Research Projects

Abstract

Doubled haploid technology is the most powerful method to accelerate the breeding of new hybrid varieties, which is especially important in breeding biennial crops such as cabbage (Brassica oleracea var. capitata L.). A variety of haploid induction techniques have been developed, however, extensive studies on haploid induction in the last decade have shown that numerous plant species and specific genotypes within species are still recalcitrant to standard haploid induction techniques. Recently published point mutations in the conserved region of the centromeric CENH3 protein resulted in haploid offspring in Arabidopsis thaliana, suggesting that plants carrying those mutations could be used as haploid inducers for nonresponsive genotypes. Genome editing techniques, such as CRISPR/Cas9, are widely used for targeted modifications in genomes of various plant species. Nevertheless, efficient induction of precise targeted point mutations remains a challenge. Base editors are recently developed CRISPR/Cas9-derived tools that enable nucleotide conversion and can be used for precise base substitutions. In order to establish an efficient CRISPR/Cas9 protocol and induce point mutations in the cenh3 gene that could lead to the development of a haploid inducer line in cabbage, the effect of different parameters on the mutation rate will first be investigated, such as the efficiency of the transformation protocol and vector construction. For this purpose, a protoplast-based transient expression system will be used, which has a high screening efficiency and offers the possibility to optimize genome editing protocols prior to development of transgenic plants. Different CRISPR/Cas9 vectors will be prepared using high-throughput Golden Gate cloning strategy and tested by protoplast assay. The target sites in the cabbage genome will be analyzed using next-generation sequencing to determine genome editing events. In addition, protocols for regeneration of edited plants will be optimized. The primary objective of the proposed research project is to establish an efficient genome editing protocol using CRISPR/Cas9 technology in cabbage, which in the future will enable both gene functional analysis as well as plant improvement not only in cabbage but also in other Brassica species. Further goal of the project is manipulation of cenh3 gene to obtain potential haploid inducer lines that could be used in DH production of recalcitrant genotypes. For this purpose, a novel base editing approach will be used, which has not been reported for B. oleracea L. before.

The phases of the project and their realization

Project will be organized into four main work packages addressing the effect of different transformation parameters on transformation efficiency of cabbage protoplasts (WP1; months 1-3), evaluation of influence of CRISPR/Cas9 vector construction on targeted mutagenesis (WP2; months 4-8), transient expression of base editing vectors in cabbage protoplasts and characterization of CRISPR/Cas9 events (WP3; months 9-13), and optimization of regeneration of transformed protoplast (WP4; months 9-23). To ensure timely fulfilment of the proposed tasks, 4 milestones have been envisioned during the project: determination of optimal transformation parameters (M1; month 3), determination of efficient CRISPR/Cas9 vector architecture (M2; month 8), evaluation of different base editors (M3; month 13), and development of efficient protocol for regeneration of transformed cabbage protoplasts (M4; month 23).

Project realization:

WP1: Establishment of efficient cabbage protoplast transformation protocol

Task 1.1: Testing different transformation parameters (completed)

Task 1.2: Evaluation of transformation efficiency (completed)

WP2: Optimization of CRISPR/Cas9-induced mutations

Task 2.1: Preparation of different CRISPR/Cas9 vectors and transformation of cabbage protoplasts (completed)

Task 2.2: Evaluation of genome editing events (completed)

WP3: Precise base editing

Task 3.1: sgRNA design and base editing vector cloning (completed)

Task 3.2: Transient expression in protoplasts and characterization of CRISPR/Cas9 events (completed)

WP4: Regeneration of transformed protoplasts

Task 4.1: Comparison of different regeneration protocols (completed)

Task 4.2: Delivery of BE constructs to protoplasts and performance analysis (completed)

SICRIS

References:

[1]  STAJIČ, Ester, KUNEJ, Urban. Uporaba tehnik tarčnega preurejanja genomov v podporo žlahtnjenju. V: ČEH, Barbara (ur.), et al. Novi izzivi v agronomiji 2023 = New Challenges in Agronomy 2023 : zbornik simpozija = proceedings of symposium : [Laško, 26. in 27. januar 2023]. Laško: Slovensko agronomsko društvo: = Slovenian Society of Agronomy, 2023. Str. 185-190. ISBN 978-961-94613-3-4. [COBISS.SI-ID 141877251]

[2]  STAJIČ, Ester, KUNEJ, Urban. Optimization of cabbage (Brassica oleracea var. capitata L.) protoplast transformation for genome editing using CRISPR/Cas9. Frontiers in plant science. 2023, vol. 14, art. 1245433, 9 str., ilustr. ISSN 1664-462X.  https://www.frontiersin.org/articles/10.3389/fpls.2023.1245433, Repozitorij Univerze v Ljubljani – RUL, DOI: 10.3389/fpls.2023.1245433. [COBISS.SI-ID 168169731]

[3]  STAJIČ, Ester. Improvements in protoplast isolation protocol and regeneration of different cabbage (Brassica oleracea var. capitata L.) cultivars. Plants. 2023, vol. 12, iss. 17, art. 3074, 12 str., ilustr. ISSN 2223-7747. https://www.mdpi.com/2223-7747/12/17/3074, Repozitorij Univerze v Ljubljani – RUL, DOI: 10.3390/plants12173074. [COBISS.SI-ID 168164355]