Production of transgenic local rice cultivars (Oryza sativa L.) for improved drought tolerance using Agrobacterium mediated transformation.

Rice being the staple food of middle and south India, there is an extensive research undertaken in protecting the species and improving the quality and yield. Several recombinations have been made to the rice genome to impart various qualities which lack in the pure breed. Oryza faces various natural stress, like temperature variance, high salinity, etc., drought is one of the major parameters affecting the growth and yield of the plant. Transgenic rice cultivars can be generated for drought tolerance using the Agrobacterium mediated transformations. The current work aims to impart the gene for drought tolerance in Oryza sativa L. using Agrobacterium mediated transformation. The gene targeted in this context is dehydration response element binding factors (DREB). DREB plays a major role in response to drought mediated stress. Sambha mahsuri (Indica type) and Cotton dora sannalu (Indica type) the two local cultivars have been transformed for the gene AtDREB1A under 35s CaMV promoters (pBIH binary vector) for which the vector used was Agrobacterium. The target plant tissue being used was calli. Optimization of the parameters was performed for a lethal dose of hygromycin, cefotaxime level, and acetosyringone level. PCR amplification was used for the confirmation of the transgenic (T 0) species in which 23% and 18% for Sambha mahsuri and Cotton dora sannalu, respectively. Southern blotting was performed for the genomic DNA. Normal growth was shown by the T 1 transgenic plants whose expression was confirmed by RT-PCR. The T 1 transgenic plants showed good tolerance to drought mediated stress for a total period of one and a half week under greenhouse condition. The study can be concluded by producing a potentially successful drought resistance T 1 species produced using Agrobacterium mediated transformation.

Introduction

Plants are seriously affected by exposure to various adverse environmental stresses such as drought, salinity, high temperature, cold, and light. Among these abiotic stresses, drought is a widespread environmental threat seriously affecting plant growth and development (Shinozaki and Yamaguchi-Shinozaki, 2000). Drought effected area is increasing worldwide and over 20% of rice growing area worldwide is suffering from drought stress. Drought stress is becoming an increasingly severe problem limiting rice productivity in many regions and even mild drought stress can significantly reduce rice yield (Swamy and Kumar, 2013). Plants have developed a complex network of stress signaling and regulation of gene expression mechanism to respond and adapt to the stress condition with suitable functional changes (Tang et al., 2012). Plants perceived specific stress signals with the onset of stress conditions resulting in the activation of various signaling modules including hormones, signal transducers and various transcription factors. The signals are further transduced by the signaling components to induce the expression of various stress-related genes and the resultant cellular damage is neutralized by the discrete product of the expressed genes through various physiologic and metabolic processes (Shinozaki and Yamaguchi-Shinozaki, 1996, Lata and Prasad, 2011). Among these, transcription factors are key regulators of changes in the gene expression and environmental stress responses (Shinozaki et al., 2003). The transcription factors involved in abiotic stress tolerance are classified into different family’s i.e. ABRE/ABF (ABA-responsive element-binding protein/ABA-binding factor), AP2/ERF (APETALA2/ethylene responsive factor), bZIP (basic leucine zipper), MYC/MYB (myelocytomatosis oncogene/myeloblastosis oncogene), CUC (NAC) and various protein kinases (Shinozaki and Yamaguchi-Shinozaki, 2000, Todaka et al., 2012). Both ABA-dependent and ABA-independent processes are involved in the activation of transcription factors during stress responses. Among the transcription factors, the DREB genes of AP2 family, have been identified for their function in the ABA-dependent pathway by recognizing ABA-responsive elements (ABREs) containing a 9 bp (TACCGACAT) cis acting core motif (DRE) (Choi et al., 2000, Yamaguchi-Shinozaki and Shinozaki, 2006). The DREB transcription factors have a main role to induce the expression of the number of genes involved in drought stress and genetic manipulation of the genes encoding DREB transcription factors displayed remarkable improvement in plant response toward drought stress (Liu et al., 1999, Udvardi et al., 2007). Over expression of DREB genes that regulate expression of multiple genes from various pathways is an important way of achieving tolerance to drought stress in plants. Several genes encoding DREB TFs have been incorporated into rice plants resulting in drought stress tolerance with either constitutive or inducible promoters (Lata and Prasad, 2011). Transgenic rice plants over-expressing various DREB genes showed strong tolerance to drought stress (Dubouzet et al., 2003, Ito et al., 2006, Datta et al., 2012, Ravikumar et al., 2014). Several transgenic rice lines were generated over-expressing OsDREB1A and OsDREB1B from rice and DREB1A, DREB1B and DREB1C from Arabidopsis under the control of constitutive promoter ubiquitin and 35s CaMV resulting in tolerance toward drought stress (Ito et al., 2006, Oh et al., 2009). Gene manipulation approaches involving varieties of genetic transformation methodologies have been employed to enhance the tolerance of crop plants toward multiple stresses (Kasuga et al., 1999). The standard rice transformation protocol for generating a large number of transformants, however, is limited to only a few cultivars, indicating that experimental parameters for rice transformation have not been fully optimized (Ozawa, 2009). Many rice varieties especially Indica type are recalcitrant to transformation and are not efficiently transformed by Agrobacterium. In the present study, AtDREB1A gene under CaMV 35s promoter was successfully transferred into Sambha mahsuri (Indica type) and Cotton dora sannalu (Indica type) local rice cultivars. The transgenic plants showed enhanced tolerance toward drought stress with normal plant growth and development.

