Engineering Chickpea Variety HC-1 with OsRuvB Gene for Salt Stress Tolerance

Various abiotic stresses like drought, salinity, flooding, heat and chilling accounts for more than 50% losses in crop yield worldwide (Rasool et al., 2013). Among various abiotic stresses, salt stress is a major threat to chickpea causing yield losses upto100%, depending upon the severity and developmental stage of plants (Mann et al., 2019). Since long conventional breeding has been widely used to develop stress-tolerant and high yielding crop plants but it is time-consuming, cost and labour intensive (Jangra et al., 2019). To overcome the barriers associated with traditional breeding, biotechnological approaches such as genetic engineering can be employed (Turan et al., 2012).
       
Recently, it has been found that various DNA helicases are involved in abiotic stress tolerance (Tuteja et al., 2012). So, transgenics has been employed to engineer crop plants with various DNA helicases to impart stress tolerance. Several helicases like PDH45, PDH47, STRS1, STRS2, MCM6 etc. are known to be activated during abiotic stress conditions (Pham et al., 2000; Vashisht et al., 2005; Sanan-Mishra et al., 2005; Kant et al., 2007; Tuteja et al., 2014) and helps in survival under stressed condition but the mechanism of stress tolerance is still unknown.
       
Chickpea (Cicer arietinum L.) is a nutrient-rich important legume crop and holds second position in total legume production after common bean (Shinde et al., 2019). Chickpea is a major legume crop of Haryana and its production and productivity are affected by the increased soil salinity. In order to address the salt stress problem associated with chickpea (var. HC-1) and to improve yield under stressed conditions, the present study was undertaken to engineer HC-1 (a popular chickpea variety of Haryana) variety of chickpea with OsRuvB gene for salt stress tolerance.

Transformation
 
Transgenic chickpea plants of HC-1 variety containing OsRuvB gene were developed using Agrobacterium-mediated transformation. Fresh bacterial cultures of Agrobacterium strain LBA4404 containing pCAMBIA1301 harboring OsRuvB gene (procured from Dr. Narender K. Tuteja, ICGEB, Delhi; Fig 1) were raised. Chickpea explants were transformed using the patented protocol of Kharb et al., (2012) (Indian Patent No.- 252590).
 


Screening of putative transgenic plants
 
PCR analysis
 
T₀ putative transgenic chickpea plants were screened for the presence of OsRuvB gene through PCR using gene-specific primers (Eurofins Genomics, India Pvt. Ltd.). Genomic DNA from the young leaves of T₀ and wild-type chickpea plants was isolated using CTAB method (Saghai-Maroof et al., 1984) and plasmid DNA from E. coli DH5α was isolated using mini-prep plasmid isolation kit (Qiagen) (Fig 2a). PCR reactions were carried out in 20 µl reaction mixture containing 50 ng DNA, 2 µl of 10X PCR buffer (G-Biosciences), 0.5 µl of 10 mM of each forward (5'-CATCTCTCAGGAGC TAGGTAGT-3') and reverse (5'-GATGTCTGTTGTCCGATC TCTC-3') primers, 0.5 µl of 10 mM dNTP (Thermo Scientific) and 2.5 U Taq DNA polymerase (G-Biosciences). PCR was performed in Benchtop thermocycler (G-Biosciences). The PCR was carried out at the following conditions: initial denaturation at 95°C for 10 min followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1.5 min, extension at 72°C for 1.5 min and finally a cycle of extension at 72°C for 10 min. The PCR products were resolved on 1.5% agarose gel and visualized on UV gel documentation system (Alpha Infotech).
 

 
Southern hybridization
 
The stable integration and copy number of transgene in transgenic chickpea plants were confirmed through Southern hybridization using Blot + nylon membrane, Biotin Deacalabel DNA Labelling Kit and Biotin Chromogenic Detection Kit (Thermo Fischer). Genomic DNA of T₀ generation plants carrying OsRuvB gene and plasmid DNA was subjected to EcoRI digestion and blotted on nylon membrane. The PCR amplified 957 bp fragments of OsRuvB were eluted from the gel using GET^™ Agarose DNA Kit (G Biosciences) and labeled with non-radioactive Biotin-labeled probe using Biotin Deacalabel DNA Labelling Kit (Thermo Fischer). The membrane was pre-hybridized with salmon sperm DNA at 42°C for 3 hours in hybridization chamber and hybridized with 957 bp biotin-labeled probe at 42°C for overnight. Biotin chromogenic detection kit (Thermo Fischer) was used to detect the hybridization signals.
 
