Articles
PROTOPLAST CO-TRANSFORMATION AND REGENERATION OF TRANSGENIC ‘HAMLIN’ SWEET ORANGE PLANTS CONTAINING A CDNA XA21 XANTHOMONAS RESISTANCE GENE AND GFP
Article number
738_23
Pages
235 – 243
Language
English
Abstract
Hamlin sweet orange (Citrus sinensis (L.) Osbeck) is one of the leading commercial cultivars in Florida because of its high yield potential and early maturity. Hamlin also has a high regeneration capacity from protoplasts and is often used in transformation experiments.
Citrus canker disease caused by the bacterial pathogen Xanthomonas axonopodis pv. citri is becoming a worldwide problem.
Xa21 gene is a member of the Xa21 gene family of rice that provides broad spectrum Xanthomonas resistance in rice.
Since the citrus canker pathogen is in the same genus, this gene may have potential to function against canker in citrus.
The wild-type Xa21 gene contains an intron, and there is some question as to whether dicot plants can process genes containing monocot introns (the cDNA is intron free). The development of canker resistant citrus has become an important research objective.
Plasmid DNA (pARS108) encoding the non-destructive selectable marker EGFP (Enhanced Green Fluorescent Protein) gene, and the plasmid cDNA of the Xa21 gene (pXa21-mtaq) were co-transformed into Hamlin orange protoplasts using polyethylene glycol.
More than 150 transgenic embryoids were recovered.
Over a thousand transgenic plantlets GFP positive were regenerated from 150 independent transformation events.
PCR analysis revealed the presence of the cDNA of the Xa21 and the GFP genes in some of the transgenic plantlets.
The recovery of transgenic plants was expedited by in vitro grafting.
The transgenic plants have shown normal growth and stable GFP expression for over a year in the greenhouse.
Southern blot analysis is showing 0-5 copies of the transgene per transgenic plant.
Citrus canker disease caused by the bacterial pathogen Xanthomonas axonopodis pv. citri is becoming a worldwide problem.
Xa21 gene is a member of the Xa21 gene family of rice that provides broad spectrum Xanthomonas resistance in rice.
Since the citrus canker pathogen is in the same genus, this gene may have potential to function against canker in citrus.
The wild-type Xa21 gene contains an intron, and there is some question as to whether dicot plants can process genes containing monocot introns (the cDNA is intron free). The development of canker resistant citrus has become an important research objective.
Plasmid DNA (pARS108) encoding the non-destructive selectable marker EGFP (Enhanced Green Fluorescent Protein) gene, and the plasmid cDNA of the Xa21 gene (pXa21-mtaq) were co-transformed into Hamlin orange protoplasts using polyethylene glycol.
More than 150 transgenic embryoids were recovered.
Over a thousand transgenic plantlets GFP positive were regenerated from 150 independent transformation events.
PCR analysis revealed the presence of the cDNA of the Xa21 and the GFP genes in some of the transgenic plantlets.
The recovery of transgenic plants was expedited by in vitro grafting.
The transgenic plants have shown normal growth and stable GFP expression for over a year in the greenhouse.
Southern blot analysis is showing 0-5 copies of the transgene per transgenic plant.
Authors
A.A. Omar, J.W. Grosser
Keywords
rice, resistance gene, citrus canker, protoplast transformation, Xa21
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