Plant transformationTransformed roo

Plant transformation
Transformed roots ofM. truncatulacv. Jemalong
were obtained usingAgrobacterium rhizogenesARqua1
according to the protocol of Boisson-Dernier et al.
(2001). About 3–6 weeks later, plants with transformed
roots were put individually into square 12-cm plastic
dishes (Greiner Labortechnik, Kremsmu¨nster, Austria)
on Fahraeus medium containing 1% agar.A. thaliana
plants (ecotype Columbia) were transformed using the
A. tumefaciens-mediated floral dip method. Cultivation
of Arabidopsis plants was performed on half MS
enriched with vitamins, 1% sucrose and 0.4% phytagel.
Microscopy
M. truncatularoots growing on agar were covered
with bioFolie 25 (Sartorius AG, Vivascience Support
Center, Go¨ttingen, Germany) and observed with a
LEICATCS 4D confocal microscope (Leica, Germany)
using a 63water-immersion objective. Four-day-old
A. thalianaseedlings were mounted in liquid half MS
medium containing 1% sucrose using a spacer of one
layer of parafilm between slide and coverslip, and
observed by confocal microscopy. Plants were adapted
to liquid medium overnight to allow application of
drugs.
For the observation of transgenicA. thalianaexpressing Ara6-GFP (Ueda et al., 2001), serial images were
obtained every 1mm using a fluorescence microscope
(Olympus, Japan) equipped with 40oil-immersion
objectives and a confocal unit, CSU10 (Yokogawa
Electric Corporation, Japan). Images and movies were
digitally processed with Image-Pro Plus 4.1 (Media
Cybernetics, L.P.), Adobe Photoshop 4.0 (Adobe Corp.,
Mountain View, CA) and VideoMach 2.7.2.
Drug treatments and FM4-64 staining
Growing root apices were exposed to the following
drugs: 2,3-butanedione monoxime (10 mM), latrunculin
B(1mM forA. thalianaand 10mM forM. truncatula),
jasplakinolide (1mM/5mM), brefeldin A(10mM for
A. thalianaand 100mM forM. truncatula), wortmannin
(10mM), and oryzalin (1mM). The drugs were diluted in
culture medium and directly added to the roots of
transgenic M. truncatulaor A. thaliana. Regarding
FM4-64, stock solution (1 mg/ml) was prepared in
DMSO and used at 17.5mM forM. truncatula. Before
FM4-64 treatment, plants were incubated for 25 min at
61C to slow down endocytosis.
Results
A tandem FYVE construct recognizes plant
endosomes
In both yeast and mammals, the phosphoinositide
PI(3)P accumulates preferentially in endosomal membranes (Gillooly et al., 2000), and binding of FYVEdomain proteins to PI(3)P is sufficient to target
endosomal proteins to these subcellular compartments.
ForA. thalianait is known that the classical FYVE
domain binds specifically to PI(3)P in vitro (Jensen et
al., 2001). Therefore, we were interested to know if the
tandem FYVE domain would be sufficient for targeting
to endocytic compartments in plants. To this end, the
FYVE domain from the mouse Hrs protein was

ผลลัพธ์ (อังกฤษ) 2:

Plant Transformation
Transformed Roots Ofm. truncatulacv. Jemalong
UsingAgrobacterium RhizogenesARqua1 were obtained
according to Boisson-Dernier et The Protocol of AL.
(2,001). About 3-6 weeks later, Plants transformed with
Roots were Individually Put Into Plastic Square 12-cm
dishes (Greiner Labortechnik, Kremsmünster, Austria)
on Fahraeus Medium containing 1% Agar.A. thaliana
Plants (ecotype Columbia) were transformed using The
A. tumefaciens-mediated floral dip method. Cultivation
of Plants Arabidopsis was performed on Half MS
enriched with vitamins, 1% and 0.4% Sucrose phytagel.
Microscopy
M. Growing on agar were Truncatularoots Covered
with BioFolie 25 (Sartorius AG, Vivascience Support
Center, Göttingen, Germany) and observed with a
confocal 4D LEICATCS Microscope (Leica, Germany)
using a 63? Water-immersion Objective. Four-Day-Old
A. Thalianaseedlings were Mounted Half MS in Liquid
Medium containing 1% Sucrose using a spacer of One
Layer of Parafilm between slide and coverslip, and
observed by confocal microscopy. Plants were adapted
to Liquid Medium Overnight to Allow Application of
Drugs.
For The Observation of TransgenicA. Thalianaexpressing Ara6-GFP (Ueda et AL., 2001), Serial images were
obtained using a fluorescence Every 1mm Microscope
(Olympus, Japan) Equipped with 40? Oil-immersion
objectives and a confocal Unit, CSU10 (Yokogawa
Electric Corporation, Japan). Images and movies were
Digitally Processed with Image-Pro Plus 4.1 (Media
Cybernetics, LP), Adobe Photoshop 4.0 (Adobe Corp.,
Mountain View, CA) and VideoMach 2.7.2.
Drug Treatments and FM4-64 staining
Growing root apices were exposed. The following to
Drugs: 2,3-Butanedione monoxime (10 mM), Latrunculin
B (1 mM forÃ. Thalianaand 10mM Form. truncatula),
Jasplakinolide (1 mM / 5MM), Brefeldin A (10mM for
Form A. Thalianaand 100mM. truncatula),. Wortmannin
(10mM), and Oryzalin (1 mM). The Drugs were diluted in
Culture Medium and directly added to The Roots of
transgenic A. thaliana M. Truncatulaor. Regarding
FM4-64, Stock Solution (1 mg / ml) was Prepared in
DMSO and Used at 17.5MM Form. truncatula. Before
FM4-64 Treatment, Plants were incubated for 25 min at
61C to Slow down endocytosis.
Results
A tandem FYVE Recognizes Construct Plant
endosomes
In Both Yeast and mammals, The phosphoinositide
PI (3) P accumulates preferentially in Endosomal membranes (Gillooly et AL. , 2000), and of FYVEdomain Proteins binding to PI (3) P is sufficient to Target
Proteins Endosomal to these subcellular compartments.
forÃ. Thalianait is Known that The Classical FYVE
Domain specifically binds to PI (3) P in vitro (Jensen et
AL., 2001). Therefore, we were interested to know IF The
tandem FYVE Domain would be sufficient for targeting
to Endocytic compartments in Plants. To this End, The
protein was Hrs FYVE Domain from The Mouse.

ผลลัพธ์ (อังกฤษ) 3:

Plant transformation
Transformed roots ofM. Truncatulacv. Jemalong
were obtained usingAgrobacterium rhizogenesARqua1
according. To the protocol of Boisson-Dernier et al.
(2001). About 3 - 6 weeks later plants with, transformed
roots were put individually. Into square 12-cm plastic
dishes (Greiner Labortechnik Kremsmu, ¨, nster Austria)
on Fahraeus medium containing 1%, agar.A. Thaliana
.Plants (ecotype Columbia) were transformed using the
A. Tumefaciens-mediated floral dip method. Cultivation
of Arabidopsis. Plants was performed on half MS
enriched, with vitamins 1% sucrose and 0.4% Phytagel.

M Microscopy. Truncatularoots growing. On agar were covered
with bioFolie 25 (Sartorius, AG Vivascience Support
Center Go ¨, ttingen Germany), and observed with. A
.LEICATCS 4D confocal, microscope (Leica Germany)
using a 63  water-immersion objective. Four-day-old
A. Thalianaseedlings. Were mounted in liquid half MS
medium containing 1% sucrose using a spacer of one
layer of parafilm between slide, and coverslip. And
observed by confocal microscopy. Plants were adapted
to liquid medium overnight to allow application drugs of
.
.For the observation of transgenicA. Thalianaexpressing Ara6-GFP (Ueda et al, 2001), serial images were
obtained every. 1mm using a fluorescence microscope
(Olympus Japan), equipped with 40  oil-immersion
objectives and a, confocal unit CSU10. (Yokogawa
Electric, Corporation Japan). Images and movies were
digitally processed with Image-Pro Plus 4.1 (Media
Cybernetics,, L.P.), Adobe Photoshop 4.0 (Adobe Corp,
Mountain View CA), and VideoMach 2.7.2.
Drug treatments and FM4-64 staining
Growing root apices were exposed. To the following
drugs: 2 3-butanedione, monoxime (10 mM), latrunculin
B (1mM forA. Thalianaand 10mM forM. Truncatula),
jasplakinolide. (1mM / 5mM), brefeldin A (10mM for
A. Thalianaand 100mM forM. Truncatula), wortmannin
(10mM), and ORYZALIN (1mM). The drugs. Were diluted in
.