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Legume Genomics and Genetics (online), 2011, Vol. 2, No.3, 14-21
http://lgg.sophiapublisher.com
20
3.5.2 Genetic transformation
Preparation of cotyledonary-node: The selected
soybean seeds were disinfected following the method
of chlorine disinfection (Liu and Wei, 2002). The
sterilized soybean seeds placed in the germination
medium (1/2 MSB (half inorganic salt ingredients of
MS medium + half organic ingredients of B5 medium),
0.7% agar, pH5.8). Taking 5~6 day cultured asepsis
seedlings, and cutting cotyledonary-node from vertical
direction to retain 3~4 mm hypocotyl as well as
removing the terminal bud and lateral bud then
placing into the pre-culture medium (B5 medium +1.7
mg/L 6-BA +0.1 mg/L IBA, 0.7% agar, pH 5.7).
Strains Preparation: Picking up single plaques of
Agrobacterium
GV3101 with pCAMBIA3300-HSF8
and LBA4404 from culture plates, Being Inoculated
with YEB liquid medium containing antibiotics (40
mg/L Rif, 50 mg/L Str and 100 mg/L Kan) at 200
r/min shaking culture for 12~24 h at 28
℃
until
OD
600
value going to about 0.6. Supernatant was
removed after 10 min centrifugation at 4 000 r/min,
then the sediment was resuspended with the same
volume of YEB ready for use.
Transformation and screening culture: Cotyledonary-n-
odes with pre-cultured 1 d were placed into the
Agrobacterium
strain liquid for infection about 25~30
min, then succeed to co-culture medium (B5 medium
+1.7 mg/L 6-BA +0.1 mg/L IBA +100 mg/L ferulic
acid, 0.7% agar, pH 5.2), cultured in the dark for 3~4
d. The infected cotyledonary-nodes were rinsed by
sterile water containing 500 mg/L Cef by 4~5 times,
then being inoculated into the sterilized medium (B5
+1.7 mg/L 6-BA +0.1 mg/L IBA +600 mg/L Cef,
0.7% agar powder, pH 5.7) for a week until he buds
were appearing. The explants with buds were
transferred into selective medium (B5 +1.7 mg/L
6-BA +0.1 mg/L IBA +600 mg/L Cef +3.5 mg/L
glufosinate-ammonium, + 0.7% agar, pH 5.7) (Tang et
al., 2008).
Growth and rooting of the plants with resistance: The
buds were placed on the growth medium (B5 +1.7
mg/L 6-BA +0.1 mg/L IBA +600 mg/L Cef +1.75
mg/L glufosinate-ammonium, 0.7% agar, pH 5.7)
after two weeks screening. Cutting the base of shoots
for immersing 1 minute on the filted IBA(1 mg/L)
once the clustering buds grew 3~4 cm in length, and
then immediately placed the treated shoots into
rooting medium MSB, the plantlets were transplanted
in the flower pots until the root system well developed
with a lateral root appearing.
3.6 Transgenic plants detected by PCR
Using leaves of resistant plants screened by
glufosinate-ammonium to extract soybean genomic
DNA by CTAB. Designing a primer that included the
F: 5'-TTCGCAAGACCCTTCCTC-3' and R : 5'-AC-
CCACGTCATGCCAGTT-3' based on CaMV35S pro-
moter and
bar
gene sequence for PCR amplification,
which was synthesized on the ABI 9700 by Shanghai
Sangon Bioengineering Co., Ltd. Amplified fragme-
nt was expected to 594 bp in length. PCR reaction
were performed following the protocols as 94
℃
for
pre-denaturation 5 min in advance, then 30 cycles
with 94
℃
for denaturation 30 s, 55
℃
for annealing
30 s, 72
℃
for extension 30 s, and finally 72
℃
for
extension 5 min.
3.7 T
1
generation transgenic resistant plants
detected by Real-time PCR
The three-week-old seedlings of T
1
generation
transgenic plant and non-transgenic reference plants
were transferred into the greenhouse culture at the
room temperature, and then total RNAs were
extracted from leaves of transgenic
hsf8
plants and
non-transgenic plants, respectively. Total RNAs were
reverse transcripted to cDNA. Using soybean
lectin
gene as an internal reference, two pairs of primers
were designed based on internal reference
hsf8
gene,
one pair of primers including F: 5'-ATAACTCGGCG
GAAACCT-3', R: 5'-TTGCTGTCGTTGCTCCAT-3'
was for detecting
hsf8
gene. And another pair of
primers was including F: 5'-CTTCGCCGCTTCCTTC
AA-3', R: 5'-GCCCATCTGCAAGCCTTTT-3' for
detecting
lectin
gene. Real-time PCR reaction was
performed on the real-time quantitative fluorescene
PCR instrument Mx3000p
TM
. Reaction conditions
were following as: 95
℃
for denaturation at 10 min in
advance and then 40 cycles as 95
℃
for denaturation
30 s, 60
℃
annealing 1 min.
Housekeeping gene was set up as Normal, leaf cDNAs
of Hajiao 5337 and hajiao 5489 as Calibrators, and
leaf cDNAs of T07
Ⅰ
and T07
Ⅱ
, the lines of T
1
generation plants, as unknown, repeated thrice ,
setting up one for both the target gene and