Agrobacterium mediated genetic transformation and regeneration in elite rice ( Oryza sativa L . ) cultivar BRRI dhan

Agrobacterium-mediated genetic transformation of rice (Oryza sativa L.) cultivar BRRI dhan56 was carried out in this study. Agrobacterium tumefaciens strain LBA 4404, which harbors the plasmid pIG121 that carries the genes for ß-glucuronidase gene, served as a reporter gene in the histochemical assay and the neomycin phosphotransferase ΙΙ (NPT ΙΙ) gene for the identification of resistance to kanamycin was used for genetic transformation. Twenty days old embryogenic calli from mature embryos of highly regenerating rice cultivar BRRI dhan56 were used to co-cultivate with 0.8 to 0.9 OD600 Agrobacterium for 25 min and the cultured was continued on agar medium for this study. The transformed colonies were selected by using 50 mg/L kanamycin and 50 mg/L rifampicin and confirmed by colony PCR. The PCR positive colonies were isolated to transform by using calli of indica rice cultivar BRRI dhan56. Putative leaf and root segments from plantlets obtained from transformation experiment with the plasmid pIG121 were GUS positive. Integration of the introduced gene into the genome was demonstrated by PCR. The maximum transformation efficiency of 32% was obtained by using 500 mg/L cefotaxime as a bacteriostatic agent to inhibit growth of Agrobacterium. In this study, 100 μM acetosyringone in co-cultivation medium and co-cultivation for 3 days were the optimum conditions for maximum transformation. The expression of GUS gene revealed that the calli were successfully transformed. The results of this study would be an effective tool for crop improvement and gene-function studies on the model monocot plant rice.


INTRODUCTION
Rice (Oryza sativa L.) is one of the most significant cereal crops of Poaceae family cultivated for more than 10,000 years (Sasaki, 2005).It was the first major cereal crop regenerated into whole plant from tissue culture (Vasil, 1983).It is an economically very important crop in the world, with more than half of the world's population depending on it as a primary staple food (Lu, 1999).Its cultivation is concentrated for the most part in Asian *Corresponding author.E-mail: kzaman63@ru.ac.bd.Tel: +880-17162448083.Fax: +880-721750064.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License countries, which together make up approximately 90% of the rice cultivation area in the world.Numbers of rice consumers are increasing at the rate of 1.8% every year (Islam et al., 2015).It is estimated that rice production has to be increased 50% by 2025 (Khush and Virk, 2000).Approximately 80% of the world rice production is based on indica rice varieties which are cultivated under subtropical and tropical conditions as long grain rice and thus securing a unique position in agriculture (Khush, 1997;Abbasi et al., 2010).
In recent years, some important agronomic traits of rice have been improved through biotechnological approaches (Hao et al., 2009;Skamnioti and Gurr, 2009;Cheng et al., 1998).Genetic transformation has become a valuable tool in targeted improvement and gene function studies in rice (Xu et al., 2012).Most indica rice varieties were adapted to by Agrobactrerium-mediated genetic transformation (Chan et al., 1992;Rashid et al., 1996;Khanna and Raina, 2002;Supertana et al., 2005;Ignacimuthu and Arockiasamy, 2006) involve regeneration of plants from transformed embryogenic calli, anther calli (Jiang et al., 2004;Arnold et al., 1995), and protoplasts.Agrobacterium-mediated transformation has significant advances of such as introduction of fewer copies of genes into the plant genome, high coexpression of introduced genes, transformation of relatively large segments of DNA and high fertility of transgenic plants (Cheng et al., 1997;Hiei et al., 1994;Datta et al., 1992).Agrobacterium tumefaciens mediated transformation has been well established, owing to its simplicity, low cost, as well as low copy number of transgene integration.However, the transformation efficiency is very low in the indica rice varieties.Most indica rice varieties also show a low rate of callus growth and low regeneration frequency in conventional culture (Nishimura et al., 2007;Larkin and Scrowcroft, 1981).Now Agrobacterium-mediated rice transformation has been successful in many cultivation of japonica, indica and javanica (Aldemita and Hodges, 1996;Hiei et al., 1994;Dong et al., 1996).
There are several factors which are involved in Agrobacterium-mediated gene transfer into rice.During co-cultivation, the addition of acetosyringone, media with 2,4-D, acidic pH and high osmotic pressure have been reported to be significant for the induction of vir gene expression on Ti plasmid (Turk et al., 1991;Usami et al., 1988).Most reports suggested to use actively growing, embryogenic calli derived from rice mature seed or immature embryos as receptors to be infected by A. tumefaciens (Hiei et al., 1997).In addition, shoot apices of tropical japonica have also been used as gene receiver (Park et al., 1996;Rogers and Bendich, 1994).In the present study, we report establishment of an efficient plant regeneration system from mature seeds of indica rice applicable to BRRI dhan56 as explants using different growth regulators and demonstrate their amenability to Agrobacterium-mediated transformation.

