Introduction

Pearl millet (Pennisetum glaucum (L.) R. Br.) is a robust, cross pollinating, diploid (2n = 2x = 14) annually grown major cereal crop. It is a high yielding, drought tolerant summer crop and can be grown in low rainfall areas where other crops such as maize and sorghum are not profitable (FAO 2004). Nutritionally, it is comparable to rice and is an excellent forage crop because of its low hydrocyanic acid content (Chowdari et al. 1998). Increased tillering to compensate for stress induced loss of yield, rapid deep root penetration, roots with specialized cell walls to prevent desiccation and an efficient C4 mechanism with potential growth rates make pearl millet an ideal crop for arid and semi arid tropics.

The development of an efficient method of genetic transformation is a pre-requisite for the application of bio-molecular techniques to the improvement of a given crop species. Cereals including millet crops have been primary targets for improvement by genetic transformation (Vasil 2005; Ceasar and Ignacimuthu 2009). In comparison to other major cereals, there have been only a few reports on successful genetic transformation of pearl millet (Taylor and Vasil 1991; Taylor et al. 1993; Lambe et al. 1995, 2000; Girgi et al. 2002, 2006; Goldman et al. 2003; O’Kennedy et al. 2004; Latha et al. 2006), and these were limited to biolistic methods. Although microprojectile bombardment has revolutionized the field of genetic transformation of cereals there were considerable variation seen in stability, integration and expression of the introduced transgene (Kohli et al. 1999). Moreover, this technique is expensive and requires specialized instruments such as biolistic/DNA gun for bombardment. Hence, there is a requirement for alternate methods such as Agrobacterium tumefaciens mediated genetic transformation, which appears more effective at regenerating transgenic plants with low transgene copy number. These transgenic plants were more stable over generations and have reduced gene silencing associated with integration of T-DNA into euchromatic regions (Barakat et al. 1997; Shou et al. 2004).

The two critical steps to be optimized for genetic transformation of plants are transfer of foreign DNA into the plant cells and regeneration of plants from transformed cells (Yookongkaew et al. 2007). In many species, transgenic plant recovery was difficult, because the cells transformed may not regenerate owing to several factors such as cells accessible for gene transfer are not suitable for plant regeneration (Komari et al. 1998). Therefore, development of a simple and effective approach for gene transfer is of major interest. We have developed an efficient in vitro plant regeneration protocol through somatic embryogenesis (from seeds, shoot apices and immature inflorescences explants) and direct shoot organogenesis (from shoot apex explant) for pearl millet (Jha et al. 2009). It has been observed that genetic mutations (methylation, albinism) and somaclonal variations were low in plants regenerated directly from shoot meristematic cultures as compared to plants regenerated from embryogenic calli (Bregitzer et al. 2002). Probable reason for this low frequency of mutation may be the absence of tissue dedifferentiation steps that are common in the initiation of callus and somatic embryo culture (Hirochika 1993). Thus, direct shoot organogenesis could be the preferred method for genetic transformation of pearl millet in order to minimise somaclonal variation and genotype dependence observed during callus-mediated regeneration. Transformation of shoot apices was first reported in 1988 (McCabe et al. 1988). Shoot apex explant can either be transformed using Agrobacterium or through biolistic methods (Gould and Magallanes-Cedeno 1998; Zapata et al. 1999; Cho et al. 2003; Goldman et al. 2003; Yookongkaew et al. 2007).

Two methods were used to obtain transgenic plants by transfer of DNA into the shoot apical meristem. In the first method transgenic progeny are directly produced from the shoot apex explants, or meristem cells, followed by development of transgenic plants having partially transformed reproductive organs. T0 plants produced by this way will always be chimeric. In the second method transformed shoot apical meristem cells are multiplied by treatment with growth regulators, then reprogrammed into the developmental stage under in vitro conditions (Zhong et al. 1996). Thus, manipulation of transgenic meristem cells by treatment with growth regulator to induce multiple shoot regeneration could result in more stably transformed plants (Yookongkaew et al. 2007). We used the second method (Multiple shoot induction) to optimize transformation protocol.

