Download Slide 1

Direct gene transfer into mature plant seeds using electrical
pulses and supersonic treatment
Takashi Hagio1, Yoshihiro Ido2, Sakihei Fujiwara2, Koushou Suzuki2, Shinichirou Kamachi1,
Kenichi Konagaya1, Mai Tsuda1, Yutaka Tabei1
1:National Institute of Agrobiological Sciences(2-1-2 Kannondai Tsukuba Ibaraki 305-8602 Japan),
2:Nepa Gene Co., Ltd. (3-1-6 Shioyaki Ichikawa Chiba 272-0114 Japan)
In our previous work, we described the new system that can directly deliver foreign genes into mature
seeds of wheat using electroporation (rectangular wave) after vacuum treatment. To further
characterize and improve the system, we investigated the GUS(β-glucuronidase) gene expression
profiles of mature seeds of some gramineous species changing the parameters of gene transfer
conditions adding supersonic treatment. Mature seeds of wheat, barley, maize, rice and sorghum were
tested. Supersonic treatment significantly increased the level of GUS gene expression in rice and barley.
The optimal electrical conditions for DNA delivery into barley and rice was estimated to be around 50
V/cm, 50ms pulse duration and 50 times of pulses. After the gene transfer treatment the seeds were
incubated in water supplemented with 0.5% PVP for two days, and they were transferred to GUS assay
buffer. Addition of cellulase into the electroporation buffer also increased the GUS gene expression.
Using the knowledge of transient analysis of GUS gene expression, we embarked on the experiment of
obtaining stably transformed plats. In rice, presence of nptⅡ gene was confirmed by Southern analysis
in T0 and T1 generations.
Basic Method
① Soak mature seeds in water overnight.
② Supersonic treatment
③ Transfer the seeds into electroporation buffer.
④ Vacuum treatment.
⑤ Electroporation on ice.
⑥ Incubate the seeds on ice for a few hours.
⑦ Supersonic treatment (Next day).
⑧ Grow the plants under the favorable conditions. Fig.1. Hexagonal
⑨ Select the transformed plants using antibiotics. electroporation chamber
Fig.3 Supersonic treatment
(score) 10
5
0
Fig.2. GUS gene expression
reference
(not transformed) (nptⅡgene introduced)
Fig.4. Geneticine selection of rice seeds and/or seedlings
wt1 2 3 4 5 6 7 8 9
GUS gene expression score
-20kb
12
Score
10
8
Kitaake
6
Koshihikari
-4.0kb
4
2
0
Electroporaion Electroporation
& Super sonoc
treatment
Fig.5. Effect of supersonic treatment
-1.0kb
Fig.6. Polarity changing
device
Fig.7. Southern hybridization
of T1(next) generation from a
single parent