Materials and methods

Plant materials

Mature seeds of two local rice cultivars namely Sambha mahsuri and Cotton dora sannalu collected from Agricultural research station, Warangal(Dist), Telangana (state), India, were used as starting material in the present study.

Bacterial strain

AtDREB1A gene was introduced into expression vector and the recombinant binary vector, pBIHCaMV35SDREB1A, was maintained in Escherichia coli DH51α strain and mobilized to Agrobacterium tumefaciens EH105 strains. The T-DNA region of the plasmid vector is given in Fig. 1.

Figure 1

Schematic diagram of the structure of the T-DNA region in the plasmid pBIHCaMV35SDREB1A. LB left border, T-G7 terminator, hpt hygromycin phosphotransferase gene, P-NOS promoter, 35s CaMV promoter, DREB1A gene, TNOS terminator of nopaline synthase gene, RB right border.

Callus induction and regeneration

Surface sterilization of both the varieties of the mature rice seedlings was performed with clorox (50%) and were transferred to the media N6 mentioned (Table 1) fortified with 2, 3 and 4 mg/l 2,4-D. The culture tubes were kept in 16/8 h photoperiod conditions at 25 ± 2 °C for 14 days. After the specific incubation period of 14 days, seed scutellum induced embryogenic callus were transferred on to the maintenance media.

Table 1

Media used for callus induction and regeneration.

Type of media	Culture time	Media composition
Callus induction medium	14 days	N6 salts, 2,4-D (2 mg/l, 3 mg/l and 4 mg/l), casein hydrolysate 0.3 g/l, myoinositol 0.1 g/l, sucrose 3%, gellan gum agar 0.3%, pH 5.8
Callus maintenance medium	06 days	N6 salts, 2,4-D 2 mg/l, Vits., casein hydrolysate 0.3 mg/l, myoinositol 0.1 mg/l, sucrose 3%, gellan gum agar 0.3%, pH 5.8
Regeneration media	3–4 weeks	MS medium, vitamins, 0.1 gm/l myoinositol, sucrose 3%, gellan gum agar 0.3%, pH 5.8, kinetin 0.5 mg/l, NAA:BAP (RM I = 1:2, RM II = 1:3 and RM III = 1:4)

After sub culturing, the callus was aseptically transferred to various regeneration media I, II and III (Table 1) which were further kept in light (16/8 h photoperiod) conditions for four weeks. Different concentrations of hygromycin B (25 mg/l, 50 mg/l and 75 mg/l) were used in three different experiments to determine the lethal dose for the transformation experiment.

