditions. Genetically modified poplars have been commercialized in China, and eucalypts and loblolly pine are expected to be released for commercial deployment in USA. Clonal forestry and transgenic forestry have to cope with rapid global climate changes in the future. Climate change is impacting species distributions and is a significant threat to biodiversity. Therefore, it is important to deploy Strategies that will assist the survival and evolution of forest tree species facing rapid climate change. Assisted migration (managed relocation) and biotechnological approaches offer prospects for adaptation of forest trees to climate change.

Efficient Genome Editing in Populus Using CRISPR/Cas12a

The ability to create targeted mutations using clustered regularly inter-spaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 in support of forest tree biotechnology is currently limited. CRISPR/Cas12a is a novel CRISPR effector protein that not only broadens the CRISPR/Cas targeting range but also enables the generation of large-fragment deletions. In this study, a CRISPR/Cas12a system was evaluated for the induction of targeted mutations in the woody tree poplar (Populus alba × Populus glandulosa). Three Cas12a nucleases, namely, AsCas12a (Acidaminococcus sp. BV3L6), LbCas12a (Lachnospiraceae bacterium ND2006), and FnCas12a (Francisella tularensis subsp. novicidain U112), were used. We knocked out multiple targets of the phytoene desaturase gene 8 (PDS) using the CRISPR/Cas12a genome-targeting system, and the results indicated that the AsCas12a system is the most efficient. We further optimized the co-cultivation temperature after Agrobacterium-mediated transformation from 22 to 28°C to increase the Cas12a nuclease editing efficiency in poplar. AsCas12a showed the highest mutation efficiency, at 70%, and the majority of editing sites were composed of large-fragment deletions. By using this simple and high-efficiency CRISPR/Cas12a system, multiple targets can be modified to obtain multigene simultaneous knockout mutants in tree species, which will provide powerful tools with which to facilitate genetic studies of forest trees.