Creation of Low Phytate Pea Lines Using CRISPR/Cas9-assisted Transformation

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F26784246%3A_____%2F22%3AN0000043" target="_blank" >RIV/26784246:_____/22:N0000043 - isvavai.cz</a>

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Creation of Low Phytate Pea Lines Using CRISPR/Cas9-assisted Transformation

Popis výsledku v původním jazyce

This work is focused on the development of pea (Pisum sativum L.) breeding lines with low phytic acid (lpa) content. Phytic acid is an antinutritional factor causing decreased bioavailability of mineral elements, inefficient feed utilization and increased phosphate content in excrement. Three different constructs suitable for CRISPR/Cas9 genome editing technology were created, each of them targeting a different region of myo-inositol-3-phosphate synthase (mips) gene. These constructs have been used for pea agrobacterial transformation with high virulent EHA105 and GV3101 strains of Agrobacterium tumefaciens. Screening of putative transformants has been performed by GUS staining during four selection cycles based on nptII gene (resistance to kanamycin). DNA samples isolated from GUS-positive transformants have been analysed by PCR amplification and subsequent restriction. According to observed transient expression of uidA reporter gene in transformed explants of tested pea varieties (Eso, Protecta and Trendy), Eso variety and pVO108 construct were selected for further transformations. In total 16 transformations using either individual constructs or a mixture of 2–3 constructs were carried out up to now. From over 7000 transformed node explants almost 400 were GUS positive, approximately 200 explants were tested by PCR and several selected samples were sequenced. By optimizing the individual steps, a protocol for successful pea agrobacterial transformation of the Eso variety was obtained. Our goal is to create transgenic lpa lines, which, however depend on the successful selection of a non-chimeric shoot in in vitro culture.

Název v anglickém jazyce

Creation of Low Phytate Pea Lines Using CRISPR/Cas9-assisted Transformation

Popis výsledku anglicky

This work is focused on the development of pea (Pisum sativum L.) breeding lines with low phytic acid (lpa) content. Phytic acid is an antinutritional factor causing decreased bioavailability of mineral elements, inefficient feed utilization and increased phosphate content in excrement. Three different constructs suitable for CRISPR/Cas9 genome editing technology were created, each of them targeting a different region of myo-inositol-3-phosphate synthase (mips) gene. These constructs have been used for pea agrobacterial transformation with high virulent EHA105 and GV3101 strains of Agrobacterium tumefaciens. Screening of putative transformants has been performed by GUS staining during four selection cycles based on nptII gene (resistance to kanamycin). DNA samples isolated from GUS-positive transformants have been analysed by PCR amplification and subsequent restriction. According to observed transient expression of uidA reporter gene in transformed explants of tested pea varieties (Eso, Protecta and Trendy), Eso variety and pVO108 construct were selected for further transformations. In total 16 transformations using either individual constructs or a mixture of 2–3 constructs were carried out up to now. From over 7000 transformed node explants almost 400 were GUS positive, approximately 200 explants were tested by PCR and several selected samples were sequenced. By optimizing the individual steps, a protocol for successful pea agrobacterial transformation of the Eso variety was obtained. Our goal is to create transgenic lpa lines, which, however depend on the successful selection of a non-chimeric shoot in in vitro culture.

Druh

D - Stať ve sborníku

CEP obor

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OECD FORD obor

40402 - GM technology (crops and livestock), livestock cloning, marker assisted selection, diagnostics (DNA chips and biosensing devices for the early/accurate detection of diseases) biomass feedstock production technologies, biopharming

Projekt

<a href="/cs/project/QK1810072" target="_blank" >QK1810072: Vývoj biofortifikovaných linií hrachu se sníženým obsahem kyseliny fytové</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2022

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název statě ve sborníku

Mendel...Mendel Genetics Conference Book of Abstracts

ISBN

978-80-11-01750-7

ISSN

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e-ISSN

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Počet stran výsledku

1

Strana od-do

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Název nakladatele

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Místo vydání

Brno

Místo konání akce

Brno

Datum konání akce

1. 1. 2022

Typ akce podle státní příslušnosti

WRD - Celosvětová akce

Kód UT WoS článku

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