Development of efficient catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants
- Quan Wang†1,
- Shihai Xing†1,
- Qifang Pan1,
- Fang Yuan1,
- Jingya Zhao1,
- Yuesheng Tian1,
- Yu Chen1,
- Guofeng Wang1 and
- Kexuan Tang1Email author
© Wang et al.; licensee BioMed Central Ltd. 2012
Received: 5 September 2011
Accepted: 11 May 2012
Published: 29 June 2012
As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus) produces many terpenoid indole alkaloids (TIAs), such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus, the establishment of an efficient genetic transformation method is required.
To develop a genetic transformation method for C. roseus, Agrobacterium tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report β-glucuronidase (GUS) gene and a selectable marker neomycin phosphotransferase II gene (NTPII). The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 μM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC) showed that overexpression of DAT increased the yield of vindoline in transgenic plants.
In the present study, we report an efficient Agrobacterium-mediated transformation system for C. roseus plants with 11% of transformation frequency. To our knowledge, this is the first report on the establishment of A. tumefaciens mediated transformation and regeneration of C. roseus. More importantly, the C. roseus transformation system developed in this work was confirmed in the successful transformation of C. roseus using a key gene DAT involved in TIAs biosynthetic pathway resulting in the higher accumulation of vindoline in transgenic plants.
KeywordsCatharanthus roseus Agrobacterium tumefaciens Deacetylvindoline-4-O-acetyltransferase Regeneration Vindoline
To date, genetic transformation of C. roseus has been mostly confined to hairy roots and suspension cells. Agrobacterium rhizogenes-mediated transformation involving productions of hairy (transgenic) roots in C. roseus had been reported [4, 7, 8]. However, the phenotypes of transgenic C. roseus plants transformed by A. rhizogenes are abnormal, such as shortened internodes, wrinkled leaves and abundant root mass . Thus this kind of transgenic C. roseus plants is not suitable for the production of TIAs. Transgenic C. roseus cell suspension cultures transformed by either Agrobacterium infection or by particle bombardment had been established and studied intensively [4, 9–13]. But these transgenic cells lines do not produce alkaloids in a stable manner and their ability to accumulate TIAs is gradually declined by long-term subculture . Recently A. tumefaciens-mediated transformation was employed in C. roseus, the transgenic callus and plants were obtained respectively [15, 16]. However, these transformation systems were not confirmed with other biochemical assays such as southern blot and high performance liquid chromatography (HPLC). To address these issues, in the present study we developed an A. tumfaciens mediated transformation and regeneration system of C. roseus. The stable regeneration plants were successfully acquired. To demonstrate this transformation system, DAT, an essential gene in TIAs biosynthetic pathway was overexpressed and the accumulation of vindoline was analyzed using HPLC in transformants.
Results and discussion
Optimization of transformation conditions for C. roseus
To develop an efficient system for producing transgenic C. roseus plants using A. tumefaciens, the association parameters were systemically investigated and optimized, including the concentration of Agrobacterium and acetosyringone, co-cultivation duration, and sonication condition. The detection was performed using the A. tumefaciens-mediated GUS gene transient expression. The transformation frequency was calculated as the number of kanamycin resistance plants/number of explants × 100.
Influence of transformation factors on the frequency of transient GUS expression (%) in hypocotyls of C. roseus
Transient expression rate (%)a (mean ± SE)c
Death rate (%)a (mean ± SE)b
A. tumefaciens infection density(OD) and duration (min)
53.33 ± 3.33 c
10.00 ± 2.89 b
78.33 ± 4.41 ab
11.67 ± 1.67 a
80.00 ± 2.89 a
11.67 ± 3.33 a
78.33 ± 3.33 b
15.00 ± 1.67 b
100.00 ± 0.00 a
15.00 ± 2.89 b
100.00 ± 0.00 a
21.67 ± 1.67 a
73.33 ± 1.67 b
18.33 ± 3.33 c
100.00 ± 0.00 a
46.66 ± 6.01 b
100.00 ± 0.00 a
68.33 ± 4.41 a
Co-cultivation period (d)
76.67 ± 4.41 b
0.00 ± 0.00 c
100.00 ± 0.00 a
5.00 ± 2.89 b
100.00 ± 0.00 a
20.00 ± 5.77 a
Acetosyringone concentration (μM)
13.33 ± 3.33 c
0.00 ± 0.00 cd
85.00 ± 2.89 ab
1.66 ± 1.66 bc
100.00 ± 0.00 a
5.00 ± 2.89 b
100.00 ± 0.00 a
46.67 ± 4.41 a
Secondly, the effects of co-cultivation duration and acetosyringone concentration on transformation frequency were optimized separately. The co-cultivation with A. tumefaciens between 1 to 3 d and the acetosyringone concentration (0, 50, 100 and 150 μM) on T-DNA delivery were tested. The results showed that the highest frequency of GUS transient expression (100%) and the relatively lower death rate (5%) were obtained for the explants with 2 d co-cultivation and 100 μM of acetosyringone in co-culture medium in dark (Table 1).