Agrobacterium mediated transformation

The plasmid construct (Fig. 1) was first maintained in E. coli and then mobilized into Agrobacterium strain EHA 105. One of the colonies was picked from the plate of recombinant Agrobacterium strain EHA105 and inoculated in LB broth enriched with kanamycin sulfate (250 mg/l). The suspension culture was incubated at 28 °C and 150 rpm for 2 days. The pellet from the culture was obtained by centrifuging at 8000 rpm. The pellet thus obtained was re suspended in a pre-induction medium (Table 1A) until it reached 0.4 OD600. The rice calli were dried for 5 min after immersion in infection and re inoculated on to the co cultivation media in the Petri plates (Table 1A). The effectiveness of three different acetosyringone concentrations (100 μM, 200 μM and 300 μM) was analyzed in the co-cultivation media. The calli were co-cultivated for 2 days at 25 ± 2 °C under light (16/8 h photoperiod) condition. Agrobacterium grown excessively were removed by growing them with cefotaxime (500 mg/l). The dried callus was transferred to selection media containing hygromycin (75 g/l) and cefotaxime. Different concentrations of cefotaxime (300 mg/l, 500 mg/l and 700 mg/l) were tested in the selection media. The plates were kept in growth chamber for one month under light (16/8 h photoperiod) condition at 25 ± 2 °C. The total number of hygromycin resistance callus was calculated in percentage to the total number of callus inoculated. The successfully proliferated hygromycin resistance cells were directly transferred to the media regeneration (Table 1A) discarding the untransformed callus. Regeneration media I (RMI) used for Sambha mahsuri and regeneration media II was used for Cotton dora sannalu. The callus produced was transferred to growth chamber under light (16/8 h photoperiod) condition at 25 ± 2 °C for about one month. The shoots thus produced were further inoculated onto a separate media for root induction (Table 1A). After the development of the roots the plantlets obtained were further inoculated into the tap water containing test tubes for acclimatization. Transfers of the transgenic plantlets to the controlled greenhouse conditions were performed and were allowed to grow further.

Table 1A

Media used for transformation.

Type of media	Culture time	Media composition
Bacterial suspension medium	2 days	LB medium, sucrose 3%, acetosyringone 100 μM, pH 7
Pre-induction medium	1 day	MS salts, sucrose 3%, acetosyringone100 μM, pH 7
Infection medium	5–10 min	Pre-induction medium (O.D600 of 0.4)
Co-cultivation medium	1–3 days	N6 salts, 2,4-D 2 mg/l, casein hydrolysate 0.3 g/l, myoinositol 0.1 g/l, sucrose 3%, acetosyringone (100 μM, 200 μM, 300 μM), gellan gum agar 0.3%, pH 5.8
Washing medium	10 min	MS basal medium, sucrose 30 g/l, cefotaxime 500 mg/l, pH 5.8
Selection medium	3–4 weeks	MS salts, 2,4-D 2 mg/l, hygromycin (75 mg/l), sucrose 30%, cefotaxime (300 mg/l, 500 mg/l, 700 mg/l), gellan gum agar 0.3%, pH 5.8
Regeneration medium	3–4 weeks	MS basal medium, sucrose 3%, BAP, NAA (RM III = SM and RM II = CDS) kinetin 0.5 mg/l, hygromycin 25 mg/l, gellan gum agar 0.3%, pH 5.8
Rooting medium	2 weeks	MS salts, sucrose 3%, NAA 1 mg/l, hygromycin 25 mg/l, gellan gum agar 3%, pH 5.8

RM = regeneration media, SM = Sambha mahsuri, CDS = Cotton dora sannalu.

Molecular analysis

PCR amplification and result analysis was performed to confirm the putative transformed species. DNA was extracted from leaves of these rice strains (DREB1A primers) targeting AtDREB1A coding sequence. The DREB1A primers 5′-CTACGCGTACGTACGATACA-3′F and 5′-TCGGCTTGGAGACTCCGAAT-3′R were used for the amplification of 632 bp fragment. The cycling parameters were pre-denaturation of 94 °C for 5 min which was followed by 1 min of denaturation and finally annealing at 50 °C for 30 s, chain extension step was performed at 72 °C for 1 min (35 cycles) and final extension stage was maintained at 72 °C for 10 min. PCR products were then analyzed by 1% gel electrophoresis in TAE buffer. The genomic DNA of putative T0 transgenic plantlets was subjected for Southern blot analysis. Approximately 10 μg of DNA from all the putatively transgenic and non-transgenic control plants were subjected for restriction digestion with the enzyme Sac1 and analyzed on 1% agarose gel. The restricted DNA was transferred to Nylon membrane and hybridized with 633 bp PCR amplified non-radioactively labeled DREB1A gene fragment. To evaluate the expression of DREB1A gene Reverse transcriptase-PCR analysis was performed for all the T1 transgenic lines. Total RNA from the plant samples was extracted using the RNA extraction kit (Invitrogen). RT-PCR analysis was carried out using Thermo script RT-PCR system (Gitam University, Department of Biotechnology, Vishakhapatnam). One of the samples was exempted from reverse transcription and was directly subjected to PCR. This sample acted as a negative control while the plasmid DNA (pBIHCaMV35SDREB1A) was used as a positive control. The same set of conditions as described earlier for PCR was used. The amplified product was purified by separating on a 1% agarose gel and analyzed under UV-Transilluminator.

