Screening and Integration Analysis of OsDREB 1 A BC 4 F 2 and BC 5 F 1 Generations of Transgenic Ciherang Rice ( Oryza sativa L . ) for High-Salinity Tolerance

Salinity is one of a number of abiotic stresses tha t reaten rice production in Indonesia. To support f od security programs, BB-Biogen has developed rice lines derive d from OsDREB1A transformed Ciherang up to BC4F2 and BC5F1 generations. To verify the salinity tolerance and the stability of transgene integration, the BC 4F2 and BC5F1 generations of CiherangOsDREB1A transgenic were screened to select high-salinity t olerant lines. Second, molecular analysis using the primers hptII-F/hptII-R and 35S-496-F/ OsDREB1A-R was conducted to identify the existence and the stability of the transgene integration in the BC4F2 and BC5F1 generations. Screening 543 BC4F2 and BC5 F1 CiherangOsDREB1A transgenic lines in a nutrient solution with a fin al electrical conductivity (EC) of approximately 18 mS/cm for 26 days yielded 134 putative transgeni c plants. Integration analysis using the ptII-F/hptII-R primers showed that 73 of the 134 putative transgenic plant s had positive PCR products, indicating the presenc e of the transgene in those plants. All the 73 plants also produced PC R products when tested with the specific primer 35S -496F/OsDREB1A-R, indicating that transgene integration was maint ained during the development of BC4F2 and BC5F1.


Introduction
Rice is the major staple crop in Asia, where approximately 90% of the world's rice is produced and consumed [1][2].Attempts to increase rice production are needed to cope with the increase in the human population.However, these attempts face many obstacles, including biotic and abiotic stresses, such as salinity [3].Salinity influences the productivity and the quality of agricultural crops [4][5][6].High concentrations of dissolved salts in agricultural land interferes with the physiological functioning of plants and leads to decreased crop production [7][8].Genetically modified crops with overexpression of transcription factor genes that increase plant tolerance to stress have been developed, and genes induced by abiotic stresses, such as high salinity, drought, and cold, have been discovered and published in various scientific journals [9][10][11].The role of the dehydration responsive element binding type 1A (DREB1A) gene in improving crop tolerance to abiotic stress has been studied [12][13][14][15][16][17][18].DREB1A plays an important role as a transcription factor in the regulation of plant responses to abiotic stress by inducing other genes associated with tolerance to abiotic stresses, such as high salinity [12][13][14], drought [15][16], and cold [17][18].
Given the important role of OsDREB1A in improving crop tolerance to abiotic stress, especially high salinity, BB-BIOGEN researchers used the gene to develop Ciherang-transgenic-OsDREB1A.They first isolated the OsDREB1A gene and constructed a pCAMBIA-OsDREB1A plasmid.The OsDREB1A transgene construct was inserted in the Nipponbare rice genome using Agrobacterium-mediated transformation [19].Although Nipponbare rice is not commonly grown by farmers in Indonesia, it was used in gene transformation because it shows good transformation and regeneration.Nipponbare-transgenic-OsDREB1A rice crossed with Ciherang offers superior quality and dominates farmland in Indonesia [20].The F1-Ciherang transgenic-OsDREB1A lines were then followed with a series of back-crosses to restore the genetic background of Ciherang.Thus far, five back-crosses the F1-Ciherang transgenic-OsDREB1A lines have been done and seeds of the BC5F1 generations have been obtained.In addition, self-pollination (selfing) of the BC4F1 generation also have been done and seeds of the BC4F2 generations have been obtained.To verify the salinity tolerance and the stability of the transgene integration, Ciherang-OsDREB1A transgenic lines that showed tolerance to high salinity were screened.Molecular analysis was then performed to identify the existence and the stability of the transgene integration in the BC4F2 and BC5F1 generations.