Real-time PCR analysis
 
A simple quantitative real-time PCR (Applied Biosystems 7500) procedure was used to determine the copy number of transgene in transgenic chickpea plants using standard curve as described by Ahmad et al., (2005). Gene-specific primers were designed to amplify an amplicon of 100 bp. Amplification was carried out in a 20 μl (Thermo Fischer) reaction mixture containing Maxima SYBR Green real-time PCR Master Mix (2X) (10 μl), forward (5'-GAAGCTAATAGT GGTGCAGTGA-3') and reverse (5'-CTCCTTTGGG AAATC GGAACATA-3') primers (0.5 μl each) (Thermo Fischer), template DNA (2.0 μl) and nuclease-free water (7.0 μl) (Thermo Fischer). Thermal cycling conditions used were as follows: initial denaturation at 95°C for 3 min followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 52°C for 40 sec and extension at 72°C for 50 sec. Standard curve for detection of transgene copy number was generated using different known concentrations of pCAMBIA1301. The template concentration was determined using ND-1000 Nano-drop spectrophotometer. The ABI 7500 Detection Software was used for data analysis and determination of cycle threshold (Ct). At the end of the elongation step of each PCR cycle the fluorescence of SYBR Green I was monitored. After amplification, a melting curve was acquired by heating at 95°C for 20 min with data collection at 0.2°C intervals, using ABI7500 software.
 
Transgene copy number calculation
 
The fluorescence detected due to accumulation of PCR products was normalized relative to established baseline level (DRn) and used to determine the amplification plot and Ct values by plotting cycle number. The transgene copy number of unknown samples was determined by interpolation from standard curve Ct values generated using known amount of starting DNA concentrations. Since 0.95 pg DNA is present in single copy of chickpea genome, total genome copies (Y) in template DNA (100 ng) used is 1.05 × 10⁵. Therefore, the transgene copy number corresponding to each Ct value of transgenic event (Z) was calculated as the ratio of amount calculated using standard curve (X) to the total amount of target DNA (Y) used in the real-time PCR (Z=X/Y).
 
Direct plant PCR analysis
 
PCR-based screening of T₁ chickpea plants for the presence of OsRuvB gene was carried out by subjecting small leaf disc to direct PCR (Phire Direct PCR kit) following above mentioned thermal cycle conditions.

Screening of putative transgenics
 
Putative transformants were tested for integration of OsRuvB gene in T₀ generation using PCR with gene-specific primers. An amplified fragment of 957 bp confirmed the presence of the transgene in the putative transgenic plants and no amplification was observed in the wild type plants. Plasmid DNA carrying OsRuvB gene was used as positive control and an amplicon of 957bp was observed (Fig 2b). PCR based screening of putative transgenics has been widely used by various researchers to confirm the incorporation of the transgene in legumes like cowpea (Mishra et al., 2014), peanut (Singh et al., 2014), pigeonpea (Jain et al., 2017) and chickpea (Mubina et al., 2018).
       
Wild type plants served as negative control and plasmid DNA served as positive control for screening of transgenics. Out of 100 plants screened, 17 plants showed a clear and sharp band of 957 bp, representing a transformation efficiency of 17% (Fig 3). Similarly, transformation frequencies of 13.4% and 41% were obtained by Khatodia et al., (2014) in the development of Bt chickpea cv. C235 and var. HC-1, respectively.
 

 
Southern hybridization analysis
 
Southern hybridization of transformed chickpea plants revealed single-copy integration of OsRuvB gene into transgenic chickpea lines studied (Line 4, 8, 11, 12, 14) (Fig 4A), Lanes 1-5. Similar results for Southern hybridization have been shown for Vigna mungo (Bhomkar et al., 2008), Cicer arietinum (Ghanti et al., 2011; Khatodia et al., 2014), Vicia faba (Hanafy et al., 2013) and Cajanus cajan (Jain et al., 2017) revealed stable integration of transgene into transgenic plant genome whereas no signal was recorded in non-transformed control (WT) plants.
 

