Various explants have been used for the genetic transformation of grass species, including Cenchrus. Shoot apical meristems have, by far, been the most sustainable explants, due to ease of in vitro culturing and obtaining transgenics which are genetically identical to their parent (Sticklen and Oraby 2005). The cells in shoot apical meristem undergo constant cell divisions, therefore, regeneration of plants after transformation is usually rapid. Two strategies are typically used to develop transformants by transfer of foreign T–DNA into the shoot meristems. One is that transformed newly shoots developed directly from the meristematic cells (i.e., shoot and floral meristem), wherein, putative transformants produced are usually chimeric. The other method primarily involves multiplication of transgenic apical meristem cells using plant growth regulators (TDZ or BA or Kinetin), which re–programme the dividing cells under in vitro environment (Zhong et al. 1996). Therefore, genetic manipulation supplemented with plant growth regulators to induce multiple shoot formation from shoot meristem regenerate potentially more positive transformants (Yookongkaew et al. 2007). Therefore, in our study the instant pathway of shoot organogenesis from shoot meristems was adopted for transformation (Shashi and Bhat 2023).
The delivery process of T–DNA into plant cells is influenced by numerous factors (Jones et al. 2005; Matthysse 2006). Standardization of these factors is essential for the establishment of a successful Agrobacterium transformation system in monocot plants. In Cenchrus ciliaris, there has been no report of Agrobacterium–mediated transformation, hence it was crucial to study the effects of numerous factors on T–DNA delivery. Several factors affecting gene transfer were determined by assessing the expression of reporter GUS gene in the leaves of putatively transformed plants as reported in Sorghum bicolor, Oryza sativa, Poa pratensis, Pennisetum glaucum (Sairam et al. 2003; Yookongkaew et al. 2007; Zhang et al. 2010; Jha et al. 2011).
Cenchrus seed was germinated on 1 mg/L TDZ rather than MS medium alone because it breaks dormancy and induces synchronous germination (Fig. 2a; Chengalrayan and Gallo-Megher 2001; Sumlu et al. 2010). Pre-culturing shoot apices for 12–14 hours on MS media with 3 mg/L TDZ promotes cell division (Fig. 2b). These induced cell division are in turn responsible for subsequent events of transformation such as T–DNA delivery and its integration (Sangwan et al. 1992), inhibition of adverse effects of Agrobacteria and selection medium on cell differentiation (McHughen et al.1989). Similar reports in cereal crops such as Hordeum vulgare, Triticum aesivum, Pennisetum glaucum, pre-culturing of explants has proven to be beneficial for increasing the transformation efficiency (Trifonova et al. 2001; Wu et al. 2003; Jha et al. 2011).
Hygromycin B sensitivity in selection for transformants
During all transformation events, only a few cells are transformed and regenerating putative transgenic lines without selection marker is a monotonous process. Hence, the selection marker has been essential for the regeneration of transformants, as this permits only the cells or tissues expressing the transgene to regenerate. Moreover, the selection marker also helps in subsequent screening and selection of the transgenic lines. A selectable marker such as hygromycin phosphotransferase (hptII) gene is usually used, which provides resistance against antibiotic Hygromycin B (Song et al. 2012; Hwang et al. 2014) and therefore allows screening of transgenic from non-transgenic tissues. A plasmid construct containing Hygromycin resistance gene in the T–DNA region was used for transformation protocol. The present investigation used different levels (0, 5, 10, 15, 20, 25, 30, 50 mg/L) of Hygromycin B and 30 mg/L was found to completely inhibit the growth of shoots whereas only 3% shoots survived at a concentration of 25 mg/L (Fig. 3a). Therefore, 30 mg/L Hygromycin B was used as optimum concentration for selection of putative transformants. Our results are in accordance with study by Jha et al. (2011) in Pennisetum glaucum and Padmanabhan and Sahi (2009) in Sesbania drummondii however, Li et al. (2011) found concentration of 20 mg/L was optimal for Saussurea involucrata.
Optimizing factors influenced in T-DNA delivery system
Agrobacterium tumefaciens DNA-delivery has turn into the most frequently used technique for introducing foreign gene into plant cells and subsequent regeneration of plantlets. Towards this, optimum conditions are required for conducting successful transformation experiments. Diverse factors such as bacterial cell density, duration of co–culture and co–cultivation period, use of Acetosyringone and effect of vacuum infiltration were optimised for Agrobacterium–mediated transformation in Cenchrus, based on transient GUS assay expression (Fig. 3). Standardization was performed using super–virulent Agrobacterium strain EHA 105 containing construct pCAMBIA 1301 and 1305.1 in shoot apices of genotype IG-3108. Subsequently, transgenic shoots were multiplied through direct shoot organogenesis.
