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http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2016) 40: 755-761© TÜBİTAKdoi:10.3906/biy-1507-102

Agrobacterium-mediated transformation of five inbred maize lines with the Brittle 2 gene

Thuy Ninh NGUYEN, Thi Luong TRAN, Duc Thanh NGUYEN*Plant Cell Genetics Laboratory, Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi, Vietnam

* Correspondence: nguyenducthanh_pcg@ibt.ac.vn

1. IntroductionMaize (Zea mays L.) is an important cereal in the global economy due to its contribution to feeding a third of the world’s population. In the world today, maize ranks third in cultivation area after wheat and rice (Gorji et al., 2011), and it is the leader in productivity and yield. Maize contributes to the stabilization of the world’s cereal productivity and has an important impact on the global economy as a food, feed, and industrial crop.

Genetically modified plants are rapidly becoming a common feature of modern agriculture in many parts of the world. In 1996, 1.7 million hectares (M ha) of genetically modified crops were grown worldwide, and the area increased to 81.0 M ha in 2004 (Chapman et al., 2006), 90.0 M ha in 2005 (Darbani et al., 2008a), and 181.5 M ha in 2014 (James, 2014). The most commonly used methods for genetically modified plants are the biolistics method (particle bombardment) and Agrobacterium-mediated transformation (Shou et al., 2004). A high frequency of Agrobacterium-mediated transformation has been reported in monocotyledonous species, mainly rice and maize (Ishida et al., 2003). The introduction of a transgene into the host cell genome by the Agrobacterium-mediated method is easier compared to other methods, and large DNA fragments can be transformed (Darbani

et al., 2008b). Moreover, the Agrobacterium-mediated gene transfer results in higher stable transgenic events (Frame et al., 2002; Travella et al., 2005; Zhang et al., 2005; Shrawat and Lorz, 2006) and inserts a low copy number of the transgene that can minimize gene silencing (Ombori et al., 2012). Furthermore, the aforementioned method is not only simple and less expensive, but it can also be widely applied in many laboratories.

An essential requirement for the regeneration of transgenic plants is an efficient regeneration system. The first regeneration of plants from an immature embryo culture was reported by Green and Philips (1975). Since then, regeneration of maize from immature embryos as starting material has been reported in several studies (Duncan et al., 1985; Bohorova et al., 1995; Ishida et al., 1996; Zhang et al., 2003; Oduor et al., 2006). In most research on transformation of a gene into maize by A. tumefaciens, immature embryos as initial material have presented the best transformation efficiencies (Frame et al., 2002; Takavar et al., 2010; Ombori et al., 2012). However, the transfer of a functional gene is still limited to a few maize genotypes.

The Brittle 2 (Bt2) gene codes for a small subunit of ADP-glucose pyrophosphorylase enzyme (AGPase), a key enzyme in starch biosynthesis. Bae et al. (1990) isolated the

Abstract: The Brittle 2 (Bt2) gene encodes a small subunit of ADP-glucose pyrophosphorylase (AGPase) enzyme, one of the enzymes that play an important role in endosperm starch biosynthesis. We carried out a study to transfer the Bt2 gene into several inbred maize lines and regenerate transgenic maize plants possessing the transgene, which can be used to further study the role of the Bt2 gene in starch biosynthesis. Agrobacterium tumefaciens strains C58 and EHA105 harboring the transformation vector pCambia2300/Ubi-Bt2-Nos were used to transfer the Bt2 gene into five inbred maize lines, H14, H26, H240, H95, and N18. Transformed calli were selected on selection medium containing 100 mg/L of kanamycin. The stable integration of the Bt2 gene into transgenic plants was confirmed by Southern hybridizations. We recorded the different transformation frequencies in the experiments with A. tumefaciens strains C58 and EHA105. Transgenic plants were regenerated from transformed calli of all five studied maize lines. Transformation frequencies varied from 1.09% to 2.33%, depending on maize genotypes. The results obtained show that our transformation system is effective for numerous maize genotypes. H95 and H240 are good lines for gene transfer in maize.

Key words: Agrobacterium tumefaciens, Brittle 2, maize, transformation frequency, transgenic plant

Received: 21.07.2015 Accepted/Published Online: 16.12.2015 Final Version: 21.06.2016

Research Article

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Bt2 gene from maize. The expression of Bt2 has affected the starch content (Wilson et al., 2004). Li et al. (2011) reported that overexpression of Bt2 led to enhanced seed weight and starch content.

