Overexpression of Choline Oxidase Gene in Three Filial Generations of Rice Transgenic Lines

Document Type : Research Paper

Authors

1 Genetic Angineering and Biosafety, Agricultural Biotechnology Research Institute of Iran, Agricultural Research, Education and Extension

2 Department of Agronomy and Plant Breeding, College of Agriculture, Isfahan University of Technology, Isfahan, 84156-83111, I.R. Iran

3 Department of Agronomy and Plant Breeding, College of Agriculture, Isfahan University of Technology, Isfahan, 84156-83111, IR Iran

Abstract

Background: Glycinebetaine (GB) accumulation in many halophytic plants, animals, and microorganisms confers abiotic 
stress tolerance to salinity, drought, and extreme temperatures. Although there are a few genetic and biochemical pathways to synthesize GB, but isolation of a single gene choline oxidase (codA) from Arthrobacter spp. have opened a new hatch to engineer the susceptible plants. 
Objectives: The effects of overexpressed codA gene, through multiple copy insertion and GB accumulation on salinity 
tolerance in rice were studied. 
Materials and Methods: Seed-derived embryogenic calli of ‘Tarom Molaie’ cultivar were targeted with two plasmids 
pChlCOD and pCytCOD both harboring the codA gene using the biolistic mediated transformation. The regenerated T0
plants were screened by PCR analysis. A line containing three copies of codA gene and harboring pChlCOD and pCytCOD was identified by Southern blot analysis. The expression of codA gene in this transgenic line was then confirmed by RTPCR. The Mendelian segregation pattern of the inserted sequences was accomplished by the progeny test using PCR. The effects of overexpression of codA on salinity tolerance were evaluated at germination and seedling stage using T2
-pChl transgenic line and control seeds in the presence of 0, 100, 200, and 300 mM NaCl. Finally, leaf growth dynamics of T2-pChlCOD transgenic line and control line under hydroponic conditions in the presence of 0, 40, 80, and 120 mM NaCl 
were assessed.
Results: The seed germination experiment results showed that the transformed seeds had a higher germination rate than 
the controls under all salinity treatments. But also, the leaf growth dynamics showed that the control plants had a more 
favorable leaf growth dynamic in all of the treatments. Although, the transgenic lines (T0, T1 and T2) exhibited lower 
performance than the wild type, the transgenic line varied for GB and choline contents and increasing codA gene copy 
number led to increased GB content. 
Conclusion: In a salinity sensitive crop such as rice, GB may not significantly contribute to the plant protection against 
salt stress. Also, insufficiency of choline resources as GB precursor might have affected the overall growth ability of the 
transgenic line and resulted in decreased leaf growth dynamics. 

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