To the best of our knowledge, there are no reports on Agrobacterium-mediated transformation of pearl millet. Herein we report a reproducible, rapid and efficient Agrobacterium-mediated transformation protocol using Agrobacterium strain EHA 105 harboring the binary vector pCAMBIA 1301. This Agrobacterium strain EHA 105 is one of the popular strains (with vector pCAMBIA 1301) used for plant transformation (Yookongkaew et al. 2007; Li et al. 2010, 2011). Binary vector pCAMBIA 1301 contain hygromycin phosphotransferase (hpt II) and β-Glucuronidase (GUS) genes in its T-DNA. hpt II gene is used as the selectable marker gene and GUS gene is used as reporter in order to optimize parameters for high frequency transformation and subsequent regeneration of transgenic pearl millet plants.

Materials and methods

Multiple shoot induction (shoot organogenesis) from shoot apical meristem cultures

Mature seeds of pearl millet inbred genotype 843B (obtained from ICRISAT, India) were surface disinfected with 70% ethanol for 1 min and then with 0.1% mercuric chloride for 5 min, followed by five washes in sterile distilled water. Surface disinfected seeds were grown on Murashige and Skoog (MS) medium (Murashige and Skoog 1962) containing 30 g/l sucrose and 8 g/l agar (Bactoagar, Qualigens). The pH of medium (used in all the shoot organogenesis experiments) was adjusted to 5.8 before agar was added. The media were autoclaved at 121°C for 15 min and 25 ml of media was dispensed into each 100 ml autoclaved conical flask (Borosil) and closed by sterile cotton plugs.

Emerging shoot apices consisting of shoot apical meristem and a part of mesocotyl were excised from 3 to 4 days old seedlings and cultured on MS medium + 17.6 μM BAP + 30 g/l sucrose + 8 g/l agar (pH 5.8) for multiple shoot induction (Jha et al. 2009). Cultures were maintained under 16 h light provided by fluorescent lamps with the light intensity of 50 μmol m−2 s−1 at 25 ± 2°C. Shoot tips were subcultured after every 20 days in the same induction medium. Multiple shoots produced from the shoot tips were carefully separated. For shoot elongation, small shoots were cultured on MS medium.

The shoot apices of 5–6 mm length, obtained from 3 to 4 day old aseptically grown seedlings, were used as explants for Agrobacterium-mediated transformation. These were precultured for 12 h on MS + 17.6 μM BAP + 30 g/l sucrose + 8 g/l agar (pH 5.8) 1 day before co-cultivation with Agrobacterium.

Sensitivity of shoot apices of pearl millet to hygromycin

Prior to genetic transformation, the amount of hygromycin required to inhibit shoot growth was tested. The non-transformed shoot apices were placed on MS + 17.6 μM BAP + 30 g/l sucrose + 8 g/l agar (pH 5.8) supplemented with 0, 5, 10, 15, 20, 25, 30, 35 and 50 mg/l hygromycin and subcultured 2 times at 15 days interval. The survival rates of the explants were evaluated. The concentration of hygromycin that killed most of the explant was used in subsequent transformation experiments.

Agrobacterium strain, binary vector and preparation of Agrobacterium suspension

Agrobacterium tumefaciens supervirulent strain EHA 105, harboring the binary vector pCAMBIA 1301 was used in the present study. The T-DNA of pCAMBIA 1301 (Fig. 1) contains CaMV 35S promoter driven GUS gene interrupted by a modified castor bean catalase intron and CaMV 35S promoter driven hpt II gene conferring resistance to hygromycin. The intron within the GUS gene prevents its expression by Agrobacterium and allows the detection of eukaryotic expression only (Vergne et al. 2010).

Fig. 1
figure 1

T-DNA region of pCAMBIA 1301 (plant transformation vector)

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A single bacterial colony was grown overnight in liquid YENB (0.75% yeast extract, 0.8% nutrient broth [pH 7.5]) medium supplemented with 50 mg/l kanamycin at 25°C with an orbital shaking of 150 rpm. Next morning the culture was resuspended in the same medium such that O.D.600 nm was approximately 0.1. It was then grown for 4–5 h under same conditions until an O.D.600 nm of 0.8–1.0 was reached. The suspension was pelleted down at 5,000 rpm for 5 min. The pellet was subsequently resuspended in MS-inf medium [MS + 68.5 g/l sucrose + 36.04 g/l glucose + 1 g/l casamino acid + 200 μM acetosyringone (pH 5.2) (modified from Ishida et al. 1996)] to be used for Agrobacterium-infection.

