Page 8 - BM 2011 Vol.2 No.5

Bioscience Methods
BM 2011, Vol.2, No.5 http://bm.sophiapublisher.com
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in this study, 30 minutes was the best infection time
by Agrobacterium containing GUS gene when the
OD600 of the suspended liquid reached to 0.8~1.0
(Table 6; Figure 4).
3 Materials and Methods
3.1 Materials
Seeds of Puccinellia chinampoensi. Puccinellia seed
will be soaked at 4℃ with distilled water for 4 to 7
days. After drying seeds, the seeds soaked in 75%
alcohol 1 min, and wash 3 to 4 times with sterile water,
and then soak them with 10% Sodium hypochlorite
for 30 minutes, then rinse 3~5 times with sterile water,
finally, dry them for later use in the clean bench.
Strains: Agrobacterium EHA105 containing plasmid
PBI121-GUS (Agrrobcrcterum tumefaciens)
3.2 Callus induction conditions
On the basic medium (MS +500 mg/L proline +500 mg/L
glutamine +30 g/L sucrose +0.8% agar (pH5.8~6.0)),
adding different concentrations of 2,4-D: 0 mg/L,
1 mg/L, 2 mg/L, 3 mg/L, 4 mg /L, 5 mg /L, 6 mg /L as
the induction medium; On the basic medium (MS
medium, N6 medium, altered medium I) (Mitsuoka et
al., 1994), adding 4 mg/L 2,4-D, 500 mg/L proline,
500 mg/L glutamine, 30 g/L sucrose, 0.8% agar
(pH5.8~6.0) as the induction medium. After the
disinfection of mature dry seeds , the callus were
inoculated into induction medium, placed in 25 ± 10 C,
80umol / (m2 • s) light intensity, illumination time was
12 h/d for 2 weeks under the conditions.
3.3 Callus subculture conditions
On the basic medium (MS medium, N6 medium,
modified medium S (Mitsuoka et al, 1994)
respectively), adding 2 mg/L 2,4-D, 500 mg/L proline,
600 mg/L hydrolyzed casein, 30 g/L sucrose, 0.8%
agar (pH5.8~6.0) as a subculture medium; and then on
the basic medium (S +2 mg/L 2,4-D +500 mg/L proline
+600 mg/L +30 g casein hydrolyzate/L sucrose +0.8%
agar (pH5.8~6.0)), adding 0 mg/L, 1 mg/L and 2 mg/L
ABA as a subculture medium. after subculturing ,
placed the plates under 25±10 C, 80 umol/ (m2 • s)
light intensity, illumination time was 12 h/d under the
conditions of culture and subculture period of 20 d. In
the course of subculture, we select the drier, more
compact growth good yellow embryogenic callus, not
the non-embryogenic callus.
3.4 The differentiation conditions of the callus
On the basic medium (MS +500 mg/L proline +600 mg/L
hydrolyzed casein +30 g/L sucrose +0.8% agar
(pH5.8~6.0)), adding the mixture (cell division: The
ratio of 10:1: Auxin Add 6-BA 4 mg/L, 2 mg/L, 1
mg/L, 0.5 mg/L, 0.4 mg/L, 0.3 mg/L and IAA 0.4
mg/L, 0.2 mg/L, 0.1 mg/L, 0.05 mg/L, 0.04 mg/L,
0.03 mg/L respectively) as the differentiation medium,
inoculated embryogenic callus twice subculturing,
placed them under (25± 10)℃, 80 μmol / (m2 • s)
light intensity, illumination time was 14 h/d.
3.5 Genetic transformation of the callus
The good growth embryogenic callus selected，afer
preculturing ， were then cultured and infected with
the Agrobacterium suspension (OD600 of 0.8 to 1.0)
containing PBI121 -GUS plasmid. The time course
was 15 min, 30 min and 45 min respectively. After
that， co-cultured process was made in the medium
for 7 d. Before GUS staining, in order to get rid of the
bacteria, we filtrate the callus with vacuum pump.
Finally， the efficiency of genetic transformation was
measured under the different infection time.
Authors’ contributions
TW, XH and MQZ designed and conducted this experiments; XXZ and TT
participated the experiment design and data analysis; SKL is the person who
takes charge of this project, including experiment design, data analysis,
writing and modifying of the manuscript. All authors have read and
approved the final manuscript.
Acknowledgements
This work was supported by the Heilongjiang Provincial Program for
Distinguished Young Scholars (JC200609) and State Forestry
Administration 948 Program of PR China (No. 2008429) to Shenkui Liu.
Authors appreciate two anonymous reviewers for their useful critical
comments and revising advice to this paper. And also we mentioned some
reagent suppliers and sequencing service providers in this work, that doesn’t
mean we would like to recommend or endorse their products and services.
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