doktorska disertacija
Ester Stajič (Author), Borut Bohanec (Mentor), Nataša Štajner (Thesis defence commission member), Jelka Šuštar Vozlič (Thesis defence commission member), Simon Horvat (Thesis defence commission member), Jana Murovec (Co-mentor)

Abstract

Tarčno preurejanje genoma z uporabo tehnologije CRISPR/Cas9 je vedno pogosteje uporabljen pristop za induciranje tarčnih mutacij tudi pri rastlinah. V naših poskusih smo uporabili deset različnih sgRNA za ciljanje štirih različnih mest v genomu zelja (Brassica oleracea var. capitata L.), med njimi tudi 6 sgRNA za spremembe centromernega proteina CENH3, ki je vključen v ločevanje kromosomov. Rastline z mutiranimi oblikami proteina CENH3 se lahko uporabijo kot opraševalske linije za indukcijo haploidov v procesu pridobivanja hibridov z visoko agronomsko vrednostjo. Za dostavo pripravljenih CRISPR/Cas9 vektorjev v celice rastlin zelja smo uporabili tri različne pristope: transformacijo protoplastov, agroinfiltracijo in stabilno transformacijo z uporabo bakterije Agrobacterium tumefaciens (A. t.). Za detekcijo tarčnih mutacij smo uporabili test z endonukleazo T7E1, sekvenciranje po Sangerju in sekvenciranje naslednje generacije. Slednje je pokazalo uspešno indukcijo tarčnih mutacij za vse testirane sgRNA pri vzorcih poskusov transformacije protoplastov in agroinfiltracije. Odstotki induciranih indel (insercije-delecije) mutacij so bili do 11,95 % po transformaciji protoplastov in do 14,42 % po agroinfiltraciji. Za regeneracijo rastlin z želenimi mutacijami smo optimizirali protokole za regeneracijo protoplastov z uporabo gojenja v alginatnih diskih in protokol za stabilno transformacijo z A. t. Protokoli, izdelani v okviru te doktorske disertacije, bodo pripomogli k uporabi tarčne mutageneze pri žlahtnjenju zelja.

Keywords

zelje;CRISPR/Cas9;tarčno preurejanje;protoplasti;genska transformacija;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL BF - Biotechnical Faculty
Publisher: [E. Stajič]
UDC: 606:602.64:582.683.211(043.3)
COBISS: 9448825 Link will open in a new window
Views: 966
Downloads: 603
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Other data

Secondary language: English
Secondary title: Genome editing of centromere protein CENH3 in cabbage (Brassica oleracea var. capitata L.) using CRISPR/Cas9 technology
Secondary abstract: Genome editing using CRISPR/Cas9 technology is a prevailing approach for the induction of target mutations also in plants. In our work, we used ten different sgRNAs for genome editing of four different sites in cabbage (Brassica oleracea var. capitata L.), including 6 sgRNAs for modification of centromere protein CENH3 involved in chromosome segregation. Plants carrying mutated versions of CENH3 have the potential to be used as haploid inducers in the process of production of hybrids with high agronomic value. We used three different approaches for delivery of prepared CRISPR/Cas9 vectors into cabbage cells: protoplast transfection, agroinfiltration and stable transformation using Agrobacterium tumefaciens (A. t.). To detect target mutations we used T7E1 assay, Sanger sequencing, and next-generation sequencing. The latter showed successful induction of target mutations for all tested sgRNAs in protoplast transfection and agroinfiltration experiments. Mutations were induced at frequencies up to 11,95 % for protoplast transfection and up to 14,42 % for agroinfiltration. For regeneration of plants carrying desired mutations, we optimized protocols for the regeneration of protoplast using cultivation in alginate layers and protocol for stable transformation with A. t. Protocols developed through this doctoral dissertation will help to further use targeted mutagenesis in cabbage breeding.
Secondary keywords: cabbage;genome editing;protoplasts;genetic transformation;
Type (COBISS): Doctoral dissertation
Study programme: 0
Thesis comment: Univ. v Ljubljani, Biotehniška fak.
Pages: XIV, 93, [5] f.
ID: 11462186