Bacterial cell density
The first and most important factor for optimization of transformation efficiency is density of Agrobacterium cell suspension culture. To study the effect of most suitable OD600 for Agrobacterium culture, six different levels were tested; 0.6, 0.8, 1.0, 1.2, 1.5, 2.0. When using Agrobacterium strain EHA 105 with pCAMBIA 1301 and 1305.1, the highest number of GUS expressing shoot apices were observed with OD600 = 1.0 (Fig. 3b) similar to the findings in Oryza sativa, Hordeum vulgare and Brachypodium distachyon (Kumar et al. 2005; Shrawat et al. 2007; Behpouri et al. 2018). The number of shoot apices expressing GUS at OD600 = 1.0 was observed to be 15 ± 2.9 and 18 ± 3.7 for pCAMBIA 1301 and pCAMBIA 1305.1, respectively. The lowest number of GUS positive shoot apices was recorded at OD600 = 2.0 (0.66 ± 1.0) indicating cell density had significant influence on transformation efficiency. Higher concentration of bacterial cell density (OD600 > 1.0) is harmful to infected tissues leading to cell death, thereby decreasing the percentage of transformation. However, some workers found high transformation efficiency even at OD600 > 1.0 such as Triticum aestivum and Pennisetum glaucum (Amoah et al. 2001; Jha et al. 2011), however, in Paspalum vaginatum, Panicum virgatum and Setaria italica (Lin et al. 2017; Wu et al. 2018; Sood et al. 2020), OD600 < 1.0 was more effective. Optimization of the Agrobacterium cell density is essential for the reason that at high OD levels, the plant tissues are almost entirely dense with bacterial colony and hence its elimination becomes very challenging throughout the transformation process.
Duration of infection and co–culture
GUS expression frequency is influenced by the period of exposure of plant tissue with Agrobacterium cells hence the effect of changing the length of co–culturing period (10, 20, 30, 40, 50, 60 min) was considered. The shoot apices co–cultured with Agrobacterium cells (OD600 = 1.0) for 30 minutes showed highest efficiency of GUS expression compared to the one exposed to less than 20 min or over 40–60 minutes (Fig. 3c). The results obtained were similar to the reports in Dactylis glomerata, Pennisetum glaucum, and Oryza sativa (Lee et al. 2006; Jha et al. 2011; Zhao et al. 2011). Prolonged co–culture period negatively affected the plant tissues due to overgrowing cell density. However, few studies have indicated longer duration, i.e., 50 min in Oryza sativa (Sarker and Biswas 2002) and some shorter duration, i.e., only 5 min in Zea mays and Sorghum bicolor (Howe et al. 2006; Ishida et al. 2007), yielded better results.
Co–cultivation period
The time of T– DNA transfer and integration into the genome through Agrobacterium tumefaciens is a prolonged time-consuming process and differs widely from limited hours to few days depending upon the genotype, explants and culturing environment (Danilova and Dolgikh 2005). We conducted a transient expression experiment after co–culturing for 30 min and assessed the duration of co–cultivation period. Co–cultivation was carried out in 16/8 h light/ dark photoperiod at 25 ˚C from 0, 1, 2, 3, 4, 5 days. Initial co-cultivation period of 30 minutes did not yield any GUS blue spots, nonetheless, GUS expression was prominent after 3 days of co-cultivation. Prolonged incubation up to 5 days, did not increase the GUS expression, instead caused necrosis and damage to the tissues (similar to Li et al. 2015 report). Our results indicated that percentage of transient expression increased from 1 to 3 days (Fig. 3d). Co–cultivation period for 3 days is mostly appropriate for DNA–delivery in Dichanthium annulatum, Zea mays, Paspalum vaginatum (Kumar et al. 2005; Chen et al. 2014; Wu et al. 2018) whereas in Dactylis glomerata, highest efficiency was achieved after 2 days of co–cultivation (Ma et al. 2010).
