Santosh Rama Bhadra Rao T, Vijaya Naresh J, Sudhakar Reddy P, Reddy MK, Mallikarjuna G.

Front Plant Sci. 2017 Apr 7;8:453. doi: 10.3389/fpls.2017.00453. eCollection 2017.

Abstract

Eukaryotic translational initiation factor 4A belong to family of helicases, involved in multifunctional activities during stress and non-stress conditions. The eIF4A gene was isolated and cloned from semi-arid cereal crop of Pennisetum glaucum. In present study, the PgeIF4A gene was expressed under the regulation of stress inducible Arabidopsis rd29A promoter in groundnut (cv JL-24) with bar as a selectable marker. The de-embryonated cotyledons were infected with Agrobacterium tumefaciens (LBA4404) carrying rd29A:PgeIF4A construct and generated high frequency of multiple shoots in phosphinothricin medium. Twenty- four T₀ plants showed integration of both nos-bar and rd29A-PgeIF4A gene cassettes in genome with expected amplification products of 429 and 654 bps, respectively. Transgene copy number integration was observed in five T₀ transgenic plants through Southern blot analysis. Predicted Mendelian ratio of segregation (3:1) was noted in transgenic plants at T₁ generation. The T₂ homozygous lines (L1-5, L8-2, and L16-2) expressing PgeIF4A gene were exhibited superior growth performance with respect to phenotypic parameters like shoot length, tap root length, and lateral root formation under simulated drought and salinity stresses compared to the wild type. In addition, the chlorophyll retention was found to be higher in these plants compared to the control plants. The quantitative real time-PCR results confirmed higher expression of PgeIF4A gene in L1-5, L8-3, and L16-2 plants imposed with drought/salt stress. Further, the salt stress tolerance was associated with increase in oxidative stress markers, such as superoxide dismutase accumulation, reactive oxygen species scavenging, and membrane stability in transgenic plants. Taken together our results confirmed that the PgeIF4A gene expressing transgenic groundnut plants exhibited better adaptation to stress conditions.

See: https://www.ncbi.nlm.nih.gov/pubmed/28439277

Figure 2: Schematic representation of T-DNA region of recombinant plant transformation vector pGreen0229-rd29A:PgeIF4A:poly A and groundnut transformation. (A) PgeIF4A gene was cloned under stress inducible promoter rd29A and poly A terminator, bar (Bialaphos amino transferase gene) as a selectable marker which driven by nos (nopaline synthase) promoter and nos-terminator. LB, left boarder; RB, right boarder. (B) De-embryonated half cotyledons (DEC) prepared from matured seeds. (C,D) DEC producing calli. (E) Phosphinothricin resistant calli producing shoots. (F–H) Shoot induction and multiple shoot formation. (I) Putative transgenic plants (T₀) growing in glass house.