In this article, we report a successful transfer of the Bt2 gene into five inbred maize lines by the Agrobacterium-mediated gene transfer method. The results of this study will be useful for further research on genetic engineering of starch in maize.

2. Materials and methods2.1. Plant materialsImmature maize embryos of five inbred maize lines, H14, H26, H240, H95, and N18, were obtained from the National Maize Research Institute, Vietnam Academy of Agricultural Science.2.2. Bacterial strains and transformation vectorThe Agrobacterium tumefaciens strains used in the transformation experiments were C58 and EHA105 harboring pCambia2300/Ubi-Bt2-Nos transformation vector (Figure 1) and were provided by the Plant Cell Genetics Laboratory, Institute of Biotechnology, Vietnam Academy of Science and Technology.

The plant transformation binary vector pCambia2300 (a gift of Dr Jefferson from the Center for the Application of Molecular Biology to International Agriculture, Canberra, Australia) with a kanamycin resistance selective gene and nopaline synthase (NOS) terminator was used to fuse the Bt2 gene (KC849388.1) with the ubiquitin promoter (JX947345.1).2.3. Culture media Modified N6 medium (Chu et al., 1975) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), 1-naphthalene acetic acid (NAA), sucrose, and glucose in different concentrations was used for callus induction, shoot regeneration, and root formation experiments.

2.4. Transformation of maize and regeneration of transgenic plantlets Ten to 13 days after pollination, maize ears containing immature embryos were harvested, and then the ear was husked and sterilized with 70% alcohol. A. tumefaciens strains carrying the pCambia2300/Ubi-Bt2-Nos transformation vector were cultured on LB medium to the mid-logarithmic phase (OD600 = 0.7) and collected by centrifugation. The cells were resuspended in MS (Murashige and Skoog, 1962) liquid medium with 100 µM acetosyringone. The immature embryos were detached in sterile conditions and immersed in an A. tumefaciens suspension for 30 min with agitation. Next, they were dried with sterile paper and cultured on A1 medium (the modified N6 medium: basal N6 medium supplemented with 2 mg/L 2,4-D, 10 mg/L AgNO3, 2.3 g/L L-proline, 100 mg/L casein hydrolysate, 20 mg/L sucrose, 10 g/L glucose, 100 µM acetosyringone, and 7.5 g/L agar; pH 5.8) for cocultivation for 3 days in the dark. After cocultivation, the embryos were transferred onto A2 medium (N6 plus 2 mg/L 2,4-D, 10 mg/L AgNO3, 2.3 g/L L-proline, 100 mg/L casein hydrolysate, 20 mg/L sucrose, 10 g/L glucose, 500 mg/L cefotaxime, and 7.5 g/L agar; pH 5.8) containing kanamycin (100 mg/L) for suppression of Agrobacterium growth and selection of calli, respectively. Selected type II calli (friable, yellow) of between 0.3 and 0.5 cm were transferred onto resting medium (A2 medium without kanamycin) for 1 week and then onto A3 regeneration medium (N6 medium plus 60 g/L sucrose, 0.2 mg/L BAP, and 7.5 g/L agar; pH 5.8) at 28 °C under a 10/14-h light/dark photoperiod. The regenerated shoots were then transferred onto selection medium (A3 + 100 mg/L kanamycin). Normal shoots were rooted on A4 medium (½ N6 medium plus 0–1.5 g/L NAA and 7.5 g/L agar; pH 5.8) at 28 °C under a 10/14-h light/dark photoperiod. The complete plantlets with healthy roots were transplanted into thin plastic pots to grow, followed by planting in a safe greenhouse.

Figure 1. Structure of transformation vector pCambia2300/Ubi-Bt2-Nos. RB: right T-DNA border; LB: left T-DNA border; Ubi: ubiquitin; Bt2: Brittle 2 gene; Nos: nopaline synthase gene; CaMV: P35S promoter from cauliflower mosaic virus; NPT II: kanamycin-resistant gene.

