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