Basic HTML Version
Molecular Soil Biology (online), 2011, Vol.3 No.1
ISSN1925-2005
http://msb.sophiapublisher.com
- 3 -
Figure 2 Analysis of algae expressed in different NaHCO
3
stresses
Note: Algae species included JB1~JB22 (except JB12) and
CK1~CK8, incubated as described in Materials and Methods.
Serial dilutions were spotted onto solid BBM medium
supplemented without or with additional NaHCO
3
(100 mmol/L,
200 mmol/L and 400 mmol/L). Growth was observed one week later.
The isolation and screening of special resistance-
related algae species as the foundation of resistance-
related genes have an important theoretical and
practical significance on soil salinity remediation,
ecological restoration and the promotion of
resistance-related genetic engineering of plants.
3 Materials and Methods
3.1 Materials
Soil samples were collected from alkali spot soil in
May and September, 2009, in Anda area, Heilongjiang
Province, In addition, we selected garden nursery soil
in Northeast Forestry University as the control.
Bold’s Basal Medium (BBM): NaNO
3
0.250 g, NaCl
0.025 g, K
2
HPO
4
0.075 g, KH
2
PO
4
0.175 g, H
3
BO
3
0.011 g, EDTA 0.050 g, KOH 0.031 g, Co(NO
3
)
2
·6H
2
O
0.490 mg, CuSO
4
1 mg, MnCl
2
·4H
2
O 1.440 mg,
ZnSO
4
·7H
2
O 8.820 mg , MoO
3
0.710 mg, MgSO
4
·7H
2
O
0.075 g, CaCl
2
·2H
2
O 0.025 g, FeSO
4
·7H
2
O 5 mg,
distilled water 1 L, and the pH of the medium adjusted
to 8.0.
3.2 Methods
3.2.1 Identification and purification of the algae
5 g of soil was weighed and placed in 150 mL
Erlenmeyer flask containing 50 mL sterile water, and
then incubated in the culture room, the light intensity
is 4 000~6 000 lux, photoperiod is 14 h: 10 h, 24 .
℃
When the algae appeared, we picked a fluid with a
inoculating loop, and then streaked on the BBM solid
medium until individual colonies grown on the plates,
and single colonies were purified more than 8 times.
3.2.2 Resistance analysis of algae
Single colonies of algae species purified was picked
into liquid BBM medium, when the algae solution was
dense, took some vigorous solution, centrifuged,
abandoned the supernatant, then added a certain
amount of sterile deionized water, make sure the
initial of the cell concentration was 100 mg/mL,
recorded as 5
-0
, and then diluted to 5
-1
, 5
-2
, 5
-3
, 5
-4
in
turn. 4 μL of serial dilutions (JB9, which is filament-
tous, was only picked filaments on the solid medium
with no concentration gradient) were spotted onto
solid BBM medium supplemented without or with
additional NaCl (BBM medium whose pH was
adjusted to 8.0 as the basic culture and the control
medium) and NaHCO
3
(BBM medium whose pH was
not adjusted as the basic culture and the control
medium). Growth was observed one week later.
Authors’ contributions
JW and WS designed and conducted this experiment; 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