Introduction

Rice, the staple food of greater than half of the world’s population (Hadiarto and Tran 2011) serves more than 90% of the Asian population (Khush and Brar 2001; Zeigler and Barclay 2008). Indica rice comprises 80% of cultivated rice in the world (Ramesh et al. 2009; Tie et al. 2012). Due to increase in population (1.6% year−1) there is an everyday increasing demand of rice production and the area under rice cultivation is expected to reduce to 40 million ha in the next 15–20 years (Shobarani et al. 2010). To feed the 700 million Asians getting 60% of their daily calorific intake from rice, yields must be increased by at least 50% over the next 40 years (Hibberd et al. 2008). Due to abiotic factors, 52% of the global production of rice is lost annually, of which almost 21% is because of the attack of insect pests (Brookes and Barfoot 2003). According to Savary et al. (2000), 24–41% of the rice yield was lost annually because of pests, diseases and weeds. Furthermore, the nutritional improvement of rice can also help in decreasing the evil of malnutrition in the developing world (Bajaj and Mohanty 2005). Hence, the use of biotechnological tools is the novel, powerful and effective way to increase the yield and productivity of rice varieties with exceptional grain quality and resistance to various biotic and abiotic stresses (Yaqoob et al. 2016).

Agrobacterium mediated genetic transformation appears more effective and is commonly used in rice (Lin et al. 2009; Xiao 2009). The embryogenic calli obtained from mature seed embryos are the best explants for indica rice transformation (Kant et al. 2007; Kumar et al. 2010; Sahoo et al. 2011; Sripriya et al. 2011; Endo et al. 2012). However, the genetic transformation of indica rice is a difficult task due to a low induction rate of embryogenic callus and plantlet regeneration (Kumar et al. 2005; Nishimura et al. 2006; Arockiasamy and Ignacimuthu 2007; Yookongkaew et al. 2007; Hiei and Komari 2008) and majority of the indica rice varieties are recalcitrant to invitro responses (Ge et al. 2006; Saika and Toki 2010; Tie et al. 2012). Furthermore, the transfer and integration of foreign DNA into plant genome are influenced by numerous factors like plant genotype, vector-plasmid design, phenolic compounds, bacterial strain, culture medium composition, the selectable marker genes and selection agents, temperature of co-cultivation medium and chemicals used in eliminating the Agrobacterium after co-cultivation (Dong et al. 1996; Hiei et al. 1997; Hellens et al. 2000; Cheng et al. 2004). Keeping all this in mind the present study has been devised for the first time to establish an efficient transformation protocol in rice varieties - Shalimar Rice 1, Jhelum and K332 using embryogenic callus cultures.

Materials and methods

Collection of plant material and callus induction

The experiment was conducted on the mature seeds of three high yielding commercial rice varieties - Shalimar Rice 1 (SR1), Jhelum and K 332 commonly grown in J&K obtained from the SKUAST-K, J&K, India. The research work was carried out in the plant tissue culture laboratory, ICGEB, New Delhi, India. The dehusked mature seeds of all the rice varieties were surface sterilized with 70% alcohol for 1 min under laminar air flow cabinet and washed thrice with double distilled water. These seeds were then surface sterilized in 4% sodium hypochlorite with Tween 20 (one drop per 50 ml volume) for 10 min with intermittent shaking, followed by five rinses in sterile water (3 min each). The seeds were dried on sterilized Whatman sheet No. 1 and then plated on callus induction medium (15 seeds per plate) incubated in darkness at a temperature of 25 ± 2 °C and relative humidity of 50–60%. The composition of the callus induction media used is shown in Table 1. The medium was prepared from stock solutions and pH was adjusted to 5.8, prior to addition of the gelrite. Embryogenic calli from 8 days old seeds were dissected with foreceps and then sub-cultured onto fresh plates. The plates were incubated in dark for another 3 d prior to transformation.

