Agrobacterium tumefaciens-Mediated Genetic Transformation of Green Gram [Vigna radiata (L.) Wilczek] - A Recalcitrant Grain Legume

Green gram [Vigna radiata (L.) Wilczek] is an important edible grain legume grown for its protein-rich edible seeds in many tropical and sub tropical countries. It is an important nitrogen fixing crop and has a short life cycle therefore is widely grown as mixed, inter crop or in rotation to improve nitrogen status of soil. It is grown mainly in arid and semi-arid situations across the country during Kharif and Rabi season and contributes nearly 15% of total production (Patra et al., 2018).

The reasons for low productivity in green gram are its susceptibility to biotic and abiotic stresses (Jaiwal and Gulati, 1995). Conventional breeding has limited scope due to less variability in germplasm. Genetic transformation through Agrobacterium and regeneration of transgenic plants has been reported in black gram (Das et al., 2016), green gram (Mekala et al., 2016).

In the past few years, some progress has been made regarding the in vitro regeneration of green gram (Mahalakshmi et al., 2006), but very little success has been reported regarding genetic transformation of green gram through Agrobacterium (Hoque et al., 2007). Though the genetic transformation of grain legumes have been difficult and challenging till now, but significant progress has been made in the recovery of transformed plants via Agrobacterium in soybean, pea, chickpea, pigeon pea (Bhatnagar-Mathur and Sharma 2016). The members of genus Vigna such as V. mungo and V. radiata have been shown to be susceptible to Agrobacterium tumefaciens and only transgenic callus lines have been generated. The present study describes the establishment of efficient A. tumefaciens based transformation protocol for production of transgenic of V. radiata plant.

Seeds of green gram [Vigna radiata (L.) Wilczek] cv. IPM-02-03 were procured from Regional Research and Technology Transfer Station, Odisha University of Agriculture and Technology (OUAT), Berhampur, Ganjam, Odisha were used in the present study. The experiment was conducted in Dept of Agril. Biotechnology, OUAT, Bhubaneswar in 2018.

Explant preparation

Seeds of Vigna radiata L. were washed thoroughly with water for 10-20 min and then treated with a solution of the tween 20 (5% v/v) for 10 min and surface sterilized with HgCl₂ (0.1% w/v) for 5 min. The seeds were rinsed thrice with autoclaved distilled water to remove any trace of contaminants. Seeds were sown in MS medium (Murashige and Skoog, 1962) supplemented with BAP 2 mg/l (8.5 μM). After germination the cotyledonary node and shoot tip explants were excised from 4 day old seedling under asceptic condition as described by Patra et al., (2018).

Culture media and conditions
 
Modified MS media used in this investigation were supplemented with 3% sucrose and adjusted to pH 5.8 before autoclaving at 15 lb pressure at 121°C for 15 min. To prepare semisolid media, 6 g/l agar (Himedia, India) was added before autoclaving. All the cultures were maintained under continuous white light (fluorescence density of 60 μmol/m²/s) at 25°C±2°C.
 
Binary vector and agrobacterium
 
The disarmed Agrobacterium tumefaciens strain EHA105 harboring a binary vector pCXSN, which contains TIP1 gene and a hygromycin phosphotransferase gene (hptII) both driven by CaMV35S promoter, was used for transformation studies (Fig 1).
 
Transformation and plant regeneration
 
Agrobacterium tumefaciens strain EHA105 harbouring plasmid vector pCXSN was cultured on 20 ml of YEM medium containing 50 mg/l kanamycin and 50 mg/l rifampicin and kept in an incubator for overnight at 28°C on a rotary shaker at 120 rpm. Bacteria were pelleted at 5000 rpm for 10 min and resuspended in liquid shoot regeneration medium (SR) containing MS salts, B5 vitamins, 3% sucrose and 2 mg/l BAP. The density of bacterial suspension was checked at 600 nm and dilutions were made for different concentrations of bacterial cells. Optical density 0.6-0.8 was found to be optimum for transformation. The cotyledonary node explants excised from 2 days old germinated seedling were inoculated for 10-60 min with different concentrations of  bacterial cells (10⁶-10⁹ cells/ml) and co-cultured on liquid MS (Shoot Regeneration) medium for 1-4 d under 16-h photoperiod (cool white fluorescent tubes, irradiance of 80 μmol/m²/s) and temperature of 25±2°C. To increase the transformation efficiency, acetosyringone (100 μM) was added to inoculation medium and efficiency was evaluated. The effect of pre-culture of explants, on SR medium for 0-3 d and mechanical injury, both the factors either alone or in combination prior to inoculation with bacterium, was also investigated. After co-cultivation, the explants were washed 3-4 times with liquid SR medium containing cefotaxim 250 mg/l to check the bacterial growth for further shoot development.  For each treatment, 40 explants were used and each experiment was repeated thrice. Hygromycin sensitivity test was carried out to find out the minimum concentration of hygromycin required to inhibit the growth of untransformed explants in order to design the selection medium. The test was carried out by culturing the explants on regeneration medium along with different levels of hygromycin concentration (0, 5.0, 10.0, 15.0, 20.0 and 25.0 mg/l). Putative transformants and percent of putative transformants were calculated on the basis of hygromycin selection.
 
