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Living Modified Organism
(LMO)
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Spring barley with a mutation in the VRN1 gene (vrn1_LOF_4del)
EN
vrn1_LOF_4del
No
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Prague,Organization:Institute of Experimental Botany, AS CR (IEB)Academic or research institute
, CzechiaPhone: + 420 225 106 455,Fax:Email: ueb@ueb.cas.cz,Website: http://www.ueb.cas.cz/en,
The spring barley (Hordeum vulgare L.) was modified by CRISPR/Cas-mediated targeted mutagenesis of the endogenous HvVRN1 (vernalization1) gene to alter flowering and vernalization responses. The vrn1_LOF_4del line contains a targeted 4 bp deletion in the first exon of HvVRN1. This deletion disrupts the reading frame, preventing production of a functional HvVRN1 protein and resulting in a loss-of-function allele. HvVRN1 encodes an APETALA1/FRUITFULL-like MADS-box transcription factor that functions as a key regulator of the transition from vegetative to reproductive growth and integrates vernalization signals to promote flowering in barley.
The term “Recipient organism” refers to an organism (either already modified or non-modified) that was subjected to genetic modification, whereas “Parental organisms” refers to those that were involved in cross breeding or cell fusion.
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BCH-ORGA-SCBD-12110-5 Organism Hordeum vulgare (Barley, HORVU)Crops
Hordeum vulgare L. (spring barley) cultivar Golden Promise
EN
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Spring barley with a mutation in the VRN1 gene (vrn1_LOF_76del)| Institute of Experimental Botany, AS CR(IEB) | Changes in physiology and/or production
CPGE_VEC00113
EN
- Gene editing (e.g. CRISPR-Cas, etc.)
Some of these genetic elements may be present as fragments or truncated forms. Please see notes below, where applicable.
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BCH-GENE-SCBD-299561-1 VRN1 | Hordeum vulgare (Barley, HORVU)Protein coding sequence | Changes in physiology and/or production (Growth rate, Reproduction, Genetic use restriction technology (GURT), Male sterility)
Information on the modified DNA sequence
The vrn1_LOF_4del barley line was generated using CRISPR/Cas-mediated genome editing following Agrobacterium-mediated transformation with the 18,084 bp vector CPGE_VEC00113.
The retained genetic modification consists of a 4 bp deletion in the first exon of the endogenous HvVRN1 (vernalization1) gene. The deletion disrupts the reading frame, preventing production of a functional HvVRN1 protein and resulting in a loss-of-function allele. HvVRN1 encodes an APETALA1/FRUITFULL-like MADS-box transcription factor that functions as a key regulator of vernalization responses and flowering in barley. The modified sequence is derived entirely from the endogenous barley VRN1 locus and does not introduce a novel coding sequence.
CPGE_VEC00113 contained Cas9, two guide RNA expression units targeting HvVRN1, the hpt selectable marker for transformed plant cells, the aadA bacterial selectable marker, and associated regulatory sequences. Following transformation and genome editing, regenerated plants were self-pollinated and progeny in which the vector-derived transgenic sequences had segregated away were selected. Their absence was repeatedly confirmed by molecular analyses in the T1 and T2 generations.
The T3 vrn1_LOF_4del plants selected for environmental release are homozygous for the 4 bp deletion and contain no introduced foreign genetic material.
Note
- The 4 bp deletion in the HvVRN1 locus was confirmed by Sanger sequencing.
- PCR and droplet digital PCR (ddPCR) were used to confirm the absence of vector-derived transgenic sequences in the T1 and T2 generations.
- CPGE_VEC00113 was used during development of the line but is not present in the final T3 plants.
- No foreign DNA or novel coding sequence is present in the final vrn1_LOF_4del line.
The 4 bp deletion in the first exon of the endogenous HvVRN1 gene disrupts the reading frame, preventing production of a functional HvVRN1 protein and resulting in a loss-of-function allele. As HvVRN1 encodes an APETALA1/FRUITFULL-like MADS-box transcription factor that regulates flowering and integrates vernalization signals in barley, loss of its function alters flowering regulation and developmental responses associated with vernalization. No novel gene expression or heterologous protein production is reported in the final line.
- Food
- Research
PCR amplification of the genomic region spanning the edited site followed by sequence analysis can be used to distinguish the vrn1_LOF_4del allele from the wild-type HvVRN1 sequence. PCR and ddPCR were additionally used during line development to verify the absence of vector-derived transgenic sequences.
HvVRN1 is an important determinant of flowering time and adaptation of barley to different climatic conditions and growing seasons. The vrn1_LOF_4del line was developed to investigate under field conditions the effects of HvVRN1 loss of function on flowering, vernalization responses, environmental adaptation and agronomic performance. The evaluation includes phenological and agronomic traits such as heading date, tillering, yield-related characteristics and grain quality parameters.
Compared with non-modified control plants, vrn1_LOF_4del plants have been reported to flower approximately 38 days later and to exhibit increased tillering.
- A set of GMO spring barley lines with a mutation in the VRN1 gene for testing potential agricultural applications [ English ]
- Large deletions within the first intron in VRN-1 are associated with spring growth habit in barley and wheat [ English ]
- Plant Physiology (2008). Low-Temperature and Daylength Cues Are Integrated to Regulate FLOWERING LOCUS T in Barley [PDF].pdf ( barley, flowering, vernalization, HvVRN ) [ English ]
- Direct links between the vernalization response and other key traits of cereal crops [DOI] [ English ]
- European Commission Food and Feed Information Portal. POL-GMOB-13842 - Spring barley with a mutation in the VRN1 gene [URL] [ English ]
- Development of the modular CasCADE vector system and its use for targeted mutagenesis [ German ]
| Record type | Field | Record(s) | |
|---|---|---|---|
| Risk Assessment generated by a regulatory process | Living modified organism(s) | 1 | |
| Living Modified Organism | Related LMO(s) | 1 | |