Plant materials
Indica rice (O.sativa L.) cultivar namely BRRI dhan56 was used as explants for study of in vitro regeneration and genetic transformation.Seeds were collected from Bangladesh Rice Research Institute (BRRI) Regional office, Rajshahi, Bangladesh.

Preparation of explants
Mature seeds of indica rice cultivars namely BRRI dhan56 were dehusked carefully and separately.Dehusked seeds were taken in beakers having distilled water and 1 or 2 drops of Twin-80 were added in the distilled water.The mixture was shacked for 5 min with orbital shaker.Seeds were then washed with distilled water for several times to remove the effect of Twin-80.Then seeds were bought in front of laminar air flow and surface sterilized with 0.1% (w/v) HgCl2 solution for 5 min by vigorous shaking twice.Treated seeds were rinsed with distilled water several times to remove mercuric chloride completely and blot dried on to a filter paper.

Callus induction
For callus induction, three seeds were inoculated per test tube on callus induction medium (MCI) and incubated at 26 ± 2°C in dark.MCI was prepared using by basal MS salts containing all vitamins (Murashige and Skoog, 1962) supplemented with 2.5 mg/l 2, 4dichlorophenoxyacetic acid (2, 4-D), 0.50 mg/l naphthalene acetic acid (NAA), then the medium was solidified 0.8% agar before autoclaving 21 min at 121°C and 1.07 kg cm -2 and pH of the medium was adjusted to 5.8 before addition of agar.After 20 days, non-embryogenic calli (compact, non-friable calli that develop root like structures) were discarded and only embryogenic calli were selected.These embryogenic calli were cut into around 3 equal halves, then sub-cultured again onto fresh MCI and kept for 4 days (dark, 26 ± 2°C) before transformation with A. tumefaciens.

Agrobacterium strain
The bacterial (A.tumifaciens) strain LBA4404 with the binary plasmid pBI121 strain was used for infection in the transformation.The binary vector pBI121 has the background of pBIN19.It contains a reporter gene GUS (β-glucurunidase) driven by a CaMV35S promoter and NOS terminator and a selectable marker gene nptII fused between promoter and terminator encoding for the encoding for the enzyme neomycin phosphotransferase conferring kanamycin resistance.

Culture of Agrobacterium strain
Primary culture of Agrobacterium strain was prepared by inoculating single colony from a freshly streaked plate which contains 5 ml of autoclaved liquid YEP medium (10 g/l bactopeptone, 10 g/l yeast extract, 5 g/l sodium chloride and pH 7.0) supplemented with 25 mg/l streptomycin, 50 mg/l rifampicin and 50 mg/l kanamycin.The culture was incubated for 20 hours on a rotatory incubator shaker at 120 rpm in dark at 28°C.Secondary culture of Agrobacterium strain was prepared in a 500 ml baffled flask containing 50 ml YEP medium (supplemented with same antibiotics as used for primary culture) by adding together 0.4% of the primary culture and grown under same environment.Once the OD600 reached ~1.0, Agrobacterium cells were pelleted by centrifugation at 8000 × g for 15 min at 4°C.The cells were re-suspended in MS re-suspension medium containing 150 μM acetosyringone (MS salts, 6 8 g/l sucrose, 36 g/l glucose, 3 g/l KCl, 4 g/l MgCl2 and pH 5.2) to adjust the OD600 of the bacterial suspension to 0.3.

