biologia plantarum

International journal on Plant Life established by Bohumil Němec in 1959

Biologia plantarum 45:359-365, 2002 | DOI: 10.1023/A:1016209431929

Transient Expression of β-Glucuronidase in Embryo Axes of Cotton by Agrobacterium and Particle Bombardment Methods

A.K. Banerjee1, D.C. Agrawal1, S.M. Nalawade1, K.V. Krishnamurthy1
1 Plant Tissue Culture Division, National Chemical Laboratory, Pune, India

Transient expression of β-glucuronidase (GUS) in zygotic embryo axes of two cotton (Gossypium hirsutum L.) cultivars NHH-44 and DCH-32 was induced by Agrobacterium mediated transformation or by particle bombardment. For Agrobacterium transformation, disarmed A. tumefaciens strain GV 2260/p35SGUSINT was used. In cv. NHH-44, the maximum frequency of transient expression (14.28 %) was achieved on spotting Agrobacterium paste on the apical regions of the split embryo axes. The method resulted in a transformed callus line, which showed strong GUS activity. Integration of NPTII gene was confirmed by Southern analysis. Transgene expression by particle bombardment was achieved with p35SGUSINT and pIBGUS plasmids independently. The maximum frequency of GUS expression in 29.16 % explants was observed in cultivar NHH-44 with gold microcarriers (1.1 µm) when bombarded once with rupture disc of 7586 kPa at target cell distance of 6 cm. A transformed callus line was obtained when explants were bombarded with p35SGUSINT and cultured on Murashige and Skoog's medium supplemented with B5 vitamins, 0.1 mg dm-3 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, 0.01 mg dm-3 α-naphthaleneacetic acid, 3 % glucose + 50 mg dm-3 kanamycin. High GUS activity was observed in callus tissue as well as in somatic embryo like structures achieved in liquid shake cultures.

Keywords: Gossypium hirsutum; micrografting; transgenic plants
Subjects: Agrobacterium tumefaciens; auxins; cotton, β-glucuronidase expression in embryo axes; cytokinins; embryo axis, β-glucuronidase expression in; β-glucuronidase; Gossypium hirsutum; in vitro culture, nutrient media modification; micrografting in vitro; particle bombardment and Agrobacterium transformation; transgenic cotton, particle bombardment and Agrobacterium transformation; transient expression of β-glucuronidase

Published: September 1, 2002  Show citation

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Banerjee, A.K., Agrawal, D.C., Nalawade, S.M., & Krishnamurthy, K.V. (2002). Transient Expression of β-Glucuronidase in Embryo Axes of Cotton by Agrobacterium and Particle Bombardment Methods. Biologia plantarum, 45(3), 359-365. doi: 10.1023/A:1016209431929
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