Drought tolerance analysis

To test the drought stress tolerance of T1 transgenic lines during the vegetative stage, the seed of both wild strains (15 plants each, three repeats) and the positive transformed strains were grown in pots under green house conditions. The growth of tested transformed and control plants were monitored for four consecutive weeks. The fresh clean weight of the wild type plants and the transgenic species (6 plants per sample) was compared after one, two, three and four weeks. Drought stress testing was conducted in the fifth week by withholding watering of transformed and control untransformed type plants for one week followed by re watering of the plants for one week. The growth of the plants was closely recorded for the period of drought stress and the survival rate of transformed and wild control type plants were recorded after drought stress condition.

Experimental design and statistical analysis

Completely randomize design (CRD) was used for undertaking the experiments (CRD) with three treatments. All the treatments were carried out thrice in the experiments and each time there were 48 and 25 explants in the tissue culture and transformation experiments respectively. The data was statistically analyzed by two factor factorial-CRD design using Minitab software. The mean competition using LSD (LSD and DMRT) was done using MSTATC. p ⩽ 0.05 was considered significant.

Results

Effect of growth regulators on callus induction and regeneration

The current study showed that the callus induction frequency was effected by the various concentrations of 2,4-D. The two genotypes showed a different response (Table 2 and Fig. 2). The ANOVA result further showed that 2,4-D × genotype has a prominent interaction effect on callus induction in both rice genotypes at 5% probability level. Organogenesis resulted in the formation of subsequent plantlets from seed derived calli. From the green patch regions of the calli shoot bud formation was identified after 2–3 weeks. Large numbers of tender green shoots were produced from the elongated green buds within a span of a week. Significant differences were observed between the responses of the two genotypes toward different regeneration media. The Sambha mahsuri genotype showed a maximum regeneration efficiency (87.09%) on RMIII (Table 2) (regeneration media). whereas the genotype Cotton dora sannalu showed a maximum regeneration efficiency (58.43%) on RM II (Table 2) Overall, the ANOVA results showed that regeneration efficiency and plantlet regeneration were mostly influences by the media (p ⩽ 0.05) (Table 2 and Fig. 2) for culturing and genotype of the calli.

Table 2

Callus induction and regeneration frequency of two rice cultivars (Sambha mahsuri and Cotton dora sannalu).

Callus induction			Regeneration (%)
2,4-D (mg/l)	SM (%)	CDS (%)	Regeneration media	SM (%)	CDS (%)
2	39.27 ± 2.16a (81.14)	31.07 ± 1.13b (64.69)	RM I	22.57 ± 1.10c (41.08)	12.36 ± 1.75e (26.09)
3	37.29 ± 1.43ab (77.66)	34.37 ± 0.73ab (71.63)	RM II	29.67 ± 0.97b (62.64)	27.84 ± 1.76b (58.43)
4	28.07 ± 1.19b (60.48)	36.76 ± 1.46a (75.93)	RM III	42.35 ± 0.88a (87.09)	14.96 ± 0.76d (30.55)

Mean values followed by the same letter with a column shows no significant diff. (p ⩽ 0.05).

Figure 2

Callus induction and regeneration. (A) Callus induction of Sambha mahsuri. (A1) Callus induction of Cotton dora sannalu. (B) Maintenance of Sambha mahsuri. (B1) Maintenance of Cotton dora sannalu and (C) Regeneration of Sambha mahsuri. (C1) Regeneration of Cotton dora sannalu.

Determination of lethal dose of hygromycin

The lethal dose of hygromycin was determined by exposing the calli to three variable concentrations of hygromycin. Different concentrations of hygromycin showed remarkable differences in the growth of calli. Calli showed a prominent survival rate at the less hygromycin (25 mg/l) concentration, while increasing the level of hygromycin to 50 mg/l showed necrosis of the calli which can be a prominent issue for the selection of the transgenic plants. Moving above this concentration (75 mg/l) resulted in the lysis of the calli (Table 3).

Table 3

Determination of lethal dose of hygromycin for calli selection.

