Use of silicon carbide fibers for Agrobacterium-mediated transformation in wheat

Neeta Singh and H. S. Chawla*

Department of Genetics and Plant Breeding, G.B. Pant University of Agriculture and Technology, Pantnagar 263 145, India

Silicon carbide fibers have been used for causing wounds in the immature wheat embryos for Agrobacterium- mediated genetic transformation. Immature embryos were put in a 5% silicon carbide fiber (SCF) suspension and vortexed for 2 or 3 min followed by co-cultivation with Agrobacterium tumefaciens strain LBA4404. The strain harboured the binary vectors pBI121 and pTOK233 which contained selectable marker genes and gus as the reporter gene. Agrobacterium-infected explants were stained for GUS activity. Without wounding the GUS expression was observed in 2.4% of the embryo, while 33.3% of the embryos showed GUS expression after wounding with SCF for 2 min. The binary vector pBI121 showed better response than pTOK233 for GUS expression. We propose that SCFs can be used for wounding to improve frequency of transformation by disarmed Agrobacterium strains.

Transformation studies were conducted on wheat with the Agrobacterium strain LBA4404 which carried the binary vectors pBI121 (Clonetech, USA) and pTOK233 (Yuko Hiei, Japan). The T-DNA of pBI121 contained the gus reporter gene controlled by 35S promoter and nptII selectable marker gene controlled by the nopaline synthase (NOS) promoter. The binary vector pTOK233 contained the gus reporter gene controlled by CaMV35S promoter, which had an intron in the N-terminal region of the coding sequence. The genes for selectable markers were hygromycin phosphotransferase (hptII) controlled by the 35S promoter conferring resistance to hygromycin and nptII controlled by the NOS promoter conferring resistance to kanamycin. SCFs were obtained from Advanced Composite Materials Corp. USA, whose size averaged 0.6 m m in diameter and 10–80 m m in length. SCFs were used for effecting wounds in the immature embryo explants for Agrobacterium-mediated transformation.

The strain LBA4404 was grown on YEP medium plates (yeast extract, 10 g/l; peptone, 10 g/l; sodium chloride, 5 g/l; pH 7.0). For binary vector pBI121, YEP medium with 50 mg/l kanamycin, 50 mg/l streptomycin and 50 mg/l rifampicin was used while for pTOK233, YEP medium with 50 mg/l kanamycin, 100 mg/l streptomycin and 50 mg/l hygromycin was used. Agrobacterium strains were grown in petri plates or liquid medium at 28 C for 2 days in the dark. Agrobacterium suspension for co-cultivation experiments was prepared by picking a single colony from the YEP media plates and inoculated in 5 ml of liquid YEP medium containing acetosyringone and antibiotics as per the binary vectors. The O.D. of the culture was measured at A-600 nm. The culture was diluted or concentrated to bring its value to 0.1 O.D. For co-cultivation, the Agrobacterium cell suspension was centrifuged at 5000 rpm for 10 min, supernatant removed and diluted to a final concentration in MSE1 medium (MS1 basic salts supplemented with 2 mg/l 2,4-D, 100 mg/l glutamine, 200 mg/l casein hydrolysate and 100 m M acetosyringone).

