Open Access
ARTICLE
Rapid delivery of Cas9 gene into the tomato cv. ‘Heinz 1706’ through an optimized Agrobacterium-mediated transformation procedure
BEEMNET MENGESHA KASSAHUN1,#, BEUM-CHANG KANG2,#, SU-JI BAE2, YE JIN NAM1, GRETEL FONSECA MUNDO1, GA-HUI KANG1, KYOUNGOOK KIM3, JEUNG-SUL HAN1,4,*
1 Department of Horticulture, Kyungpook National University, Daegu, 41566, Korea
2 Center for Genome Engineering, Institute for Basic Science, Daejeon, 34047, Korea
3 School of Water, Energy, and Environment, Cranfield University, Cranfield, MK43 0AL, UK
4 Institute of Agricultural Science and Technology, Kyungpook National University, Daegu, 41566, Korea
* Address correspondence to: Jeung-Sul Han,
# These authors contributed equally to this work
BIOCELL 2021, 45(1), 199-215. https://doi.org/10.32604/biocell.2021.012353
Received 27 June 2020; Accepted 24 August 2020; Issue published 26 January 2021
Abstract
Solanum lycopersicum ‘Heinz 1706’ is a pioneer model cultivar for tomato research, whose whole genome
sequence valuable for genomics studies is available. Nevertheless, a genetic transformation procedure for this cultivar
has not yet been reported. Meanwhile, various genome editing technologies such as transfection of clustered regularly
interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) ribonucleoprotein complexes into cells are
in the limelight. Utilizing the Cas9-expressing genotype possessing a reference genome can simplify the verification of
an off-target effect, resolve the economic cost of Cas9 endonuclease preparation, and avoid the complex assembly
process together with single-guide RNA (sgRNA) in the transfection approach. Thus, this study was designed to
generate Cas9-expressing ‘Heinz 1706’ lines by establishing an
Agrobacterium tumefaciens-mediated transformation
(ATMT) procedure. Here, we report a rapid and reproducible transformation procedure for ‘Heinz 1706’ by finetuning various factors:
A. tumefaciens strain, pre-culture and co-culture durations, a proper combination of
phytohormones at each step, supplementation of acetosyringone, and shooting/rooting method. Particularly, through
eluding subculture and simultaneously inducing shoot elongation and rooting from leaf cluster, we achieved a short
duration of three months for recovering the transgenic plants expressing Cas9. The presence of the Cas9 gene and its
stable expression were confirmed by PCR and qRT-PCR analyses, and the Cas9 gene integrated into the T
0 plant
genome was stably transmitted to T
1 progeny. Therefore, we anticipate that our procedure appears to ease the
conventional ATMT in ‘Heinz 1706’, and the created Cas9-expressing ‘Heinz 1706’ lines are ultimately useful in gene
editing via unilateral transfection of sgRNA into the protoplasts.
Keywords
Cite This Article
KASSAHUN, B. M., KANG, B., BAE, S., NAM, Y. J., MUNDO, G. F. et al. (2021). Rapid delivery of
Cas9 gene into the tomato cv. ‘Heinz 1706’ through an optimized
Agrobacterium-mediated transformation procedure.
BIOCELL, 45(1), 199–215. https://doi.org/10.32604/biocell.2021.012353