Transformation of shoot apices with Agrobacterium

Precultured explants (shoot apices) were immersed in MS-inf medium for different time durations (10–60 min). The O.D.600 nm of Agrobacterium in MS-inf medium was adjusted from 0.4 to 3.0 before Agrobacterium-infection. To evaluate the effect of negative pressure (0.5 × 105 Pa), a vacuum pump was used during Agrobacterium-infection (for 30 min). Following Agrobacterium-infection, shoot apex explants were blotted dry on sterile filter paper to remove excess Agrobacterium and then transferred to fresh co-cultivation medium [MS + 17.6 μM BA + 20 g/l sucrose + 10 g/l glucose + 0.7 g/l proline + 0.5 g/l MES + 0, 100, 200 or 400 μM acetosyringone + 0.5 g/l casamino acid + 8 g/l agar (pH 5.8) (modified from Ishida et al. 1996)] for 1–6 days. Co-cultivation was carried out at 25°C in 16 h light. Different concentrations of acetosyringone (Acs) (0, 100, 200 and 400 μM) were tested in co-cultivation medium. The co-cultivated shoot apices were first washed with cefotaxime solution (250 mg/l) for 5 min, then washed four times with sterile distilled water and finally blot dried and transferred onto recovery medium [MS + 17.6 μM BA + 30 g/l sucrose + 8 g/l agar + 500 mg/l cefotaxime (pH 5.8) (modified from Jha et al. 2009)] for 7 days to inhibit the growth of Agrobacterium. After recovery phase, shoot apices were transferred on to the selection medium [MS + 17.6 μM BA + 30 g/l sucrose + 8 g/l agar + 250 mg/l cefotaxime + 30 mg/l hygromycin (pH 5.8) (modified from Jha et al. 2009)] for 30 days, with 15 days subculture intervals, to stimulate the production of transgenic shoots. Surviving hygromycin-resistant shoots were transferred onto pre-regeneration medium [MS + 17.6 μM BA + 30 g/l sucrose + 8 g/l agar + 250 mg/l cefotaxime (pH 5.8) (modified from Jha et al. 2009)] for 4–6 weeks. Surviving shoots were regenerated on regeneration medium [MS + 30 g/l sucrose + 8 g/l agar + 30 mg/l hygromycin (pH 5.8) (modified from Jha et al. 2009)] for 2–3 weeks and were rooted on rooting medium [½ MS + 0.4% w/v activated charcoal + 15 g/l sucrose + 8 g/l agar + 30 mg/l hygromycin (pH 5.8) (modified from Jha et al. 2009)].

Analysis of reporter gene expression

Transformed tissues (shoot apices after co-cultivation and leaves after selection) were analyzed for β-glucuronidase expression by using X-Gluc as the substrate (Jefferson 1987). The histochemical reaction was allowed to proceed at 37°C overnight. Subsequently, the tissues were cleared in 70% ethanol. To serve as control against any background GUS staining, untransformed tissues were included at all staining occasions.

PCR and Southern blot analyses

Genomic DNA was isolated from leaves of untransformed and putatively transformed plants using Qiagen DNeasy Plant Medi Kit. PCR analysis was carried out in a 50 μl reaction mixture containing DNA (500 ng), 200 μM of each dNTP, 1 μl of each primer (10 μM), 1 unit of Taq DNA polymerase, 2.0 mM MgCl2 and 1 × Taq Buffer. PCR conditions used were 94°C for 5 min for initial melting followed by 30 cycles of amplification with each cycle consisting of the following steps: 94°C for 30 s, 60°C for 1 min and 72°C for 1 min with a final extension at 72°C for 7 min. GUS gene specific primers were used for detection of GUS gene. These primers amplify a product of 1,029 bp from pCAMBIA 1301 plasmid. The sequences of primers used were 5′-GCC ATT TGA AGC CGA TGT CAC GCC-3′ (forward primer), 5′-GTA TCG GTG TGA GCG TCG CAG AAC-3′ (reverse primer). Primers used for amplification of hpt II gene were 5′-CTATCGGCGAGTACTTCTACAC-3′ (forward primer), 5′-GTGTCACGTTGCAAGACCTA-3′ (reverse primer). These primers amplify an internal hpt II sequence of 694 bp.

