【Objective】 We generated CRISPR-based mutants of PtrbHLH106 to explore its function in the wood formation of Populus trichocarpa. 【Method】 Based on the previous RNA-seq data of various cell types (cambium, xylem and phloem cells) of P. trichocarpa, we cloned a bHLH transcription factor, PtrbHLH106, which is highly expressed in the cambium and xylem. The CRISPR/Cas9 gene editing system was used to generate ptrbhlh106 mutants. Phenotype observations of ptrbhlh106 and wild-type (WT) plants grown for 60, 90 and 120 d were carried out. Paraffin sections of stem internodes of ptrbhlh106 and WT plants grown for 120 days were created, and toluidine blue staining was used for observation and statistical analyses of different wood cells. 【Result】 Phenotype observation showed that there were no significant differences in plant height and ground diameter. Compared to that in WT plants, the internodes length of 8th stem reduced, total number of stem internodes increased, and the layer of cambium cells increased in mutants. However, the differences in the above indexes were not significant. In addition, the lumen area of per vessel increased, and the number of fiber cells decreased significantly in ptrbhlh106. 【Conclusion】 The phenotypic differences between ptrbhlh106 mutants and WT plants showed that PtrbHLH106 is involved in the regulation of secondary xylem development in P. trichocarpa.
Long breeding periods and scarce germplasm resources have become the main bottlenecks in woody plant improvement. Recently, the CRISPR/Cas system has been developed into a precision site-directed editing technology that has been widely used in the breeding of woody plants such as Populus spp., Malus spp., Citrus spp., Vitis vinifera and Cassava spp.. The yield, quality, biotic- and abiotic-stress resistance, and other traits of woody plants have been significantly improved by genome editing, achieving the fixation of excellent genotypes within a single generation. This approach has accelerated the breeding process of woody plants and improves their breeding efficiency. Here, we summarize the application of CRISPR/Cas technology to woody plants, analyzing existing problems and future development trends. This review aims to provide a useful reference for gene function research and improvement of woody plants.
【Objective】 We used the CRISPR/Cas9 system to generate PtrHBI1 loss of function mutants of Populus trichocarpa in order to investigate the function of the PtrHBI1 transcription factor in wood formation. This investigation provides new clues for creating superior varieties of trees suitable for pulp and papermaking. 【Method】 Based on RNA-seq data of different cell types (cambium, xylem and phloem) of wild-type P. trichocarpa by laser capture microdissection, the bHLH transcription factor PtrHBI1 was identified. Subcellular localization of PtrHBI1 was analyzed using the stem-differentiating xylem (SDX) protoplast system of P. trichocarpa, and tissue-specific expression of PtrHBI1 was analyzed by in situ hybridization. We then used the CRISPR/Cas9 system to generate ptrhbi1 mutants of P. trichocarpa and analyzed the growth traits of ptrhbi1. Meanwhile, we performed a morphological analysis using paraffin sections and analyzed the wood composition of the mutant using the Klason acid hydrolysis method and high-performance liquid chromatography. 【Result】 According to the results, we found that PtrHBI1 was localized in the nucleus of P. trichocarpa SDX cells. The results of in situ hybridization showed that PtrHBI1 was mainly expressed in the cambium and xylem regions. The height of ptrhbi1 mutants created by CRISPR/Cas9 was significantly increased, and the internode number and stem basal diameter were significantly larger than those of the wild type during a certain period. In addition, the ptrhbi1 mutant showed a significant increase in the lumen area of per vessel cells and a significant decrease in the number of fiber cells. However, the number of vessel cells and layers of cambium cells in the ptrhbi1 mutant were similar to those in the wild type. The wood composition analysis showed that the glucose content of the ptrhbi1 mutant increased. Compared with the wild type, although the total lignin content was not significantly changed, the mass ratio of syringly to guaiacy (S/G) ratio of the mutant was significantly decreased. 【Conclusion】 PtrHBI1 was involved in regulating the growth and secondary xylem development of P. trichocarpa, which may play an important role in wood formation.