Table 1 Composition of the different media used
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Agrobacterium tumefaciens strain and transformation

The binary plasmid pCAMBIA1301 having gus and hpt as reporter and plant selection marker genes respectively, driven by CaMV 35S promoter and npt II gene outside the T-DNA region as bacterial selection marker was mobilized into Agrobacterium strain EHA105. Primary culture was initiated by inoculating a single colony in 10 ml YEM overnight incubated at 28 °C with appropriate antibiotics like 15 mg l−1 rifampicin and 50 mg l−1 kanamycin on a rotatory incubator shaker (Kuhner, Switzerland) at 200 rpm. About 1% of this primary culture was inoculated in a 250 ml flask containing 100 ml YEM broth. The culture was then grown at 28 °C till O.D600 reached up to 1. The cells were pelleted down by centrifugation at 4000 rpm for 30 min at 4 °C. After decanting the supernatant, the pellet was dissolved in 1–2 ml of the liquid infection medium (Table 1) using a micropipette. Using liquid infection medium, the cells were diluted to desired final O.D600 of 0.5.The bacterial pellet was suspended in 100 ml of infection medium at room temperature along with 100 µM of acetosyringone. The 11 days old embryonic calli (~100) were placed in the 500 ml Erlenmeyer flask containing 100 ml bacterial suspension for 30 min with 20 min intermittent shaking followed by 10 min 25 Hg vacuum (Millipore). The embryonic calli of all the rice varieties were then dried on sterile tissue paper and co-cultured on plates containing co-cultivation medium (Table 1). To standardize the optimum requirement, 100–200 µM of acetosyringone was added to the co-cultivation medium. The plates were incubated in dark for 48–72 h at 22–28 °C. After different experimental durations, co-cultured calli were washed with 100 ml of double distilled water containing cefotaxime (250 mg l−1) and carbenicillin (250 mg l−1) for 4–5 times, 12 min for each wash. The embryonic calli were then dried on sterile tissue paper and cultured on plates containing resting media (Table 1). The plates were incubated again in the dark for 5–7 days at 25 °C.

Selection and regeneration

The calli were then transferred to plates containing selection medium (Table 1). The plates were incubated under continuous dark conditions at 25 °C for 10 days; the resistant calli was sub-cultured for an additional 10 days in the same medium. The hygromycin resistant calli, which survived after two rounds of selection were transferred to regeneration medium (Table 1) kept in the dark for one week. The regenerated rice shoots were separated and transferred to fresh tubes containing half MS B5 media supplemented with 1% sucrose. For rooting, the explants were transferred to the rooting medium after which the plantlets were hardened and transferred to controlled glasshouse conditions. These plants were used for further transgenic analysis.

GUS histochemical staining assay

The histochemical assay for gus gene expression was performed with the putative transformant calli of the three rice varieties according to Jefferson (1987) using 5-bromo-4-chloro-3-indoxyl-β-d-glucuronide (X-Gluc - Duchefa, The Netherlands) as the substrate. The GUS assay buffer contained 0.1 M sodium phosphate buffer (pH 7.0), 0.1% Triton X-100, 10 mM EDTA, 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide and 0.1 M X-Gluc. The samples were incubated overnight at 37 °C and cleared in 50% ethanol for 4 h to clean the tissue before observation. The transient transformation frequency was calculated by the blue spots which were observed 3 day’s post-transformation. The frequency of transient transformation is expressed as the percentage of calluses showing GUS spots over the total number of callus kept for staining (calculated following De Clercq et al. 2002). To determine the optimum conditions for transformation, the conditions were changed each time and the effects on the percentage transient gus expression was calculated. The calli were observed under a stereo microscope (Stereo Discovery V8 - Carl Zeiss) and were photographed by the attached digital camera (Carl Zeiss Axio Cam).

Genomic DNA isolation and Southern blot analysis

Genomic DNA was isolated from young leaves of the putative transgenic plants following the Cetyl Trimethyl Ammonium Bromide (CTAB) protocol described by Doyle and Dickson (1987). 15 µg of genomic DNA from putative transgenic plants and negative controls (non-transformed plants) were digested by Xho I for hpt gene confirmation and by Hind III for identifying the independent transformation events in southern positive plants by overnight incubation at 37 °C. The digested samples were run on 0.8% agarose gel prepared in 1X TBE overnight at 30 V and transferred onto positively charged nylon membrane (Hybond N+, Amersham Pharmacia) by capillary blotting. The membrane was UV cross-linked by exposure to ultraviolet light using Stratalinker UV crosslinker (Stratagene, UK). A 1.1 kb Xho I digested fragment of hpt gene was used as probe. Probe was prepared as the manufacturer’s instructions (Roche Diagnostics Inc., Mannheim, Germany). Hybridization was carried out at 55 °C. All other steps like hybridization, washing, detection of the blot etc. were carried out according to the manufacturer’s instructions.

