Journal
PHYSIOLOGIA PLANTARUM
Volume 173, Issue 3, Pages 993-1007Publisher
WILEY
DOI: 10.1111/ppl.13499
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Funding
- Federal Ministry of Education and Research (BMBF), Germany [FKZ 031B0536]
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The study introduced a new technique for DNA-free genome editing in plants by using pulsed laser to generate cavitation bubbles for efficient transfection of walled cells. By targeting endogenous genes PDS and ADF in tobacco, genome-edited plants were produced with high efficiency. The physiological, cellular, and molecular effects of ADF mutations in T2 mutant plants under drought and salinity stress suggest that ADF acts as a key regulator of osmotic stress tolerance in plants.
DNA-free genome editing involves the direct introduction of ribonucleoprotein (RNP) complexes into cells, but this strategy has rarely been successful in plants. In the present study, we describe a new technique for the introduction of RNPs into plant cells involving the generation of cavitation bubbles using a pulsed laser. The resulting shockwave achieves the efficient transfection of walled cells in tissue explants by creating transient membrane pores. RNP-containing cells were rapidly identified by fluorescence microscopy, followed by regeneration and the screening of mutant plants by high-resolution melt analysis. We used this technique in Nicotiana tabacum to target the endogenous phytoene desaturase (PDS) and actin depolymerizing factor (ADF) genes. Genome-edited plants were produced with an efficiency of 35.2% for PDS and 16.5% for ADF. Further we evaluated the physiological, cellular and molecular effects of ADF mutations in T2 mutant plants under drought and salinity stress. The results suggest that ADF acts as a key regulator of osmotic stress tolerance in plants.
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