Screening of tolerance of rice lines to high salinity.
Screening of high-salinity tolerance was done in a hydroponic system using a salinized nutrient solution.The methods following the standard of the International Rice Research Institute (IRRI) used by Gregorio et al. [21] with a modification of using a conventional greenhouse instead of a Phytotron chamber.Styrofoam seedling floats were used in the setup (Figure 1).They comprised rectangular Styrofoam 40 × 30 × 2.5 cm with 66 holes (6 holes × 11 holes ) and a nylon net bottom.The float was placed in a rectangular 42 × 32 × 16 cm plastic tray with a 17 L capacity.
The seeds were placed in petri dishes with moistened filter papers for 5 days to germinate.The seedlings were then transferred to the seedling float in a tray containing normal nutrient solution adapted from Yoshida et al. [22] for up to 14 days after germination to allow sufficient growth before salinization.Salinization began 14 days after seeding.The nutrient solution was salinized by adding 3 and 6 g of NaCl to give an electrical conductivity (EC) of 6 and 12 mS/cm, respectively.The initial salinity was EC = 6 mS/cm.Three days later, the salinity was increased to EC = 12 mS/cm by adding NaCl to the nutrient solution.The nutrient solution was not aerated and renewed every 8 days, and a pH of 5.0 was maintained daily.The test entries were assessed at 10 and 16 days after the initial salinization.The IRRI standard evaluating score was used to rate the visual symptoms of salt toxicity (Table 1).
Transgene integration analysis.Analysis of transgene integration was performed on the plants categorized as salinity tolerant (putative transgenic).DNA was isolated   by following the CTAB extraction method [23].The hptII-F/hptII-R primer and the primer combination 35S-496-F/OsDREB1A-R were used to analyze the transgene integration of OsDREB1A.The HptII-F primer (5' GAT CGA GCT GCC TCC AGC AGT G 3') and the hptII-R primer (5 'GCA TCT GCC ATG GTG CAC 3') were used for amplification of the hygromycin sequence, which is a selection marker of the OsDREB1A transgene inserted into the plant genome.The composition of PCR was as follows: 2 µL of DNA template (100 ng/µL), 2 µL of PCR buffer (10× conc.), 1.2 µL of MgCl 2 (25 mM), 0.4 µL of dNTP (each 10 mM), 1 µL of hptII forward primer (10 µM), 1 µL of hptII reverse primer (10 µM), 0.16 µL of Taq DNA polymerase (5U/µL), and 12.24 µL of nuclease-free water for a final volume of 20 µL.The PCR conditions were an initial denaturation at 94 °C for 5 minutes, followed by 35 cycles of a denaturing step at 94 °C for 30 seconds, an annealing step at 67 °C for 30 seconds, and an extension step at 72 °C for 30 seconds.A final extension step at 72 °C for 5 minutes was included after the 35 cycles.
Plants that tested positive in the PCR analysis of hptII-F/hptII-R were used to analyze the transgene integration using the gene-specific primer OsDREB1A combined with the 35S promoter-496 primer (35S-496-F/OsDREB1A-R primer).The OsDREB1A-R primer (5' ACA GTC GAC ACT TGT TCC ATC ACA TTA CCG A 3') was attached to the OsDREB1A sequence.The 35S-496 primer (5' CCA CTA TCC TTC GCA AGA CC 3') was attached to the base 496 of the 35S promoter sequence.The 35S promoter was not present in the nontransgenic plants, and these plants did not test positive in the PCR analysis.The composition of PCR was: 4 µL of DNA template (100 ng/µL), 2 µL of 10× PCR buffer with MgCl 2 , 0.4 µL of dNTP (each 10 mM), 2 µL of GC-Rich (5× conc.), 1 µL of 35S-496-forward primer (10 µM), 1 µL of 35S-496 reverse primer (10 µM), 0.16 µL of Taq DNA polymerase (5 U/µL), and 10.24 µL of nuclease-free water for a final volume 20 µL.The PCR conditions were an initial denaturation at 95 °C for 3 minutes, followed by 35 cycles of a denaturing step at 94 °C for 1 minute, an annealing step at 55 °C for 1 minute, and an extension step at 72 °C for 1 minute 30 seconds.A final extension step at 72 °C for 7 minutes was included after the 35 cycles.