About 10 μg of genomic DNA isolated from independently transformed plants was digested with NcoI and Eco72I to release ~2 kb GUS gene from the T-DNA region of pCAMBIA 1301. The digested DNA was electrophoresed on 1% agarose gel and transferred to Hybond-N+ membrane (Amersham Pharamacia Biotech, UK) using standard protocols (Sambrook et al. 1989). These DNAs were hybridized against a random primed radiolabelled probe of 1,029 bp fragment obtained by PCR amplification of pCAMBIA1301 with GUS specific primers (forward primer 5′-GCC ATT TGA AGC CGA TGT CAC GCC-3′, reverse primer 5′-GTA TCG GTG TGA GCG TCG CAG AAC-3′). Scanning and recording of images were performed in phosphorimager (FUGIFILM, FLA 5000).

Data analysis

Explants showing GUS blue spots were recorded as positive. Each experiment was replicated 3 times and each set with a replicate consisted of at least 55 explants. The transformation frequency was calculated as the total number of transgenic plantlets produced relative to the total number of explants infected by Agrobacterium. The data was analyzed using SPSS software version 10 (www.spss.en.softonic.com). Analysis of variance (ANOVA) was used to test the statistical significance, and the significance of differences among means was carried out using LSD (Least significant difference) at a significance of P = 0.05.

Results and discussion

The T-DNA transfer from Agrobacterium into host plant genome is known to be influenced by several factors (reviewed by Cheng et al. 2004; Jones et al. 2005). As Agrobacterium-mediated transformation is a multi factor, complex interaction process (Liu and Pijut 2010), it is very difficult to analyze that which factor (such as inoculation, co-culture, co-cultivation, selection, acetosyringone etc.) contributes the most to the transformation efficiency. Step by step optimization of such factors proved to be of considerable importance for the establishment of successful transformation systems in monocotyledonous crops. Since there has been no report of successful transformation of pearl millet using Agrobacterium tumefaciens, it was considered important to investigate the effects of some of the factors on T-DNA delivery. In the present study, various factors affecting gene delivery were determined by assaying the activity of GUS gene in leaf tissues of putatively transformed plants by histochemical assays after selection. Most of the studies of Agrobacterium-mediated transformation of cereals use tissues consisting of actively dividing meristematic cells, such as immature embryos and callus induced from scutella. There are only a few reports on Agrobacterium-mediated cereal transformation using shoot apices or shoot apical meristem as explant (Sairam et al. 2003 in maize; Park et al. 1996; Arockiasamy and Ignacimuthu 2007; Yookongkaew et al. 2007 in rice; Zhang et al. (2010) in Kentucky blue grass). In pearl millet, direct shoot organogenesis without visible intervening callus phase was developed by us (Jha et al. 2009) using shoot apices as explants, which was a rapid and reproducible regeneration protocol. In the present study, pearl millet shoot apices were transformed with Agrobacterium and transformed shoot apices were propagated by multiple shoot formation.

Shoot organogenesis

For shoot organogenesis without any intervening callus phase (i.e. multiple shoot induction), aseptically grown shoot apices were cultured on MS medium containing 17.6 μM BA and subcultured every 20 days onto the same induction medium. Multiple shoots (Fig. 2a) produced within 4–6 weeks were carefully separated for shoot elongation. Small shoots were cultured for 2–3 weeks on MS medium for shoot elongation. As reported previously, a maximum of 22 shoots formed in 9 weeks on MS + 17.6 μM BA (Jha et al. 2009). Shoots were rooted on ½ strength MS containing 0.4% (w/v) activated charcoal.

