Aims – Fast growing world global population and rapid progressing climate change are significant challenges for future scientific research. Besides ensuring sufficient amounts of high-quality food, also adequate amounts of renewable and bio-based material resources have to be produced. One important renewable material resource is wood, harvested from trees either grown in forests or in short rotation coppices (SRC). Fast growing trees grown in SRCs like poplar (Populus spec.) and willow (Salix spec.) offer the opportunity to combine the rising demand of wood for material and energy use along its value chain in frame of a bioeconomy strategy. Unfortunately, forest tree breeding is very slow due to tree’s characteristics like longevity and long vegetative phases. Therefore, the new set of novel breeding techniques, in particular CRISPR/Cas9, offers the opportunity to significantly accelerate tree breeding. This project aimed to methodically test two different kinds of CRISPR/Cas9 systems in poplar leading to non-transgenic genome edited plants.

Work plan – Two different kinds of CRISPR/Cas9 systems will be tested in poplar: (i) plasmid containing Cas9 gene and guideRNA (gRNA), and (ii) DNA-free Cas9/gRNA ribonucleinprotein complex. Both systems will contain gRNA for PHYTOENE DESATURASE (PDS) gene. The PDS gene is not of commercial interest, however, in this proof-of-concept project lasting two years, it serves as an easily detectable marker gene for successful PDS gene editing. In case of success, this gene can easily be replaced by any gene of commercial interest.

The two systems will transiently be transferred to both poplar protoplasts and germinating pollen. In a previous BMBF-funded project, we developed protocols to successfully isolate, stably as well as transiently transform poplar protoplasts. CRISPR/Cas9-treated protoplasts will be cultivated until the callus stage. Obtained calli will be screened for heterozygous and homozygous edited sites in the PDS candidate gene, and regenerated to plants. Possible occurrence of “somaclonal variation” in plants regenerated from protoplasts will be checked for SNPs inactivating the PDS gene that are not attributable to the CRISPR/Cas9 system by sequencing the down- and upstream regions of the PAM sequence.

Germinating pollen treated with CRISPR/Cas9 plasmid DNA or ribonucleinprotein complex will be used to pollinate female poplar. Due to previous work, we developed an early flowering protocol in poplar under application of the heat-shock controlled FLOWERING LOCUS T (FT) system. Using this approach, we are able to perform crossings in six to eight month-old poplar instead of 7 to 10 years old plants. Obtained seedlings will be screened for heterozygous edited sites in the PDS candidate gene, for presence of the early-flowering gene and, in case of the plasmid approach, for absence of the Cas9 gene. Selected seedlings will be cultivated for 6-8 months, and thanks to the early flowering protocol, heat-treated to induce flowering. Male plants will be used to pollinate female ones. Obtained F2-seedlings will be screened for homozygous edited sites in the candidate gene and absence of the FT gene. Plants produced by this technology are still transgenic in regard to European legislation. In this proposal, the early flowering technology is applied just to test the proof-of-concept approach and will later not be considered in practical applications.

Expected results – The application of both systems will lead to poplar plants that don’t carry any foreign DNA sequences but only reveal a mutation in the selected PDS candidate gene. While the protoplast approach will be applicable only in poplar clones with sufficiently developed protoplast regeneration systems, the transient pollen approach could easily be transferred to any other tree species. Thus, adopting this novel proof-of-concept approach to other tree species like ash, elder, beech or oak, could significantly accelerate tree breeding to encounter the biotic and abiotic threats ongoing with a rapid progressing climate change.