Plant Materials and Growth Conditions
Thirteen fragrant and six non-fragrant cultivars from across China were used to determine the allelic variation of OsBADH2 locus. The plants were grown in farm's field at the Sichuan University. Primary transformants (T0) were first planted in the artificial climate incubators (BINDER, Tuttlington, Germany) under standard conditions (28°C day, 20°C night; 12 h light, 12 h dark), and transplanted into the field 5 weeks later. Wild type and the transgenic progeny plants were grown side by side in farm's field at the Sichuan University. After harvesting, the T1 stubs were transferred into pots and grown in a growth chamber (25°C, 12/12 h photoperiod at 200 μmol photons m-2 s-1). Tillers were regenerated from the stubs. To investigate the germination and seedling phenotypes, wild type and T2 seeds derived from the T1 transgenic plants were germinated under various concentrations of NaCl in the growth chamber.
Plasmid Construction and Rice Transformation
DNA manipulations were carried out by using standard procedures (Molecular Cloning). Sequences from OsBADH2 cDNA (GenBank accession no. AK071221) were amplified by RT-PCR for construction of CaMV35S-OsBADH2-RNAi vector. An inverted-repeat fragment was constructed into vector pSK int  and transferred into pHB (driven by 2 × CaMV35S promoter ) at the BamH I and Xba I restriction sites by using PCR with primers (B2IF1: 5' GCTCTCGAGTCTAGACCAATGGCCAGATTTGCAGT 3'; B2IR1: 5' GCACAAGCTTTGCGAGCAGTTCACCCAGAT 3'; B2IF2: 5' GGTGGATCCCCAA TGGCCAGATTTGCAGT 3'; B2IR2: 5' CACGAATTCTGCGAGCAGTTCACCCAGAT 3') introducing unique restriction sites at the product ends.
Transgenic plants were generated by Agrobacterium tumefaciens-mediated transformation according to the described procedure , and transformed lines were first selected for hygromycin (50 mg L-1) resistance and then analyzed by PCR to determine the presence of T-DNA integration. Primers designed to the hygromycin phosphotransferase gene (hpt) of pHB for confirmation of integration are 5' TAGGAGGGCGTGGATATGTC 3' and 5' TACACAGCCATCGGTCCAGA 3' (GenBank accession No.E00777). PCR was performed by using Taq DNA Polymerase (Takara, Dalian, China) in MJ Mini™PCR (BIO-RAD, Hercules, California, USA), following the instruction given by the manufacturer.
Genomic DNA was extracted from mature rice leaves to determine the allelic variation of OsBADH2 locus in different varieties (Table 1). A 463 bp portion of OsBADH2 gene was amplified by PCR with primers designed for the OsBADH2 (5' ACATAGTGACTGGATTAGGTTCTG 3' and 5' CATCAACATCATCAAACACCACT 3'). This region of the gene includes the previously identified mutations putatively responsible for the aroma production . PCR products were cloned into the pMD18-T Vector (Takara, Dalian, China). Cycle sequencing was performed with the ABI Prism BigDye Terminators v2.0 cycle sequencing reaction kit (Applied Biosystems, Foster City, California, USA). Sequences were determined with an ABI Prism 377 genetic analyzer (Applied Biosystems). The sequence analysis was performed using the software DNAMEN v5.0 (Lynnon Biosoft Inc., Vandreuil, Quebec, Canada).
For semi-quantitative RT-PCR, total RNAs were extracted using Trizol reagent following the protocol provided by the manufacturer (Invitrogen, Carlsbad, CA) and treated with DNase (TaKaRa, Dalian, China). About 1 μg of total RNA from each sample was used for first-strand cDNA synthesis (Toyobo, Osaka, Japan). ACTIN gene was employed as positive internal control . Primers used for the RT-PCR analysis were designed for the ACTIN (5' AAGATCCTGACGGAGCGTGGTTAC 3' and 5' CTTCCTAATATCCACGTCGCACTTC 3'), and OsBADH2 (5' CCAATGGCCAGATTTGCAGT 3' and 5' TGCGAGCAGTTCACCCAGAT 3'), OsBADH1 (5' TGCGAACGCTGGTCAAGTCT 3'; 5' ATCACAGCGCCAGCTAGACC 3'). For real-time quantitative RT-PCR, total RNAs were treated with DNase (TaKaRa, Dalian, China) and about 1 μg of total RNA from each sample was used for first-strand cDNA synthesis (Toyobo, Osaka, Japan). Primers for real-time quantitative RT-PCR were designed for OsBADH2 (5' TGAAGCCGGTGCTCCTTTGT 3'; 5' CACTATAGGACTTTTTCCACCAAG 3'), OsBADH1 (5' GGTCAAGCCTGTTTCGTTAGAG 3'; 5' CAACCAACCTATCCAAGAATCGCT 3'), and the control ACTIN (5' ACCTTCAACACCCCTGCTAT 3'; 5' CACCATCACCAGAGTCCAAC 3'). The real time quantitative PCR was carried out in a total volume of 25 μL containing 12.5μL of SYBR® Premix Ex Taq TM (TaKaRa, Dalian, China), 0.2 μM of each primer, and 7.5 μL of 1:65 diluted cDNA. Thermal cycling consisted of a hold at 95°C for 40 seconds followed by 40 cycles of 95°C for 5 seconds, 57°C for 10 seconds and 72°C for 15 seconds. After amplification, samples were kept at 95°C for 1 min and 55°C for 1 min. Then the temperature was raised gradually by 0.5°C every 10 seconds to perform the melt-curve analysis. Each sample was amplified in triplicate and all PCR reactions were performed on the iCycler®PCR system (BIO-RAD, Hercules, California, USA). REST software  was used to quantify the OsBADH2 and OsBADH1 mRNA levels with ACTIN normalization by the 2-Ct method. To confirm the specificity of the PCR reaction, PCR products were electrophoresed on 1% agarose gel to verify accurate amplification product size.
