Articles
Novel breeding strategies for tackling present and future challenges in Prunus species: the case of peach
Article number
1352_58
Pages
419 – 426
Language
English
Abstract
CRISPR-Cas9-based genome editing has been used in peach [Prunus persica (L.) Batsch] and a peach x almond hybrid (rootstock GF677) to address some major agronomic issues.
For the recessive resistance to Plum pox virus (Sharka) disease, a single guide RNAs was designed targeting the host susceptibility gene eIFiso4E, known to be involved in Prunus spp. in the host-virus interaction. PpeTAC1 is the gene responsible for the tree architecture in peach, due to its role on defining the branches angle: its silencing, leading to more vertical branches, can promote yield in high density orchard through a more efficient interception of the light by minimizing interference with other shoots.
For PpeTAC1 knockout, two sgRNAs were designed on the gene with the aim to obtain a deletion of 400 bp and cloned into a vector carrying the hCAS9 cassette and NptII as the marker gene.
Lastly, for the modulation of the flower biology in Prunus, the strategy followed involves the knockout of the PpeTFL gene, to induce early flowering, through the excision of a 1100 bp fragment by a double guide CRISPR/hCAS9 construct.
Adult explants (leaves with petiole, nodes and basal calli) and immature embryonic tissues from peach (Independence’, Rich Lady’ and Royal Glory’) and rootstock GF677 were infected with the Agrobacterium tumefaciens strains (LBA4404 or GV3101) carrying the different constructs.
Regeneration response in mature tissues of controls was influenced by type of explant and genotype.
Epicotyls development always occurred both in control and infected ones.
However, regardless of the type of explant and the selection strategy adopted (antibiotic concentration and/or application time), molecular analyses have not detected editing events.
Further experiments are in progress to increase the regeneration response and the transformation efficiency of the infected explants.
For the recessive resistance to Plum pox virus (Sharka) disease, a single guide RNAs was designed targeting the host susceptibility gene eIFiso4E, known to be involved in Prunus spp. in the host-virus interaction. PpeTAC1 is the gene responsible for the tree architecture in peach, due to its role on defining the branches angle: its silencing, leading to more vertical branches, can promote yield in high density orchard through a more efficient interception of the light by minimizing interference with other shoots.
For PpeTAC1 knockout, two sgRNAs were designed on the gene with the aim to obtain a deletion of 400 bp and cloned into a vector carrying the hCAS9 cassette and NptII as the marker gene.
Lastly, for the modulation of the flower biology in Prunus, the strategy followed involves the knockout of the PpeTFL gene, to induce early flowering, through the excision of a 1100 bp fragment by a double guide CRISPR/hCAS9 construct.
Adult explants (leaves with petiole, nodes and basal calli) and immature embryonic tissues from peach (Independence’, Rich Lady’ and Royal Glory’) and rootstock GF677 were infected with the Agrobacterium tumefaciens strains (LBA4404 or GV3101) carrying the different constructs.
Regeneration response in mature tissues of controls was influenced by type of explant and genotype.
Epicotyls development always occurred both in control and infected ones.
However, regardless of the type of explant and the selection strategy adopted (antibiotic concentration and/or application time), molecular analyses have not detected editing events.
Further experiments are in progress to increase the regeneration response and the transformation efficiency of the infected explants.
Publication
Authors
C. Miccoli, G. Gambacorta, G. Urbinati, M. Santiago-Reyes, A. Gentile, S. Monticelli, E. Caboni, H. Prieto, I. Verde, V. Decroocq, E. Vendramin, S. Micali
Keywords
adventitious regeneration, CRISPR-Cas9, genome editing, Prunus persica (L.) Batsch
Groups involved
Online Articles (87)