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2.5. PCR analysis and Southern blot analysis of transgenic plants 2.5.1. PCR amplification of genomic DNAGenomic DNA from young maize leaves was extracted by the cetyltrimethylammonium bromide (CTAB) protocol (Saghai-Maroof et al., 1984). The Ubi promoter was fused with the Bt2 gene as shown in the transformation vector (Figure 1). To verify the Bt2 gene in transgenic plants, we checked for the presence of the Ubi promoter by PCR using specific primers for the Ubi promoter: UbiSF 5’- TACTGCAGGTGCAGCGTGACCCG-3’ and UbiSR 5’-TAGGATCCTGCAGAAGTAACACCAAACAA-3’. The presence of the Bt2 gene in the genome of transgenic plants was analyzed by Southern blot. Reaction conditions for PCR with the Ubi promoter’s specific primers were as follows: initial denaturing at 94 °C for 3 min, followed by 94 °C for 1 min, 55 °C for 50 s, and 72 °C for 1 min for a total of 35 cycles, with a final extension at 72 °C for 10 min. PCR products were electrophoresed in 1% agarose gel and stained with ethidium bromide for visualization under UV light.2.5.2. Southern blottingThe Southern analysis was performed to verify the integration of the Bt2 gene into the genome of transgenic maize plants. Genomic DNA samples from PCR-positive transgenic maize plants with the Bt2 gene were digested overnight at 37 °C with restriction enzymes BamHI and SacI, or only SacI. Plasmid DNA of pCambia2300/Ubi-Bt2-Nos was digested with BamHI and SacI to obtain a 1.5-kb Bt2 gene fragment for preparation of the probe. The probe was labeled with the Biotin DecaLabel DNA Labeling Kit (Thermo Scientific, USA). The digested genomic DNA from transgenic maize plants was separated on 1% agarose gel and transferred to the Hybond-N nylon membrane (Magna Charge Nylon, USA). Hybridization

was conducted at 42 °C. Hybridization and detection were performed according to manufacturer’s instructions using the Biotin Chromogenic Detection Kit (Thermo Scientific).

3. Results3.1. Selection of transgenic calliAfter infection of immature embryos (30 min) by Agrobacterium, followed by production and culture of calli on selection medium (3–4 weeks), many calli showed necrosis, browning, and death. This phenomenon has been reported as the reason for reduction of transgenic plant regeneration in a variety of crops (Hansen, 2000; Karthikeyan et al., 2012). In our study, we used two Agrobacterium strains, C58 and EHA105, for transformation and the transformed calli were selected on selection medium A2 containing 100 mg/L kanamycin. The ratios of selected calli were different between inbred maize lines in the two Agrobacterium strains used. The ratio of selected calli from C58-treated embryos (11.98%) was lower than that from EHA105-treated embryos (13.16%). In the case of C58 treatment, the number of selected calli was highest in the H95 line (12.79%), while in the case of EHA105, the highest ratio of selected calli (17.44%) was recorded in the H240 line (Table 1). The lowest ratios (6.04% and 8.84%) of selected calli were obtained in the N18 line. 3.2. Regeneration of transgenic plantsAfter selection on A2 medium and culturing on resting medium (A2 medium without kanamycin), transgenic calli were transferred onto A3 regeneration medium. Shoots were regenerated after 6 weeks of culture. Results obtained showed that shoot regeneration ratios of H14, H26, H240, and H95 were quite high and varied from 52% to 84% when transformation was performed with the

Table 1. Number of selected calli from inbred maize lines transformed by C58 and EHA105 Agrobacterium strains.

Maize linesC58 EHA105

EC SC RSC (%) EC SC RSC (%)

H14 150 16 10.67 150 21 14.00

H26 165 18 10.91 165 23 13.94

H240 172 17 11.33 172 30 17.44

H95 150 22 12.79 150 24 16.00

N18 215 13 6.04 215 19 8.84

Average 170.4 17.2 11.98 170.4 23.4 13.16

EC: embryogenic calli; SC: selected calli; RSC: ratio of selected calli.

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According to the obtained results (Table 3), we found that H95 had a higher transformation efficiency (based on Southern analysis) than the remaining lines. Figure 5 illustrates regeneration of transgenic plants from the inbred maize line H95. Most of the regenerated transgenic plants from the investigated maize lines had normal morphology and set seeds.

