Genetic Transformation in Peach (Prunus persica L.): Challenges and Ways Forward

Ricci, A.; Sabbadini, Silvia; Prieto, Humberto; Padilla, Isabel; Dardick, Chris; Z, Li; Scorza, R.; Limera, Cecilia; Mezzetti, Bruno; Pérez‐Jiménez, Margarita; Burgos, L.; Petri, César

doi:10.3390/plants9080971

Cited by 33 publications

(15 citation statements)

References 93 publications

(134 reference statements)

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“…Reducing the rate of chimerism in transformed shoots is a similar bottleneck in genetic engineering for strawberry and other Rosaceous plants [63]. To dissociate chimeras and recover completely transformed shoots in a transformation without selection is a great challenge.…”

Section: Discussionmentioning

confidence: 99%

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A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry

Zhao²,

et al. 2021

Plants

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Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. ‘Shanghai Angel’ by optimizing the dose of Thidiazuron and identifying the most suitable explants. The expression cassette was assembled with all DNA fragments from F. vesca, harboring a sugar transporter gene FvSTP8 driven by a fruit-specific FvKnox promoter. Transformed strawberry was developed through an Agrobacterium-mediated strategy without any selectable markers. Other than PCR selection, probe-based duplex droplet digital PCR (ddPCR) was performed to determine the T-DNA insert. Four independent transformed shoots were obtained with a maximum of 5.3% efficiency. Two lines were confirmed to be chimeras, while the other two were complete transformants with six and 11 copies of the intragene, respectively. The presence of a vector backbone beyond the T-DNA in these transformants indicated that intragenic strawberries were not obtained. The current work optimized the procedures for producing transformed strawberry without antibiotic selection, and accurately determined the insertion copies by ddPCR in the strawberry genome for the first time. These strategies might be promising for the engineering of ‘Shanghai Angel’ and other cultivars to improve agronomic traits.

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Section: Discussionmentioning

confidence: 99%

“…The transformation of a plant is actually a war of cell survival, where the plant's defense system interacts with the intruding pathogen Agrobacterium [63]. To obtain a desirable transgenic or intragenic plant with a single copy of a novel DNA is a matter of the two organisms.…”

Section: Discussionmentioning

confidence: 99%

A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry

Zhao²,

et al. 2021

Plants

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“…Whereas a remarkable cultivar/variety-dependence has been observed in these successful experiences, reliable and reproducible systems are yet to be developed for most Prunus spp. [2,5].…”

Section: Tissue Culture Systems In Prunus Sppmentioning

confidence: 99%

“…Although the first descriptions for in vitro propagation were Gene Editing in Prunus Spp. : The Challenge of Adapting Regular Gene Transfer Procedures… DOI: http://dx.doi.org/10.5772/intechopen.98843 described as early as 1982 [28], all the considerable research published to date pursuing the development of a reproducible regeneration system based on adult tissue as starting explants has shown this to be more complex than originally thought [2].…”

Section: Regeneration In Prunus Persicamentioning

confidence: 99%

“…In Prunus spp., a huge amount of research has been carried out regarding regeneration and gene transfer protocols using different explants and strategies to improve these procedures [2]. From that research, current gene transfer procedures in the genus include some technical pipelines for whole plant production in species such as P. salicina, P. domestica, and certain genotypes in P. avium.…”

Section: Introductionmentioning

confidence: 99%

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Gene Editing inPrunusSpp.: The Challenge of Adapting Regular Gene Transfer Procedures for Precision Breeding

Vergara¹,

Olivares²,

Olmedo³

et al. 2022

Prunus - Recent Advances

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Successfully gene editing (GE) in Prunus spp. has been delayed due to its woody nature presenting additional difficulties in both, proper regeneration protocols and designing efficient gene transfer techniques. The availability of adequate, single cell culture techniques for GE such as protoplast regeneration, is a limiting step for the genus and for this reason, the improvement of regular regeneration protocols and finding more efficient techniques for the delivery of the “editing reagents” seem to be a reasonable strategy to incorporate GE in the genus. During the last 10 years, we have focused our efforts optimizing some previous regeneration and gene transfer procedures for Japanese plum (P. salicina), sweet cherry (P. avium) and peach (P. persica) to incorporate them into a GE technology on these species. In parallel, delivery techniques for the CRISPR/Cas9 editing components, i.e., guide RNA (gRNA) and Cas9, have been developed with the aim of improving gene targeting efficiencies. In that line, using DNA virus-based replicons provides a significant improvement, as their replicational release from their carriers enables their enhanced expression. Here, we make a brief overview of the tissue culture and regeneration protocols we have developed for P. salicina, P. avium and P. persica, and then we proceed to describe the use of Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express the editing reagents in vivo and to evaluate their editing capability on individuals derived from Agrobacterium-mediated gene transfer experiments of these species. We show part of our characterization assays using new BeYDV-derived vectors harboring multiple gRNAs, the Cas9 gene, and the green fluorescent protein reporter gene. We also describe a dedicated genome analysis tool, by which gRNA pairs can be designed to address gene deletions of the target genes and to predict off-target sequences. Finally, as an example, we show the general results describing GE of the peach TERMINAL FLOWER 1 gene and some preliminary characterizations of these materials.

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A simple and efficient protocol for transient transformation of sliced grape berries

Liu¹,

Ampomah-Dwamena²,

Wei³

et al. 2022

Protoplasma

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Genetic Transformation in Peach (Prunus persica L.): Challenges and Ways Forward

Cited by 33 publications

References 93 publications

A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry

A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry

Gene Editing inPrunusSpp.: The Challenge of Adapting Regular Gene Transfer Procedures for Precision Breeding

A simple and efficient protocol for transient transformation of sliced grape berries

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