The regeneration mediums used in this study
The components of the medium
MS + 150 mg/L casein hydrolysate + 250 mg/L L-proline + 30 g/L sucrose + 3 g/L gelrite
MSCP + 1.0 mg/L 2,4-D + 1.0 mg/L NAA + 0.1 mg/L ZT + 250 mg/L carbenicillin
MSCP + 5.0 mg/L BA + 0.5 mg/L NAA + 250 mg/L carbenicillin
MSCP + 1.75 mg/L BA + 0.55 mg/L IAA + 250 mg/L carbenicillin
Experimental design for kanamycin concentration optimization
Kan concentration of MSCP1 (mg/L)
Kan concentration of MSCP2 (mg/L)
Kan concentration of MSCP3 (mg/L)
Regeneration of transgenic C. roseus
Molecular characterization of transgenic C. roseus plants with GUS
To determine the presence and the integration of transgene in kanamycin resistant plants, PCR and DNA blot hybridization were performed with genomic DNA. First the putative transgenic plants were screened by PCR using GUS gene-specific primers to detect the presence of target gene in host. The representative results are shown as in Figure 6E. It indicates the 400 bp fragments of GUS genes in kanamycin-resistant transformed plants were amplified, which is the same as positive control. No specific amplification product was detected in non-transgenic control plants.
The transgenic plant integrated with foreign gene was further verified by southern blot with the fragment of GUS as probe. The genomic DNA was obtained from two independent PCR-positive and GUS-positive transgenic C. roseus plants. The southern results indicate that the T-DNA was inserted into genome of both transgenic plants. One transgenic plant has a single copy and another has two copies, while no signal is detected in the untransformed control (Figure 6F).
Altogether, the results of molecular analyses confirm that these regenerated plants are stably integrated with GUS gene. In this study we obtained 16 putatively transformed plants by transformed the explants three times using same method. The percentage of explants producing regenerates was 19% and the frequency of explants producing independent transgenic plants was established at 11%.
Analysis of transgenic C. roseus plants with overexpressed DAT
The DAT mRNA level was analyzed using real-time PCR in 9 independently DAT transformed C. roseus plants. The results reveal that the expression level of DAT was increased in all the transgenic plants (Figure 7B). Especially for d31, d35 and d42 transgenic plants, the expression of DAT was significantly increased by 7.64, 6.14 and 7.58-fold respectively.
Because the transformation of C. roseus at whole plant level had no report before, the functions of genes in TIAs were investigated using hair root or suspension cell transformation so far. Here the established transformation system provides a potential possibility to investigate the effect of gene expression upon the alkaloids yields on C. roseus whole plant, and would contribute to the successful modification of the medicinal plants for higher natural product yields.
Here we report an Agrobacterium-mediated transformation and regeneration system for C. roseus. The parameters influencing the transformation frequency are systematically investigated, including the concentration of Agrobacterium and acetosyringone, co-cultivation duration, sonication condition and selection pressure of kanamycin. The results show that the transformation frequency arrived at 11%. In order to validate the established transformation system, the key gene, DAT, in TIAs biosynthetic pathway was overexpressed. The HPLC results reveal that the production of vindoline was enhanced in transgenic plants with DAT overexpression.
In conclusion, all the results obtained show that the C. roseus transformation protocols developed in this work has great potential to be used in the discovery of genes function in TIAs biosynthetic pathway, in addition to improve the productions of TIAs.
Seeds of C. roseus cultivar Pacifica Cherry Red were purchased from PanAmerican Seed Company (USA). Seeds were sterilized with 75% (v/v) ethanol for 1 min and 10% (v/v) NaClO for 10 min, and then were washed three times with sterile distilled water. Finally, seeds were transferred onto Petri plates containing MS  basal medium. Cultures were germinated 16 h light and 8 h dark photoperiod at 25 ± 2°C. Hypocotyls, about 5 mm, were excised from 5 d-old germinated seedlings.
A. tumefaciensstrain and vector used for transformation
For transformation, A. tumefaciens strain EHA105 harbouring the binary pCAMBIA2301 (CAMBIA Company, Australia) was used for transformation. The vector contains a GUS reporter gene and a selection marker gene NPTII (neomycin phosphotransferase gene conferring resistance to kanamycin) which are inserted between the CaMV35S promoter and the A. tumefaciens nos terminator separately. An intron inside the coding sequence is included in the GUS reporter gene to ensure that expression of glucuroidase activity is derived from eukaryotic cells.
