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Bioscience Methods 2012, Vol.3, No.4, 27
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1.6 The adventitious buds rooting
The adventitious buds about 1 to 2 cm in length were
transferred to rooting medium for inducing roots, it
was normal to generate roots after 3 to 5 days
culturing. After two weeks it can see that the 1/2 MS
medium adding the additional of IAA (0.1 mg/L,
0.2 mg/L, 0.3 mg/L) was the best to induce normal
milky white and health roots with more lateral roots,
the average length of the roots was longer; thereinto,
the 1/2MS medium adding IAA 0.1 mg/L is the best
for tomato callus rooting resulting in health and
thicken roots (Figure 1E). Whereas the 1/2 MS
medium adding the additional of NAA (0.1 mg/L,
0.2 mg/L, 0.3 mg/L) for inducing roots was slower to
generate roots. In comparison of three treatments,
1/2MS medium adding NAA 0.2 mg/L is better but the
root color appeared gray and short and weak roots, not
suitable for tomato rooting induction. Definitely, the
control medium without adding any hormone can
induce the callus rooting, but the induced roots were
week, thin and long even long time cultured. It
suggested that adding IAA for inducing roots should
be more effective than that of adding NAA; and the
best rooting medium should be 1/2MS+IAA 0.1 mg/L.
Contamination control is the key factor to success of
the trial in plant tissue culture. Therefore, complete
disinfection of the explants is the premise of
contamination control. According to the different plant
species and explant, it should adjust the concentration
of disinfectant and treating time accordingly. Seed
surface of tomato has tiny villi, incomplete disinfection
will result in failure in the whole trial. In this research
we thought that the disinfection for tomato seed
surface should adopt the procedure as 70% ethanol
immersing for 30 s and then rinsing 20% sodium
hypochlorite with Tween
-
20 for 20 min, which was
proved to achieve very good disinfection effect.
Plant tissues as explants used for transformation are
usually hypocotyl, petiole, cotyledon and stem segments
and so on; it is more commonly used for hypocotyl,
cotyledon, and petiole. Cotyledons and hypocotyls of
tomato are the most employed explants for regeneration
in vitro
. The study of Sun et al (2006) indicated that
two kinds of tomato explants, cotyledons and
hypocotyls, could induce callus and adventitious buds,
obviously the conclusion of this research is consistent
with this.
Plant hormones are indispensable key substance in the
medium, which play an important and significant roles
in regulating in explant callus induction and root, bud
and organ differentiation. In tomato tissue culture,
plant hormone, IAA, NAA, 2, 4-D, ZT and 6-BA and
so on are commonly used to inducing the adventitious
buds. Wang et al (2007) had shown that the
combinations of ZT and IAA was the best to induce
buds of tomato callus than other combinations, which
budding under the same conditions at least was as
early as 2~5 days, This studies have shown that ZT
and IAA combination for inducing buds was the best
combination that was good for callus growth.
The explants placed in the different ways would result
in differentiation rate of cotyledon bud. This study has
shown that back-up placing of the cotyledon is the
best way. Regarding the leaves, it is generally believed
that the leaf back of dicotyledon plants has lots of
stoma, so leaf the back of leaf placing pre-cultured
would be conducive to nutrient absorption, therefore,
this would be the reason that the explant leaf were
commonly exposed to the medium with the leaf back.
However, Shi et al (1999) showed that face leaves
exposed to the medium had the best effects than that
of the back leaves exposed to the medium. Definitely,
the placement of leaf explants would affect the
induction of adventitious buds but there may also be
differences due to different plant species.
Rooting is the key to establish the regeneration system
in plant tissue culture; the quality and the rooting rate
would determine whether or not that tissue culture
technique could be applied in the production. There
are many factors affecting the rooting, such as
physiological and biochemical status of plantlets itself,
primary medium, growth regulators, and other
external factors, but plant hormones may be the most
important internal regulatory factor. Most of the
rooting cases would be achieved with auxin alone
(Zhao et al., 2009). However, our experiment suggested
that the 1/2MS added IAA was good for inducing
roots, indicating that different hormones should be
differenced to rooting.
3 Materials and Methods
3.1 Experimental materials and site
This experiment was carried out at tissue culture
lab in College of Horticulture of Shanxi Agricultural