Explant material for transformation
Seeds were obtained from the J. curcas (Jc-MD) elite plants which were pre-selected by Y Hong and C Yi . The seeds were germinated on1/2Murashige and Skoog salt medium. Cotyledons were harvested from five- to seven-day-old seedlings, cut into small pieces (5 × 5 mm) and used as explants.
The detailed transformation protocol has previously been described . In brief, the protocol consisted of four steps; co-cultivation, shoot regeneration, shoot elongation and rooting.
Small cotyledons pieces were incubated with Agrobacterium cells harboring the target expression cassette in 20 mL of medium II for 10 to 20 minutes at 25°C. Explants were then transferred to the co-cultivation medium for two to three days at 22°C in the dark. Then, the co-cultivation explants were rinsed several times with sterile water followed by one wash with 300 mg/L cefotaxime. The cotyledon tissues were blotted dry on a pad of sterilized paper to remove excess surface water. The explants were then placed on a callus formation medium and transferred to darkness at 25 ± 1°C for three weeks. Under this condition, the untransformed explants usually turned brown.
Explants with newly emerged hygromycin-resistant callus were transferred into shoot regeneration medium I for three weeks at 25°C under 16-hour light (100 μmol/m2/s)/8-hour dark cycles. During this period, any shoots regenerated from callus (about 35% to 40%) would be transferred to shoot regeneration medium II. The transformed hygromycin-resistant regenerated shoots about 2 to 3 mm were transferred to β-estradiol induction medium without hygromycin to induce marker excision. After two weeks of induction, shoots were transferred back to regeneration medium II without hygromycin. Any callus with no regenerated shoots were transferred to shoot regeneration medium III for further regeneration.
After four weeks, the regenerated shoots were transferred into shoot elongation medium for elongation and bud multiplication
Elongated shoots of about 2.5 cm were rooted on a rooting medium. Normally, more than one month was required to obtain roots. An alternative method was to use grafting to increase the plant survival rate. Elongated shoots were used as scions for grafting onto non-transgenic rootstocks. Healthy and vigorously growing Jatropha plants were chosen to be rootstocks. Both scions and rootstocks were cut into the cambium region so that phloem tissues from both parts connected after joining. The graft joint was then wrapped with parafilm and secured with tape. The grafted Jatropha plants were maintained under low light intensity (10 to 20 μmol/m2/s) and 85% humidity for seven days.
Transgenic plasmid construction and materials
The pCAMBIA1300-derived vector which carried a 35S-GFP gene cassette was used for Jatropha transformation. Transgenic lines (called 35S:GFP lines) were confirmed using gene markers, fluorescence and protein gel analysis.
To generate the β-estradiol chemical-regulated inducible JcFAD2-1 RNAi lines, we used a gene-specific 862-bp fragment corresponding to the coding region (nt 85 to 946) of the JcFAD2-1 cDNA. This cDNA fragment was PCR-amplified with forward primer 5'-ATCACTCGAGCCACCATTCACACTTGGTCAG-3' and reverse primer 5'-GTATAAGCTTCATGAGTGTCTGTAATGTTATG-3'. The PCR fragment was inserted in the sense orientation into the XhoI/HindIIII sites of a pSK-int vector as described previously . Another fragment, amplified with forward primer 5'-CAATAACTAGTACCATGGGTGCCGGTGGCAGAATG-3' and reverse primer 5'-TATTGGATCCGGAAACTTGTTTTTGTACCAGAACAC-3', was subsequently placed in the antisense orientation into the BamHI/SpeI sites of pSK-int-sense FAD2-1 to form pSK-int-FAD2-1 RNAi. Finally, the entire RNAi cassette comprising the sense and antisense fragments interspersed by the actin II intron was excised from pSK-int using the flanking XhoI/SpeI sites and inserted into the XhoI/SpeI site of pX7-GFP vector, yielding the construct pX7-FAD2-1i.
To generate seed-specific JcFAD2-1 RNAi lines, we replaced the G10-90 promoter in pX7-GFP with the soybean seed storage protein 7S gene promoter. The resulting vector was called pX8-GFP. The entire FAD2-1 RNAi cassette in pSK-int vector was inserted into pX8-GFP to substitute the GFP coding region and hence the construct pX8-FAD2-1i was formed.
