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Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato
Year:
2022
Source of publication :
Planta
Authors :
אשל, דני
;
.
בוקובזא, שמואל
;
.
טפר-במנולקר, פאולה
;
.
קומאר, מנוג'
;
.
Volume :
Co-Authors:

Gulzar A Rather 
Dana Ayzenshtat
Paula Teper-Bamnolker
Manoj Kumar
Zohar Forotan
Dani Eshel
Samuel Bocobza  

Facilitators :
From page:
0
To page:
0
(
Total pages:
1
)
Abstract:

An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments, UBIQUITIN10 promoters of several plant species, kanamycin selection, and transient overexpression of the BABYBOOM transcription factor. Plant protoplasts represent a reliable experimental system for the genetic manipulation of desired traits using gene editing. Nevertheless, the selection and regeneration of mutated protoplasts are challenging and subsequent recovery of successfully edited plants is a significant bottleneck in advanced plant breeding technologies. In an effort to alleviate the obstacles related to protoplasts' transgene expression and protoplasts' regeneration, a new method was developed. In so doing, it was shown that linearized DNA could efficiently transfect potato protoplasts and that UBIQUITIN10 promoters from various plants could direct transgene expression in an effective manner. Also, the inhibitory concentration of kanamycin was standardized for transfected protoplasts, and the NEOMYCIN PHOSPHOTRANSFERASE2 (NPT2) gene could be used as a potent selection marker for the enrichment of transfected protoplasts. Furthermore, transient expression of the BABYBOOM (BBM) transcription factor promoted the regeneration of protoplast-derived calli. Together, these methods significantly increased the selection for protoplasts that displayed high transgene expression, and thereby significantly increased the rate of gene editing events in protoplast-derived calli to 95%. The method developed in this study facilitated gene-editing in tetraploid potato plants and opened the way to sophisticated genetic manipulation in polyploid organisms.

Note:
Related Files :
BABYBOOM
genome editing
potato
protoplast
regeneration
Transfection
UBIQUITIN10 promoter
עוד תגיות
תוכן קשור
More details
DOI :
10.1007/s00425-022-03933-z
Article number:
0
Affiliations:
Database:
PubMed
Publication Type:
מאמר
;
.
Language:
אנגלית
Editors' remarks:
ID:
60003
Last updated date:
28/06/2022 17:43
Creation date:
28/06/2022 16:32
Scientific Publication
Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato

Gulzar A Rather 
Dana Ayzenshtat
Paula Teper-Bamnolker
Manoj Kumar
Zohar Forotan
Dani Eshel
Samuel Bocobza  

Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato .

An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments, UBIQUITIN10 promoters of several plant species, kanamycin selection, and transient overexpression of the BABYBOOM transcription factor. Plant protoplasts represent a reliable experimental system for the genetic manipulation of desired traits using gene editing. Nevertheless, the selection and regeneration of mutated protoplasts are challenging and subsequent recovery of successfully edited plants is a significant bottleneck in advanced plant breeding technologies. In an effort to alleviate the obstacles related to protoplasts' transgene expression and protoplasts' regeneration, a new method was developed. In so doing, it was shown that linearized DNA could efficiently transfect potato protoplasts and that UBIQUITIN10 promoters from various plants could direct transgene expression in an effective manner. Also, the inhibitory concentration of kanamycin was standardized for transfected protoplasts, and the NEOMYCIN PHOSPHOTRANSFERASE2 (NPT2) gene could be used as a potent selection marker for the enrichment of transfected protoplasts. Furthermore, transient expression of the BABYBOOM (BBM) transcription factor promoted the regeneration of protoplast-derived calli. Together, these methods significantly increased the selection for protoplasts that displayed high transgene expression, and thereby significantly increased the rate of gene editing events in protoplast-derived calli to 95%. The method developed in this study facilitated gene-editing in tetraploid potato plants and opened the way to sophisticated genetic manipulation in polyploid organisms.

Scientific Publication
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