Results and Discussion
Screening of high-salinity tolerance.Screening the BC4F2 and BC5F1 generations of the OsDREB1A-Ciherang lines for salinity tolerance for 26 days resulted in 134 tolerant plants of a total 543 tested plants at a final level of EC of about 18 mS/cm (Table 2).The screening period was extended to 26 days, and the EC was increased to 18 mS/cm because the putative transgenic rice plants were not selected properly after 16 days at an EC of 12 mS/cm.At day 16, susceptible control (IR29) with score 3 still observed, so the tested plants with score 3 at day 16 suspected were not properly selected.The screening period was extended until the susceptible control plant observed had score 7 or 9 based on IRRI standard evaluating score.
A score of 3 indicates plants that are tolerant to salinity stress according to the IRRI standard evaluating score of visual symptoms due to salt toxicity at the seedling stage.At the end of the screening period, IR 29 (susceptible controls) had a score of 7-9, Pokali Putative transgenic HT = Highly tolerant; T = Tolerant; M = Moderately tolerant; S = Susceptible; HS = Highly susceptible (tolerant control) had a score of 3-5, and the Ciherang wt.control (nontransgenic) had a score of 5-9 (Figure 2).The BC4F2-K14-35-4 lines (64.9%) showed the highest tolerance to high salinity, and the BC5F1-K13-47-3 lines (3.5%) showed the lowest tolerance.The percentage of tolerant rice lines to high-salinity stress after screening is shown in Table 2.
The BC4F2 generation of the OsDREB1A transgenic rice lines tended to have a higher percentage of salinitytolerant plants compared to the BC5F1 generation.The greater salt tolerance is probably due to the greater presence of the OsDREB1A construct in the BC4F2 generations (progeny from self-pollination) compared to the BC5F1 generation (progeny from back-cross) (Figure 3).
According to Mendel's laws, in self-pollinating heterozygous plants (F1), three-quarters of the progeny plants of the F2 generation should have genes inserted.When heterozygous plants (F1) are back-crossed with Ciherang wt., one-half of the progeny of the resulting population should have the gene inserted [24].

Figure 2. Conditions of Plants at the End of the High-Salinity Screening
Selfing Back-cross (AA) (Aa) (aa) (Aa) (aa)