4. Discussion4.1. Selection of transgenic calliIn our study, the ratio of selected calli from C58-treated embryos was lower than that from EHA105-treated embryos (11.98% compared to 13.16%). The ratios of selected calli also varied among investigated maize lines: high frequencies of selected calli were obtained in the H95 and H240 lines, while H18 had a low frequency. Different ratios of transformed calli in different maize lines by A.

Agrobacterium EHA105 strain and 56% to 76% when using the C58 strain (Table 2). The lowest plant regeneration ratio was obtained in the N18 maize line (only 4% to 8%).

As shown in Table 2, the numbers of regenerated plants were also different in the investigated maize lines. One to three plants were regenerated from the selected calli. Regenerated plants were then transferred onto A4 medium and root induction was observed after 4 weeks of culture. 3.3. Analysis of transgenic plants by PCR and Southern blotIn the pCambia2300/Ubi-Bt2-Nos vector, the Bt2 gene is driven by the ubiquitin (Ubi) promoter. Hence, to verify the genetic transformation in the plants, we checked for the presence of the Ubi promoter. The products of PCR using specific primers for the Ubi promoter showed that many transgenic plants from the H14, H26, H240, H95, and N18 inbred lines have bands of approximately 2 kb, corresponding to the size of the Ubi promoter (Figure 2).

A total of 1515 embryos were transformed, of which 36 T0 transgenic plants (2.97%) possessed the Ubi promoter. Among the maize lines used, H240 had the highest transformation frequency at 2.97% while H14 showed the lowest transformation frequency at 1.8% (Table 3).

Southern blot hybridization showed that the Bt2 gene was successfully transferred into the maize plants. Upon Southern blotting, we observed the presence of the 1.5-kb Bt2 fragment (Figure 3) in several transgenic maize lines.

Out of the 36 plants from 5 maize lines that were positive for the Bt2 gene upon PCR analysis, 26 plants were confirmed for integration of the Bt2 gene by Southern blot and the transformation frequencies were from 1.09% to 2.33% depending on the maize line. When genomic DNA of transgenic plants was digested with SacI, an enzyme that cuts once in the T-DNA region, Southern analysis revealed that the transgenic plants possessed 1 to 3 copies of the Bt2 gene (Figure 4).

Table 2. Regeneration of transgenic plants from inbred maize lines transformed by C58 and EHA105 Agrobacterium strains.

Maize linesC58 EHA105

No. of SC No. of RC PRR (%) No. of RP No. of SC No. of RC PRR (%) No. of RP

H14 25 14 56.00 17 25 13 52.00 20

H26 25 18 72.00 20 25 17 68.00 21

H240 25 17 68.00 23 25 19 76.00 56

H95 25 19 76.00 28 25 21 84.00 39

N18 25 1 4.00 2 25 2 8.00 3

SC: selected calli; RC: regenerated calli; PRR: plant regeneration ratio; RP: regenerated plants.

Figure 2. PCR identification of Ubi promoter in transgenic plants. M: DNA marker, 1 kb; 1: negative control (nontransformed plant); 7: positive control (DNA plasmid); 2–6: transgenic plants from H14 (2), H26 (3), H240 (4), H95 (5), and N18 (6) inbred maize lines.

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tumefaciens have been reported previously (Surekha and Arundhati, 2007; Sharma et al., 2011; Ombori et al., 2012; Song et al., 2012). This may occur due to the genotypic specificity of the maize lines and the effectiveness of different Agrobacterium strains.4.2. Regeneration of transgenic plantsThis study showed that four maize lines, H14, H26, H240, and H95, had high plant regeneration frequencies from the transformed calli with values ranging from 52% to 84%. Only one line (H18) had a low regeneration frequency. High frequency of plant regeneration from transformed calli shows that the plant regeneration system developed by us is suitable for maize transformation. Most published articles have not mentioned plant regeneration frequency from selected transformed calli; however, there was one publication reporting high regeneration frequency from transformed calli of the popular Hi-II maize line with a value of 93% (Zhao et al., 2001).4.3. Transformation frequencyBased on PCR analysis, transformation frequencies in this study varied from 1.8% to 2.97% depending on investigated maize lines. After Southern analysis, transformation frequencies were 1.09% to 2.33%. Overall, in our work the transformation efficiency of 5 inbred maize lines was 1.71%. Our results are higher than those of Ombori et al. (2012), who reported an efficiency of 1.4%, and lower than that of Takavar et al. (2010), who obtained an efficiency of 6.45% in the S61 inbred maize line. 4.4. Copy number of transgeneThe copy number of the transgene can affect transgene expression positively or negatively. Multiple copy integration of a transgene can cause gene silencing (Li et al., 2002). Li et al. (2002) reported no expression of the transgene (GUS) in transgenic tobacco plants with 3–4 copies of the transgene, while high GUS activity

