Regeneration of chickpeas under in vitro conditions has previously been optimized (Sarmah et al., 2004). However, a further improvement in transformation efficiency and regeneration of stable transgenic chickpeas has proved a challenge. Half embryos (one cotyledon plus axis) were found to be the most promising explants to produce stable transgenic chickpeas (Sarmah et al., 2004). The current study aims to further increase the transformation efficiency by reducing chimerism, which is a major bottleneck for gene transmission to subsequent generations. To achieve this, we modified several important steps of the existing protocol. These included the concentration of kanamycin, type of light, a method of explants preparation including co-cultivation with Agrobacterium, duration of subculturing, acclimatization of T₀ plantlets and early PCR screening. These changes are outlined in the protocol flow diagram (Figure 1).

To demonstrate the robustness of the protocol and stability of gene transmission in subsequent generations, chickpeas were transformed with five different gene constructs based on the binary vector pOPT-EBX (Figure 2). The binary vector pOPT-EBX was modified from pCAMBIA 2300 in Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Queensland, Australia. These modifications included replacing the NOS promoter with the S1 promoter from subterranean clover stunt virus DNA segment (Schünmann et al., 2003) to drive the neomycin phosphotransferase II gene (NPTII) and inserting the CaMV35S promoter between Stu I and Sma I sites to drive genes of interest. Details of all the binary vectors used here and their transformation into Agrobacterium tumefaciens strain AGL1 were described previously (Tan et al., 2018). The genes in the different T-DNAs are shown schematically in Figure 2. The GUS (uidA) gene is a reporter gene that encodes the beta-glucuronidase enzyme. The BAG (Bcl-2 associated athanogene) functions as adapter proteins forming complexes with signaling molecules and molecular chaperones and is involved in programmed cell death pathways. The BAG genes in this study have been isolated from the model plant, Arabidopsis thaliana (AtBAG4) and the resurrection grass Tripogon loliiformis (TlBAG). The NAS genes were isolated from chickpea (CaNas2) and rice (OsNas2) where they catalyze the biosynthesis of nicotianamine (NA) and are involved in Fe uptake and translocation in plants. The ferritin gene was isolated from soybean (GmFerritin) and is an iron storage protein that allows for safe sequestration of iron in a soluble and bioavailable form.

The chickpea cv HatTrick was used for transformation because it is a widely cultivated modern variety in Australia. Approximately 200g of chickpea seeds was rinsed with sterile MilliQ (SMQ) water in 500 mL tissue culture jar followed by disinfecting in 70% (v/v) ethanol for 2 min shaking by hand. The ethanol was replaced with a freshly prepared 1.5% (v/v) sodium hypochlorite solution (diluted with SMQ) and agitated for 7–8 min by hand. Following sterilization, the sodium hypochlorite was decanted, and the seeds were washed 5–7 times with SMQ water. Damaged and discolored seeds were removed, and the remaining seeds were imbibed overnight in SMQ water at ambient room temperature and light conditions (Figure 3A).

A starter Agrobacterium culture was prepared by inoculating approximately 5 mL of Luria-Bertani (LB) medium (Supplementary Table 1) containing 25 mg/L rifampicin and 50 mg/L kanamycin with 100 μL of AGL1 Agrobacterium glycerol stock harboring the gene(s) of interest. This culture was shaken (200 rpm) overnight at 28°C. To prepare the final working culture, 2.5 mL of the starter culture was used to inoculate 500 mL of LB media containing 50 mg/L kanamycin. The final culture was shaken (200 rpm) overnight at 28°C until an absorbance (at 600 nm) of 1.0–1.2 was reached. One hour prior to transformation, 0.5 mL acetosyringone (100 mM) was added to the culture.

Approximately 1 mm was trimmed off the ‘beak’ of the overnight-imbibed chickpea seeds (Figure 3C). The seed coats were removed, and the resulting embryos were bisected along the longitudinal axis with a scalpel to yield two explants each containing half the axis attached to one cotyledon (Figure 3B,C). These explants were used in different co-cultivation conditions (Figure 1). Explants were either uninjured or injured (micro-injury was delivered with a needle five times along the axis as shown in Figure 3C and inset) and incubated in Agrobacterium culture at 22°C under 70 μmol s⁻¹ m⁻² of red LED (Valoya L30 spectrum AP67 Conviron Finland) or fluorescent (OSRAM L18W/77 G13 Fluora Germany) lights for 1 h. Four sets of experiments were performed as outlined in Figure 1. To minimize experimental error due to unequally cut half embryos (Supplementary Figure 1B) only regenerable half-embryo explants obtained after 5 days of co-cultivation were counted as the initial explants (250 explants/experiment, repeated thrice for statistical calculations) (Supplementary Figure 1A).

