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Bioscience Methods
BM 2011, Vo.2, No.5
http://bm.sophiapublisher.com
- 34 -
media, which can be more conducive to the formation
of embryogenic callus and improve the quality of
callus, useful to the differentiation of the next step.
1.4 Impact of hormone ABA on the callus subculture
Abscisic acid (ABA) is a plant hormone with high
activities. It plays an important role in increasing
induction rate and improving the quality of callus.
Although a certain concentration of ABA can reduce
callus growth rate, it is very useful to improve the
status of callus to be conducive to the formation of
embryogenic callus. Our studies indicated that the
growth rate and the state of callus reached to the best,
and it is prone to form the embryogenic callus with
dense texture and dry appearance when the callus was
cultivated on the medi um supplemented 1 mg/L ABA
(10.54±0.81) %. However, as the concentration of
supplemented ABA increasing, the induced callus will
grow to be brown and a large number of hairy roots
will grow up (Table 4). Therefore, it is very useful for
the callus growth to supplement 1 mg/L ABA into the
subculture medium.
Table 4 Impact of different concentrations of ABA on callus subculture after 20 days’ cultivation
Medium
ABA (mg/L)
W
0
(g)
W
1
(g)
Growth rate (%)
Modified medium S
0
1.172 7
3.452 7
11.40±3.73
Modified medium S
1
0.897 1
3.005 6
10.54±0.81
Modified medium S
2
0.900 2
2.834 6
9.67±3.25
Note: W
0
: Referred to the weight at the beginning of inoculation; W
1
: Referred to the weight after 20 days’ of cultivation on the
different subculture media
1.5 Effect of different hormone concentrations on
the callus differentiation
In this study, we discussed the effects of different
hormone
combination
ratios
and
hormone
concentration supplemented into the differentiation
medium. We found that when the hormone
combination ratio (C
6-BA
: C
IAA
) is 10:1, different
contents of hormone containing in the medium will
have different effects on the callus differentiation.
High concentration of hormone will negatively affect
the differentiation, increase the browning rate, and
even directly lead to the death of callus cells. When
the concentrations of 6
-
BA and IAA supplemented is
0.4mg/L and 0.04 mg/L, the callus differentiation rate
reaches up to 53.73% (Table 5; Figure 3). To prevent
browning, 500 mg/L proline was supplemented into
the induction, subculture and differentiation media.
1.6 Infection time of
Agrobacterium
have an impact
on the callus genetic transformation
Infection time of
Agrobacterium
have an obvious
effect on the transformation efficiency. Long time of
infection will inhibit the growth of the receptor, hard
to get the tranformants, but short time of infection go
against the attachment of
Agrobacterium
to the
receptor and decrease the transformation rate. In this
study, the result showed that the transient expression
Table 5 Callus differentiation at different concentrations when
hormone ratio (6
-
BA:IAA) is 10:1
Medium
Differentiation rate (%) Browning rate (%)
1
12.56±1.84
80.34±1.66
2
25.42±1.76
70.23±1.42
3
42.35±1.68
50.45±1.59
4
47.42±1.87
15.14±1.68
5
53.73±3.34
12.23±1.28
6
39.53±1.79
10.12±1.24
Note: 1~6 means to the different amounts of hormone adding
into the basic medium; 1, 6
-
BA 4 mg/L and IAA 0.4 mg/L; 2,
6
-
BA 2 mg/L and IAA 0.2 mg/L; 3, 6
-
BA 1 mg/L and IAA
0.1 mg/L; 4, 6
-
BA 0.5 mg/L and IAA 0.05 mg/L; 5, 6
-
BA
0.4 mg/L and IAA 0.04 mg/L; 6, 6
-
BA 0.3 mg/L and IAA
0.03 mg/L
Figure 3 Callus differentiation at different concentrations of
hormone when the ratio of C
6-BA
: C
IAA
is 10:1
Note: RD refers to the rate of callus differentiation. Note: 1
-
6
means to the different amounts of hormone adding into the
basic medium as Table 5.