Co-cultivation and selection of transformed calli
Sub-cultured (four days) embryogenic calli were collected and Agroinfected by immersing them in the Agrobacterium culture (LBA4404) for 20 to 25 min with blinking gentle shaking at 50 rpm.The Agroinfected calli were dried on sterile filter paper for 5 to 6 min.Calli were then transferred to the co-cultivation medium containing 10 g/l glucose, pH 5.2, 150 μM acetosyringone (Ali et al., 2007) and incubated at 26 ± 2°C in the dark for around 48 h.Once slight growth of Agrobacterium appeared around most of the calli, the calli were rinsed 8 to 10 times with 500 mg/l cefotaxime in sterile distilled water, dried on sterile filter paper and transferred onto first selection medium (MCI containing 500 mg/l cefotaxime and 100 mg/l kanamycin) and incubated for 13 days at 26 ± 2°C in dark.After the first selection, brown or black calli were removed and only creamish healthy calli were shifted to the fresh MS media for second selection and maintained at 26 ± 2°C in dark.After second selection for 11 days, micro calli could be observed which were finally transferred to fresh MS media for third selection and allowed to proliferate for five days at 26 ± 2°C in dark.

Regeneration of transformed calli
After third selection, black or brown microcalli were discarded and only granular 'macrocalli' were transferred onto MS media containing two or three growth regulators viz.6 benzylaminopurine (BAP), kinetin, naphthalene acetic acid (NAA), pH 5.8; 250 mg/l cefotaxime and 50 mg/l kanamycin added after autoclaving.These microcalli were incubated at 26 ± 2°C in dark for seven days for the first phase of regeneration.During the second phase of regeneration, these were shifted to fresh same regeneration medium and incubated in light for four days.The regeneration frequency was calculated as per the formula: Regeneration frequency (%) = [(Number of microcalli regenerating shoots) / (Number of microcalli incubated)] x 100.
For development of vigorous roots, the regenerated shoots were shifted to rooting media and finally transfer to soil pot for hardening.

Histo-chemical GUS assay
Infected calli after co-cultivation as well as regenerated shoots of putative transformants were assayed for transient GUS activity according to the procedure described by a researcher (Jefferson, 1987).Tissue segments were immersed overnight in assay buffer containing 20% methanol, 500 µM potassium ferrocyanide, 500 µM potassium ferricyanide, 5 mM EDTA, 50 mM sodium phosphate buffer (pH 7), 0.5% triton X-100 and 20 mM X-Glu.Then treated explants were transferred to 95% ethanol for preservation and the explants were observed for visualization of color products under zoom stereomicroscope.

DNA isolation and PCR analysis
Genomic DNA was collected from transformed and untransformed calli by using the CTAB method.PCR analyses were carried out by using two GUS primers namely; forward 5' CCTGTAGAAACCCCAACCCG 3' and reverse 5' TGGCTGTGACGCACAGTTCA 3' for amplification of GUS gene transformants.The reaction mixture (20 μl) of PCR consists of 1.0 μl DNA template, 2.0 μl 10× buffer, 1.0 μl (2.5 mM) dNTPs, 2.0 μl (25 mM) MgC12, 1.0 μl of each primer (F/R), 0.4 μl Taq DNA polymerase and ddH2O 13 μl.Reaction procedures were carried out at 94°C for 4 min and followed by 25 cycles at 94°C for 1 min, 56°C for 45 s and 72°C for 1 min.After the final cycle, the reactions were maintained at 72°C for 5 min before completion.Finally, PCR products were analyzed on 1% agarose gel with 0.5× TBE buffer.

