Multiple traits of agronomic importance in transgenic indica rice plants: Analysis of transgene integration patterns, expression levels and stability

Shahina B. Maqbool, Paul Christou

Research output: Contribution to journalArticle

110 Citations (Scopus)

Abstract

We cotransformed indica rice (Oryza sativa L. cvs. Basmati 370 and M7) with three plasmids, carrying a total of four genes, by particle bombardment. The Bacillus thuringiensis (Bt) δ-endotoxin genes cryIAc and cry2A were carried on separate vectors, while the gna (snowdrop lectin) and hpt (hygromycin phosphotransferase) genes were linked on the same, cointegrate vector. We evaluated the molecular and expression profiles of 29 independently derived transgenic lines over two generations. The gna and hpt genes cointegrated with a frequency of 100% as expected. Furthermore, 60% of the transgenic plants carried all four genes. Southern blot analysis showed that transgene copy number ranged from 1 to 15. We used western blots to determine protein expression levels in R0 and R1 plants. We observed wide variation in the expression levels of the nonselected transgenes among independently-derived lines, but expression levels within lines derived from the same clone were similar. Consistent with previous reports, we observed no correlation between transgene copy number and the level or stability of protein expression. We show that the introduction of multiple agronomically valuable genes into the rice genome by cotransformation is a practical strategy for engineering elite rice varieties.

Original languageEnglish (US)
Pages (from-to)471-480
Number of pages10
JournalMolecular Breeding
Volume5
Issue number5
DOIs
StatePublished - 1999
Externally publishedYes

Fingerprint

Transgenes
agronomic traits
transgenes
hygromycin-B kinase
Genes
genetically modified organisms
rice
genes
phosphotransferases (kinases)
protein synthesis
Bacillus thuringiensis
Protein Stability
Genetically Modified Plants
endotoxins
Southern Blotting
Endotoxins
lectins
Southern blotting
Bacilli
Oryza

Keywords

  • δ-endotoxins (Cry1Ac and Cry2A)
  • Basmati 370 and M7 varieties
  • Particle bombardment
  • Snowdrop lectin (GNA)
  • Transgenic rice

ASJC Scopus subject areas

  • Agronomy and Crop Science
  • Horticulture
  • Plant Science
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology

Cite this

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abstract = "We cotransformed indica rice (Oryza sativa L. cvs. Basmati 370 and M7) with three plasmids, carrying a total of four genes, by particle bombardment. The Bacillus thuringiensis (Bt) δ-endotoxin genes cryIAc and cry2A were carried on separate vectors, while the gna (snowdrop lectin) and hpt (hygromycin phosphotransferase) genes were linked on the same, cointegrate vector. We evaluated the molecular and expression profiles of 29 independently derived transgenic lines over two generations. The gna and hpt genes cointegrated with a frequency of 100{\%} as expected. Furthermore, 60{\%} of the transgenic plants carried all four genes. Southern blot analysis showed that transgene copy number ranged from 1 to 15. We used western blots to determine protein expression levels in R0 and R1 plants. We observed wide variation in the expression levels of the nonselected transgenes among independently-derived lines, but expression levels within lines derived from the same clone were similar. Consistent with previous reports, we observed no correlation between transgene copy number and the level or stability of protein expression. We show that the introduction of multiple agronomically valuable genes into the rice genome by cotransformation is a practical strategy for engineering elite rice varieties.",
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T2 - Analysis of transgene integration patterns, expression levels and stability

AU - Maqbool, Shahina B.

AU - Christou, Paul

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N2 - We cotransformed indica rice (Oryza sativa L. cvs. Basmati 370 and M7) with three plasmids, carrying a total of four genes, by particle bombardment. The Bacillus thuringiensis (Bt) δ-endotoxin genes cryIAc and cry2A were carried on separate vectors, while the gna (snowdrop lectin) and hpt (hygromycin phosphotransferase) genes were linked on the same, cointegrate vector. We evaluated the molecular and expression profiles of 29 independently derived transgenic lines over two generations. The gna and hpt genes cointegrated with a frequency of 100% as expected. Furthermore, 60% of the transgenic plants carried all four genes. Southern blot analysis showed that transgene copy number ranged from 1 to 15. We used western blots to determine protein expression levels in R0 and R1 plants. We observed wide variation in the expression levels of the nonselected transgenes among independently-derived lines, but expression levels within lines derived from the same clone were similar. Consistent with previous reports, we observed no correlation between transgene copy number and the level or stability of protein expression. We show that the introduction of multiple agronomically valuable genes into the rice genome by cotransformation is a practical strategy for engineering elite rice varieties.

AB - We cotransformed indica rice (Oryza sativa L. cvs. Basmati 370 and M7) with three plasmids, carrying a total of four genes, by particle bombardment. The Bacillus thuringiensis (Bt) δ-endotoxin genes cryIAc and cry2A were carried on separate vectors, while the gna (snowdrop lectin) and hpt (hygromycin phosphotransferase) genes were linked on the same, cointegrate vector. We evaluated the molecular and expression profiles of 29 independently derived transgenic lines over two generations. The gna and hpt genes cointegrated with a frequency of 100% as expected. Furthermore, 60% of the transgenic plants carried all four genes. Southern blot analysis showed that transgene copy number ranged from 1 to 15. We used western blots to determine protein expression levels in R0 and R1 plants. We observed wide variation in the expression levels of the nonselected transgenes among independently-derived lines, but expression levels within lines derived from the same clone were similar. Consistent with previous reports, we observed no correlation between transgene copy number and the level or stability of protein expression. We show that the introduction of multiple agronomically valuable genes into the rice genome by cotransformation is a practical strategy for engineering elite rice varieties.

KW - δ-endotoxins (Cry1Ac and Cry2A)

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KW - Particle bombardment

KW - Snowdrop lectin (GNA)

KW - Transgenic rice

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