Data analysis

All data were subjected to analysis of variance (ANOVA) and caparisons of the mean were made with the Tukey’s least significant difference test at P ≤ 0.05.

Results and discussion

Callus induction

The optimum conditions for the callus induction and plant regeneration from mature seeds in the three rice varieties - Shalimar Rice 1, Jhelum and K 332 have been already standardised in our lab (Fig. 1A). Maximum callus induction was observed in MS B5 media containing 3% maltose, 500 mg l−1 l-proline, 500 mg l−1 casein hydrolysate, 5 mg l−1 l-glutamine, 40 mg l−1 cystiene, 5 mg l−1 l-asparagine, 100 mg l−1 ascorbic acid, 4 mg l−1 AgNO3, 600 mg l−1 MgCl2 supplemented with 4 g l−1 gelrite, 0.2 mg l−1 BAP and 2.5 mg l−1 2,4-D. The initiation of callus formation started just after 5–7 days on the scutellum region. In rice, maltose as a carbon source has been recommended for the embryogenic calli induction (Lin and Zhang 2005; Zaidi et al. 2006; Jadhav et al. 2011). 2,4-dichlorophenoxyacetic acid (2,4-D) is very effective in callus induction (Shahsavari et al. 2010) and l-proline has a positive impact on calli and its regeneration ability (Shahsavari 2010).

Fig. 1
figure 1

Callus induction, transformation and regeneration of transgenic rice. Three rice varieties, VIZ., SR 1, Jhelum and K 332 were used: (A) 6 days old scutellar-derived calli with seed, (B) calli on the co-cultivation medium, (C) blue spots showing transient GUS expression

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Agrobacterium mediated transformation

Since co-cultivation with Agrobacterium decreases embryogenesis and regeneration frequency (He et al. 2010), we optimized the conditions of co-cultivation. The embryonic calli dried on sterile tissue paper were co-cultured on plates containing co-cultivation medium (Fig. 1B). The plates were incubated in dark for 48–72 h at 22–28 °C. The effect of different concentrations of acetosyringone and temperature during co-cultivation period on transient GUS expression was observed (Fig. 1C). The GUS expression was more at 22 °C as compared to 28 °C in all the rice varieties (Fig. 2). The Agrobacterium overgrowth in the calli kept at 22 °C and 48 h was less as compared to co-cultivation at 28 °C and 72 h in all the rice varieties. Furthermore, co-cultivation at 28 °C and 72 h resulted in necrosis of the calli. The overall transformation efficiency of Shalimar Rice 1, Jhelum and K 332 were 5.7, 9.25 and 7.69% respectively. Temperature during co-cultivation affects the T-DNA transfer during Agrobacterium-mediated transformation (Subramaniam and Rathinam 2010). Co-cultivation in presence of 100 μM acetosyringone resulted in substantial GUS expression at pH 5.2 in all the rice varieties (Fig. 3). Similar results have been reported by several workers in the genetic transformation of indica rice cultivars (Kumar et al. 2005; Švábová and Griga 2008; Karthikeyan et al. 2012). Addition of acetosyringone in the co-cultivation medium has been reported to induce vir genes, extend host range to Agrobacterium strains and was found to be essential for rice transformation (Mohanty et al. 1999; Hoque et al. 2005; Tyagi et al. 2007). The induction of vir genes and initiation of T-DNA transfer during Agrobacterium-mediated transformation occur better at acidic pH of co-cultivation medium (Huang and Wei 2005). The use of reductants such as ascorbic acid or l-cysteine in the co-cultivation medium increased the frequency of transformation (Enríquez-Obregón et al. 1999; Olhoft and Somers 2001).

