3 Biotech

, 9:10 | Cite as

Pea p68, a DEAD-box helicase, enhances salt tolerance in marker-free transgenic plants of soybean [Glycine max (L.) Merrill]

  • Sivabalan Karthik
  • Narendra Tuteja
  • Andy Ganapathi
  • Markandan ManickavasagamEmail author
Original Article


Protein p68 is a prototype constituent of DEAD-box protein family, which is involved in RNA metabolism, induced during abiotic stress conditions. In order to address the salinity stress faced by economically important soybean crop, we have transformed soybean cv. PUSA 9712 via direct organogenesis with marker free construct of p68 gene by Agrobacterium-mediated genetic transformation. The putative transgenic plants were screened by Polymerase chain reaction (PCR), Dot blot analysis and Southern blot hybridization. Reverse transcriptase-PCR (RT-PCR) and Quantitative real-time PCR (qRT-PCR) established that the p68 gene expressed in three out of five southern positive (T1) plants. The transformed (T1) soybean plants survived irrigation upto 200 mM of NaCl whereas the non-transformed (NT) plants could not survive even 150 mM NaCl. The transgenic soybean (T1) plants showed a higher accumulation of chlorophyll, proline, CAT, APX, SOD, RWC, DHAR and MDHAR than the NT plants under salinity stress conditions. The transformed (T1) soybean plants also retained a higher net photosynthetic rate, stomatal conductance and CO2 assimilation as compared to NT plants. Further analysis revealed that (T1) soybean plants accumulated higher K+ and lower Na+ levels than NT plants. Yield performance of transformed soybean plants was estimated in the transgenic green house under salinity stress conditions. The transformed (T1) soybean plants expressing the p68 gene were morphologically similar to non-transformed plants and produced 22–24 soybean pods/plant containing 8–9 g (dry weight) of seeds at 200 mM NaCl concentration. The present investigation evidenced the role of the p68 gene against salinity, by enhancing the tolerance towards salinity stress in soybean plants.


Soybean Agrobacterium tumefaciens EHA105 p68 gene Salinity CaMV 35S promoter Binary vector qRT-PCR 



Sodium chloride

CaMV 35S

Cauliflower mosaic virus 35S promoter


Ascorbate peroxidase




Superoxide dismutase


Dehydroascorbate reductase


Monodehydroascorbate reductase




Relative water content



Jawaharlal Nehru Scholarship (Ref no: SU-1/88/2016-17/79) for doctoral studies awarded by Jawaharlal Nehru Memorial Fund, New Delhi, India is thankfully acknowledged by the first author (Sivabalan Karthik).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13205_2018_1553_MOESM1_ESM.docx (325 kb)
Supplementary Fig. 1 Schematic representation of binary vector pCAMBIA1300 with pea p68 gene (marker free construct). (DOCX 325 KB)


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Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  • Sivabalan Karthik
    • 1
  • Narendra Tuteja
    • 2
  • Andy Ganapathi
    • 1
  • Markandan Manickavasagam
    • 1
    Email author
  1. 1.Department of BiotechnologyBharathidasan UniversityTiruchirappalliIndia
  2. 2.Plant Molecular Biology GroupInternational Centre for Genetic Engineering and Biotechnology (ICGEB)New DelhiIndia

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