Acetosyringone dosage on T–DNA insertion
Acetosyringone, a phenolic compound is well–known to enhance the virulence of Agrobacterium cells, to facilitate the T–DNA integration and also increase the percentage of transformation (Stachel et al. 1985; Dalton 2020). In monocotyledonous plants, these compounds are not naturally synthesized, hence addition of acetosyringone in the media during plant–bacterial interface supports the delivery of T–DNA (Cheng et al. 1997; Koichi et al. 2002). Using the optimal conditions (i.e., OD600 = 1.0, incubation period of 30 min), for transient expression of GUS, varying concentration of acetosyringone (0, 100, 200, 300, 400, 500 µM) in co–cultivation medium was assessed, after co–culturing with bacterial cells. It was observed that GUS expression efficiency was higher with increasing concentrations of acetosyringone (100 to 400 µM) and the maximum frequency of 23 ± 1.1 and 32.3 ± 1.4 for pCAMBIA 1301 and pCAMBIA 1305.1 respectively was obtained on co–cultivation medium with 400 µM acetosyringone (Fig. 3e). Addition of acetosyringone in co–cultivation medium significantly influenced the expression of GUS and was vital for successful C. ciliaris transformation but, did not improve the transformation efficiency with or without 200 µM in MS–infection medium (data not shown; also reported by Wu et al. 2018). This phenolic compound has been described to be a crucial constituent of transformation in Sorghum bicolor, Brachypodium distachyon, Setaria italica and Setaria viridis (Jeoung et al. 2002; Chen et al. 2019; Nguyen et al. 2020; Sood et al. 2020). The variation in the effect of acetosyringone on plant transformation of graminaceous plants may be due to the differences in the cell density inoculum, co–cultivation period and the proficiency of target cells.
Infection under negative pressure using vacuum pump
Vacuum infiltration is one of the additional steps in Agrobacterium-mediated transformation to enhance transformation efficiency. In this experiment, shoot apices were immersed in MS-infection liquid medium with Agrobacterium cells and placed in vacuum desiccator, a negative pressure of 0.5 x 105 Pa was applied with vacuum pump for 30 minutes and incubated at 25 ºC and 80 rpm. This resulted in an increased GUS expression efficiency for Cenchrus transformation. Similar studies using vacuum infiltration for transformation have been reported in other graminaceous species such as Festuca arundinacea and Panicum virgatum (Wang and Ge 2005; Xi et al. 2009; Li and Qu 2011; Lin et al. 2017). Applying negative pressure to shoot apices immersed in Agrobacterium cells for more than 30 min, results in explants entirely inhabited by Agrobacterium, making it harder to remove in the pre-regeneration medium, which subsequently impairs shoots growth. In Cenchrus, the highest transformation percentage (17.3 ± 1.4) was obtained with a negative pressure of 0.5 x 105 Pa compared to 13.6 ± 1.3 without the pressure (Fig. 3f). Vacuum infiltration at 0.5 x 105Pa have been widely used by many workers (Quan et al. 2003; Yang et al. 2005; Zhang et al. 2010; Jha et al. 2011) for maximising transformation efficiency. In Seashore paspalum, Wu et al. (2018) reported that vacuum infiltration along with sonication helped increase the transformation rates.