Callus induction
Transformation of rice using Agrobacterium mediated methods and subsequent regeneration are dependent on several factors such as the choice of explants, hormonal concentrations of the medium used and nutritional supplements highly affects the callusing and regeneration (Lin et al., 1995;Katiyar et al., 1999).A reporter (Hiei et al., 1994) reported that scutellum derived callus was the most amenable explants for Agrobacterium mediated transformation.In the present investigation, seeds of elite indica rice cultivar BRRI dhan56 was used as explants for Agrobacterium mediated transformation.Calli were produced by using different types of hormonal concentrations of 2, 4-D (1.5 to 3.0 mg/l), NAA (0.5 to 2.0 mg/l), 2, 4-D + NAA (1.5+1.0 to 3.0+1.0mg/l), 2, 4-D + BAP (1.50+0.10 to 2.50+.50 mg/l) and NAA + BAP (1.0+0.20 to 2.0+1.50 mg/l) were used and 2, 4-D + NAA (2.0+1.0 mg/l) was showed the highest result where 95.22% explants produced calli in BRRI dhan56 (Figure 1A and B).All the recorded data are given in Table 1.With the increase of concentration of 2, 4-D above 2.0 mg/L the callus induction efficiency was reduce in rice cultivar.This indicate that the use of low concentration of 2, 4-D was enough for production of high amount of callus in rice.It was revealed from the results of tissue culture experiments that BRRI dhan56, showing the best callus induction with combinations of 2, 4-D and NAA.
Similar result for the callus induction in rice were also reported by others (Rashid et al., 2003;Islam et al., 2013;Roly et al., 2013Roly et al., , 2014;;Islam et al., 2014a;Islam et al., 2014b).After 12 days, calli of BRRI dhan29 was sub cultured in MS medium containing different concentrations of 2, 4-D for development of Embryogenic calli (Table 2).The highest Embryogenic calli (95.22%) showed MS media with 2, 4-D + NAA (2.0+1.0 mg/l).Different days (10, 15, 20, 25 and 30 days) of calli were tested as suitable for transgenic rice BRRI dhan56.For Agrobacterium-mediated genetic transformation, twenty days of old calli are suitable for transgenic rice BRRI dhan29 (Figure 1C and D).Regeneration responses of embryogenic calli derived from mature seeds were influenced with the concen-trations and combinations of 2,4-D and NAA present in the regeneration media.Similar results were reported by some researcher (Pandey et al., 1994;Islam et al., 2013) who observed a high regeneration frequency for the medium containing high levels of 2, 4-D and NAA and after a certain levels 2, 4-D and NAA, callus induction frequency decreased.

Genetic transformation of rice callus
In our present investigation, Agrobacterium strain LBA4404 harboring the plasmid pBI121 contains kanamycin resistance gene (nptII) as the selectable marker gene.Kanamycin was tested as a selective agent for transgenic rice BRRI dhan56.Infected calli from mature seeds of BRRI dhan56 were cultured on shoot induction medium containing various concentrations (50, 75, 100, 125 and 150 mg/l) of kanamycin.Shoot regeneration was greatly inhibited by the increased concentration of kanamycin (Figure 2).The highest response was 32.0% of the explants regenerated shoots in the presence of 100 mg/l kanamycin and there was no shoot regeneration with 150 mg/l or greater concentrations of kanamycin.The effects of different incubation periods (15, 20, 25, 30 and 35 min) with the Agrobacterium strain on the calli were studied at a constant optical density (OD 600 of ~0.9).In every case, 10 calli were infected and the highest 32% GUS positive result was found with 25 min incubation period.On the other hand, the lowest 10% GUS positive result was found with 15 min incubation period (Figure 3).Optical density (OD) of Agrobacterium strain is an important factor for genetic transformation in rice.In this study, five different optical density levels were tested viz., 0.6, 0.8, 0.9, 1.0 and 1.1 (Figure 4).The highest number of GUS expressing calli was observed at OD 0.8 to 0.9.It was clearly confirmed that the bacterial strain LBA4404 (pBI121) showed highest peak of performance in lower  range of OD and it gradually decreases with the increase of OD taken.Duration of co-cultivation, the temperature of incubation, with Agrobacterium, concentration and the composition of bacteriostatic agent and duration of selection and concentration of antibiotic selection marker are the other important factors reported which affect Agrobacterium mediated transformation in rice (Okkels and Pederson, 1998;Katiyar et al., 1999).To find the optimum conditions for co-cultivation, different concentration of acetosyringone and the duration of cocultivation were tested very carefully.The frequency of kanamycin resistant calli obtained following each variation in co-cultivation conditions was taken as the transformation frequency.For optimum transformation, the presence of 100 µM acetosyringine in co-cultivation medium, 500 mg/L cefotaxime to inhibit Agrobacterium growth and co-cultivation for three days were found to be the most suitable.

