Open Access

ARTICLE

Rapid delivery of Cas9 gene into the tomato cv. ‘Heinz 1706’ through an optimized Agrobacterium-mediated transformation procedure

BEEMNET MENGESHA KASSAHUN^1,#, BEUM-CHANG KANG^2,#, SU-JI BAE², YE JIN NAM¹, GRETEL FONSECA MUNDO¹, GA-HUI KANG¹, KYOUNGOOK KIM³, JEUNG-SUL HAN^1,4,*

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 T₀ plant genome was stably transmitted to T₁ 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.

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