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Bioscience Methods 2012, Vol.3, No.4, 27
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University; the tested tomato variety named Zhongshu
Si Hao was purchased from Shanxi Academy of
Agricultural Sciences.
3.2 Treatment of seed disinfection
Tomato seeds were rinsed with fresh water, and then
immersed in 70% alcohol for 30 s, transferred to 0.1%
mercuric chloride, 20% sodium hypochlorite and 20%
sodium hypochlorite with Tween-20. of which treated
with 0.1% mercuric chloride for 20 s, 30 s, 1 min, 3 min,
respectively; treated with 20% sodium hypochlorite
for 10 min, 15 min, 20 min and 30 min, respectively;
treated with 20% sodium hypochlorite with Tween-20
same as that without tween
-
20. The 50 seeds were
used for each treatment, repeated three times. Seed
germination number and contamination rate were
investigated after seed germination.
3.3 Induced bud medium screening
8 days after seed germination, unfolding cotyledons
were cut into pieces with the size of 0.5 cm × 0.5 cm
to be as explants, some pieces with a petiole and or
some pieces from the middle of the leaves. The media
for adventitious bud differentiation are the MS (N &
N organic) medium adding the different combinations
of BA and IAA, BA and NAA, ZT and IAA, with
different concentrations. 5 bottles for each combination,
four explants placed in a bottle, repeated three times.
The induced callus rate and induced bud rate were
investigated after 20 days culture.
3.4 The effects of different explants on bud
differentiation
Steriled tomato cotyledon, leaf, stem and root
segments were placed on the selected best bud
inducing medium Vaccine of true leaves, cotyledons,
stems and roots (size: 5 mm×5 mm), 20 pieces per
explant were placed in the four bottles, repeated three
times. The type with highest rate of induced bud was
chosen.
3.5 The effects of the different parts of cotyledon
on morphogenesis
Employing the selected optimum medium described
by 3.3 section, the cotyledons cut into three parts
(Figure 2), 20 cuttings of each par were flat placed on
the differentiation medium, repeated by three times,
the part with highest rate of the bud differentiation
was determined.
Figure 2 The cotyledon was cut into three parts
3.6 The effects of cotyledons placing ways on the
bud differentiation
Employing the selected optimum medium described
by 3.3 section, cotyledon was placed in the medium in
two ways, face up and back up, each trial sets five
bottles, four explants per bottle, repeated 3 times,
callus inducing rate and budding inducing rate were
investigated after 20 days culture.
3.7 The adventitious buds rooting
when the adventitious shoots grow up to about 2 cm,
the bud at the base was cut to insert in the rooting
medium, the culture medium was 1/2 MS medium
supplemented with different concentrations of IAA
(0, 0.1 mg/L, 0.3 mg/L, 0.5 mg/L) and different
concentration of NAA (0, 0.1 mg/L, 0.3 mg/L, and
0.5 mg/L). After two weeks we start to determine the
best medium for rooting by the observation and
comparison.
3.8 Light conditions
Photoperiod conditions for tissue culture were set as
16 h/8 h (light/dark). Culture shelves for light culture
with illumination intensity was from 2 000 to 3 000 Lx
and temperature at (25±1)
℃
; Biochemical incubator
for dark culture with the temperature at (25±1)
℃
.
References
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exploitation, Anhui Nongye Kexue (Journal of Anhui Agricultural
Sciences), 34(9): 1864-1865
He X.X., Lu Y.M., Bai J.Y., and Li Y.F., 2003, The establishment of tissue
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