To get fresh cells, a single colony of A. tumefaciens with pCAMBIA2301 vector was inculcated in liquid Luria Bertani (LB) medium containing 100 mg/L kanamycin and 100 mg/L rifampicin (Sigma, USA), and grown at 28°C for 36 h with shaking (150 rpm). The initial culture was diluted 1:1000 with liquid LB medium and grown on a shaker (250 rpm) until the OD600 reached to 0.5, 0.8 and 1.0. Then the cells were centrifuged (2,000 × g, 10 min) and the supernatant was removed. The bacterium was re-suspended in liquid MS medium containing 100 μM acetosyringone and the OD600 was adjusted to 0.5. At last, the bacterium was shaken (100 rpm) again for 2 h in dark at 28°C.
Genetic transformation and co-cultivation
The explants of C. roseus were immersed in liquid MS medium with 100 μM acetosyringone in tissue culture tubes. Then these tubes were sonicated for 5/10/15 min (40 Hz, 0/60/80/100 W) with the sonicator DL-60D (Shanghai hengxin, China) separately. After that, the explants were transferred into pre-sterilized flasks containing bacterial suspension, and were shaken gently at 25 rpm for 30 min at room temperature. Explants were then blot-dried with sterile paper towels and transferred onto petri dishes containing 1/2 MS medium with 100 μM acetosyringone. The co-cultivation period was 1 ~ 3 d in the dark at 28°C.
After co-cultivation, the explants were transferred into MSCP callus induction medium containing 1.0 mg/L 2, 4-D, 1.0 mg/L NAA, 0.1 mg/L ZT, 250 mg/L carbenicillin and 40 mg/L kanamycin (MSCP1 medium) for 10 d. Then the explants were transferred to the MSCP medium supplemented with 5.0 mg/L 6-BA, 0.5 mg/L NAA, 250 mg/L carbenicillin and 70 mg/L kanamycin (MSCP2 medium), which was effective on shoot initiation for 10 d. The resulting explants were transferred to shoot elongation MSCP medium supplemented with 1.75 mg/L 6-BA, 0.55 mg/L IAA, 250 mg/L carbenicillin and 90 mg/L kanamycin (MSCP3 medium) for 20 d, and subcultured every week. The elongated shoots were separated and transferred to root initiation medium (1/2 MS) containing 250 mg/L carbenicillin for 1 ~ 2 months. The regeneration seedlings were removed from the growth cabinet and were transferred to gardening soil. All the culture conditions were performed as pre-described for the plant seeding.
Analysis of putative transformants
Histochemical GUS activities in leaf and root segments of the control and putatively transformed plantlets were investigated according to Jefferson . Tissues were immersed in a buffer containing 2 mM X-Gluc, 50 mM phosphate, 50 mM potassium ferrocyanide and 5% Trition X-100 at pH 7.0. The reaction mixture was placed in a mild vacuum for 10 min, and then incubated overnight at 37°C. Tissues containing chlorophyll were repeatedly soaked in 95% ethanol until chlorophyll was removed. Transient expression of GUS gene was examined after 5 d of co-cultivation, while stable expression of the reporter gene was scanned after the C. roseus regeneration plants were transplanted to greenhouse for 3 months.
Genomic DNA was isolated from the young leaves of putative transgenic plants using CTAB method . Putative transgenic plants were initially screened by PCR using the GUS gene-specific primers GUS FI (5′-GGGTGAAGGTTATCTCTATGAAC-3′) and GUS RI (5′-CACTGATACTCTTCACTCCACAT-3′) to detect the presence of target gene in the host.
The integration of the GUS gene in the transgenic C. roseus was examined by southern hybridization. Approximately 50 μg of genomic DNA per sample was digested with HindIII which was unique site in the plasmid. Then the digested DNA were fractionated by 1.0%-agarose-gel electrophoresis, transferred onto a positively charged Hybond-N+ nylon membrane (GE Heathcare, USA), and hybridized with an alkaline-phosphatase-labelled partial cDNA sequence of GUS. The probe (402 bp) was generated by PCR with primers GUS FII (5′-CAGTCTTACTTCCATGATTTCTTTA-3′) and GUS RII (5′-AGTAAAGTAGAACGGTTTGTGGTTA-3′). Hybridization and signal detection were performed using Amersham Gene Images AlkPhos Direct Labelling and Detection System (GE, UK). The hybridized signals were visualized by exposure to Fuji X-ray film at room temperature for 4 h.