Regenerate stable putative transformant by using Agrobacterium T–DNA delivery system
By using above optimal conditions, approx. 90 shoot apices for each plasmid were immersed in Agrobacterium MS- infection medium with OD600 = 1.0 for 30 min under vacuum with constant stirring at 80 rpm at 25ºC (Fig. 2c). Excess agrobacterial suspension was removed from surface of shoot apices by absorbing on sterile Whatman™ filter paper and after that infected shoot apices were cultured for 3 days on co–cultivation medium with 400 µM acetosyringone for 16/8 h light/dark photoperiod at 25 ˚C (Fig. 2d). If the infected shoot apices were transferred into first selection medium directly after co–cultivation stage, these shoot apices suffered from the biotic stress caused by Agrobacterium cells on selection medium. To avoid this, shoot apices were sub–cultured onto recovery medium after co-cultivation. In this medium, shoots meristem multiplied without selection agent but contained Cefotaxime to remove excess Agrobacterium growth. Shoot apices were rinsed with 250 mg/L Cefotaxime for 10 min, washed thoroughly with sterile double distilled water for 2–3 times, subsequently dry-blotted on filter paper and sub–cultured onto recovery medium for 1 week (Fig. 2e). After recovery stage, infected shoot apices were sub–cultured to first selection medium with 30 mg/L Hygromycin B for 2 weeks (Fig. 2f) and after that again sub–cultured on II selection medium for 2 week to permit the better multiplication of transformed shoot meristem (Fig. 2g, h). Transformed shoot apices alive after II selection (Fig. 2i) were cultured on pre–regeneration medium for 2 weeks to enhance the proliferation of putative transgenic shoots (Fig. 2j). Hygromycin B–resistant shoots and leaves was tested with GUS stain and indicated strong GUS expression (indicated by blue colour in the tissues). Of the transformed shoots, 14 plants showed GUS expression with pCAMBIA 1301 and 17 with pCAMBIA 1305.1 (Fig. 2s). On the other hand, leaves from non–transformed plantlets, used as control, did not show any blue colour. GUS as a reporter gene has been extensively used by several worker for confirming regenerants using Agrobacterium transformation (Luo et al. 2004; Li et al. 2005; Wang et al. 2011; Song et al. 2012). These transformed shoots were then multiplied on regeneration medium for 2 weeks (Fig. 2k, l). Healthy grown elongated shoots with adventitious shoots were separated (Fig. 2m) and single shoots were transferred into rooting medium (Fig. 2n, o). Surviving plantlets were hardened in pots filled with sterilized vermiculite and soilrite for 3 weeks (Fig. 2p). These transformed plants were covered with autoclaved transparent polythene to retain humidity and nutrients were provided Hoagland's solution through capillary system (Hoagland and Arnon 1950; Fig. 2q, r).
Overall data from 10 independent experiments are summarised in Table 2. The data shows a total of 706 explants were co–cultivated with Agrobacterium and 47 transferred to II selection medium, 14 survived from selection and 6.34 ± 0.86% multiplied on regeneration medium (Table 2). Stable transformation efficiency in these experiments for pCAMBIA 1301 averaged to 1.42 ± 0.34 and ranged between 1.3 to 2.8%. Similarly 699 shoot apices were co–cultured with Agrobacterium containing pCAMBIA 1305.1 and 46 plants were transferred to II selection medium and 17 survived the selection and 6.97 ± 1.2% plants regenerated on regeneration medium (Table 2). We obtained a transformation efficiency of 1.37 ± 0.54% with pCAMBIA 1305.1 and percentage ranged between 1.2 to 4.3% (Table 2). This efficiency of Agrobacterium mediated transformation in comparable to the efficiencies attained in other lower grass species such as Festuca arundinacea (8%), Festuca pratensis (2%), Poa pratensis (1.42%), Eleusine coracana (3.8%), Eremochloa ophiuroides (8%) and Paspalum vaginatum (9%) (Dong and Qu 2005; Gao et al. 2009; Zhang et al 2010; Ceasar and Ignacimuthu 2011; Liu et al. 2012; Wu et al. 2018). Regenerated plantlets were confirmed positive from screening by the transient expression of GUS assay (Fig. 2t) which were further used for PCR analysis.