 
Real-time PCR analysis
 
Standard curve, which was generated using real-time PCR represented Ct values against log10 (plasmid copy number). A linear curve was observed with correlation coefficient (R²) of 0.927 (Fig 4B). Using standard curve amount of DNA in transgenic chickpea was obtained through regression equation. A single peak for each sample in the dissociation curve was obtained represents amplification of specific product. In the present study, qPCR results showed single-copy insertion of transgene into transgenic chickpea genome and non-significant values were recorded in wild type plants. Single copy insertion was obtained in transgenic lines (line 4, 8, 11, 12 and 14) whereas no insertion was recorded in non-transgenic chickpea plants (Fig 4C).
 
The real-time PCR copy number results were compared with the results obtained with Southern hybridization analysis. It was found that real-time PCR analysis was significantly correlated with Southern blot analysis and both showed single copy insertion in respective transgenic chickpea plants. Similarly, single copy insertion was observed in transgenic chickpea (Khatodia et al., 2014) and pigeonpea (Jain et al., 2017).
 
Screening of T₁ transgenic chickpea plants carrying OsRuvB gene
 
T₁ generation plants (10 plants from each T₀ line) were screened using direct PCR kit (Phire plant direct PCR kit) using gene-specific primers. PCR analysis showed the amplification of 957 bp fragment (Fig 5a, 5b and 5c) in the transgenic plants.
 

 

 

 
Hypothetical mechanism exhibited by transgenic chickpea plants for salt stress tolerance
 
Helicases are known as transcriptional activators or as molecular motors that are involved in several cellular processes at transcriptional and translational level including nucleic acid metabolism and has emerged as potential molecules to engineer abiotic stress tolerance in plants (Sarwat and Tuteja, 2018). There are many reports which support the role of DNA helicases in counteracting the adverse effects of abiotic stresses (Kant et al., 2007). The exact mechanism of helicase action during stress has not been understood yet, but possible mechanism of helicase  mediated salt stress tolerance can be elucidated from some studies. There could be two possible sites of action for the helicases: (a) at the level of transcription and translation to enhance or stabilize protein synthesis or (b) in an association with DNA subunit protein complexes to alter gene expression. It is evident that mRNA and protein synthesis are very sensitive to stress, so factors involved in transcription and translation are potential targets of salt toxicity in plants. In response to stress, the extra secondary structures could be formed in the 5’ UTR region in mRNA of genes, which could be inhibitory for translation. In order to proceed with protein synthesis, these inhibitory secondary structures need to be resolved. Stress-induced DNA helicase may resolve these inhibitory structures, not only remove these structures but also protect mRNA from degradation. Overall, these stress-induced helicases help in recovering the functions of the genes which have role in stress adaptations and overexpression of DNA helicases can provide the exploitation of DNA/RNA metabolic pathways for engineering crops. This is suggested that helicases found to work in conjunction with other stress-induced transcriptional factors. This hypothesis seems to support the role of DNA helicases in salt stress tolerance mechanism in plants.

In the present study, transgenic chickpea plants (var. HC-1) carrying OsRuvB gene for salt stress tolerance have been developed. The transgenic plants with stable and single-copy insertion and can be further undertaken for physiological studies at controlled as well as at field level to develop transgenic chickpea plants with improved salt stress tolerance.
       
The study also shows the utility of SBYR green in real-time PCR to determine the transgene copy number in transgenic chickpea plants. The transgene copy number detection method described in the present study is simple, efficient and cost-effective and detection can be done at early growth stages. This SYBR-based assay is highly accurate, sensitive and uses gene specific primers to determine the copy number in a short time period and can be used as an alternative to Southern hybridization.

The authors are highly thankful to Dr. Narendra K. Tuteja for providing the vector construct and to Dr. Meenakshi Prasad for providing the real-time PCR facility.

The authors declare no conflict of interest.