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Molecular Plant Breeding 2011, Vol.2 No.2
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selected plant, which was consistent with the target
band size, whereas no bands appeared in the
non-transgenic control plants (Figure 5).
Figure 4 GUS active transgenic plant by histochemical staining
Note: 1: GUS active transgenic leaf; 2: GUS activie transgenic
root; 3: Negative control of untransformed leaf; 4: Negative
control of the untransformed root
Figure 5 The transgenic plant identified by PCR
Note: 1: DL2000 marker; 2-3: Plasmid pCAMBIA0390; 4:
Blank (ddH
2
O); 5: The non-transformed control; 6: Transgenic
plant
2 Discussions
Definitely, the ideal material for rice transformation is
immature embryos, but immature embryo is quite
limited with harvest season, geographical limit,
timeconsuming for operations and specific facility etc.
The advantages of mature embryos used for
transformation are limitless material sources and
simple operation procedures. Therefore, the use of
mature embryos in rice transformation of rice become
more common.
Regarding on issues of distilled water immersion and
removal of endosperm processing, Lin et al., (1996)
considered that regulation system for dormant and
buffer action may exist in the mature rice seed.
Therefore, induction expression is low when faced
with external high-hormone environment. Removal of
the endosperm part of the rice mature seed can broke
the system stability of buffering capacity, resulting in
absorbing much more growth hormone in the culture
medium to promote cellular dedifferentiation into callus.
Using single-distilled water immersion might promote
cells of the mature seed to transform from the original
hibernation status to the active differentiation stage. In
addition, the authors believe that the explant sterilized
by mercuric mercury method could be certain poisoned
to affect the callus induction of explants in the later
stage, while singledistilled water soaking and removal
of endosperm treatment could alleviate explant toxicity
to increase callus induction rate.
Light culture prior to differentiation is one of the
methods to improve the rate of callus differentiation
(Duan et al., 2001). In this study, subculture stage was
removed during genetic transformation due to the
reports on the rate of callus differentiation reduction
following subculture. Our results confirmed the
transformed plants can be acquired in short period
even removal of subculture stage. Selecting embryonic
callus with bright-yellow color, granular and dry
surface for infection and callus culture with green
spots will be easy to be differentiation.
Dehydration processes such as starvation and drying
treatment will facilitate to callus differentiation (Lu et
al., 2008; Masayoshi et al., 1992). The process for
dehydration may lead to a callus cell physiological
and biochemical changes, which might promote the
absorption of the exogenous hormone. Dehydration by
using natural drying on the clean bench may be suitable
time period within the 30 miinutes based on the
judgement of the status of callus, because callus
dehydrated very quickly in environment of clean
benches. If the drying time be longer, the vast part of
the callus will be getting to die. In addition, there are
some reports that indicated hypertonic environment in
the internal of medium can be effective on rice callus
dehydration, for example, increasing the agarose
concentration in the medium, or adding a certain
concentration of mannitol, sorbitol and so on.
In this study, the selectable marker Bar gene from soil
Streptomyces (
Streptomyces hygroscopieus
), encoding
phosphine chloramphenicol acetyltransferase (Phosp-
hoinothricin), acetyltransferase was known to inhibit
the activity of Gln synthetase in nitrogen metabolism
of plant and to destruct photosynthesis of plant, which