Hygromycin (mg/l)	Genotype	Calli survived (14 days)	Calli survived (24 days)	Range (5%)
25	SM	19.46 ± 0.47	14.83 ± 1.07	a
	CDS	15.72 ± 0.78	09.96 ± 1.23
50	SM	13.08 ± 0.49	09.80 ± 1.03	b
	CDS	09.06 ± 1.32	06.32 ± 1.13
75	SM	04.31 ± 1.00	01.36 ± 0.16	c
	CDS	02.13 ± 0.13	00.00 ± 000

Mean values followed by the same letter within a column shows no significant differences (p ⩽ 0.05).

Effect of different levels of acetosyringone

100 μM concentration of acetosyringone was used in co-cultivation media resulting in better transformation and variant regeneration in both Samba mahsuri (47%) and Cotton dora sannalu (32.73%). A decrease in the hygromycin resistance calli was prominently observed due to the elevated concentration of AS (300 μM) resulting in a decrease in the percentage of calli resistance to hygromycin at the rate of 4% and 7% decrease observed in Samba mahsuri and Cotton dora sannalu, respectively. Both genotypes of the rice responded differently to three concentrations of acetosyringone with Sambha mahsuri having the highest transformation efficiency compared to Cotton dora sannalu. ANOVA results demonstrated a significant effect on transformation efficiency and plantlet regeneration in both acetosyringone and variety while their interaction showed non-significant effect (Fig. 3).

Figure 3

Percentage of hygromycin resistant calli recovered after co-cultivation on selection media. Means that do not share a letter are significantly different.

Elimination of Agrobacterium in selection media using cefotaxime

Various concentrations of cefotaxime were tested for the elimination of bacterial proliferation in selection media. A considerable increase in the efficiency of transformation and regeneration potential were observed, when transformed calli were subjected to less concentrations (300 mg/l) of cefotaxime. The Agrobacterium was fully eliminated at the cefotaxime concentration of 300 mg/l and no overgrowth was recorded throughout the experiment (see Table 4).

Table 4

Determination of effective dose of cefotaxime for elimination of Agrobacterium.

Cefotaxime (mg/l)	Genotype	Agrobacterium contamination (%)	Range (5%)
25	SM	39.20 ± 1.34	A
	CDS	23.39 ± 0.31
50	SM	09.97 ± 0.23	B
	CDS	06.54 ± 0.37
75	SM	00	C
	CDS	00

Mean values followed by the same letter within a column shows no significant differences (p ⩽ 0.05).

Analysis of putative transgenic plants for regeneration and genetic characterization

The regenerated plantlets possessing well-developed roots and shoots were transferred into tap water containing test tube which was incubated for 4 days which was subsequently shifted to glass house for acclimatization (Fig. 4). PCR was performed to confirm the transformation in the plants (T0) followed by Southern blotting. Net transformation frequency of 33% and 19% for Sambha mahsuri and Cotton dora sannalu (Fig. 5) was confirmed by polymerase chain reaction confirmation of the transgenic plantlets. The calculation of transformation efficiency was made by checking the PCR positive plantlets’ percentage to the initial calli number inoculated in the co-cultivation experiment. The size of amplified AtDREB1A gene in transgenic plantlets was found to be 632 bp, thereby confirming the incorporation of the target gene (Fig. 6). Hybridization using Southern blot of the transformed lines showed one and two copy of the chimeric gene incorporated into the genome (Fig. 7). The expression of AtDREB1A transgene under 35s CaMV promoter (Fig. 8) was confirmed by analyzing the results of Reverse transcriptase-PCR of the T1 transgenic lines.

Figure 4

Different phases of transgenic plant development after co-cultivation. (A) Calli selection after co-cultivation. (B) Transformed calli with green spots. (C) Putative transgenic plantlet regeneration. (D) Acclimatization of transgenic plantlets under greenhouse condition.

Figure 5

Transgenic plant recovered from each hygromycin resistant calli. Means that do not share a letter are significantly different.

Figure 6

PCR Lane 1: marker, Lane 1–7: transgenic plants (SM), Lane 7–13 transgenic plants (CDS),C−ve = −VE: control plant and C+ve = +VE: positive sample (Gene construct).

Figure 7

Southern blotting of the T0 transgenic rice plants with each lane is representing the copy number of the transgenic lines. Lane 1: control, Lane 2: transgenic lines, Lane 3: transgenic lines of CDS of SM, Lane 4 and Lane 5: positive control Lane 6: negative control.