After 1 h incubation, the bacterial cultures were drained, and the explants were dried on sterile Whatman filter paper. Explants were placed, cut-side-down, on filter paper over Gamborg’s B5 (Gamborg et al., 1976) medium supplemented with 1 mg/L 6-benzylaminopurine (BAP) and 1 mg/L naphthalene acetic acid (NAA) along with 1 ml of acetosyringone (100 mM) (Supplementary Table 1) in a Petri plate and co-cultivated for 5 days at 22 ± 1°C with 70 μmol s⁻¹ m⁻² LED or fluorescent light in a growth room with a 16 h light/ 8 h dark cycle.

After co-cultivation the explants were transferred to regeneration and selection (RS1) basal MS (Murashige and Skoog, 1962) medium supplemented with 3% sucrose, 10 mM MES,) for shoot 0.5 mg/L BAP, 0.5 mg/L kinetin, 0.05 mg/L NAA, 100 mg/L kanamycin, 25 mg/L meropenem (a member of the family of carbapenems which inhibits the growth of Agrobacterium by interfering with the synthesis of the bacterial cell wall) (Ranbaxy Australia Pty Ltd induction (Supplementary Table 1). The explants were incubated under LED or fluorescent light at 22 ± 1°C, and a 16 h light/ 8 h dark cycle for 19–20 days. Roots and any dead or dying tissues were removed and the surviving shoot clumps were sub-cultured onto fresh regeneration and selection (RS2) medium supplemented with 0.5 mg/L BAP, 0.5 mg/L Kinetin, 100 mg/L kanamycin and 25 mg/L meropenem (Supplementary Table 1). The shoot clumps were sub cultured for one more round for 19–20 days on RS2. At the completion of a third round of selection, clumps of secondary shoots (Figure 3E) that emerged from the base of the explants were ready for PCR testing and grafting. Small shoots from the PCR-positive clump that was not large enough for grafting were further elongated by subculturing on RS3 medium (Figure 3F and Supplementary Table 1).

Histochemical GUS staining was performed according to Jefferson (Jefferson et al., 1987) on 5-day old explants immediately after co-cultivation. Similarly, putative transgenic shoots at the third round of regeneration and selection were stained for GUS expression. The shoots that stained positively for GUS across all tissues were considered to be non-chimeric. In contrast, shoots with blue and non-blue sections were considered to be chimeric. GUS expression in vegetative parts of 2-week-old seedlings of the T₂ progeny of stable transformants was assayed by dipping seedlings in GUS stain for 24 h at 37°C. Non-transgenic seedlings were stained as controls. Chlorophyll was removed from stained tissues by incubation in 70% ethanol for 24 h.

A single in vitro shoot from putative transgenic clumps was harvested and DNA was isolated as described (Thomson and Henry, 1995). PCR was done using GoTaq (Promega) and GUS (uidA) gene-specific primers (Fw TGAACATGGC ATCGTGGTGA and Rv GCTAACGTAT CCACGCCGTA). The resulting products were separated via electrophoresis in a 0.8% agarose gel and made visible by staining with SYBR^® Safe (Life Technologies).

In vitro shoots from putative transgenic shoot clumps obtained after the third round of selection were grafted onto non-transgenic rootstocks under in vitro conditions as described earlier (Sarmah et al., 2004) except that the rootstock culture medium was replaced with ½ MS without sucrose. The grafted plantlets were incubated at 22 ± 1°C, with 16 h light/8 h dark cycle, for 2 weeks with frequent removal of side shoots until the graft had healed.