DNA extraction
 
The total genomic DNA was extracted from young fresh leaves of the putative transformants (T₀) and non-transformed (control) plants by CTAB method (Doyle and Doyle, 1990).
 
Polymerase chain reaction analysis
 
Putative transformants (T0) were screened by the polymerase chain reaction (PCR) for the presence of the TIP1 gene. The genomic DNA of plants obtained from co-cultivated explants, control plants and plasmid DNA (positive control) were used as template for PCR confirmation of the targeted transgene with primers TIP1 gene.
Nucleotide sequence of specific primer TIP1 gene is as follows:
Forward- 5'-GATTGA AGA TGC CGA TCA GAA ACA TCG CCA-3'
Reverse- 5'-CTG GGT CTA GTAGTCAGT GGT TGG GAG CTG-3'
 
PCR was carried out with Taq DNA polymerase following modified PCR protocol. To ensure that reagents were not contaminated, DNA from non-transformed (control) plants was included in the experiments. The amplified products were separated by electrophoresis on a 1% agarose gel and visualized with ethidium bromide in Gel Documentation Unit.

Cotyledonary node explants excised from four days old in-vitro raised seedlings were tested on MS medium and transformed by infecting with Agrobacterium tumefaciens strain LBA4404 (Fig 2A) containing the binary vector pCXSN-TIP1 (Fig 1). The multiple shoots (Fig 2B and Fig 2C), developed from cotyledonary node were transferred to fresh MS semi-solid culture with 50 mg/l kanamycin after 4 -6 weeks under standard cultural conditions. In the present study, transformation frequency was highest when co-cultivation was performed at 24°C. These shoot buds were transferred into MS basal medium supplemented with 0.5 mg/l NAA ad developed roots in 4 weeks from (Fig 2D) basal part of shoots. These roots elongated (10-15 cm) and became sturdy and branched (Fig 2E) in 4 weeks and developed plantlets. Significant survivability was found from the explants co-cultivated for 72 hours 81.45%. Survivability percentage was reduced to 61.92% in co-cultivation period of 96 hours. Hence 72 hours found to be suitable for co-cultivation period.





Selection of transformants

Kanamycin and hygromycin have been successfully employed as a selectable marker in transformation of various legume crops. It is compulsory to determine the highest concentration of the selective agent that suppresses growth and proliferation of untransformed cells. Hygromycin at 25 mg/l in shoot regeneration medium drastically reduced the survival as well as the regeneration frequency of the explants and completely bleached the non transformed shoots. Hygromycin concentrations higher than 25 mg/l were lethal causing necrosis of explants and complete inhibition of regeneration. Therefore, 25 mg/l Hygromycin was chosen for the selection of the transformed shoots in the transformation experiments. Root induction was completely inhibited in non-transformed (control) shoots cultured on MS medium containing 0.5 mg/l IAA medium containing 25.0 mg/l hygromycin.

The stable transformed shoot buds were selected by periodic increase in hygromycin concentration. Initially after 20 days on medium containing 5.0 mg/l hygromycin, a high number of shoot buds were obtained which later developed roots and develop plantlets (Fig 2F). When transferred to medium containing higher concentrations of the antibiotic (15.0, 20.0, 25.0 mg/l), many of the shoots became yellowish, and were discarded after 45 days. Out of the developing hygromycin resistant transgenic plants, about 20% showed normal shoot and root development compared to a conversion frequency of 67% in the control plantlets. The putative transgenic plants had no phenotypic abnormalities in comparison to the untransformed control plants (Fig 2G). The results of the current work showed that the use of cotyledonary node explants is more efficient compared for genetic transformation of green gram (Vigna radiata L.) varieties.

Optimization of transformation protocol

Optimization of transformation protocol is required for efficient transformation by A. tumefaciens. Bacterial concentration (cells/ml), inoculation time, co-cultivation period, addition of acetosyringone, temperature and pH in co-cultivation medium are significant factors affecting competence of tissue(s) and Agrobacterium virulence for achieving the maximum transformation frequencies. These factors were optimized to improve the transformation efficiency on the basis of transient TIP1 expression using cotyledonary node explants (Table 1).



Molecular analysis of putative transformants

Putative transformants were screened by the PCR in presence of hptII genes. For this purpose the total genomic DNA was isolated from the leaf tissue of transformants survived on the selection medium (Hygromycin B 25 mg/l) as well as control. Then the PCR reaction was carried out using hptII gene specific primer and plasmid DNA used as a positive control.