Transformants were selected using PCR-based genotypic analysis with either P1 and P4 primers (for X7-FAD2-1 RNAi lines) or P7S and P4 primers (for X8-FAD2-1 RNAi lines), together with primer pairs for the HPT gene. Additional file 5 shows the sequence information of these primers. Independent lines (X7#79, X7#170 from X7-FAD2-1i; X8#34, X8#291 from X8-FAD2-1i) were confirmed by the analysis of the expression of FAD2-1 and fatty acid composition in endosperms of individual seeds. Plants were grown in a greenhouse under natural photoperiods and ambient temperature (ranged from 25 to 35°C) in Singapore.
Fatty acid analysis
Total lipid was extracted from 100 mg fresh Jatropha leaves as previously described . The outer seed coat was removed from dried Jatropha seeds. The seeds were surface sterilized for 1 minute with 75% (v/v) ethanol, immersed in 10% (v/v) H2O2 for 1 hour, rinsed with sterile water two times, and finally immersed in sterile water overnight at 28°C in darkness for 24 hours. The seed endosperm was carefully separated from the embryo. The dry endosperm part was ground to fine powder and the lipids were extracted with hexane three times. The combined supernatant was transferred to a glass vial and the hexane was evaporated with a flow of dry nitrogen gas at 50°C. The weight of the raw oil was determined and the oil content was recorded as the ratio of raw oil to dried endosperm weight.
About 10 mg of lipid was transmethylated with 3N methanolic-HCl (Sigma, St. Louis, MO, USA) plus 400 μL 2,2, dimethoxypropane (Sigma). The resultant FAMEs were separated by GC and detected using GC Agilent 6890 (Agilent, Santa Clara, CA, USA) employing helium as the carrier gas and DB-23 columns for components separation. The GC analysis was performed at 140°C for 50 seconds and 30°C per minute ramp to 240°C, and the final temperature was maintained for 50 seconds. Peaks were identified based on their retention times compared with a FAME reference mixture (Sigma). The fatty acid composition value included in the analyses was calculated based on the peak area percentage of total fatty acids in three biological replicates. The data were presented as means ± standard deviations.
RNA extraction and analysis
The 100-mg leaf or endosperm samples were ground to fine powder in liquid nitorigen and extracted with plant RNA purification reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration was measured by Nanodrop (Thermo Scientific, Wilmington, DE, USA). Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Madison, WI, USA) was used for RT reactions. Real-time PCR was performed with Power SYBR® Green PCR Master mix (Applied Biosystems, Foster City, CA, USA) and run in ABI7900HT. All samples were run in triplicate and the data were analyzed with RQ manager at a pre-set threshold cycle value (Applied Biosystems). The Jatropha rbcL transcript served as an internal control for leaf RNA samples while the Jatropha α-tubulin transcript served as an internal control for seed RNA samples. Threshold cycle values included in the analyses were based on three biological replicates, with three technical replicates for each biological sample. Standard deviation was calculated based on the three biological replicates. See Additional file 5 for PCR primer sequences.
Southern blot analysis
Total genomic DNA was isolated from the leaves of glasshouse-grown transgenic or control plants by the cetyltrimethylammonium bromide method . Genomic DNA was digested with restriction enzymes and separated on 0.8% agarose gels. The gels were processed and transferred to a nylon Hybond-N+ membrane (GE Biosciences, Buckinghamshire, UK) following standard procedures . Membranes were hybridized with 7S promoter, HPT or FAD2-1 open reading frame probes. The probes were labeled with [α-32P]-deoxycytidine triphosphate by random prime synthesis using Amersham Rediprime II Random Primer Labelling System (GE Biosciences). Hybridization was performed overnight at 42°C using the ULTRAHyb-Oligo hybridization buffer (Ambion, Austin, TX, USA) and signals were detected by autoradiography.
Plant growth condition, agronomic traits collection and statistical analysis
All the transgenic or control plants were grown in a biosafety level 2 greenhouse according to standard practices. The transgenic and control plants were transplanted into pots (diameter 30 cm) and randomly placed in a greenhouse with a space of 1 m × 1 m for each tree in Temasek Life Sciences Laboratory, Singapore. Plant management, such as fertilization, pesticides spraying, watering and artificial fertilization, was carried out according to normal practices. Half a year after transplanting, the plants were pruned at the height of 50 cm from the ground. The tree height, diameter of main stem and number of primary branches were measured and recorded.
Every fruit and seed of these plants was collected and counted and their weight was taken over the whole experiment. Dry T1 seeds were weighed and endosperms were further analyzed for oil content and oil profile. Four T0 plants with > 70% oleic acid content were grouped as high-oleic-acid plants and the other 16 T0 plants with normal oleic acid content (37% to 42%) were grouped as non-high-oleic-acid plants. The data were analyzed with Student's t-test and presented in Table 1 as means ± standard errors.