Efficient Regeneration of Transgenic Rice from Embryogenic Callus via Agrobacterium-Mediated Transformation: A Case Study Using GFP and Apple MdFT1 Genes

Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants,...

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Published inPlants (Basel) Vol. 13; no. 19; p. 2803
Main Authors Do, Van Giap, Kim, Seonae, Win, Nay Myo, Kwon, Soon-Il, Kweon, Hunjoong, Yang, Sangjin, Park, Juhyeon, Do, Gyungran, Lee, Youngsuk
Format Journal Article
LanguageEnglish
Published Switzerland MDPI AG 01.10.2024
MDPI
Subjects
Agrobacterium
Agrobacterium tumefaciens
Agrobacterium-mediated transformation
Callus
Cloning
Corn
Crops
Cultivars
Efficiency
eGFP reporter gene
embryogenic callus
Environmental aspects
Explants
Expression vectors
Flowering
FLOWERING LOCUS T gene
Flowers & plants
Fluorescence
Fluorescence microscopy
Gene expression
Gene manipulation
Genes
Genetic analysis
Genetic aspects
Genetic transformation
Genetically altered foods
Genetically modified crops
Genomes
Green fluorescent protein
Growth
Hygromycin
MdFT1 gene
Methods
Phenotypes
Phenotypic variations
Physiological aspects
Plant bacterial diseases
Plant tissue culture
Plants (botany)
Plasmids
Polyethylene glycol
Regeneration
Rice
Seedlings
Testing
Transgenes
Transgenic plants
transgenic rice
Vectors (Biology)
α-Amylase
South Korea
eGFP reporter gene
Agrobacterium-mediated transformation
MdFT1 gene
heterologous expression
embryogenic callus
genetic transformation
transgenic rice
Online AccessGet full text
ISSN2223-7747
2223-7747
DOI10.3390/plants13192803

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Abstract Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species.
AbstractList Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice ( L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein ( ) and the apple ( )-like gene ( ). Antisense was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using -mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense -expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species.
Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice ( Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein ( eGFP ) and the apple FLOWERING LOCUS T ( FT )-like gene ( MdFT1 ). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium -mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1 -expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species.
Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species.
Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species.Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species.
Audience Academic
Author Park, Juhyeon
Win, Nay Myo
Kwon, Soon-Il
Yang, Sangjin
Do, Gyungran
Lee, Youngsuk
Kweon, Hunjoong
Kim, Seonae
Do, Van Giap
AuthorAffiliation 2 Postharvest Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju-gun 55365, Republic of Korea; microdo@korea.kr
1 Apple Research Center, National Institute of Horticultural and Herbal Science, Rural Development Administration, Daegu 43100, Republic of Korea; seonaekim@korea.kr (S.K.); naymyowin@korea.kr (N.M.W.); topapple@korea.kr (S.-I.K.); kweonhj@korea.kr (H.K.); yangsangjin@korea.kr (S.Y.); wngus1113@korea.kr (J.P.)
AuthorAffiliation_xml – name: 2 Postharvest Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju-gun 55365, Republic of Korea; microdo@korea.kr
– name: 1 Apple Research Center, National Institute of Horticultural and Herbal Science, Rural Development Administration, Daegu 43100, Republic of Korea; seonaekim@korea.kr (S.K.); naymyowin@korea.kr (N.M.W.); topapple@korea.kr (S.-I.K.); kweonhj@korea.kr (H.K.); yangsangjin@korea.kr (S.Y.); wngus1113@korea.kr (J.P.)
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Issue 19
Keywords eGFP reporter gene
Agrobacterium-mediated transformation
MdFT1 gene
heterologous expression
embryogenic callus
genetic transformation
transgenic rice
Language English
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Snippet Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic...
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SubjectTerms Agrobacterium
Agrobacterium tumefaciens
Agrobacterium-mediated transformation
Callus
Cloning
Corn
Crops
Cultivars
Efficiency
eGFP reporter gene
embryogenic callus
Environmental aspects
Explants
Expression vectors
Flowering
FLOWERING LOCUS T gene
Flowers & plants
Fluorescence
Fluorescence microscopy
Gene expression
Gene manipulation
Genes
Genetic analysis
Genetic aspects
Genetic transformation
Genetically altered foods
Genetically modified crops
Genomes
Green fluorescent protein
Growth
Hygromycin
MdFT1 gene
Methods
Phenotypes
Phenotypic variations
Physiological aspects
Plant bacterial diseases
Plant tissue culture
Plants (botany)
Plasmids
Polyethylene glycol
Regeneration
Rice
Seedlings
Testing
Transgenes
Transgenic plants
transgenic rice
Vectors (Biology)
α-Amylase
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Title Efficient Regeneration of Transgenic Rice from Embryogenic Callus via Agrobacterium-Mediated Transformation: A Case Study Using GFP and Apple MdFT1 Genes
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