@Article{biocell.2021.012353,
AUTHOR = {BEEMNET MENGESHA KASSAHUN, BEUM-CHANG KANG, SU-JI BAE, YE JIN NAM, GRETEL FONSECA MUNDO, GA-HUI KANG, KYOUNGOOK KIM, JEUNG-SUL HAN},
TITLE = {Rapid delivery of <i>Cas9</i> gene into the tomato cv. ‘Heinz 1706’ through an optimized <i>Agrobacterium</i>-mediated transformation procedure},
JOURNAL = {BIOCELL},
VOLUME = {45},
YEAR = {2021},
NUMBER = {1},
PAGES = {199--215},
URL = {http://www.techscience.com/biocell/v45n1/41413},
ISSN = {1667-5746},
ABSTRACT = {<i>Solanum lycopersicum</i> ‘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 <i>Agrobacterium tumefaciens</i>-mediated transformation
(ATMT) procedure. Here, we report a rapid and reproducible transformation procedure for ‘Heinz 1706’ by finetuning various factors: <i>A. tumefaciens</i> 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<sub>0</sub> plant
genome was stably transmitted to T<sub>1</sub> 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.},
DOI = {10.32604/biocell.2021.012353}
}