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Research Article

CRISPR/Cas9-Mediated Improvement of Major Rice Variety TBR225 for Low Cadmium Accumulation

Phuong Duy Nguyen1, Van Thi Pham1, Xuan Hoi Pham1, Mai Quynh Le2,3,*
Plant Breeding and Biotechnology 2025;13:71-83.
Published online: April 25, 2025

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*Corresponding to Mai Quynh Le TEL. +84-9-4748-5588, E-mail. lequynhmai80@vnu.edu.vn
• Received: December 5, 2024   • Revised: March 6, 2025   • Accepted: April 7, 2025

Copyright © 2025 by the Korean Society of Breeding Science

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CRISPR/Cas9-Mediated Improvement of Major Rice Variety TBR225 for Low Cadmium Accumulation
Plant Breed. Biotech.. 2025;13:71-83.   Published online April 25, 2025
DOI: https://doi.org/10.9787/PBB.2025.13.71
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CRISPR/Cas9-Mediated Improvement of Major Rice Variety TBR225 for Low Cadmium Accumulation
Plant Breed. Biotech.. 2025;13:71-83.   Published online April 25, 2025
DOI: https://doi.org/10.9787/PBB.2025.13.71
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CRISPR/Cas9-Mediated Improvement of Major Rice Variety TBR225 for Low Cadmium Accumulation
Image Image Image Image
Fig. 1 PCR analysis of transgene integration in T0 (a) and T1 (b) generations using specific primer pairs for OsActin (endogenous control), HPT (marker gene), gRNA, and Ubiquitin::Cas9 expression cassette. C: positive control (pCas9/sgRNA-OsNRAMP5 in T0; parental DNA in T1); WT: wild-type plant; M: 1.0 kb ladder.
Fig. 2 Analysis of CRISPR/Cas9-induced mutations in TBR225 OsNRAMP5. (a) Schematic diagram of OsNRAMP5 gene structure showing two target sites on exon 1. Black rectangles: exons; lines: introns and UTRs. (b) OsNRAMP5 allele alignment of T0 lines (L1, L4, L22) and their T1 progeny (L1.59, L4.28, L22.21) in the target region. (c) Predicted amino acid sequences of OsNRAMP5 mutant alleles. Boxes: gRNA target sites; arrows: predicted CRISPR/Cas9-induced DSBs; asterisks: stop codons. Left column: rice line names (a1, a2: allele 1, 2; WT: wild-type). Right column in (b): genotype (+/-: insertions/deletions; numbers: modified nucleotide count; capital letters: altered nucleotides; wt: wild-type). Right column in (c): polypeptide length. []: base pair or amino acid counts.
Fig. 3 Metal accumulation in OsNRAMP5-edited TBR225 rice lines under different metal treatments. Plants were exposed to various metal concentrations for 2 months (a-d) or 3 months (e). (a) Fe concentrations in grain after treatments with 0, 0.1, 1.0, and 10.0 mM FeCl2. (b) Zn concentrations in grain after treatments with 0, 0.01, 0.1, and 1.0 mM ZnCl2. (c-e) Cd concentrations in root (c), shoot (d), and grain (e) after treatments with 0, 30, and 300 µM CdCl2. Bars represent mean±SD from 3 biological replicates. WT: wild-type plant. Asterisks indicate significant differences compared to WT (Tukey's HSD test; p<0.05).
Fig. 4 Agronomic trait analysis of OsNRAMP5 mutants. Three mutant lines (L1.59, L4.28, and L22.21) and WT plants were cultivated in green-house condition. (a) Plant morphology. (b) Time duration. (c) Plan height. (d) Number of tilters per plant. (e) Number of filled grains per panicle. (f) Individual grain yield. (g) Amylose content in grain. Data are means±SD. There is no significant difference between WT and mutant lines (Tukey's HSD test; p<0.05).

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Fig. 4

CRISPR/Cas9-Mediated Improvement of Major Rice Variety TBR225 for Low Cadmium Accumulation

Result of TBR225 rice transformation

Experiment No. sample Ratio (%)z
Agrobacterium-innoculated callus 5063 -
Hygromycin-resistant callus 1598 36.71
Regenerated plant 134 3.08
Plant with positive OsActin PCR 134 3.08
Plant with positive HPT PCR 49 1.13
Plant with positive gRNA PCR 49 1.13
Plant with positive Cas9 PCR 49 1.13

Frequency of OsNRAMP5 mutant genotypes in T0 transgenic plants

Location Mutant genotype ratiosz
Heterozygous Homozygous Bialellic wt
gRNA1 1/20 2/20 4/20 13/20
gRNA2 3/20 2/20 1/20 14/20
gRNA1-gRNA2 1/20 1/20 5/20 13/20

Frequency of OsNRAMP5 mutation types in T0 transgenic plants

Location Mutant type ratiosz
Deletion Insertion Substitution
gRNA1 12/14 0/14 1/14
gRNA2 3/12 6/12 0/12

Segregation of T-DNA in T1 generation

T0 plant Genotypez No. of T1 plants tested T-DNA inheritance Mutation inheritance No. of T-DNA-free mutant plantsx
No. of T-DNA-free plants χ2 (3:1)y χ2 (15:1)y No. of homozygous plants χ2 (1:1)y
L1 Bialellic 60 11 1.422 14.951 37 3.267 7
L4 Bialellic 60 19 1.422 66.151 24 2.400 2
L9 Bialellic 60 3 12.8 0.16 31 0.067 2
L10 Heterozygous 37 8 0.225 14.921 13w 3.270 0
L22 Bialellic 60 11 1.422 14.951 35 1.667 6
Table 1 Result of TBR225 rice transformation

zNumber of positive samples/total Agrobacterium-inoculated calli)×100%. HPT: Hygromycin phospho transferase marker gene.

Table 2 Frequency of OsNRAMP5 mutant genotypes in T0 transgenic plants

zNumber of genotypes/total number of tested plants.

Table 3 Frequency of OsNRAMP5 mutation types in T0 transgenic plants

zNumber of alleles with a specific mutation type/total number of alleles in all mutant plants

Table 4 Segregation of T-DNA in T1 generation

zGenotype notation of OsNRAMP5 mutation: (+/-) indicates nucleotide insertion/deletion; capital letters represent substituted nucleotides; (wt) denotes wild-type allele; (sub) indicates nucleotide substitution; numbers represent the length of insertion or deletion in base pairs; (||) separates the two alleles; (/) separates the two gRNA mutation sites.

yχ2 critical value (df=1, p<0,05)=3,841.

xNumber of T-DNA-free homozygous mutant plants.

wIncludes both mutant (5) and wild-type plants (8).

Table 1

Table 2

Table 3

Table 4

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