biologia plantarum

International journal on Plant Life established by Bohumil Němec in 1959

Biologia plantarum 54:265-271, 2010 | DOI: 10.1007/s10535-010-0046-7

Production and selection of marker-free transgenic plants of Petunia hybrida using site-specific recombination

R. S. Khan1, I. Nakamura1, M. Mii1,*
1 Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University, Chiba, Japan

MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic plants. Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Petunia hybrida 'Dainty Lady' employing isopentenyl transferase (ipt) gene as the selection marker gene. β-glucuronidase (GUS) gene was used as model gene of interest. Infected explants were cultured on Murashige and Skoog (MS) medium without plant growth regulators (PGR) and antibiotics. Shoots showing extreme shooty phenotype (ESP) were produced from the adventitious shoots separated from the explants. Visual selection was carried out until production of morphologically normal shoots (approximately 4 months after infection). Histochemical GUS assay detected GUS gene in both ESP and normal shoots. PCR analysis confirmed the presence of model gene (GUS gene) and excision of the selection marker (ipt) gene in the normal transgenic plants. The insertion sites (1-3 for ipt gene and 1-2 for GUS gene) were detected by Southern blot analysis using DIG-labeled probes of both genes. These results show that ipt-type MAT vector can be used successfully to produce marker-free transgenic Petunia hybrida plants on PGR- and antibiotic-free MS medium.

Keywords: extreme shooty phenotype; GUS gene; ipt gene; MAT vector; PCR; Southern blot; transformation
Subjects: Agrobacterium tumefaciens; β-glucuronidase; isopentenyltransferase; multi-auto-transformation (MAT) vector; petunia; Petunia hybrida; polymerase chain reaction (PCR); Southern blot analysis; transgenic plants

Received: May 7, 2008; Accepted: January 5, 2009; Published: June 1, 2010  Show citation

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Khan, R.S., Nakamura, I., & Mii, M. (2010). Production and selection of marker-free transgenic plants of Petunia hybrida using site-specific recombination. Biologia plantarum, 54(2), 265-271. doi: 10.1007/s10535-010-0046-7
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