Optimisation of regeneration parameters improves transformation efficiency of recalcitrant tomato

Prihatna, Cahya; Chen, Ruolan; Barbetti, Martin J.; Barker, Susan J.

doi:10.1007/s11240-019-01583-w

Cited by 10 publications

(9 citation statements)

References 31 publications

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“…These results clearly indicated the significant role of hormonal composition and genotypes on shoot induction and regeneration. Similar effects of these two variables on shoot regeneration were also reported for several tomato cultivars such as Micro-Tom ( Pino et al, 2010;Cruz-Mendívil et al, 2011), Rio Grande (Khoudi et al, 2009;Prihatna et al, 2019), and Pusa Ruby (Sarker et al, 2009). Several other regeneration studies from leaves and cotyledons of tomato cultivars such as Castle Rack (Devi et al, 2008), Pusa Uphar (Kaur and Bansal, 2010), Smart 18 (Kalyani and Rao, 2014), and PKM-1 (Sherkar and Chavan, 2014) have resulted in 65-94% shoot regeneration capacity with different hormonal combinations.…”

Section: Optimization Of Tissue Culture and Regeneration Parameters Osupporting

confidence: 58%

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

Seçgin¹,

Kavas²,

Yıldırım³

2021

Turk J Agric For

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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.

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Section: Optimization Of Tissue Culture and Regeneration Parameters Osupporting

confidence: 58%

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

Seçgin¹,

Kavas²,

Yıldırım³

2021

Turk J Agric For

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“…Cytokinins in plants stimulate cell division, bud initiation, and proliferation ( Van Staden et al, 2008 ). In vitro regeneration of tomato has been significantly affected by the explants, genotypes, and PGR concentrations and combinations used in the culture medium ( Alatar et al, 2017 , Prihatna et al, 2019 , Saeed et al, 2019 , Vinoth et al, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

“…Among various concentrations of hygromycin, 20 mg l −1 was observed as the most suitable concentration for the successful recovery of transformants of tomato ( Koul et al, 2014 , Li et al, 2013 ). Diverse hygromycin concentrations have been successfully applied as a selection agent to recover transgenic plants in tomato ( Koul et al, 2014 , Prihatna et al, 2019 , Gadir et al, 2018 ). Pre-culturing of explants was successfully enhanced the transformation efficiency in different explants of several tomato genotypes ( Koul et al, 2014 , Shah et al, 2016 , Gadir et al, 2018 , Stavridou et al, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

“…Transgenic tomato plants were successfully produced from various explants like cotyledon, hypocotyl, and leaf explants using Agrobacterium -mediated genetic transformation ( Koul et al, 2014 , Sun et al, 2015 , Gadir et al, 2018 ). Various transformation efficiencies and transgenic plants recovery were observed from different tomato explants and genotypes ( Koul et al, 2014 , Gadir et al, 2018 , Prihatna et al, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

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Highly efficient Agrobacterium-mediated transformation and plant regeneration system for genome engineering in tomato

Sandhya

Jogam

Venkatapuram

et al. 2022

Saudi Journal of Biological Sciences

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“…F. hindsii is a model species for citrus studies and has complete genome sequencing and assembly [ 18 ]. However, due to the limitation of genotype dependence, the transformation efficiency of F. hindsii is much lower than that of other crops, which increases the workload and further hinders research on citrus genetics and functional genomics [ 35 , 36 ]. In this study, we confirmed that the CRISPR/Cas9 system with pK7WG2D as the binary vector can efficiently edit the genome of F. hindsii , while the pMDC32 vector was not suitable for F. hindsii ( Figs 2 and 3 ).…”

Section: Discussionmentioning

confidence: 99%

An efficient CRISPR/Cas9 system for simultaneous editing two target sites in Fortunella hindsii

Zhang

et al. 2022

Horticulture Research

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The CRISPR/Cas9 system is a revolutionary genome editing technique, that has been widely used in numerous plants. For plants (e.g. citrus) with very low transformation efficiency, how to optimize gene editing efficiency and induce large fragment deletion has been the focus of research. Here, we reported that CRISPR/Cas9 induces efficient deletion of 16–673 bp fragments in the genome of Fortunella hindsii (F. hindsii). The ability of two binary vectors, pK7WG2D and pMDC32, to introduce specific mutations into the genome of F. hindsii was evaluated. Double sgRNAs were designed to achieve precise editing of two sites of a gene and deletion of fragments between two sites. The construction of vectors based on Golden Gate assembly and Gateway recombination cloning is simple and efficient. pK7WG2D is more suitable for F. hindsii genome editing than the pMDC32 vector. The editing efficiency using the pK7WG2D vector reached 66.7%. The allele mutation frequency was 7.14%–100%. The plants with 100% allele mutations accounted for 39.4% (13 100% allele mutation plants/33 mutants). The proportion of mutant plants with fragment deletion induced by this editing system was as high as 52.6% (10 fragment deletion mutants/19 FhNZZ mutants). Altogether, these data suggest that our CRISPR/Cas9 platform is capable of targeted genome editing in citrus and has broad application in research on the citrus functional genome and citrus molecular breeding.

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Optimisation of regeneration parameters improves transformation efficiency of recalcitrant tomato

Cited by 10 publications

References 31 publications

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

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

Highly efficient Agrobacterium-mediated transformation and plant regeneration system for genome engineering in tomato

An efficient CRISPR/Cas9 system for simultaneous editing two target sites in Fortunella hindsii

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