Figure 8

RT-PCR analysis of T1 independent transgenic lines: M: ladder, Lane 1–9: transgenic plants of SM and CDS (AtDREB1A amplified fragment of 632 bp), 10 and Lane 11: control plant.

The growth of both transformed and native wild type strains were normal by the measurement of fresh weight of the plants for four consecutive weeks. The fresh weight (6 plants/sample) was found similar for both the strains of plants after one, two, three and fourth weeks .The four week old transgenic lines (SM-1, SM-2, SM-3, SM-4, SM-5, CDS-1, CDS-2, CDS-3, CDS-4) and the non-transgenic wild strains of the plants were subjected to drought stress by withholding water for a period of 7 days which was later followed by re-watering for one week. Under the drought stress condition of one week, the non-transformed wild strain plants started leaf rolling and wilting on the 3rd day of drought exposure, whereas the transformed plant strains remained healthy and showed a normal growth. The Complete wilting and drying was shown by the wild strains after one week of water withhold whereas the transgenic plants showed minor symptoms of drought induced damage. After re-watering, the plantlets were recovered after one week of stress while the controlled non transgenic plants died (Table 6). The AtDREB1A over expressing plants showed a significantly increased drought tolerance in contrast to the wild type plants. The survival rate of transgenic plants were significantly high (100%) compared to the wild-type control (0%) after drought stress. These results suggest that AtDREB1A over expression under the 35s CaMV constitutive promoter enhanced the tolerance rate among rice and were found stably integrated as apparent from the RT-PCR results.

Table 6

Reverse transcriptase-PCR analysis of transgenic rice lines and the survival rate of transgenic rice lines after one week of drought stress.

Transgenic lines	Reverse analysis	Transcriptase-PCR Survival rate of transgenic rice lines after drought stress
Control	−	Died
SM (1)	+	Survived
SM(2)	+	Survived
SM(3)	+	Survived
SM (4)	+	Survived
SM (5)	+	Survived
CDS (1)	+	Survived
CDS (2)	+	Survived
CDS (3)	+	Survived
CDS (4)	+	Survived

+ Represents RT-PCR positive transgenic plants and − represents RT-PCR negative control plants.

