The inability of a plant to produce pollen, male gametes, or functional anthers is known as male sterility (MS). The MS introduction into parental lines is an important task in breeding programs for many horticultural crops, allowing reduced costs for F1 hybrid seed production. In ornamentals, MS lines and F1 hybrids have not received much attention because propagation is usually agamic. However, vegetative propagation allows the direct commercialization of MS lines, which could be very beneficial, e.g., for the removal of pollen allergens. Recent advances in genome editing (GE) technology based on the CRISPR/Cas system have initiated a new era, which is also associated with progress in the acquisition of first-generation non-transgenic modified plants and the development of foreign DNA-free editing techniques. In this context, we describe a proof of concept in the development of a CRISPR/Cas9-based breeding strategy for producing MS into Petunia x hybrida, which is a model system of the Solanaceae family and is consistently used as a point of reference in ornamental research. The general objective of this study is to create a temporary protoplast transfection system to obtain CRISPR/Cas-edited DNA-free plant material, by the direct delivery of a preassembled ribonucleoprotein (RNP) complex consisting of purified Cas9 protein and vitro-synthesized single guide RNA (sgRNA). In particular, we report the results and ongoing work related to the targeting of a petunia MYB candidate gene. Kinetic analysis of target gene expression in vegetative tissues and flower buds at different phenological stages suggested its potential importance in the formation and development of related tissues and pollen. In addition, several sgRNAs were identified and selected based on their ability to target the investigated sequence in the coding sequence and the predicted transcription starting site region.
New insights into a candidate MYB gene for the induction of male sterility in Petunia × hybrida through a CRISPR-edited DNA-free transient transformation system
Farinati S.;Betto A.;Barcaccia G.
Funding Acquisition
2025
Abstract
The inability of a plant to produce pollen, male gametes, or functional anthers is known as male sterility (MS). The MS introduction into parental lines is an important task in breeding programs for many horticultural crops, allowing reduced costs for F1 hybrid seed production. In ornamentals, MS lines and F1 hybrids have not received much attention because propagation is usually agamic. However, vegetative propagation allows the direct commercialization of MS lines, which could be very beneficial, e.g., for the removal of pollen allergens. Recent advances in genome editing (GE) technology based on the CRISPR/Cas system have initiated a new era, which is also associated with progress in the acquisition of first-generation non-transgenic modified plants and the development of foreign DNA-free editing techniques. In this context, we describe a proof of concept in the development of a CRISPR/Cas9-based breeding strategy for producing MS into Petunia x hybrida, which is a model system of the Solanaceae family and is consistently used as a point of reference in ornamental research. The general objective of this study is to create a temporary protoplast transfection system to obtain CRISPR/Cas-edited DNA-free plant material, by the direct delivery of a preassembled ribonucleoprotein (RNP) complex consisting of purified Cas9 protein and vitro-synthesized single guide RNA (sgRNA). In particular, we report the results and ongoing work related to the targeting of a petunia MYB candidate gene. Kinetic analysis of target gene expression in vegetative tissues and flower buds at different phenological stages suggested its potential importance in the formation and development of related tissues and pollen. In addition, several sgRNAs were identified and selected based on their ability to target the investigated sequence in the coding sequence and the predicted transcription starting site region.
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