Sensory aroma evaluation
Determination for the presence or absence of aroma was made according to previously described methods . Basmati370 was used as a control for the sensory aroma evaluation. At tillering stage of transgenic plants, 2 g fresh leaves were excised from individual plants, cut into 5 mm long pieces and kept in petri dishes mixed with 10 ml of 1.7% potassium hydroxide (KOH) solution. The petri dishes were kept under room temperature for about 10 minutes. They were then opened one by one, and the samples were sniffed and scored for presence or absence of aroma emission. To confirm the presence or absence of aroma, leaf tissue from tillers regenerated from stubs of T1 plants were soaked in KOH solution and evaluated for the aroma emission. In addition, about 20 grains harvested from T1 plants were placed into 5-ml screw-cap tube containing 1 ml of fresh water and were then incubated at 65°C for 2 hours. The aroma was evaluated after storage at 4°C for 20 min . All the samples were sniffed by three well-trained panelists. The samples were classified into two categories in presence or absence of aroma.
Gas chromatography-mass spectrometry (GC-MS)
The hulled rice grains and/or mature leaves from the OsBADH2-RNAi transgenic and wild-type japonica cv. Nipponbare were collected, and Thai Hom Mali 105, a commercial aromatic variety from Thailand purchased from a retail store, was used as control . The 2, 4, 6-trimethylpyridine (TMP, Sigma Aldrich Chemical Co., Germany) was used as an internal standard [39, 40]. It was dissolved in a precisely measured volume of 0.1 M HCl to give an internal standard solution with 2.00 ppm concentration of TMP. The milled rice grains/leaves (30 g) were added to a 250 mL flask containing 120 mL of internal standard solution. The mixture was stirred for 2 h before filtration. 9 mL of 1.0 M NaOH was added to the filtrate to make the solution slightly basic, then transferred to two 50 mL centrifuge tubes and centrifugated at 8000 rpm for 10 min. About 80 mL of the supernatant liquor was transferred to a 250 mL pear-shaped separatory funnel. Then 120 mL of dichloromethane was immediately added as an organic solvent. The extraction was conducted twice, resulting in 240 mL of dichloromethane solution. After drying with anhydrous sodium sulfate, the extract was concentrated to 1 mL using a rotary evaporator under reduced pressure and a temperature of 26°C. The concentrated extract was transferred to a tube and left open to the air at room temperature until its volume decreased to 0.1 mL, from which 1 μL was taken for qualitative analysis.
Samples were determined on a GC/MS system (GC-MSQP2010W, Shimadzu, Japan). Helium gas (purity 99.999%) at a pressure of 80 Kpa was used as the GC carrier gas. The injector and the GC/MS interface temperatures were set at 170°C and 250°C, respectively. The temperature of Rtx-wax capillary colum (30 m × 0.25 mm id, film thickness 0.25 μm, Restek, Bellefonte, PA) was programmed starting at 40°C after injection of samples. With the initial temperature of 40°C held for 2 min, it was ramped to 80°C at 6°C/min. After hold for 1 min in 80°C, it increased to 120°C at a rate of 4°C/min, then mounted up to 200°C at 8°C/min and held there for 20 min. The effluent from the capillary column went directly into the masspectrometer, operated in the electron impact (EI) mode with an ionization voltage of 70 eV, and the ion source temperature was 200°C [39, 40].
Agronomical trait measurements
To investigate the effects of the disrupted OsBADH2 on crop performance, agronomical traits including plant height and 1000-grain weight were measured by using T2 fragrant/non-fragrant segregated progeny plants derived from four independent primary transformants.
Evaluation of germination and seedling growth rates
To compare grain sprouting rates between wide type and OsBADH2-RNAi repression lines, 50 grains from each of three wild type controls (WTa, b and c) and four OsBADH2-deficient lines (OsB2-Ra, b, c and d) were placed and incubated in Petri dishes containing two pieces of filter paper moistened with 0, 50, 100 mM NaCl for 8 days. Sprouting index (∑RGT/t, RGT, the number of spouted grains in the t day) and germination rates (G/N × 100%, G, the number of spouted grains; N, the total number of grains) were measured.
For seedling growth rate experiments, 30 grains from each of three wild type controls (WTa, b and c) and four OsBADH2-deficient lines (OsB2-Ra, b, c and d) were incubated in distilled water for 7 days, and subsequently subjected to different salt concentrations (0, 50, 100 mM NaCl) for another 7 days. The length of shoots and roots, the number of roots and the fresh weight of shoots and roots were examined, respectively.