Fig. 2
figure 2

Agrobacterium-mediated transformation and plant regeneration in P. glaucum. a Multiple shoot induction from shoot apex explants on MS medium containing 17.6 μM BA (scale bar 10 mm). b, c Transient GUS expression in shoot apex explants co-cultivated with A. tumefaciens strain EHA105 harbouring the plasmid pCAMBIA1301 (scale bar 5 mm). d Shoots growing on hygromycin containing selection medium (scale bar 10 mm). e GUS expression in transformed and control leaves (scale bar 5 mm). f Regeneration of transformed plants on regeneration medium (scale bar 10 mm). g, h Rooting and hardening of transgenic plants (scale bar 10 mm). i Transgenic plants 6 weeks after transfer to pots

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This kind of meristematic shoot culture resulting in the formation of multiple shoots by differentiating axillary and adventitious buds by the use of BA have been described for maize (Zhong et al. 1992), oat (Zhang et al. 1996), barley (Zhang et al. 1998), sorghum (Zhong et al. 1998), pearl millet (Devi et al. 2000; Jha et al. 2009), finger millet (Kumar et al. 2001) and wheat (Ahmad et al. 2002). All cereal crops tested have shown a similarity of response to medium containing different concentrations of BA. The medium containing lower concentrations of BA (2.2 or 4.4 μM) promoted more axillary buds than adventitious buds even after 3 months of cultures. The medium with 8.8 μM or 17.6 μM BA mostly stimulated shoot apical domes to enlarge, followed by differentiation of both axillary and adventitious buds from the leaf axils and enlarged apical domes within the first 2–3 months of incubation (reviewed by Sticklen and Oraby 2005). Kinetin was also reported to induce multiple shoots in finger millet (Kumar et al. 2001) and pearl millet (Jha et al. 2009).

Sensitivity of shoot apex explants to different concentrations of hygromycin

The effects of various concentrations of hygromycin were evaluated on shoot apex explants to determine the appropriate selection dose. Our analysis revealed that fewer than 5% of the shoot apices were able to survive in the presence of 30 mg/l hygromycin and that no shoots survived in the presence of higher concentrations of hygromycin (Fig. 3). Therefore, 30 mg/l hygromycin was used in subsequent transformation experiments.

Fig. 3
figure 3

Effect of hygromycin on survival rate of shoot apices. The survival rate of shoot apices was defined as: survival rate (%) = No. of shoot apices survived/no. of shoot apices evaluated

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Influence of Agrobacterium cell density, inoculation period, co-cultivation period, acetosyringone concentration and vacuum condition on transformation frequency

Cell density

A critical factor in the shoot apices transformation system is the density of the Agrobacterium inoculum in the inoculation medium (Li et al. 2007). We obtained the best transformation frequency (3.34%) using Agrobacterium inoculum at O.D.600 1.2 (Table 1). A reduction in the mean transformation frequency was observed following inoculation with higher densities of Agrobacterium cells, possibly because of increased production of toxins to the receptor cells (Gu et al. 2008). Several workers reported the optimum cell density for Agrobacterium-infection in O.D. < 1 [Zhao et al. (2000) in sorghum; Kumria et al. (2001) in rice; Sarker and Biswas (2002) in wheat; Gasparis et al. (2008) in oat] while several reported O.D. = 1 as best suited for Agrobacterium-infection [Kumar et al. (2005a) in rice; Shrawat et al. (2007) in barley; Yookongkaew et al. (2007) in rice]. Our results are in accord with that of Amoah et al. (2001), where they reported O.D. between 1 and 1.5 as best suited for producing highest GUS expression frequency in wheat callus. Determining the optimal inoculation density was important because at high O.D. levels, the explant tissues were almost wholly colonized by the bacteria, elimination of which becomes more difficult following the co-cultivation and, subsequently, during selection and regeneration stages. Usually this would necessitate the use of antibiotics at higher levels which, in itself, has detrimental effects on plant tissue development.

Table 1 Effect of Agrobacterium concentration (O.D.600 nm values) on the transformation frequency of shoot apex explants of pearl millet
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Inoculation period

The duration of the exposure time of explant to Agrobacterium cells also influences the transformation frequency. Pearl millet shoot apex explants incubated for 30 min with Agrobacterium cells at O.D.600 = 1.2 showed significantly increased frequency of transformation compared to those transformed for 10 and 20 min, while exposure to Agrobacterium for more than 45 min resulted in a decline in transformation frequency (Table 2). Prolonged inoculation time adversely affected the explant because of overgrowth of Agrobacterium. Different inoculation periods were reported by different workers. Zhao et al. (2000); Howe et al. (2006) in sorghum and Ishida et al. (2007) in maize, reported 5 min of inoculation period as optimal, while Sarker and Biswas (2002) reported 50 min of inoculation period as optimal along with 5 min vacuum treatment in rice. Our results are in accord with Lee et al. (2006) in orchardgrass and Zhao et al. (2011) in chinese upland rice, where they recommended an inoculation period of 30 min.

