Collection of seeds and sterilization
Seeds of horse gram var. Paiyur 1 and Paiyur 2 were purchased from Regional Research Station, Paiyur, Tamil Nadu, India. Initially, the seeds were washed with Tween 20 solution for 5 min and rinsed using running tap water. Surface sterilization was done with 4% sodium hypochlorite for 5 min, followed by 70% ethanol washes for 2 min. Further, the seeds were properly washed thrice in sterile double-distilled water followed by surface sterilization with 0.1% HgCl2 (w/v) for 5 min. Finally, the seeds were thoroughly washed for five to six times in sterile water and germinated on Whatman filter papers.
Analysis of seed germination percentage
Seed germination refers to the initial appearance of radicle length, and nearly 2 mm was measured. To determine the germination percentage, healthy seeds were selected and placed in Petri plates with moistened Whatman filter papers in dark conditions at 25 ± 2 °C. Germination rate was counted every day (up to the 7th day). Germination percentage was calculated by using the following formula:
$$ \mathrm{Germination}\ percentage=\frac{\mathrm{number}\ of\ seeds\ germinated}{\mathrm{total}\ number\ of\ seeds}\times 100 $$
Gene, strain, binary vector, and Agrobacterium culture
For transformation studies, Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA2301 was used. The T-DNA region carries uidA or GUS–A reporter gene and was initiated by CaMV35S promoter and NOS terminator sequences, and the kanamycin-resistant gene, NPTII, was governed by the CaMV35S promoter and terminator sequences (Fig. 1). A loop of a colony from the mother culture was inoculated in a 50 mL of yeast extract mannitol (YEM) medium supplemented with kanamycin 50 mg/L and rifampicin 20 mg/L for 16–18 h at 28 °C with 180 rpm. The cells of 200 μL culture at log phase were taken and added into a fresh 100-mL medium containing the above antibiotics at 28 °C. Afterwards, the Agrobacterium culture with various cell densities of 0, 0.3, 0.6, and 0.8 OD was harvested by centrifugation at 8000 rpm for 10 min. The supernatants were discarded and the resulting pellet was dissolved in an infiltration medium comprising 3 mM MES, 1/2 strength MS medium supplemented with 3% sucrose, and 100 μM acetosyringone (final pH 5.4).
In planta transformation
The horse gram, var. Paiyur 2, was used to study the in planta transformation efficiency. Several factors were optimized such as Agrobacterium cell density, infection time, and sonication combined vacuum infiltration. Initially, seeds were surface-sterilized and germinated for 24 h in dark condition on sterilized moistened paper towels in Petri dishes at 25 °C. (Fig. 2a). Plumules of 1 cm in length devoid of seed coats were used for transformation. Agrobacterium cells were inoculated in the YEM liquid medium with respective antibiotics and cultured overnight at 28 °C with 180 rpm. Agrobacterium cells were harvested at different cell densities and resuspended in a 50-mL infiltration medium containing 100 μM acetosyringone.
The seeds were gently pricked two to three times using a sterile needle and immediately transferred into the Agrobacterium infiltration medium at different time (0, 10, 15, and 30 min) intervals followed by sonication for 0, 2.5, 5.0, and 7.5 min (temperature 27 °C; 8 s on, 2 s off; 17% power) by using an ultrasonic homogenizer (SKL–150 DN model, frequency 45 kHz). Further, sonicated seeds were placed in a desiccator and vacuum infiltrated for 0, 2.5, 5.0, and 7.5 min (300 mmHg) using a vacuum pump instrument (GAST DOA–P704–AA). Subsequently, seeds were blotted on a sterile filter paper for 15 min for the removal of excess Agrobacterium cells. The seeds were further transferred to Petri dishes for co-cultivation in the dark at 25 °C for 48 h. The co-cultivated seeds have been rinsed thrice in sterile double-distilled water supplemented with 250 mg/L of cefotaxime to remove Agrobacterium contamination. The seedlings were transferred to the pots containing autoclaved vermiculite soil for further germination and maturity (Fig. 2b–f) whereas the seeds without Agroinfection were used as control. The seeds were collected from agroinfected T0 plants and germinated on the half-strength MS medium supplemented with 100 mg/L kanamycin for the screening of putative transgenic plants. Subsequently, the T1 plants were used for the GUS histochemical staining assay and molecular confirmation.
GUS histochemical analysis for stable gene expression
This method was carried out to analyze the stable GUS gene expression in T1 germinated seedlings and leaf samples according to the described procedure [24]. Initially, the seedlings and leaf samples were washed in 50 mM sodium phosphate buffer (pH 7.0) and incubated in a GUS histochemical reagent (50 mM sodium phosphate buffer (pH 7.0), 0.5 M EDTA (pH 7.0), 100 mM K3[Fe(CN)6], 100 mM K4[Fe(CN)6], 1% Triton X–100) for 30–45 min at 37 °C. Afterwards, the samples were transferred into a X-gluc staining solution (2 mM X-gluc, 50 mM sodium phosphate buffer, 20% methanol) and placed in dark condition for 24–48 h at 37 °C. After incubation, the chlorophyll pigments were completely destained with 100% ethanol and the samples were observed for the GUS expression.
Isolation of plant genomic DNA and PCR analysis
Plant genomic DNA was isolated from leaf samples of selected T1 GUS-positive and wild-type control plants using a modified cetyltrimethylammonium bromide (CTAB) method [25]. Polymerase chain reaction (PCR) was executed to find the presence and integration of both GUS and NPTII gene fragments using genomic DNA isolated from T1 transgenic and non-transgenic (negative control) leaf samples along with plasmid pCAMBIA2301 control (positive control). The standard PCR procedure was followed using primers specific to the NPTII gene (FP: 5′-TCAGAAGAACTCGTCAAGAAGGCGATA-3′; RP: 5′-GGGGATTGAACAAGATGGATTGCACGC-3′) and GUS gene (FP: 5′-TTATGCGGGCAACGTCTGGTATCA-3′; RP: 5′-ACGCTTGGGTGGTTTTTGTCA-3′). The amplification of respective genes was performed in a PCR thermal cycler–200TM (MJ Research Inc., Waltham, Mass, USA). The PCR conditions were programmed with 94 °C for 5 min of initial denaturation, repeated for 32 cycles at 94 °C for 1 min of denaturation, 60 °C for 1 min of annealing, and 72 °C for 1 min of extension, followed by 72 °C for 10 min of final extension. Finally, the amplified PCR fragments were confirmed qualitatively by electrophoresis in 0.8% agarose gel and gel image captured using a Syngene gel documentation system (G-Box).
Total RNA isolation and semi-quantitative RT-PCR analysis
Total RNA was extracted from GUS and genomic PCR-positive transgenic and wild-type control plants by using spectrumTM plant total RNA isolation kit (Sigma-Aldrich, USA). Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was performed to study the stable gene expression of NPTII and GUS transcripts. One microgram of total RNA was reverse-transcribed to single-stranded cDNA using AffinityScript QPCR cDNA Synthesis Kit according to the manufacturer’s guidelines (Stratagene, USA). PCR cycling parameters were set as initial denaturation of 94 °C for 3 min, repeated by 30 cycles of 94 °C for 40 s, 60 °C for 40 s, and 72 °C for 60 s, and a final extension at 72 °C for 10 min. Amplified fragments were analyzed by 0.8% agarose gel electrophoresis and then visualized using Syngene gel documentation system (G-Box).
Statistical analysis
The data were analyzed using SPSS software (Version 17.0, SPSS Inc, USA). The experiments were performed thrice and subjected to one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) at P < 0.05 significance level.
