Production of transgenic diploid Cucumis melo plants

García-Almodóvar, R.C.; Gosálvez, Blanca; Aranda, Miguel A.; Burgos, L.

doi:10.1007/s11240-017-1227-2

Cited by 13 publications

(11 citation statements)

References 38 publications

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“…Cotyledon transformation was used for melon transformation using Agrobacterium strain AGL-0 as described by Castelblanque et al (2008) except that the cotyledons were cut as in García-Almodóvar et al (2017). In brief, half of the proximal parts of the cotyledons from 1-day-old seeds were cut and co-cultured with transformed Agrobacterium for 20 min in the presence of 200 μM acetosyringone.…”

Section: Melon Transformationmentioning

confidence: 99%

Knock-Out of CmNAC-NOR Affects Melon Climacteric Fruit Ripening

et al. 2022

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Fruit ripening is an important process that affects fruit quality. A QTL in melon, ETHQV6.3, involved in climacteric ripening regulation, has been found to be encoded by CmNAC-NOR, a homologue of the tomato NOR gene. To further investigate CmNAC-NOR function, we obtained two CRISPR/Cas9-mediated mutants (nor-3 and nor-1) in the climacteric Védrantais background. nor-3, containing a 3-bp deletion altering the NAC domain A, resulted in ~8 days delay in ripening without affecting fruit quality. In contrast, the 1-bp deletion in nor-1 resulted in a fully disrupted NAC domain, which completely blocked climacteric ripening. The nor-1 fruits did not produce ethylene, no abscission layer was formed and there was no external color change. Additionally, volatile components were dramatically altered, seeds were not well developed and flesh firmness was also altered. There was a delay in fruit ripening with the nor-1 allele in heterozygosis of ~20 days. Our results provide new information regarding the function of CmNAC-NOR in melon fruit ripening, suggesting that it is a potential target for modulating shelf life in commercial climacteric melon varieties.

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Section: Melon Transformationmentioning

confidence: 99%

Knock-Out of CmNAC-NOR Affects Melon Climacteric Fruit Ripening

et al. 2022

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“…The methodology used for the regeneration and stable transformation of melon was that described in García‐Almodóvar et al . (2017). Briefly, the A .…”

Section: Methodsmentioning

confidence: 99%

Editing melon eIF4E associates with virus resistance and male sterility

Pechar

Donaire

Gosálvez

et al. 2022

Plant Biotechnology Journal

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Summary The cap‐binding protein eIF4E, through its interaction with eIF4G, constitutes the core of the eIF4F complex, which plays a key role in the circularization of mRNAs and their subsequent cap‐dependent translation. In addition to its fundamental role in mRNA translation initiation, other functions have been described or suggested for eIF4E, including acting as a proviral factor and participating in sexual development. We used CRISPR/Cas9 genome editing to generate melon eif4e knockout mutant lines. Editing worked efficiently in melon, as we obtained transformed plants with a single‐nucleotide deletion in homozygosis in the first eIF4E exon already in a T0 generation. Edited and non‐transgenic plants of a segregating F2 generation were inoculated with Moroccan watermelon mosaic virus (MWMV); homozygous mutant plants showed virus resistance, while heterozygous and non‐mutant plants were infected, in agreement with our previous results with plants silenced in eIF4E. Interestingly, all homozygous edited plants of the T0 and F2 generations showed a male sterility phenotype, while crossing with wild‐type plants restored fertility, displaying a perfect correlation between the segregation of the male sterility phenotype and the segregation of the eif4e mutation. Morphological comparative analysis of melon male flowers along consecutive developmental stages showed postmeiotic abnormal development for both microsporocytes and tapetum, with clear differences in the timing of tapetum degradation in the mutant versus wild‐type. An RNA‐Seq analysis identified critical genes in pollen development that were down‐regulated in flowers of eif4e/eif4e plants, and suggested that eIF4E‐specific mRNA translation initiation is a limiting factor for male gametes formation in melon.

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“…Agrobacterium tumefaciens (strain AGL0) cells were transformed with the binary CRISPR/Cas9 construct. Plant transformation was performed by co-cultivation of the Agrobacterium culture with one-day-old cotyledons of VED as previously described (Castelblanque et al ., 2008) including the following modifications: cotyledons were dissected as in (García-Almodóvar et al ., 2017), regeneration media was supplemented with 6-bencylaminopurine (BA) and Indole-3-acetic acid (IAA) and Agrobacterium was co-cultured with the explants during a period of three days. Transgenic plants containing the bar gene were selected by L-Phosphinothricin (PPT) resistance and were grown in a growth room under a 12-h light/12-h dark cycle at 28 °C.…”

Section: Methodsmentioning

confidence: 99%

CRISPR/Cas9 gene editing uncovers the role of CTR1 and ROS1 in melon fruit ripening and epigenetic regulation

Giordano

Domingo

Quadrana

et al. 2022

Preprint

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Melon (Cucumis melo L.) has emerged as an alternative model to study fruit ripening due to the coexistence of climacteric and non-climacteric varieties. The previous characterization of a major QTL ETHQV8.1 sufficient to trigger climacteric ripening in a non-climacteric background allowed the identification within the QTL interval of a negative regulator of ripening CmCTR1-like (MELO3C024518), and a putative DNA demethylase CmROS1 (MELO3C024516), the orthologue of DML2, a DNA demethylase regulating fruit ripening in tomato. To understand the role of these genes in climacteric ripening, we generated homozygous CRISPR knockout mutants of CmCTR1-like and CmROS1 in a climacteric genetic background. The climacteric behavior was altered in both loss-of-function mutants in two summer seasons with an advanced ethylene production profile compared to the climacteric wild type, suggesting a role of both genes in climacteric ripening in melon. Single cytosine methylome analyses of the CmROS1 knockout mutant revealed DNA methylation changes in the promoter regions of key ripening genes as ACS1, ETR1 and ACO1, and ripening associated-transcription factors as NAC-NOR, RIN and CNR, suggesting the importance of CmROS1-mediated DNA demethylation for triggering fruit ripening in melon.

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Production of transgenic diploid Cucumis melo plants

Cited by 13 publications

References 38 publications

Knock-Out of CmNAC-NOR Affects Melon Climacteric Fruit Ripening

Knock-Out of CmNAC-NOR Affects Melon Climacteric Fruit Ripening

Editing melon eIF4E associates with virus resistance and male sterility

CRISPR/Cas9 gene editing uncovers the role of CTR1 and ROS1 in melon fruit ripening and epigenetic regulation

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