Illing, Luca James Champley (2019) The optimisation of CRISPR-Cas9 transformation in brassica species using the eIEF4E resistance gene as a model target. PhD thesis, University of Warwick.

Preview

PDF WRAP_Theses_Illing_2019.pdf - Submitted Version - Requires a PDF viewer. Download (13Mb) | Preview

Request Changes to record.

Abstract

Genome editing in plants has provided many new breakthroughs due to the development of different methods, such as CRISPR/Cas9, by which this can now be carried out. Beneficial traits should now be more quickly integrated into breeding lines. The Potyvirus TuMV, affects a wide variety of crop species including Brassicas, causing losses in yield and increasing production costs, therefore broad-spectrum resistance to TUMV is needed. Previous work identified a B. rapa variety, RLR22, which had resistance to TuMV due to a single mutation in an isoform of eIF4E, eIF(iso)4E, truncating the protein. This stops the virus utilising the eIF4F translation initiation complex, which consists of three subunits eIF4E, eIF4A, and eIF4G. This represents an ideal target for developing CRISPR/Cas9 knock outs as equivalent resistance has not yet been found in B. oleracea. Further investigation into the genetic makeup of the eIF4F complex enabled the targeting of specific paralogs that might confer resistance. Bioinformatic analysis of the genes involved identified several novel paralogs of eIF(iso)4e, eIF4A, eIF(iso)4g. This allowed for the design of paralog specific PCR primers and guide RNAs. RNAseq analysis also revealed alternative splicing taking place in all the gene families involved. CRISPR modification of B. oleracea proved difficult, resulting in only one heterozygous edited plant. This is potentially due to lethality of the knock-out of the target gene. In B. rapa, novel transformation techniques were employed to overcome the obstacles of recalcitrance to being transformed in this species. Protoplast protocols were optimised for use in Brassica and these increased transformation rates significantly. However, the use of embryogenesis transcription factors to increase regeneration of plants, and the use of Carbon Nanodots to aid in transformation were unsuccessful. CRISPR modification in Brassicas remains difficult, however, these complications stress the obstacles that must be overcome for industry to implement such technologies into their breeding programs.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QK Botany Q Science > QR Microbiology > QR355 Virology
Library of Congress Subject Headings (LCSH):	Plants -- Virus resistance -- Genetic aspects, Brassica -- Genetics, Potyvirus diseases, Gene editing
Official Date:	September 2019
Dates:	Date Event September 2019 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Barker, Guy C. ; Walsh, John A.
Sponsors:	Biotechnology and Biological Sciences Research Council (Great Britain) ; Great Britain. Agriculture and Horticulture Development Board
Format of File:	pdf
Extent:	x, 98 leaves : illustrations
Language:	eng

Request changes or add full text files to a record

Repository staff actions (login required)

View Item

Downloads