The PCR amplified product was electrophoresed on 1.2% agarose gel. Expected band of about 752 bp was obtained only in six samples out of 17 samples tested. The transformation efficiency found to be about 4.41% (Table 3 and Fig 4). Six T0 plants produced 44 seeds which on germination produced T1 plants were tested for presence of the TIP1 gene.






From the PCR analysis, 28 numbers of plants were found to be positive for presence of TIP1 transgene (Fig 3, 4 and Table 3).

The expression of TIP1 gene through Agrobacterium-mediated transformation and tolerant to moisture and salinity stress was detected in Arabidopsis (Li et al., 2015). The natural ability of A. tumefaciens to introduce DNA into plant cells is being widely exploited for the genetic transformation of plants (VanWordragen and Dons, 1992). Therefore, this system is considered to be suitable for the transformation of green gram. Transformation and successful regeneration procedures were based on a direct shoot organogenesis protocol previously described by Eissa et al., (2002).





The sensitivity of plant cells to the selective agent depends upon the genotype, the explant type, size and developmental stage of tissue(s). Hence it is compulsory to determine the lowest concentration of the selective agent that suppresses growth and proliferation of untransformed cells. The survival, regeneration frequency and the average number of shoots per explant decreased with increasing hygromycin concentration.

The maximum transformation frequency was observed at a concentration of 107 cells/ml with a constant increase up to 107 cells/ml and decrease their off, as reported in tobacco, Arabidopsis thaliana (Lin et al., 1994) and other grain legumes (Bean et al., 1997).

Co cultivation duration directly affect the transformation efficiency where as longer incubation with Agrobacterium, led to bacterial leaching. Longer period of co-cultivation resulted in negative effects due to the overgrowth of the bacterium in V. radiata (Sonia et al., 2007). Higher transformation efficiency could be achieved by 2-3 days of incubation period. Transformation efficiency can further be enhanced by adding acetosyringone at various concentrations to the co-cultivation medium (Table 1). Acetosyringone was applied to improve the Agrobacterium infection which is a phenolic compound produced by the wounding of plant tissues that induces the transfer of T-DNA from Agrobacterium to the plant genome (Lee et al., 1995). This might be due to vir gene activation which facilitated the transgene integration via wound of the explant under the influence of acetosyringone. In this present investigation, addition of 100 μM acetosyringone to freshly excised cotyledonary nodes tissues could increase the transformation efficiency (Table 1). The increase in the transformation efficiency of freshly cut tissues in the presence of acetosyringone suggests that the synthesis of vir inducers during incubation and the accumulation of vir inducing compounds in the wounded and pre incubated plant tissues are an important factors contributing to the increased competence of the incubated common bean cotyledonary node tissues to Agrobacterium mediated transformation. Stimulation of plant cell division and activation of the DNA replication machinery during the incubation period may play an important role in the integration of plasmid DNA leading to stable transformation.

Droste et al., (2000) used 100 µM of acetosyringone to increase the virulence of Agrobacterium tumefaciens in Glycine max. Similarly, acetosyringone has been used to achieve higher transformation efficiency in several Vigna species, such as V. radiata (Sonia et al., 2007) and V. mungo (Sainger et al., 2015). Higher transformation frequency has been reported at low pH of bacterial inoculation and co-culture media (Solleti et al., 2008). Temperature plays an important role in co cultivation process affecting T-DNA transfer. Co-cultivation at low temperature has been reported in several instances (Popelka et al., 2006). It has also been found that the temperature dependence profile of T-DNA transfer does not parallel with that of vir gene induction, indicating that another factor possibly the formation of a conjugal pilus is causing temperature sensitivity of T-DNA transfer (Fullner et al., 1996).

In the present study, the hptII gene coding was tested by selection of hygromycin stable transgenic shoots. The transgenic character of hygromycin resistant shoots was confirmed by detection through PCR in resistant tissues. Thus, these studies provide strong confirmation for a hygromycin resistant gene transferred to plant cells. Similar results were achieved by Jaiwal et al., (2001) who regenerated transformed shoots directly from cotyledonary node explants of mungbean cultured on medium containing 75 mg/l kanamycin after co-cultivation with LBA4404 (pTOK233).

Transgenics technology supplements the breeding programme for genetic improvement of crop plants where conventional breeding cannot be achieved due to sexual barrier and limited availability of genes within the germplasm.

The authors wish to acknowledge to the Department of Biotechnology, Govt of India for providing financial assistance for the present investigation. The authors also acknowledge Vice Chancellor, Orissa University of Agriculture and Technology, Bhubaneswar for proving necessary infrastructure and laboratory facilities for carry out the research work. The authors also acknowledge Dr. S.K. Panda, Professor and Head, Department of Biochemistry, Central University of Rajasthan for providing gene construct for the experiment.

The authors declare that there is no conflict of interest in the present investigation.