The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.

CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.

This study focuses on the role of the qualitative leaf wax composition in modulating the cuticular water loss using a Populus × canescens cer6 mutant line, which accumulates C34–C46 wax ester dimers and is reduced in wax monomers >C24. The two literature‐based hypotheses to be tested were the importance of the amount of wax esters and the weighted mean carbon chain length in restricting cuticular water loss. The main results were acquired by chemical analysis of cuticular wax and gravimetric cuticular transpiration measurements. Besides additional physiological measurements, the leaf surface properties were characterised by scanning electron microscopy and spectrophotometric light reflectance quantification. Mutation of the CER6 gene resulted in striking changes in qualitative wax composition but not quantitative wax amount. Based on the strong accumulation of dimeric wax esters, the relative proportion of esters increased to >90%, and the weighted mean carbon chain length increased by >6 carbon atoms. These qualitative alterations were found to increase the cuticular transpiration of leaves by twofold. Our results do not support the hypotheses that enhanced amounts of wax esters or increased weighted mean carbon chain lengths of waxes lead to reduced cuticular transpiration.

DNA isolation is a fundamental technique for all molecular biology laboratories. Depending on the plant species, DNA isolation can be challenging. In particular, adapted protocols rarely exist for tree species which are not used as standard model organisms. Here, we describe a flexible DNA isolation protocol that works for 59 tree species in a modular system. It is based on an ATMAB-containing extraction buffer to which proteinase K and/or boric acid are added, depending on the plant species. Subsequent purification steps include one or two precipitations with dichloromethane and, depending on the tree species, an optional sodium acetate precipitation. Using leaf material of a hybrid poplar clone from in vitro culture, it was determined that higher amounts of DNA could be isolated with this material than from field leaves. Starting from leaf material, DNA isolation for difficult cases was achieved with cambium or root tissue. This protocol was used to extract DNA for subsequent PCR amplification. Markers for cpDNA, mtDNA, and genomic DNA were used for standardized testing.