Grafted plantlets were removed from the medium and the roots were washed to remove all attached agar. The seed coat was removed to reduce the risk of fungal contamination. The plantlets were transferred to 150 mm diameter pots half-filled with autoclaved mixture (potting mix: perlite, 3:1) and the roots were covered with additional autoclaved mixture. The mixture was gently compressed around the roots and the plantlets were irrigated with 25 mL of tap water. During the transfer, care was taken to keep the plantlets upright and that the graft was above the mixture. The plantlets were covered with a transparent sterilized 500 mL plastic jar and placed in a growth cabinet (CONVIRON model Adaptis A1000 Finland) set at 22 ± 1°C, with 90 μmol s⁻¹ m⁻² LED light for 16 h light/8 h dark cycle. The transparent plastic jar was wiped free of condensation daily and the pots were watered every 3 days. After 10 days or when the plants were nearly touching the walls of the jar, the jar was left partially open for 8–9 days for further acclimation, and then completely removed. The plants were transplanted to 400 mm × 250 mm pot for completion of the life cycle.

Seeds (T₁ generation) were harvested from T₀ plants and sown in a commercial potting mix (Searles Australia) in 100 mm tube stocks inside a growth cabinet with 90 μmol s⁻¹ m⁻² LED light for 16 h light/8 h dark cycles. The potting mix was kept moist, but not saturated. After emergence, leaf samples were collected from fully expanded leaves for PCR screening. PCR-positive progeny was transplanted to 400 mm × 250 mm pots containing potting mix supplemented with a slow-release fertilizer (Osmocote, Scotts, Australia) for further growth and seed production. Seeds were collected from dried pods and allowed to dry further at room temperature for at least 3 days before being stored at 4°C. The process of harvesting, screening and transplanting was repeated to the 2nd generation, by which stage, homozygous lines had been selected for seed bulking. The time taken from sowing to harvest was 60–63 days.

Early PCR screening using gene-specific primers (Supplementary Table 2) for detecting positive shoots of AtBAG, TlBAG, CaNas2, and OsNas2 was used to validate transgenic events (data not presented). Southern blot analysis was performed to demonstrate transgene integration and to estimate gene copy number (Tan, 2018). Two single digestions, EcoRI and NcoI, were used to test the copy number of AtBAG4 and TlBAG lines. BamHI digestion was used for testing the integrity of the AtBAG4 insertion and StuI/NcoI double digestion was used for testing TlBAG insertion. KpnI digestion was used to estimate copy number of biofortification genes.

All data were subjected to one-way ANOVA (MINITAB 17 Statistical Software, 2010). Differences among means for treatments were evaluated by Fischer LSD test at 0.05 probability levels.

Transformed half embryo explants (THEE) grown under LED light (70 μmol s⁻¹ m⁻² light intensity) accumulated anthocyanins in the cotyledons (dark purple) which accompanied the development of healthy green shoot primordia (34.8 ± 1.4%) during the 5 days of co-cultivation (Supplementary Figures 2A,B). Meanwhile, cotyledons of THEE grown under fluorescent light (70 μmol s⁻¹ m⁻² light intensity) accumulated less anthocyanin (cream colored) and developed fewer shoot primordia (31.3 ± 1.6%) (Table 1 and Supplementary Figures 2C,D).

We observed that half embryo chickpea explants with micro-injury, grown under fluorescent light (70 μmol s⁻¹ m⁻² light intensity) produced fewer (15.7 ± 4.8%) shoots than the non-micro-injured (31.3 ± 1.6%) (Table 1). However, micro-injured THEE grown under LED light generated 2.5-fold more shoots than those grown under fluorescent light (39.2 ± 1.4% and 15.7 ± 4.8% respectively) (Table 1). These results indicate that micro-injury negatively affected shoot regeneration of THEE grown under fluorescent light but not under LED light.

To investigate whether micro-injury also played a role in chickpea transformation efficiency, in vitro shoots were screened by PCR at the end of the third round of selection. Micro-injured THEE grown under LED lights produced the highest number of transgenic shoots (5.3 ± 0.72) compared to other treatments (Table 1 and Figure 3E,F). Non-micro-injured THEE grown under LED light produced slightly more transgenic shoots (3.6 ± 0.66) than micro-injured THEE grown under fluorescent light (2.6 ± 0.54) (Table 1) although the difference was not statistically significant. When comparing the mean of PCR positive shoots obtained from micro-injured THEE grown under fluorescent light and that of non-micro-injured THEE grown under the same condition (fluorescent light), a significantly higher mean of PCR positive shoots was recorded for the micro-injured THEE. These results suggest that (i) micro-injury enhances transformation efficiency of chickpea and (ii) the efficiency of chickpea transformation can be further improved by the combined effects of LED lights and micro-injury.

