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

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 RT-PCR. 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.

Keywords: Glycine betaine (GB); Rice; Salt stress; Transformation; Oryza sativa L.