An Efficient -Mediated Genetic Transformation Method for Cav. Embryogenic Callus

Overview

Journal Plants (Basel)

Date 2023 Mar 11

PMID 36904062

Authors

Daniela Cordeiro

Ana Alves

Ricardo Ferraz

Bruno Casimiro

Jorge Canhoto

Sandra Correia

Affiliations

Soon will be listed here.

Abstract

Somatic embryogenesis in (tamarillo) has proven to be an effective model system for studying morphogenesis, since optimized plant regeneration protocols are available, and embryogenic competent cell lines can be induced from different explants. Nevertheless, an efficient genetic transformation system for embryogenic callus (EC) has not yet been implemented for this species. Here, an optimized faster protocol of genetic transformation using is described for EC. The sensitivity of EC to three antibiotics was determined, and kanamycin proved to be the best selective agent for tamarillo callus. Two strains, EHA105 and LBA4404, both harboring the p35SGUSINT plasmid, carrying the reporter gene for β-glucuronidase () and the marker gene neomycin phosphotransferase (), were used to test the efficiency of the process. To increase the success of the genetic transformation, a cold-shock treatment, coconut water, polyvinylpyrrolidone and an appropriate selection schedule based on antibiotic resistance were employed. The genetic transformation was evaluated by GUS assay and PCR-based techniques, and a 100% efficiency rate was confirmed in the kanamycin-resistant EC clumps. Genetic transformation with the EHA105 strain resulted in higher values for insertion in the genome. The protocol presented provides a useful tool for functional gene analysis and biotechnology approaches.

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