Figure 3. Variations in Genetic Make-up Following Selfing and Back-crossing
Transgene integration analysis.The polymerase chain reaction technique was used to determine the stability of the transgene integration in the transgenic plants.Individual BC4F2 and BC5F1 plants that can tolerate high salinity (score of 3) were then subjected to molecular analysis to detect the presence of the OsDREB1A transgene.Isolation of genomic DNA from 134 putative transgenic rice plants was performed as a first step in the molecular analysis.The purity and the concentration of the DNA were checked using a NanoDrop 2000 Spectrophotometer.A DNA sample with a concentration of 100 ng/µL was used as a PCR template to ensure that differences in the thickness of the bands formed from the PCR were not due to variations in the concentration of the DNA.
Gene integration in the transgenic plants was confirmed by PCR analysis using the hptII (hygromycin phosphotransferase) primer.Hygromycin is a selection marker gene on plasmid pCAMBIA.It is located in the T-DNA region and is inserted in tandem with the OsDREB1A gene in the plant genome.The results of the PCR using the hptII primer indicated that not all tolerant plants were positive for the hptII gene.Positive amplification using the hptII primer was indicated by the formation of bands of approximately 500 bp.Integration analysis using the hptII-F/hptII-R primers showed that 73 of the 134 salt-tolerant plants had positive PCR products.The presence of individual BC4F2 and BC5F1 putative transgenic generations with negative results on the PCR analysis using hptII showed that some nontransgenic plants were not detected in the high-salinity screening stage.The percentage of (+) hptII-F/hptII-R plants is shown in Table 3.
Various reasons may explain the escape of nontransgenic plants in the high salinity-screening in the current study.First, the use of a conventional greenhouse in this study likely led to fluctuations in the day/night temperature and the minimum relative humidity during the day.Fluctuations in these two factors throughout the time of screening cannot be controlled in a conventional greenhouse, and they can result in suboptimal screening of high salinity in plants [25].According to Gregorio et al. 1997 [21], adverse effects of salinity decrease with low temperature and high humidity.Research conducted by BB-BIOGEN in 2010 on salinity screening of the transgenic rice Nipponbare-OsDREB1A in a conventional greenhouse produced similar results, with putative transgenic rice plants testing negative for hptII.Second, the nontransgenic plants may have escaped detection due to the ability of the plant to induce an internal defense against abiotic stress during their vegetative growth phase.Several studies showed that rice plants are relatively more tolerant to salinity at germination, become more sensitive during transplanting as young seedlings (2-3 leaves), increase tolerance during the vegetative growth phase, become intolerance during the flowering phase, and then return to be more tolerant to salinity during the adult phase [21,[26][27][28].In consideration of the sensitive period, the screening of high salinity in this study began 14 days after the rice had germinated when the plants were about 15 cm high and had 2-3 leaves (Figure 4).
Observations showed that some of the nontransgenic plants thought to have been affected by high-salinity stress at the seedling stage when they had 2-3 leaves survived and underwent normal growth during the vegetative phase and until the end of the extended period of high-salinity screening.As noted earlier, this may be due to an increase in the plant's internal defense to salinity stress.Third, the nontransgenic plants may have escaped detection because the high-salinity screening period of 26 days was not sufficient to suppress the growth of some of the plants, especially those that had progressed beyond their sensitive period.
The plants that tested positive for hptII can also be expected to show overexpression of the OsDREB1A construct because the hygromycin selection marker gene (hptII) and the OsDREB1A gene were included in a single T-DNA construct (Figure 5).Further analysis of transgene integration was performed using the OsDREB1A gene-specific primer and the 35S-496 primer developed from the promoter (primer 35S-496-F/OsDREB1A-R).This strategy was used because a The analysis of transgene integration using primer combinations was performed for 73 individual hptIIpositive plants.The percentage of (+) 35S-496-F/OsDREB1A-R plants is shown in Table 4. Positive amplification using the 35S-496-F/OsDREB1A-R primers was indicated by the formation of bands of approximately 1000 bp (Figure 6).
Reconfirmation by PCR was performed using the same primers on individual PCR-negative plants to confirm the transgene integration (plant no.62, 86, 96, 111, 122, and 123).The results showed that all six plants possessed the 1000 bp region of 35S-496-F/OsDREB1A-R. Based on this result, it appears that all transgenic plants that have a PCR band when amplified using hptII-F/hptII-R primers also produce a PCR band using 35S-496-F/OsDREB1A-R primers.It can be concluded that the stability of the transgene is maintained during selfing and back-crossing.

Conclusions
Screening of the high-salinity tolerance of transgenic Ciherang-OsDREB1A BC4F2 and BC5F1 lines successfully selected 134 salt-tolerant plants from a total of 543 test plants.However, not all tolerant plants harbored the transgene, with 73 of the 134 tolerant plants showing a positive PCR band using hptII-F/hptII-R primers.All the transgenic plants that showed a positive PCR band using the hptII-F/hptII-R primer also produced a PCR band when using the 35S-496-F/OsDREB1A-R primer, indicating that the stability of the transgene was maintained during the development of BC4F2 and BC5F1.

Figure 1 .
Figure 1.Seedling Floats for Salinity Screening at the Seedling Stage.