Stable integration of foreign gene and confirmation
After inoculation with Agrobacterium, the explants were co-cultivated for 3 days on normal MS media.The explants were then sub-cultured on regeneration medium (BAP 2.0 mg/l + 1.0 mg/l NAA + 1.5 mg/l KIN) containing carbenicillin.With 2 to 3 weeks of culture, the calli developed shoot buds (Figure 5A and B) and were subcultured on the same medium for shoot development (Figure 5C).After proliferation, shoot were transferred on selection medium with kanamycin and the same hormonal combination (Figure 5D) first cycle and second cycle selection.At the same time control plants were subsequently cultured on selection medium first cycle and second cycle selection and 90% shoots died after 21 days.A bacteriostatic agent namely cefotaxime was used to inhibit Agrobacterium growth after co-cultivation.It has been recorded that the use of high concentration of bacteriostatic agents may reduce the regenerability of the calli as they structurally resemble auxins.Further, in combination with other callus inducing hormones such as 2, 4 D may cause loss in regeneration potential (Lin et al., 1995;Okkels and Pederson, 1998).Regenerated shoots harvested from selection medium and transfer in the normal MS medium for vigorous root development (Figure 5E) before micro plant transfer in pot for establishment.After six days, a well-developed root system was observed in the plantlets.Then the plantlets transferred to soil survived under the normal environmental conditions and grew to maturity.With the purpose of confirm the presence of the transgene, calli were subjected to histochemical staining.GUS assay was performed according to Jefferson (1987) in two stages, first, After 3 days of co-cultivation in calli and second the leaves and roots from putative plants were taken and incubated in X-gluc buffer.Indigo blue coloration was observed in calli (Figure 6A), section of calli (Figure 6B), leaves and root (Figure 6C and 6D).Appearance of blue color following overnight incubation at 37°C with GUS assay revealed the presence of the transgene.GUS gene was amplified in transformed plant DNA and plasmid DNA where forward and reverse primer amplified 880 bp GUS gene segment (Figure 7).Compared to the nontransformed calli with regeneration frequency of 96% and transformed calli showed very low regeneration frequency (32%).A researcher (Kumar et al., 2005) recorded a transformation efficiency of 4.6 to 5.5% and 6.4 to 7.3% for two recalcitrant elite Indica rice cultivars, which were lower transformation frequencies than these results.

Conclusion
In conclusion, we have established rapid multiple shoot induction and efficient plant regeneration method from seeds elite indica rice cultivar BRRI dhan56 in a genotype independent manner.The combination of 2, 4- D + NAA (2.0+1.0 mg/l) was shown to enhance the highest callus induction, where 95.22% explants produced calli in BRRI dhan56.Compared to the nontransformed calli with regeneration frequency of 96% and transformed calli showed very low regeneration frequency (32%).For optimum transformation, the presence of 100 µM acetosyringine in co-cultivation medium, 500 mg/L cefotaxime to inhibit Agrobacterium growth, OD 600 0.8-0.9 of Agrobacterium strain and cocultivation for 3 days were found to be the most suitable.The method described was simple, inexpensive and does not require any advanced equipment.This study would be an effective tool for crop improvement and gene-function studies on the model monocot plant rice.

Figure 1 .
Figure 1.Different types of calli from mature embryo in elite rice cultivar BRRI dhan29: (A) and (B) Ten days old callus, (C) and (D) Embryogenic calli derived from mature embryo on callus induction medium.

Figure 6 .
Figure 6.(A) Expression in transformed callus and (B) section of transform callus after 3 days of co-cultivation, (C) GUS expression in leaf segment and (D) root segment of putative plant.

Figure 7 .
Figure 7. Confirmation of GUS gene through PCR analysis.