Discussion

In the present study, optimization of an effective protocol for genetic transformation using Agrobacterium for the gene AtDREB1A of two rice genotypes was performed. Further, the transformed rice strains obtained were tested for drought stress tolerance. The optimization of tissue culture conditions is the preliminary step for an efficient Agrobacterium mediated transformation experiment (Hiei and Komari, 2008). In this regards, different factors like type of explants, genotype and nutrient media supplemented with different concentrations of hormones play a crucial role in the success of transformation (Yookongkaew et al., 2007, Sahoo et al., 2011). The two rice genotypes showed different callus induction frequencies on media containing three different concentrations of 2,4-D i.e. Sambha mahsuri and Cotton dora sannalu showed better callus induction frequency at a concentration of 2 mg/l and 4 mg/l, respectively. Previous studies revealed that the response of the individuals toward callus induction depends on its genotype (Lin and Jhang, 2005, Ge et al., 2006). Subsequently, the callus obtained from the seeds was subjected to different hormonal combinations represented by RMI, RMII and RMIII (NAA:BAP) for regeneration. The regeneration media RM III was found effective for Samba mahsuri derived calli with regeneration efficiency of 89% and maximum plantlets were produced. The results of the current research were acceptable with the previous studies (Lee et al., 1999, Yang et al., 2000). Cotton dora sannalu genotype showed a maximum regeneration efficiency of 36% and high numbers of plantlets were produced on RM II. The regeneration/production of complete plantlets from transformed callus proves to be a major constrain in Agrobacterium mediated genetic transformation of rice (Rachmawati et al., 2004, Kumar et al., 2005). The stringency of the selection process in which minimum calli escaped, lies in the hygromycin concentration at 50 mg/l which was proved to be optimum for the selection process but it has a less negative effect on plantlet regeneration. Hygromycin at 50 mg/l was also used for the first successful Agrobacterium-mediated transformation of Indica and Javanica rice cultivars (Dong et al., 1996, Rashid et al., 1996). Our team was successful in eliminating Agrobacterium at a lower dose of 500 mg/l after infection. The regeneration of plantlets was found to be inhibited at a high concentration of cefotaxime (Nauerby et al., 1997, Rashid et al., 1996). The importance of acetosyringone to Agrobacterium co-cultivation procedures was inhibited at high concentrations of shown in various studies. (Rashid et al., 1996, Gould, 1997, Ali et al., 2007). The success in the Agrobacterium mediated transformation differs greatly at various concentrations of acetosyringone and the interaction of it with various genotypes. A maximum transformation efficiency was found in co-cultivation media containing 100 μM acetosyringone for both the rice genotypes i.e. 47% for Sambha mahsuri and 32.73% for Cotton dora sannalu (Fig. 3). The current results are comparatively similar and in association with the previous studies (Ali et al., 2007, Bernal et al., 2009). A low transformation efficiency was observed under high concentrations of acetosyringone in agreement with the previous study (Amoah et al., 2001). Analysis based on amplicon of PCR led to the confirmation of the transgenic population with 19% for Cotton dora sannalu cultivars and 33% efficiency for Samba mahsuri. The effectiveness of transformation differs with the genotype, type and age of explants (Hiei and Komari, 2008). One or two copy numbers were found in the Southern blot analysis of all the PCR positive transgenic plants. We have successfully introduced DREB1A gene in two rice cultivars Sambha mahsuri (Indica) and Cotton dora sannalu (Indica) by Agrobacterium mediated transformation. There is a high dependency on the genotype of the plantlets for the successful transformations particularly in Indica type cultivars and development of protocol for gene transformation techniques using Agrobacterium will pave the way for transgenic rice development in the future. The active expression of chimeric transformed gene in T1 transgenic plants was confirmed by RT-PCR analysis. The T1 transgenic plants showed normal growth and both transformed and control species were similar with the same growth rate that was confirmed by weighing their fresh weight for four consecutive weeks (one week interval) (Table 5). Oh et al., 2009 revealed a growth in stunted form in rice plants after the overexpression of the transgene with 35s CaMV promoter. DREB transcription factors play an important role in the stress tolerance pathways independent of ABA gene which induces the expression of a large number of stress genes in plants (Lata and Prasad, 2011). We have developed transgenic plants of two rice cultivars Sambha mahsuri (Indica) and Cotton dora sannalu (Indica) overexpressing DREB1A gene. The T1 generation was tested at their vegetative stage for successful transformation and drought stress survival. Several studies conducted on drought stress of the transgenic plants at their vegetative stage (Tang et al., 2012, Datta et al., 2012). Overexpression of transcription factors (AP37 and AP59) in rice under constitutive promoter control resulted in an increased tolerance to drought at the vegetative stage (Oh et al., 2009). Some transcription factors (AREBs), of drought stress, function mostly in vegetative stage (Kim et al., 2004, Fujii et al., 2011). The transgenic rice lines of 28 growth days were subjected to drought stress condition for one week and then re-watered for the upcoming 7 days. The transgenic lines showed a complete recovery after drought stress and showed a high tolerance rate. Comparatively the wild strains showed high wilting and completely died (Table 5). The transformation experiment also revealed that the survival rate was drastically increased in the transformed strains than the wild type. Previously, over-expression of several DREB genes in various plant species like rice (Oh et al., 2005, Chen et al., 2008, Datta et al., 2012, Ravikumar et al., 2014), Arabidopsis (Liu et al., 1998, Qin et al., 2007, Matsukura et al., 2010), wheat (Pellegrineschi et al., 2004, Shen et al., 2003) etc. has resulted in drought resistant plant strains.

Table 5

Fresh weight of transgenic and controlled plants (2 plants/sample) for four consecutive weeks in glass house at normal condition.

Time period sowing (weeks)	After seed fresh weight in gm (control plants)	Fresh weight in gm (transgenic plants)
1	0.47 ± 0.037	0.44 ± 0.016
2	1.12 ± 0.027	0.98 ± 0.029
3	2.03 ± 0.042	1.97 ± 0.031
4	3.74 ± 0.029	3.29 ± 0.028

Conclusions

The current study concluded by optimization of Agrobacterium mediated genetic transformation system in two different local rice cultivars i.e. Indica rice cultivars. Stable transgenic line with control normal plants growth and development were produced. The transgenic rice lines with strong constitutive expression of AtDREB1A transcription factor resulted in enhance tolerance toward drought stress. After fixing the trait in next generations, these lines might prove to be better candidates for developing drought resistant local rice cultivars in the future.