Fig. 2
figure 2

Effect of temperature during co-cultivation period on transient GUS expression. The experiments were carried out with 50 calli and repeated three times in all the rice varieties. Different small letters mean that they significantly differ from each other at P = 0.05. Vertical bars indicate standard deviation

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Fig. 3
figure 3

Effect of different concentrations of acetosyringone during co-cultivation period on transient GUS expression. The experiments were carried out with 50 calli and repeated three times in all the rice varieties. Different small letters mean that they significantly differ from each other at P = 0.05. Vertical bars indicate standard deviation

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The co-cultivated calli at 22 °C were then washed with 100 ml of double distilled water containing 250 mg l−1 cefotaxime and carbenicillin for 4–5 times. Similar concentration of cefotaxime and carbenicillin (250 mg l−1) proved effective in preventing Agrobacterium overgrowth in rice (Kumar et al. 2005). 200–500 mg l−1 cefotaxime has been used for genetic transformation of indica rice cultivars (Kumria and Rajam 2002; Nazim-Ud-Dowla et al. 2008). Carbenicillin possesses auxin-like structural features thereby increasing the regeneration potential of the explants (Nauerby et al. 1997; Ling et al. 1998).

Selection and regeneration

The selection media containing 250 mg l−1 of cefotaxime and carbenicillin prevented Agrobacterium overgrowth and the use of 50 mg l−1 hygromycin resulted in killing of the untransformed calli (Fig. 4A). The hygromycin resistant calli, which survived after two rounds of selection were transferred to the regeneration medium containing MS B5 medium supplemented with 4% sucrose, 3 g l−1 phytagel, 2.5 mg l−1 BAP, 1 mg l−1 zeatin, 0.2 mg l−1 NAA and 0.5 mg l−1 TDZ. BAP and NAA were found essential to facilitate regeneration in rice callus cultures (Rashid et al. 2004; Karthikeyan et al. 2009; Hussain et al. 2010; Karthikeyan et al. 2012). Thidiazuron (TDZ) has been reported to induce high-frequency somatic embryogenesis and organogenesis in monocots (Ganeshan et al. 2006; Cheruvathur et al. 2010; Deroles et al. 2010). After 15–25 days, the calli started to turn green in colour and the appearance of shoot primordial was noticed (Fig. 4B). The regenerated rice shoots were separated and transferred to glass tubes containing half MS B5 medium supplemented with 1% sucrose for elongation and rooting (Fig. 4C). After rooting, the plants were transferred to the pots containing 50% vermiculate mixed with soil for hardening. Pots were kept in the humidity chamber for 3–5 days and then transferred to green house (Fig. 4D).

Fig. 4
figure 4

(A) Calli on the selection medium containing 50 mg l−1 hygromycin, (B) calli showing initiation of regeneration of shoots, (C) plantlets in the glass tubes for elongation and rooting, (D) plants regenerated in the green house

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Molecular analysis of the transgenic plants

DNA from the regenerated transgenic plants and control plants in all the three rice varieties were digested with Xho I enzyme to confirm for the hpt gene (1.1 kb) by Southern hybridization (Fig. 5A). DNA of the southern positive plants were subsequently digested by single cutter enzyme (Hind III) to identify the independent transformation events (Fig. 5B). Most of the plants had single copy T-DNA insertion but a few had multiple copy T-DNA insertions. All the confirmed transgenic plants were fertile and set seed normally. The overall regeneration frequency of Shalimar Rice 1, Jhelum and K 332 were 64, 75 and 77% respectively. This is the first report on Agrobacterium mediated transformation in these rice varieties. This protocol can be used further for genetic modifications in these rice varieties and might help in the transformation of other related rice genotypes.

Fig. 5
figure 5

Southern hybridization of transgenic plants. About 15 μg of genomic DNA from transformed and non-transformed plants was digested with (A) Xho I; Lanes 1 to 4—SR 1, 5 to 8—Jhelum, 9 to 12—K 332, 13—Plasmid pCAMBIA1301 and 14 to 16—Non-transformed control DNAs of SR 1, Jhelum and K 332 respectively and (B) Hind III; 1 to 4—SR 1, 5 to 8—Jhelum, 9 to 11—K 332 and 12 to 14—Non-transformed control DNAs of SR 1, Jhelum and K 332 respectively

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Conclusions

The present protocol provides an efficient and rapid transformation system, which could be conveniently used for producing genetically modified plants.