Optimization of Agrobacterium-mediated transformation and regeneration for CRISPR/ Cas9 genome editing of commercial tomato cultivars

Abstract

Tomato (Solanum lycopersicum) is the second most important horticultural crop worldwide that is widely used as a model plant in genetic manipulation of Solanaceae. CRISPR/Cas9 system has been successfully utilized in several studies for genome edition of model tomato cultivars. However, these genome editing systems should be also optimized for commercial tomato cultivar for direct application of genome editing in field conditions. In this study, we have optimized an Agrobacterium-mediated gene transfer and regeneration system for CRISPR/Cas9 genome editing in two commercial tomato cultivars for the first time. The effect of explant type, genotype, pre-transformation time, Agrobacterium concentration, infection time, and different co-culture periods of bacteria were evaluated to optimize the regeneration and transformation parameters. The highest regeneration capacity of 83 % was obtained from cotyledons of Crocker incubated in a medium supplemented with BA (3 mg/L) and IAA (0.1 mg/L). The maximum transformation frequency was obtained by using the following parameters: cotyledon explants of commercial Crocker cultivar that were left for 2 days of pre-transformation incubation, infected with Agrobacterium for 10 minutes at a concentration of OD 600 of 0.6 and co-cultivated with Agrobacterium cells for 48 hours. CRISPR/Cas9 system was tested with two gRNAs targeting the phytoene desaturase gene.Fully albino and chimeric plants were successfully produced with optimized transformation and culture conditions in up to 71% of all regenerated plants. In the current study, we optimized the implementation of the CRISPR/Cas9 technique in a commercial tomato cultivar and our method will enable breeders to make necessary changes in traits of interest to improve tomato crop for commercial applications.

Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System

“…The transformed single colonies were selected by growing them on a solid selective LB media containing 50 mg/L kanamycin, 50 mg/L gentamicin, and 10 mg/L rifampicin under 28°C for two days. After colony PCR and sanger sequencing, recombinant plasmids were isolated from E. coli using Bacterial Plasmid Isolasyon kit (FavorGen, Wien, Austria) and transformed into Agrobacterium tumefaciens (GV3101) by electroporation according to the protocol described in Seçgin et al [ 37 ]. After the growth of Agrobacterium cells on the selection media, single colonies were incubated in liquid selective media until the bacterial density reached OD 600 0.8.…”

Genome-wide Identification of the Aconitase Gene Family in Tomato (<em>Solanum lycopersicum</em>) and CRISPR-based Functional Characterization of <em>SlACO2</em> on Male-sterility

“…The Cas9/gRNA constructs harboring the gRNA1 and gRNA2 targeting the SlACO2 gene were transformed into Agrobacterium tumefaciens strain Gv3101 using electroporation separately. Agrobacterium-mediated transformation of tomato (Solanum lycopersicum) cultivar 'Crocker' were performed according to Secgin et al (2021) [41]. All tomato lines were grown in standard glasshouse conditions of 16-h day length and 24-26°C, with a night temperature of 18°C.…”

Genome editing for healthy crops: traits, tools and impacts

“…The dsDNA nature of DNA viruses, Geminiviridae and Caulimoviridae , make them good targets for CRISPR/Cas and this has become a popular approach to antiviral engineering in crops ( Table 1 ). CRISPR-mediated resistance against DNA viruses was developed for Cauliflower mosaic virus ( Liu et al., 2018 ), Cotton leaf curl Multan virus ( Yin et al., 2019 ), Tomato yellow leaf curl virus (TYLCV), ( Ali et al., 2015a ; Ali et al, 2015b ; Seçgin et al., 2021 ) Beet severe curly top virus ( Baltes et al., 2015 ) and Bean yellow dwarf virus ( Ji et al., 2015 ) in Arabidopsis and tobacco plants. gRNA/Cas9 constructs were designed to target and cleave viral replication (REP), coat protein and noncoding stem-loop sequences [TAATATTAC] common to all geminiviruses.…”