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Bioscience Methods 2012, Vol.3, No.4, 27
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28
low regeneration frequency, instability, and high
proportion of deformity bud are still existing (Liang et
al., 2004). Therefore, In this research we attempted to
study the factors related to tomato cotyledon regeneration
in order to establish a stable regeneration system, and
to lay the foundation for the genetic transformation of
tomato in future.
1 Results and Analysis
1.1 Screening seed disinfection methods and obtaining
aseptic seedlings
The results showed that the seeds treated with
mercuric chloride had low seed germination rate, long
germinating time and irregular germination, whereas
the seeds treated with sodium hypochlorite had higher
seed germination rate, short germination time and
identical germination. If tween
-
20 was added into
sodium hypochlorite, the effects of sterilization will be
better and the germination rate can reach 90% up
(Figure 1A). Summing up the above results, treating
tomato seed with 70% ethanol for 30 s and then 20%
sodium hypochlorite with tween
-
20 for 20 min
would be more appropriate method for seed surface
sterilization (Table 1).
Figure 1 Establishment of regeneration system from cotyledon
of Chinese tomato
Note: A: Aseptic seeding; B: Induction of calli; C: Induction of
adventitious buds; D: Buds induced from the stem; E: Induction
of adventitious roots; F: Plant regeneration
Table 1 Effects of different sterilizing methods on tomato seed surface disinfection
Code
Soaking time (min)
No. of
germination
Ratio of
germination (%)
No. of
contamination
Ratio of
contamination (%)
70%
ethanol
0.1%
HgCl
20%
NaClO
20%
NaClO+Tween
-
20
1
2
4
6
11
12
13
14
15
16
17
18
30 s
30 s
30 s
30 s
30 s
30 s
30 s
30 s
30 s
30 s
30 s
30 s
20 s
30 s
1
3
-
-
-
-
-
-
-
-
-
-
-
10
15
20
30
-
-
-
-
-
-
-
-
-
-
-
-
10
15
20
30
25.2
23.1
14.7
8.0
4.5
39.5
42.8
29.6
10.9
42.3
46.9
25.5
50.4±0.35fF
46.2±0.56gG
29.5±0.65hH
16.0±0.33lL
9.2±0.12nM
79.0±0.24cC
85.6±0.51bB
59.2±0.48eE
21.8±0.22jJ
84.6±0.85bB
93.8±0.81aA
51.0±0.32fF
2.8
0
0
0
50.0
4.6
1.7
1.2
42.3
1.6
0.8
0
4.0±0.25dD
0.0
0.0
0.0
100.0±0.00aA
9.2±0.56cC
3.4±0.24eE
2.4±0.36gF
84.6±0.34bB
3.2±0.75fE
1.6±0.33hG
0.0
1.2 The effects of different hormone combinations
on tomato bud differentiation
Using MS medium (N & N-organic) as primary medium
combining the different hormones, the regeneration
frequency of the combinations (Table 2) showed that
tomato cotyledon differentiation rate was higher at the
level of ZT 2.0 mg/L. Almost every tested explants
could differentiate callus tissues and adventitious
buds; and the robust combination was ZT 2.0 mg/L
and IAA 0.1 mg/L together, which can induce strong
buds and less blank buds and deformed buds, and the
average budding number reached 5.5 (Figure 1C). The