Table 2 Effect of length of exposure time to Agrobacterium inoculums on transformation frequency of shoot apex explants of pearl millet
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Co-cultivation period

The co-cultivation duration is a crucial factor influencing Agrobacterium-mediated gene transfer in pearl millet. Using the optimal conditions described above (O.D.600 = 1.2 and inoculation period of 30 min), the effect of varying the length of co-cultivation period was investigated. Co-cultivation duration for 2–3 days was generally considered suitable for Agrobacterium-mediated transformation as reported for many plant species such as wheat (Amoah et al. 2001; Mitic et al. 2004), rice (Al-Forkan et al. 2004; Hoque et al. 2005; Yookongkaew et al. 2007); buffel grass (Kumar et al. 2005b) and switchgrass (Somleva et al. 2002). Lee et al. (2006) and Ma et al. (2010) conducted co-cultivation for 1–7 days on orchardgrass callus and bast fiber plant ramie callus, respectively. Lee et al. (2006) found co-cultivation of 3 days as optimal, while Ma et al. (2010) reported 2 days of co-cultivation as optimal. In the present study, co-cultivation was carried out in the 16 h light from 0 to 6 days. Table 3 shows that the transformation frequency increased from 0.57 ± 0.13% at 1 day to 4.07 ± 0.63% at 3 days. Extension of co-cultivation period from 1 to 3 days significantly enhanced the frequency of transformation. Although co-cultivation for more than 2 days resulted in slight Agrobacterium growth around the explant, GUS expression was more intense when compared to shoot apices cultured for 2 days in transient GUS expression studies (Fig 2b, c). Extending the co-cultivation to longer than 3 days resulted in an abundant proliferation of Agrobacterium, tissue necrosis and subsequent cell death. Our results are in agreement with the observation of Wu et al. (2003) and Shrawat et al. (2007) where they reported 3 days co-cultivation as optimal for barley callus. Yang et al. (2010) optimized several factors such as Agrobacterium cell density, inoculation period and co- cultivation period for transformation of boston ivy and reported that co-cultivation period was critical for transformation.

Table 3 Effect of length of the co-cultivation period on the transformation frequency of shoot apex explants of pearl millet
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Acetosyringone

Plant specific phenolic compounds that induce the expression of Agrobacterium vir genes are important for gene transfer (Stachel et al. 1985). In monocots, where such compounds are not synthesized, addition of phenolic compounds such as acetosyringone during plant and bacterial interaction supports the gene transfer (Stachel et al. 1985; Hiei et al. 1994). Using the optimal conditions described above (O.D.600 = 1.2, inoculation period of 30 min and co-cultivation period of 3 days), the effect of varying concentration of acetosyringone (0–400 μM) in co-cultivation medium was investigated. No GUS expression was observed when acetosyringone was excluded from the co-cultivation medium (Table 4). It was found that increasing the concentration of acetosyringone from 100 to 400 μM significantly enhanced the transformation frequency, the highest frequency 4.66 ± 0.64% being obtained on medium containing 400 μM acetosyringone. These results suggest that inclusion of acetosyringone in co-cultivation medium significantly influences the transformation frequency and therefore is essential for successful transformation of pearl millet. This observation supports earlier reports in a number of species showing that the addition of acetosyringone during co-cultivation increases the number of transformed cells in the target tissue in rice (Hiei et al. 1994), wheat (Wu et al. 2003) and barley (Shrawat et al. 2007). Although acetosyringone has not been found to be essential for Agrobacterium-mediated transformation of barley (Tingay et al. 1997; Fang et al. 2002) it has been reported to be a key component to successful transformation of rice (Hiei et al. 1997), maize (Ishida et al. 1996) and wheat (Cheng et al. 2003; Wu et al. 2003). The difference in the requirement of acetosyringone for successful transformation of cereals may be because of the differences in the inoculation and co-cultivation duration and also in the competence of target tissues (Shrawat et al. 2007).

