Limits...
Expression of Pennisetum glaucum Eukaryotic Translational Initiation Factor 4A ( PgeIF4A ) Confers Improved Drought, Salinity, and Oxidative Stress Tolerance in Groundnut

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5383670&req=5

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.

Mentions: The Agrobacterium tumefaciens (LBA4404) carrying vector, pGreen0229: rd29A: PgeIF4A: poly A with nos-bar gene as selectable marker (Figure 2A) was used to infect 742 DECs (Figure 2B) prepared from matured groundnut seeds. Out of these 225 explants induced calli in the medium supplemented with phosphinothricin (PPT), BAP and 2, 4-D with a frequency of 30.3% (Figures 2C–E; Table 1). Subsequently these proliferated resistant calli differentiated into shoots in 2 weeks. About 38 green shoots from resistant calli (16.8%) differentiated into multiple shoots in 30–40 days (Figures 2F–H). The PPT resistant shoots were continuously grown and maintained on selective media. Roots generated on NAA supplemented medium. About 30 rooted plants were acclimatized under controlled greenhouse conditions (Figure 2I). The hardened plants grew normally and set flowers and pods. These plants were used for further molecular analysis.


Expression of Pennisetum glaucum Eukaryotic Translational Initiation Factor 4A ( PgeIF4A ) Confers Improved Drought, Salinity, and Oxidative Stress Tolerance in Groundnut
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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383670&req=5

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.
Mentions: The Agrobacterium tumefaciens (LBA4404) carrying vector, pGreen0229: rd29A: PgeIF4A: poly A with nos-bar gene as selectable marker (Figure 2A) was used to infect 742 DECs (Figure 2B) prepared from matured groundnut seeds. Out of these 225 explants induced calli in the medium supplemented with phosphinothricin (PPT), BAP and 2, 4-D with a frequency of 30.3% (Figures 2C–E; Table 1). Subsequently these proliferated resistant calli differentiated into shoots in 2 weeks. About 38 green shoots from resistant calli (16.8%) differentiated into multiple shoots in 30–40 days (Figures 2F–H). The PPT resistant shoots were continuously grown and maintained on selective media. Roots generated on NAA supplemented medium. About 30 rooted plants were acclimatized under controlled greenhouse conditions (Figure 2I). The hardened plants grew normally and set flowers and pods. These plants were used for further molecular analysis.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus