Articles
Establishing a W. Murcott (C. reticulata × C. sinensis) protoplast editing platform for precision breeding of commercial traits
Article number
1448_33
Pages
261 – 268
Language
English
Abstract
Citrus is the most important genus in the Rutaceae family.
Climate change and population growth are putting pressure on agricultural systems, which must respond with more resilient and productive cultivars, generating them faster than through traditional genetic improvement.
New breeding techniques based on gene editing are potentially able to improve specific plant traits without side effects, introducing new stable, heritable and targeted modifications into already commercially successful cultivars.
At our company located in Chile, the objective of our work has been to develop a platform for editing, regenerating modified materials, and managing juvenility to accelerate the process of obtaining new Citrus cultivars.
The model has been tested in W. Murcott mandarin.
Protocols to obtain friable embryogenic callus from ovule culture, protoplast isolation (5 to 10 million protoplasts g‑1 with 85 to 92% viability), transient transformation with plasmids and/or proteins (10 to 20% transformation efficiency with 70% cell viability), transgene-free editing (0.4-1% efficiency), embryo regeneration (cotyledonary embryos in 4-6 months) have been adjusted.
Obtained materials are screened by high-throughput genotyping, using automated DNA extraction, and PCR amplification followed by Illumina sequencing, to detect the correctly edited plantlets.
In addition, speed breeding using controlled conditions (light, heat, cold, irrigations and others) will be performed to reduce juvenility.
We have already obtained biallelic PDS knockout embryos as a proof of concept and currently are working to improve two commercially interesting traits testing >5 genes for each trait.
Climate change and population growth are putting pressure on agricultural systems, which must respond with more resilient and productive cultivars, generating them faster than through traditional genetic improvement.
New breeding techniques based on gene editing are potentially able to improve specific plant traits without side effects, introducing new stable, heritable and targeted modifications into already commercially successful cultivars.
At our company located in Chile, the objective of our work has been to develop a platform for editing, regenerating modified materials, and managing juvenility to accelerate the process of obtaining new Citrus cultivars.
The model has been tested in W. Murcott mandarin.
Protocols to obtain friable embryogenic callus from ovule culture, protoplast isolation (5 to 10 million protoplasts g‑1 with 85 to 92% viability), transient transformation with plasmids and/or proteins (10 to 20% transformation efficiency with 70% cell viability), transgene-free editing (0.4-1% efficiency), embryo regeneration (cotyledonary embryos in 4-6 months) have been adjusted.
Obtained materials are screened by high-throughput genotyping, using automated DNA extraction, and PCR amplification followed by Illumina sequencing, to detect the correctly edited plantlets.
In addition, speed breeding using controlled conditions (light, heat, cold, irrigations and others) will be performed to reduce juvenility.
We have already obtained biallelic PDS knockout embryos as a proof of concept and currently are working to improve two commercially interesting traits testing >5 genes for each trait.
Publication
Authors
M.J. Montañola, I. Fuentes, A. Galaz, C. Valenzuela, S. Ríos, I. Ríos, J. Jimenez, K. Olivos, T. Norambuena, B. Pollak
Keywords
protoplasts, citrus editing, plasmid, protoplast transformation, citrus protoplasts
Groups involved
Online Articles (103)