Abstract
AbstractFor many important crops including sorghum, use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but also by the ability to regenerate a fertile plant from the engineered cell through tissue culture. Here, we report that Wuschel2 (Wus2)-enabled transformation increases not only the transformation efficiency, but also the CRISPR/Cas-targeted genome editing frequency in sorghum (Sorghum bicolor L.). Using Agrobacterium-mediated transformation, we have demonstrated Wus2-induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased the regeneration capacity that resulted in higher transformation efficiency across different sorghum varieties. Subsequently, advanced excision systems and “altruistic” transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrate up to 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using Wus2-enabled transformation, compared to without Wus2, across various targeted loci in different sorghum genotypes.
Funder
Corteva Agriscience generously provided funding for this project.
Publisher
Springer Science and Business Media LLC
Subject
General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology,Medicine (miscellaneous)
Reference45 articles.
1. Gao, C. & Nielsen, K. K. Comparison between Agrobacterium-mediated and direct gene transfer using the gene gun. Methods Mol. Biol. 940, 3–16 (2013).
2. Altpeter, F. et al. Advancing crop transformation in the era of genome editing. Plant Cell 28, 1510–1520 (2016).
3. Lowe, K. et al. Rapid genotype “independent” Zea mays L. (maize) transformation via direct somatic embryogenesis. Vitr. Cell Dev. Biol. Plant 54, 240–252 (2018).
4. Mookkan, M., Nelson-Vasilchik, K., Hague, J., Zhang, Z. J. & Kausch, A. P. Selectable marker independent transformation of recalcitrant maize inbred B73 and sorghum P898012 mediated by morphogenic regulators BABY BOOM and WUSCHEL2. Plant Cell Rep. 36, 1477–1491 (2017).
5. Lowe, K. et al. Morphogenic regulators Baby boom and Wuschel improve monocot transformation. Plant Cell 28, 1998–2015 (2016).
Cited by
39 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献