Abstract

Proline plays important role in osmo-adjusment and prevention of osmotic stress in plants. In order to analyze osmotic stress resistance, germination and growth pattern in vitro and in vivo, the Arabidopsis thalianaδ-1-pyrroline-5-carboxylate synthetase (P5CS) which is the regulatory enzyme of proline biosynthesis, was reverse transcribed, amplified into double stranded cDNA, cloned and transformed into tobacco using Agrobacterium tumefaciens mediated transformation. Seeds of chimeric T₀ transgenic plants were grown; T₁ and the succeeding T₂ transgenic plants were obtained. Subsequently, wild type and binary vector transformed control plants along with T₂ transgenic lines were exposed to salt concentrations from 0 to 250 mM during germination and growth stages. Results showed a significant difference (P < 0.0001) between T₂ and control plants, while the control plants germinated in the presence of salt concentrations up to 50 mM and tolerated 100 mM salt during growth phase. Transgenic T₂ lines were able to germinate in 200 mM NaCl and tolerated up to 250 mM salt during growth phase. Assessing proline contents, we observed 7.5 times more proline in T₂ transgenic plants than control plants in normal condition which increased up to 3.3 folds in stress condition. This osmotic stress resistance was further assessed for T₃ generation against drought and salt stresses in vivo. Results indicated osmotic stress resistance of T₃generation as well as stable expression of P5CS along with achievement of homozygous transgenic lines.

Key words: δ-1-Pyrroline-5-carboxylate synthetase (P5CS), osmotic stress, transgenic plants, germination, growth phase.

Abbreviation

P5CS, δ-1-Pyrroline-5-carboxylate synthetase; NADPH,nicotinamide adenine dinucleotide phosphate; P5CR, δ-1-pyrroline-5-carboxylate reductase; PRODH, proline dehyrogenase; P5CDH, pyrroline-5-carboxylate dehydrogenase; PCR, polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; TBE, tris-borate-ethylenediaminetetraacetic acid; SSC,saline sodium citrate; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; EDTA, ethylenediaminetetraacetic acid.