Chimerism in legume transformation is fairly common and a prime factor in the non-transmission of genes to subsequent generations. Minimizing chimerism in transgenic plants is, therefore, an important target to obtain transmission of transgenes to the T₁ generation. We investigated whether the combination of LED light and micro-injury can minimize chimerism in THEE. Transient expression of GUS in micro-injured THEE incubated under LED light after 5 days co-cultivation showed intense blue coloration of explants (Figure 4C) as compared to the faint blue color observed in non-micro-injured THEE (Figure 4E). The intensities of blue color on THEE were still lower in micro-injured as well as in non-injured THEE co-cultivated under fluorescent lights (Figure 4G, I). The non-transformed explants (control) grown under both LED and fluorescent light did not develop any blue color (Figure 4A). GUS staining of healthy in vitro shoots randomly picked from each set of experiments after the third round of regeneration and selection showed that the entire shoots from micro-injured THEE grown under LED light were intensely blue (Figure 4D). No signs of chimerism were observed (Figure 4D and Supplementary Figure 3C) whereas, shoots obtained from non-micro-injured THEE grown under LED light developed only patches of blue (Figure 4F). Almost all the shoots from fluorescent light-grown THEE with or without micro-injury were chimeric (Figure 4H,J, Supplementary Figure 3B, and Supplementary Table 1). The control shoots grown under LED and fluorescent light were not blue (Supplementary Figure 3A).

The overall efficiency of any transformation protocol depends on the number of plantlets that survive acclimatization and in our initial attempts we found that chickpea acclimatization was difficult. This appeared to be mainly due to the fact that chickpea is very sensitive to excess moisture. When maintained under high humidity, grafted chickpea leaves and shoots became extremely susceptible to fungal infection. We found that perlite in the autoclaved mixture (potting mix: perlite, 3:1) that slowly released water to the plantlet prevented infection. It was also important to pre-sterilize soil mixtures and pots and to remove moisture regularly from the pot covers. In summary, about 70% of the plantlets could be successfully acclimatized in 18–19 days (Figure 3H,I; data not shown). As soon as the transgenic chickpea plantlets were acclimatized they could be transferred to bigger pots and grown under high-intensity LED to produce 10–12 mature T₁ generation seeds within about 9 weeks. Using this improved protocol, a transgenic event can be taken to the 2nd generation within 8 months after transformation (Figure 1). By this stage, homozygous lines had been selected. It was essential to examine whether the T₀ transgenic events generated using LED light plus micro-injury treatment transmitted the transgene stably in subsequent generations. All the vegetative parts such as leaves, shoots and roots and cotyledons of T₂ progeny turned blue. The reproductive tissues such as corolla, petals, stamen and pistil were also blue (Figure 6D,F). The Vegetative and reproductive parts of non-transgenic control plants remained white (Figure 6A–D).

To assess the transformation efficiency quantitatively, we monitored the entire process during regeneration of in vitro shoots under selection using the uidA gene and observed a 2.1 ± 0.35% transformation efficiency (Figure 7). The improved protocol was used to produce transgenic chickpea lines with TlBAG and AtBAG4 genes as well as OsNas2 and CaNas2 genes. Southern blot analysis of T₁ plants showed that two out of three AtBAG4 lines (#25 and #32) contained a single copy of the gene. For TlBAG lines (#15, #16, #35 and #36) three out of four lines had single copies (Figure 7A,B). Similarly, three of the five CaNas2 lines (#3, #4, and #5) had single copies of the NPTII gene (Figure 7C). Segregation of the transgene in the first generation (T₁) progeny of TlBAG (TP-35, TP-36), CaNas2 (TP-5), as well as the two copies contained in AtBAG4 (TP-32), was determined by qPCR (Supplementary Table 2). Thus, micro-injured explants and growth under LED not only produced stable GUS transgenics but also produced chickpeas with a range of other genes that were inherited in subsequent generations (Table 2).