Table 2
Summary of Agrobacterium– mediated transformation of 3 days old shoot apex explants of Cenchrus ciliaris genotype IG-3108
Binary Vector | Number of Experiment | Number of shoot apex evaluated | After co-cultivation period | Recovery medium | 2 week after I selection | 2 week after II selection | Percentage of regeneration | PCR analysis | Southern positive plants | Transformation frequency (%) |
GUS | Hygromycin |
pCAMBIA 1301 | 1 | 70 | 64 | 62 | 40 | 6 | 8.5 | 2 | 2 | 2 | 2.8 |
2 | 65 | 61 | 59 | 45 | 5 | 7.6 | 2 | 2 | 1 | 1.5 |
3 | 87 | 83 | 80 | 64 | 10 | 11.4 | 3 | 3 | 2 | 2.2 |
4 | 56 | 52 | 50 | 35 | 3 | 5.3 | 0 | 0 | 0 | 0 |
5 | 90 | 82 | 80 | 45 | 7 | 7.7 | 2 | 2 | 2 | 2.2 |
6 | 60 | 54 | 53 | 30 | 2 | 3.3 | 1 | 1 | 0 | 0 |
7 | 74 | 69 | 66 | 42 | 4 | 5.4 | 1 | 1 | 1 | 1.3 |
8 | 72 | 68 | 67 | 38 | 5 | 6.9 | 2 | 2 | 2 | 2.7 |
9 | 63 | 60 | 60 | 32 | 1 | 1.6 | 1 | 1 | 1 | 1.5 |
10 | 69 | 65 | 63 | 29 | 4 | 5.7 | 0 | 0 | 0 | 0 |
| Total | 706 | 685 | 640 | 400 | 47 | 6.34 ± 0.86 | 14 | 14 | 11 | 1.42 ± 0.34 |
pCAMBIA 1305.1 | 1 | 65 | 63 | 60 | 31 | 7 | 10.7 | 1 | 1 | 0 | 0 |
2 | 55 | 50 | 49 | 35 | 9 | 16.3 | 1 | 1 | 0 | 0 |
3 | 83 | 78 | 76 | 30 | 4 | 6.0 | 6 | 6 | 3 | 3.6 |
4 | 72 | 67 | 65 | 25 | 5 | 6.9 | 1 | 1 | 1 | 1.3 |
5 | 69 | 65 | 63 | 27 | 3 | 4.3 | 3 | 3 | 3 | 4.3 |
6 | 90 | 81 | 79 | 15 | 4 | 4.4 | 0 | 0 | 0 | 0 |
7 | 60 | 56 | 52 | 20 | 2 | 3.3 | 2 | 2 | 2 | 3.3 |
8 | 85 | 80 | 76 | 17 | 4 | 4.7 | 2 | 2 | 1 | 1.2 |
9 | 50 | 48 | 45 | 12 | 3 | 6.0 | 0 | 0 | 0 | 0 |
10 | 70 | 66 | 63 | 22 | 5 | 7.1 | 1 | 1 | 0 | 0 |
| Total | 699 | 654 | 628 | 234 | 46 | 6.97 ± 1.2 | 17 | 17 | 10 | 1.37 ± 0.54 |
Shoot apex were co–cultured with EHA 105 harbouring pCAMBIA 1301 and 1305.1 at OD600 = 1.0 and an infection time of 30 minutes with vaccum and co–cultivated for 3 days on co–cultivation medium supplemented with 400µM acetosyringone |
Molecular analysis of putatively transformants
The putatively transformed plants obtained after selection and regeneration were analyzed for the nptII (GUS) and hptII (Hygromycin) gene integration using PCR (Fig. 4). Genomic DNA from leaves of putatively transformed and non–transformed (control) were extracted following the Qiagen Kit method. Expected GUS PCR product (1029 bp for pCAMBIA1301 and 406 bp for pCAMBIA 1305.1) were amplified using DNA from 14 and 17 transformant lines while no amplified PCR product was observed in non–transformed plant (Table 2), thus verifying successful transgenics. Out of 31 GUS positive transgenic plants, 14 (pCAMBIA 1301; Fig. 4a, only 11 lines shown) and 17 (pCAMBIA 1305.1; Fig. 4c shows only representative 10 lines) transgenic plants were again tested using hptII gene primers and showed a fragment of size 694 bp (Fig. 4b shows only 9 lines) and 355 bp (Fig. 4d, shows only 8 lines) respectively. Contrastingly, genomic DNA of non–transformed plant used as negative control indicated no amplification with hpt II gene-specific primers.
Stable T–DNA integration of the transgene in PCR–positive transgenic plants was also established by Southern blot analysis. Genomic DNA was digested with EcoR1 restriction enzyme which knows a single site within the T–DNA region of the pCAMBIA 1301and hybridized to a 1029 bp probe specific to GUS gene. Out of 14 plants identified through PCR, 11 plants contained single copy of GUS transgene in Southern Blot, thus confirming their stable transgenic status (Table 2 Fig. 4e represents 6 out of the 11 transgenic lines). On the other hand, in case of pCAMBIA 1305.1, genomic DNA was digested with enzyme EcoRI and 355 bp probe for hpt II gene coding region was used. No signals found in non–transformed plants after hybridization. However, out of 17 plants identified through PCR, 10 plants of hptII transgenic lines (Table 2; Fig. 4e shows representative 6 out of 10 transgenic line), indicated integration of hptII gene into the plant genome (Fig. 4e). This acquisition of low copy number is similar to the results obtained from other grasses transformed (Han et al. 2005; Bajaj et al. 2006; Gao et al. 2008; Lee et al. 2010; Xu et al. 2011; Lin et al. 2017).