Immature embryos (1 to 1.5 mm diameter) of two Indian varieties, UP2338 and PBW226 were used as explants for co-cultivation with Agrobacterium. Twelve immature embryos were taken in eppendorf tubes containing 5% SCF suspension (2.5 mg dissolved in 50 m l of sterile distilled water) and 20 m l of MSE1 liquid medium. This mixture was vortexed (Genei vortex, Bangalore) for 2 to 3 min to injure the explants. Agrobacterium co-cultivation was done: (i) after wounding of explants with SCF; and (ii) without wounding with SCF. Both the explants were transferred to tubes containing acetosyringone-induced Agrobacterium culture and were incubated at 25 C for 2 h on a platform shaker at 100 rpm. The explants were then blot dried on a sterile filter paper and then transferred to MSE1 medium plates without selection agent in the dark at 25 C for 3 days. After co-cultivation, the explants were transferred to MSEC medium (MSE1 with 400 mg/l cefotaxime) for 3 days. The explants were then transferred to a selection medium of MSK121 (MSEC with 100 mg/l kanamycin) or MSK233 (MSEC with 100 mg/l kanamycin and 100 mg/l hygromycin). Histochemical GUS assay was performed on explants taken from selection media, i.e. after 6 to 10 days of co-cultivation. X-Gluc stain was prepared by dissolving 30 mg X-Gluc in 500 m l DMSO and then 10 ml solution is made up with 5 mM each of potassium ferricyanide and potassium ferrocyanide, 0.1 M phosphate buffer (pH 7.0) and 0.3% Triton X-100. The co-cultivated embryos were put in wells of ELISA plates with X-Gluc stain and incubated at 37 C for 36 h.

The GUS expression frequency in the explants without SCF treatment in both the varieties with both the vectors was very low (2.4%) whereas with SCF treatment the overall frequency was 20.7% (Table 1, Figure 1). Wounding of embryos with SCF for 2 min was more effective (33.3%) than treatment with SCF for 3 min (12.5%) in both the varieties with both the binary vectors. GUS activity was reproducibly observed when maize cell suspension cultures were vortexed for 60 s on vortex Genei II along with plasmid DNA2. High transient expression of gus gene 1484.gif (15916 bytes)was observed when dry embryos of wheat were vortexed in a suspension of SCFs and vector DNA for 10–30 min3. The exact mechanism for SCF-mediated transformation is not known. Silicon carbide has great intrinsic hardness and fractures to give sharp cutting edges4. It has been observed in maize cell suspension cultures that SCF pierces the walls of cells during vortexing and along with the fibers the DNA adhering to it might enter the cells5. But later reports suggest that SCFs do not carry the DNA into the treated cells but function as numerous needles facilitating 1485.jpg (25692 bytes)DNA delivery into the cells during the mixing process2. In our experiments also, SCF only created injury to the cells, which facilitated Agrobacterium-mediated transformation. Serik and coworkers3 reported that GUS expression appeared in the form of small patches in SCF-treated mature embryos in a direct delivery approach. In the present study also GUS expression was observed as patches on the embryo surface. It seems that SCF whiskers have created injury on the immature embryos which resulted in good infection.

On comparison, binary vector pBI121 was found to give better transformation frequencies than pTOK233. When both the varieties were considered UP2338 was found to be better with pBI121 while PBW226 responded only when co-cultivated with pTOK233 vector.

Generally, in monocots, co-cultivation with Agrobacterium requires wounding of the tissue for the infection process to take place. Wounding by bombardment with naked gold particles using a particle gun followed by Agrobacterium co-cultivation has been used6. But this technique is expensive and needs skillful handling. Workers have used SCFs for delivery of DNA as a physical direct method of transformation in Nicotiana7, maize2,5 and wheat3.

Preculture of rice embryos for 2–5 days followed by co-cultivation with Agrobacterium for 2–3 days and then transfer to a medium with antibiotic selection agent resulted in efficient production of transgenic plants8. High GUS expression frequency in wheat has been observed with the addition of Silwet surfactant in the inoculation medium9. Our studies showed that the efficiency of Agrobacterium-mediated transformation can be improved by using SCF for wounding. This can be considered as a practical alternative requiring no expensive equipment or consumables, especially in laboratories which have no access to sophisticated equipment.

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 ACKNOWLEDGEMENTS. We thank Dr Yuko Hiei, Japan Tobacco Inc, Japan, for providing the binary vector pTOK233 and Dr Foroughi-Wehr, Gruenbach, Germany, for providing silicon carbide fibers. N.S. is grateful to ICAR for fellowship assistance during this study.

 Received 10 September 1998; revised accepted 3 March 1999