Table 4 Effect of acetosyringone concentration in the co-cultivation medium on the transformation frequency of shoot apex explants of pearl millet
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Vacuum infiltration of shoot apices with Agrobacterium

Vacuum treatment to infiltrate tissues with Agrobacterium has been successfully used in transformation of cereals (Dong et al. 2001; Amoah et al. 2001). In our system, a negative pressure of 0.5 × 105 Pa, created by the vacuum pump in the vacuum desiccator, resulted in increased transformation frequency (5.79 ± 0.43%) in comparison to that obtained at atmospheric pressure (Fig. 4). It has been suggested that a vacuum pump creates a negative pressure environment that results in an increase in effective Agrobacterium volatilization, a condition conducive to the transfer of a foreign gene into plant cells (Gu et al. 2008). Vacuum treatment of shoot apices with Agrobacterium for more than 30 min resulted in explant tissues being completely colonized by the Agrobacterium, making it more difficult to eliminate it in the recovery and subsequent stages resulting in loss of shoot apices and subsequent growth (data not shown). In the present study, when combined with optimized parameters, vacuum infiltration assisted transformation of shoot apices of pearl millet significantly enhances the transformation frequency. Our results are in agreement with Shrawat et al. (2007) who reported that vacuum infiltration (along with sonication) assisted in Agrobacterium-mediated transformation of barley.

Fig. 4
figure 4

Effect of vacuum infiltration treatment on transformation frequency in pearl millet. Shoot apex explants were inoculated with A. tumefaciens strain EHA 105 harbouring pCAMBIA 1301 at O.D.600 = 1.2 with and without negative pressure of 0.5 × 105 Pa created by vacuum pump for infection time of 30 min and co-cultivated for 3 days on co-cultivation medium containing 400 μM acetosyringone. Transformation frequency was calculated as number of transgenic plants/total number of explants inoculated ×100%

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Regeneration of stably transformed plants

Using the optimal transformation procedure, the shoot apices immersed in Agrobacterium suspension (MS-inf medium) of O.D.600 = 1.2 for 30 min under vacuum treatment, and then co-cultivated for 3 days on co-cultivation medium gave the maximum transformation frequency. If the shoot apices were cultured on the selection medium immediately following co-cultivation these shoot apices appeared to suffer from the combined stress of Agrobacterium and selection. To alleviate this problem, the explants were transferred to recovery medium (as described in materials and methods). After the recovery phase, shoot apices were transferred to selection medium containing hygromycin for 30 days, with 15 days subculture intervals, to allow the growth of transformed shoots. The shoot apices that survived on selection medium (Fig. 2d) were transferred onto pre-regeneration medium for 4–6 weeks to stimulate the production of transgenic multiple shoots. These transformed plants showed GUS expression in leaves (Fig. 2e). Transgenic shoots were then separated individually and regenerated on regeneration medium for 2–3 weeks (Fig. 2f). The well grown shoots with leaves were rooted on rooting medium (Fig. 2g, h) and then hardened in pots containing autoclaved agropeat. The transgenic plants were covered with polythene bags to retain moisture and nutrients were provided by Hoagland’s medium (Hoagland and Arnon 1950) (Fig. 2i).

