49. Application of enhanced lipofection to rice transformation

National Institute of Genetics, Mishima 411-8540, Japan

The transformation of rice has been demonstrated usually by Agrobacterium-mediated transfection. However, the method has certain disadvantages in unexpected rearrangement of large size and repeated DNA during Agrobacterium culture. Lipofection is one of the useful methods for animal cell transformation especially with huge size DNAs such as YAC clones, while it has been applied to plant protoplasts only in a few reports because of its low transformation frequency. Recently, enhancing reagent has been developed to improve the lipofection efficiency in mammalian cell lines (Shih et al. 1997). Here, Plus reagent, which is an enhancing reagent available from Invitrogen, was examined to determine whether it could improve lipofection efficiency in rice protoplasts or not.

Calli induced from mature seeds of Oryza sativa cv. Nipponbare were transferred in liquid medium and cultured three months. The calli were washed in a buffer composed of 1.47 mg CaCl2, 985.0 mg MgSO4-7H2O, 85 mg KH2PO4, 600 mg MES and 100 g mannitol per liter, pH 5.7. Then, the cell walls of calli were digested with 4% Cellulase Onozuka RS and 1% Macerozyme R-10 in wash buffer for 2 hours at 28C. After filtered with a 20- micro m-nylon screen, the protoplasts were washed and suspended to a final density of 2 x 106 protoplasts/ml in the wash buffer.

A plasmid containing modified green fluorescence protein sGFP(S65T) gene was kindly provided by Prof. Y. Niwa (Univ. Shizuoka, Japan). The plasmid DNA in 10 micro l of 10 mM Tris (pH 7.5) were mixed with 40 micro l of diluted Plus reagent (Invitrogen Co., Carlsbad, CA, USA) in 10 mM Tris and placed at room temperature for 15 min. Four to eight micro l Plus reagent were used for 1 micro g DNA. Then, Lipofectin reagent (Invitrogen) diluted up to 50 micro l with wash buffer was added to the Plus reagent-DNA mixture and placed at room temperature for 15 min to form liposome-DNA complex. The liposome-DNA solution was added to 2 ml of protoplast solution and incubated for 30 min at room temperature. The solution was mixed with one volume of 27% (w/v) PEG 6000 in wash buffer and allowed to stand for 15 min. The protoplasts were rinsed with wash buffer once and with R2 medium (Ohira et al. 1973) containing 100 g/l of sucrose and 2 mg/l of glycine twice. They were resuspended in 1 ml R2 medium and mixed with an equal volume of 2% (w/v) low melting agarose at 40C to form agarose beads. The beads were incubated in R2 medium in the dark at 28C overnight, and the cells were observed under a fluorescence microscopy.

Although the combination of cationic liposome with PEG treatment increased the transfection frequency in the other plants (Sporlein and Koop 1991; Sawahel 2002), the PEG treatment without liposome was enough for efficient transfection in this study (Table 1A). However, it required a large amount of DNA, more than 20 micro g, for higher efficiency. The addition of Plus reagent with Lipofectin marked the peak frequency of 1.4 x 10-3, which was 6-9 times higher than that obtained without Plus reagent, despite the amount of plasmid DNA was reduced. Plus reagent also improved the frequency of PEG-mediated transfection without Lipofectin to more than 1 x 10-4. In the case of without PEG treatment, the Plus reagent also increased transformation frequency in the lipofection as shown in Table 1B. The peak frequency of 1.0 x 10-3 was higher than that of the standard PEG method and comparable to the highest performance in Table 1A. These results showed that the use of Lipofectin and enhancing reagent together promoted transformation efficiency of rice protoplasts. Interestingly, the precomplexed DNA with Plus reagent, without Lipofectin and PEG, could be transferred into rice protoplasts (Table 1B). Therefore, it was suggested that the Plus reagent was a major to enhance the efficiency of DNA introduction, the Lipofectin might have a minor and the PEG a little effect.

This research was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences, Japan.


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