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Constitutive Overexpression of the Plasma Membrane Na+/h+ Antiporter for Conferring Salinity Tolerance in Rice

Farida Yasmin
Plant Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh

Sudip Biswas
Plant Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh

Sabrina M. Elias
Plant Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh

Zeba I. Seraj*
Plant Biotechnology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

At the cellular level, the Salt Overly Sensitive (SOS) signaling pathway comprising SOS3, SOS2, and SOS1 has been proposed to mediate cellular signaling under salt stress to maintain ion (Na+) homeostasis. In this regulatory pathway, both OsSOS1 encoding plasma membrane and OsNHX1 encoding vacuolar Na+/H+ antiporters are regulated by SOS3-SOS2 protein kinase complex. In the present study, the rice variety BRRI dhan28 which is popular with farmers and high yielding, but salt sensitive, was transformed with the OsSOS1 gene isolated from salt tolerant Pokkali rice and driven by the constitutive promoter, CaMV35S. The construct was transformed through a tissue culture independent Agrobacterium mediated in planta transformation method that circumvents the problems associated with tissue culture based indica rice transformation methods. Integration of the foreign genes (OsSOS1) into the genome of transgenic plants was confirmed by gene-specific PCR and Southern blot analysis. The level of transgene expression (SOS1) was also quantified by semi-quantitative RT PCR and real time PCR. Genetic segregation ratio for T₁ progenies was calculated and found to follow the Mendelian law of inheritance in case of positive transformants. The transformants were shown to be salt tolerant compared to wild type in molecular analysis as well as physiological screening. Future work will involve transformation of both the OsSOS1 and OsNHX1 genes together; with the expectation for enhancing the tolerance level compared to currently available transgenic rice.

Keywords: In planta transformation, Transformation efficiency, Salinity tolerance, Salt overly sensitive 1 (SOS1) gene

Location:

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Rice

Objectives:

In the current study, a tissue culture independent transformation method (in planta) was applied to transform farmer popular high yielding but salt-sensitive BRRI dhan28 rice in order to enable higher transformation efficiency.

Methodology:

The OsSOS1 gene in the clone used for transformation was obtained from the rice landrace Pokkali as described in detail in Razzaque et al. 2014. Briefly, the complete cDNA from Pokkali RNA using gene specific primers was prepared using the RT superscript kit from Invitrogen and cloned into the directional pENTR vector. The direction was ensured by adding CACC in the 5 position to the forward primer. Positive clones confirmed by sequencing were then inserted into the destination vector, pH7WG2 by LR recombinase following the manufacturer’s instructions (In Vitrogen). Transformed Agrobacterium strain (LBA4404) containing pH7WG2.0_CaMV OsSOS1 constructs was cultured and prepared for transformation following the standard protocol described in Lin et al. (2009) with some modifications. Densely grown bacterial cells were first inoculated into liquid YM (Yeast extract mannitol broth) (Boumahdi et al. 2001) medium and incubated at 28°C overnight, then centrifuged and re-suspended into the liquid AB medium (sucrose, glucose, AB buffer and AB salt) (Hiei et al. 2008). For improving the transformation efficiency, acetosyringone was added to both liquid YM medium and bacterial re-suspension medium (Parvin et al. 2015). Agrobacterium was selected using streptomycin (20 mg/l) and spectinomycin (20 mg/l). Finally, bacterial density was measured at OD600 and held at 0.6 (Lin et al. 2009, Parvin, et al. 2015). Following the protocols described by (Amin et al. 2012), mature seeds of BRRI dhan28 were sterilized. For Agrobacterium inoculation, two-day-old germinated seeds were used. During transformation, the embryonic apical meristem of approximately 40 germinated seeds of BR28 was infected with Agrobacterium containing the pH7WG2.0_CaMV-OsSOS1 construct as described in detail in Lin et al. (2009). Briefly, two-day-old germinating seedlings were inoculated with Agrobacterium solution near the plumule with a 0.5 mm injection needle using a 450 angle for piercing. These seeds were then vacuum infiltrated while immersed in the Agrobacterium inoculum solution. Infected seeds were transferred onto Petri dishes containing wet filter paper and incubated in the dark at 28°C for 6 -7 days. Then, the seedlings were treated with carbenicillin solution (250 mg/l) for 1 h to kill and remove the remnants of Agrobacterium. After that, seedlings were washed well with ddH₂O and transferred to new Petri dishes containing wet filter paper. Seedlings were then kept in light for 16 hrs and in dark for 8 hrs at 28°C. Seedlings were subsequently transferred to hydroponic solution (Yoshida et al. 1976) when they appeared green and healthy. After 2 - 3 days, the hydroponic pots were transferred to the net house (confined area with cemented floor for growing the rice plants in pots, completely covered in netted mesh, to keep out animals and insects. There is no other rice growing near the vicinity). Mature seedlings of the T₀ line (18 - 21-days-old) were transferred to soil and allowed to pollinate naturally to set seeds (T₁) (Parvin, et al. 2015). Plants that showed positive results in both leaf disk senescence (LDS) assay and PCR analysis were chosen for generation advancement to T₁. T₁ seeds were sown from 4 panicles whose flag leaves showed good score after senescence tests (P-1-1, P-1-2, P-1-3 and P-2-3). When the plants were mature, leaf disk senescence (LDS) assays were performed. The flag leaf was taken from T₀ transgenic lines and the 2nd leaf was taken for T₁ transgenic lines for this assay because these are the likely ones transformed according to the original protocol (Lin et al. 2009). Leaf squares of 1.0 × 1.0 cm were excised from healthy and fully expanded rice leaves of similar age both from the transgenic lines and wild type plants. The disks were floated in a 20 ml solution of NaCl (150 mM or 15 dS/m) for T₀ transgenic lines and 0, 100, 200 mM NaCl (10 and 20 dS/m) for T₁ (the lower concentrations for T₀ transgenic lines was because the transgene is hemizygous) for 3 days and then scored for damage. The treatment was carried out at 25°C. The analysis was carried out with three biological replicates in three independent experiments.

Source of Information: Plant Tissue Cult. & Biotech. 25(2): 257-272, 2015 (December)

Article Link (Online Journal): http://www.banglajol.info/index.php/PTCB/article/view/26259/17640

Funding Source:

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Achievement/Findings:

Significant results from this study can be applied to the development of high yielding salt tolerant rice varieties. BRRI has already invented moderately salt tolerant rice varieties (BRRI dhan40, BRRI dhan41, BRRI dhan47, BRRI dhan53, BRRI dhan54, BRRI dhan55 and BRRI dhan61), which can withstand moderate level of salinity stress (60 to 140 mM) at seedling stage (Rice knowledge bank, Bangladesh Rice Research Institute). But the present study showed an improved salinity tolerance level of 150 to 200 mM in the high yielding but salt sensitive BRRI dhan28. In the future, comparative studies could be done at reproductive stage to further analyze the effect of OsSOS1 gene overexpression with respect to constitutive and inducible promoters. Simultaneous overexpression of the plasma membrane and vacuolar Na+/H+ antiporter encoding genes might have a cumulative and hence more enhanced effect compared to currently cultivated lines.

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