Direct gene transfer into mature plant seeds using electrical pulses and supersonic treatment

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 1National Institute of Agrobiological
Sciences(2-1-2 Kannondai Tsukuba Ibaraki 305-8602
Japan), 2Nepa 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(ß-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 npt? gene was
confirmed by Southern analysis in T0 and T1
generations.
Basic Method ? Soak mature seeds in water
overnight. ? Supersonic treatment ? Transfer the
seeds into electroporation buffer. ? Vacuum
treatment. ? Electroporation on ice. ? Incubate
the seeds on ice for a few hours. ? Supersonic
treatment (Next day). ? Grow the plants under the
favorable conditions. ? Select the transformed
plants using antibiotics.
(score) 10 5 0 Fig.2. GUS gene
expression reference
Fig.1. Hexagonal electroporation chamber
(not transformed) (npt?gene introduced) Fig.4.
Geneticine selection of rice seeds and/or
seedlings
Fig.3 Supersonic treatment
wt
1 2 3 4 5 6 7 8 9
-20kb
-4.0kb
-1.0kb
Fig.5. Effect of supersonic treatment
Fig.6. Polarity changing device
Fig.7. Southern hybridization of T1(next)
generation from a single parent