In this work, the parameters of A. tumefaciens cell density, acetosyringone concentration, sonication condition and kanamycin concentration were investigated. Each treatment has three flasks replicates and each flask contains at least 20 explants. The data was analyzed with SAS software. The values are expressed as the mean of triplicate and analyzed by Duncan's New Multiple Range Test (DNMRT) with P value of 0.05.
Validation of the established transformation system
After 3 months of the regenerated C. roseus growing in greenhouse, three pair leaves at the top of the transgenic C. roseus plants and control were collected and mixed, which were used for the analysis of real-time PCR and HPLC. DAT cDNA was cloned by RT-PCR using RNA extracted with Plant (Leaves) Total RNA Isolation Kit (Watson Biotechnologies, Inc, Shanghai, China). In RT-PCR, DAT-F (5′-CCCATGGGATGGAGTCAGGAAAAATATCG-3′, with NcoI site) and DAT-R (5′-CGGTAACCTTAATTAGAAACAAATTGAAGTAGC-3′, with BstEII site) were used as the forward and reverse primer respectively, and the parameters used during PCR reaction were as follows: 94°C, 5 min; 32 cycles: 94°C, 30 s; 52°C, 30 s, 72°C, 30 s; 72°C, 5 min. The amplified fragment was cloned into pMD18-T vector (TakKaRa, Japan) and sequenced. After confirmation by sequencing, the pMD18-DAT was double-digested by NcoI and BstEII, and then ligated into the NcoI and BstEII sites of pCAMBIA2301. The resultant plasmid pCAMBIA2301-35 S::DAT::NOS was then transferred into A. tumefaciens strain EHA105, and the resulting strains were used in the transformation of C. roseus. Transgenic DAT C. roseus plants were obtained using the same transformation procedure.
Southern blot analysis was carried out using a biotin-labelled 335 bp DAT fragment (DAT FII: 5'-GCTATTGTTCAACTAAGTCAT-3' and DAT RII: 5'-GCAGTCAAAACCTCTACTCGAG-3') as probe. Genomic DNA of randomly selected transgenic plants and a control plant were digested with BamHI. The probe labelling and hybridization was also performed as previously described. The expression level of DAT was detected by real-time PCR in which the detail procedures were shown as in our previous study . Briefly, the specific primers for their corresponding genes were analyzed, which include DAT-FIII (5'-CTTCTTCTCATCACGTACCAACTC-3') and DAT-RIII (5'-ATACCAAACTCAACGGCCTTAG-3') for DAT gene, and Rps9-F (5'-TCGCAACTATGGTAAGACCT-3’) and Rps9-R (5'-CTGTTCATCCTCCTCAAAAG-3') for Rps9 gene. The cDNA for real-time PCR was synthesized from RNA samples using Prime ScriptTM Reverse Transcriptase Reagent with oligo (dT) as primer according to the manufacturer’s instruction (TaKaRa, Japan). The real-time PCR analysis was performed in Peltier Thermal Cycler PTC200 (Bio-Rad, USA) using the cDNAs as templates. SYBR Premix Ex Taq (TaKaRa, Japan) was used in PCR reactions to quantify the amount of dsDNA. The relative Ct, the threshold of cycle value, was used to estimate the initial amount of template in reactions.
To prepare the samples for HPLC, the young leaves of transgenic C. roseus were dried at 45°C for 48 h and pulverized in a mortar . Then 200 mg of each sample were immersed with 1 mL methanol for 10 min. The tubes were dipped into ultrasonic bath with the power of 80 W for 60 min. The mixture was centrifuged at 12,000 rpm for 10 min and the supernatant was filtered with 4 μm filter membrane. The extracts were stored at 4°C.
For HPLC analysis, the commercialized vindoline (Sigma-Aldrich, USA) was prepared at a concentration of 1 mg/L in methanol and used as standard . The HPLC analysis was performed using a Sapphire-C18 (4.6 mm × 250 mm, 5 μm) column at a column temperature of 35°C and Hitachi L-2000 series HPLC system. This system consists of an L-2000 Organizer, an L-2130 Pump, an L-2200 AutoSampler, an L-2301 Column Oven and an L-2455 Diode Array Detector. The injected samples (10 μL) were detected at 220 nm by L-2455 Diode Array Detector. The mobile phase (acetonitrile and diethylamine buffer solution; 1:1) was used at a constant flow rate of 1 mL per min. The UV absorbance of standards and alkaloids were acquired. At last, the amount of vindoline was determined by using regression equation of calibration curve . The TIAs level was determined by the areas of peak in chromatographic profile at 14.41 min for vindoline.
Research project funding was from China National High-Tech “863” Program (grant number 2011AA100605), Shanghai Science and Technology Committee (grant number 08391911800) and Shanghai Leading Academic Discipline Project (Project Number: B209).
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