Establishment of high-efficiency Agrobacterium-mediated transformation has greatly facilitated the widespread application of transformation in cereals (Repellin et al. 2001; Cheng et al. 2004). This technique has been widely used to introduce genes of interest into cereal genomes and also as a common means of testing gene function by enhancing or inhibiting expression of target genes (Kobayashi et al. 2001; Nagasaki et al. 2001; Mori et al. 2002). There are no reports of Agrobacterium-mediated transformation in pearl millet. All the reports have used biolistics for transformation of pearl millet (Taylor and Vasil 1991; Taylor et al. 1993; Lambe et al. 1995, 2000; Girgi et al. 2002, 2006; Goldman et al. 2003; O’Kennedy et al. 2004; Latha et al. 2006) and have used precultured immature zygotic embryos and embryogenic callus as a transformation target. Transgenic plants regenerated by a more or less long term callus phase are at enhanced risk of somaclonal variations and problems in transgene inheritance and stability of transgene expression (Bregitzer and Tonks 2003). In our study, a system in which shoot apices having shoot apical meristems was used as the material for gene transfer in pearl millet inbred genotype 843B. Transgenic plants regenerated directly from shoot apical meristems will always be chimeric. However, a feasible technique for avoiding this phenomenon was to multiply transgenic shoot apical meristem cells by treatment with plant growth regulator (Zhong et al. 1996). We used BA to multiply the transformed shoot apices containing shoot apical meristems and produced multiple shoots from single transformed shoot tips. These shoots were regenerated after selection and used for transgenic analysis. In recent years, several reports on use of multiple-shoot apical meristem as (Agrobacterium-mediated) transformation targets are available (Zhang et al. 2010 in Kentucky blue grass; Zhang et al. 2005 and Sairam et al. 2003 in corn; Yookongkaew et al. 2007 in rice, Gu et al. 2008 in winter jujube), indicating great potential of this technique.

Genomic DNA was extracted from the leaves of untransformed control plants and transformed plantlets obtained after selection and regeneration. PCR amplification of the GUS region was performed to detect the presence of the transgene in the genome of T0 generation transformants (named as (T01–T012).The GUS gene (1,029 bp) was amplified by using genomic DNA from transformant lines tested (Fig. 5a). Out of ten GUS primer positive transgenic lines, six lines (T02, T03, T04, T05, T08 and T010) were again cross checked by hpt II gene specific primers and showed amplification of a 694 bp (Fig. 5b). Stable integration of the transgene in PCR positive plants (T02, T03, T04, T05, T08 and T010) was confirmed by Southern analysis. Genomic DNA digested with NcoI and Eco72I released ~2 kb GUS gene internal to the T-DNA region and hybridized to a 1,029 bp radiolabelled probe specific for the GUS gene from pCAMBIA 1301. Out of six, five lines were shown to contain the GUS transgene, thus confirming stable transgenics (Fig. 5c).

Fig. 5
figure 5

PCR analysis and Southern blot analyses of transgenic plants of Pennisetum glaucum. a PCR amplification of 1,029 bp GUS fragment gene in hygromycin resistant plants after selection (lane 1 1 kb marker, lane 2 positive control plasmid pCAMBIA1301, lanes 3–14 (named as T01–T012): Putative GUS positive transgenic plants of pearl millet, lane 15 untransformed control plant). b PCR amplification of 694 bp hpt II gene in 6 transformed plants (T02, T03, T04, T05, T08 and T010) (lane 1 1 kb marker, lane 2 positive control plasmid pCAMBIA1301, lane 3 untransformed control plant, lanes 4–9: hpt II gene positive transgenic plants). c Southern blot analysis of transgenic plants (T02, T03, T04, T05, T08 and T010). Genomic DNA was digested with NcoI and Eco72I and hybridized with alpha p32 dCTP labeled GUS probe. (lane 1 positive control plasmid pCAMBIA 1301, lane 2 untransformed control plant, lanes 3–8 transgenic plants analysed)

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In conclusion, we have effectively accomplished multiple shoot regeneration from shoot apex explant containing shoot apical meristems in pearl millet, which served as an explant source for Agrobacterium-mediated transformation. Establishment of an easy, rapid, and widely applicable transformation system for pearl millet is very important for crop improvement and for study of gene function. Our results showed that optimization of optical density, inoculation duration, co-cultivation period, acetosyringone concentration and vacuum infiltration assisted inoculation improved the probability of T-DNA delivery. By using this procedure, multiple shoots resistant to hygromycin were obtained and transgenic plants have been regenerated. It was shown by PCR and Southern blot hybridization analysis that the GUS gene was integrated into the genome of the T0 generation transgenic plants. This is a pioneering report on Agrobacterium-mediated transformation of pearl millet inbred genotype 843B using shoot apices as the target tissue. Successful transformation requires a balance between the different factors affecting the transformation frequency, in order to achieve maximum number of T-DNA transfer events to occur in the target tissue, while maintaining the regenerability of recipient plant cells.