Figure 3. Southern analysis of T0 transformed plants. Genomic DNA was cut by BamHI and SacI enzymes and probed with the Bt2 gene. M: DNA ladder, 1 kb; Lane 1: negative control (nontransformed plant); Lanes 2, 4, 5, 6, 7: transgenic plants from H240, H26, H14, H95, and N18 maize lines, respectively; 3: positive control (PCR product of the Bt2 gene).

Table 3. PCR and Southern analysis results of T0 transgenic plants from five inbred maize lines.

Maize lines No. of IE* No. of PCR-PTP No. of Southern-PTP TF (%)

H14 276 5 (1.8%)** 3 1.09

H26 357 7 (1.96%) 6 1.68

H240 101 3 (2.97%) 2 1.98

H95 429 12 (2.80%) 10 2.33

N18 352 9 (2.57%) 5 1.42

Total 1515 36 26 1.71

* IE: infected embryos; PCR-PTP: PCR-positive transgenic plants; Southern-PTP: Southern-positive transgenic plants; TF: transformation efficiency.**Values in brackets are ratios of PCR-positive transgenic plants.

Figure 4. Southern analyses of T0 transformed plants. Genomic DNA was cut by the SacI enzyme and probed with the Bt2 gene. M: DNA ladder, 1 kb; Lane 1: negative control (nontransformed plant); Lanes 2–6: transgenic plants of H14 (2), H95 (3), H26 (4), N18 (5), and H240 (6); 7: positive control (PCR product of the Bt2 gene).

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was observed in plants that had a lower transgene copy number. We have obtained transgenic maize plants with 1 to 3 copies of the Bt2 gene and most of them had 1 or 2 copies. The works of Freme et al. (2002) and Ombori et al. (2012) reported 1 to 5 and 2 to 6 copies of the transgene, respectively.

Since the first maize transformation by the biolistics bombardment method (Godon-Kamm et al., 1990) and the Agrobacterium-mediated method (Ishida et al., 1996), maize transformation has only been successful for a few genotypes in laboratories around the world (Ji et al., 2013). The results obtained here show that our transformation system is effective for numerous maize genotypes. In addition to the five maize lines in this study, the system was successfully used for transformation of other inbred maize lines such as H4 and CML161 with similar transformation efficiencies (Tran et al., 2014). Most of the generated transgenic maize plants have a lower transgene copy number. Genotype plays an important role in the regeneration of transgenic plants in maize (Gorji et al., 2011; Seth et al., 2012). In our study,

the H240 and H95 lines performed with high regeneration and high transformation efficiency. Thus, these two lines are good material for future gene transfer experiments in maize. The results reported in this study will contribute to further research on the production of transgenic maize plants. Moreover, the transgenic maize lines possessing the Bt2 gene hence generated will be a valuable tool for the study of starch biosynthesis, not only in maize, but in plants in general.

AcknowledgmentsThe authors thank Chitra C Iyer from the Ohio State University, Ohio, USA for her English editing and valuable comments. This research was supported by the project “Production of maize parent lines with enhanced starch biosynthesis by gene transfer technology” under the Program for Development and Application of Biotechnology in Agriculture, time period 2010–2020, from the Ministry of Agriculture and Rural Development, Vietnam.

Figure 5. Regeneration of transgenic plants from inbred maize line H95. A) Immature embryos after infection by A. tumefaciens on A1 medium. B) Callus induction after 1 week of coculture on A1 medium. C) Type II friable calli after 4–6 weeks of selection on kanamycin-containing medium. D) Regenerated plants on selective medium. E) Transgenic plants in the pots after 5 days. F) Transgenic plants with emerged ears. G) Ears from transgenic plants.

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