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This thesis was conducted to explore the possible mechanisms of shoot rege
neration from rice callus induced by osmotic stress. The relationships among
osmotic stress, endogenous phytohormone levels, carbohydrate metabolisms,and s
hoot regeneration were studied. In addition, the "regeneration-related factor
s" were discussed in this thesis. The calli were induced from immature embr
yos of two rice cultivars (Oryza sativa L.) and used for regeneration experime
nts. One is Ai-Nan-Tsao 39, ANT39, whose callus showed high shoot regeneratio
n ability. The other is Tainan 5, TN5, whose callus showed greatly increased
regeneration frequency only after being exposed to an osmotic stress exerted b
y either metabolizable (ex. sucrose) or non-metabolizable (ex. mannitol) sacch
arides. At first, the callus growth, shoot regeneration frequency, water and
osmotic potentials of culture mediumand callus under different osmotic stress
treatment were determined. Then, the cellular contents of soluble sugars and
starch, and the cellular localization and activities of enzymes related to the
ir metabolism, including Sol-IT, Bound-IT, SS, AGPase, SPase, α-Amy, and β-A
my of rice calli and shoot regenerating tissues were systematically analyzed.
Finally, changes of carbohydrate metabolism was further investigated in TN5-An
A treatment, in whichosmotic stress was replaced by 2mM anthranilic acid and 1
00μM ABA. The results showed, highly regenerable callus, ANT39-Dry, TN5-Su
c18, and TN5-M6, have lower callus growth rate, water content, cellular water
and osmotic potentials than un-regenerable callus, TN5. It suggested that sho
ot regeneration is closely related to cellular osmotic status. In addition, h
igher starch and sucrose contents at callus induction stage and a sudden incre
ase or retaining higher glucose content at the first day after the callus bein
g transferred to regeneration medium were found in highly regenerable callus.
The starch and sucrose contents were decreased during shoot regeneration. Th
e phenomenon of starch accumulation and disappear were also observed from hist
ochemical analysis. On the other hand, there are no significant changes in ca
rbohydrate contents and histochemical appearance in un-regenerable callus. We
concluded, therefore, the higher starch content at callus induction stage and
higher glucose content at the initiation of shoot regeneration stage were bot
h the important "regeneration-related factors" in rice callus. The carbohyd
rate metabolic pathway was different between ANT39-Dry and TN5-M6 although the
changes of carbohydrate contents were similar. In ANT39, the lower Bound-IT
and SS, higher Sol-IT activity at callus induction stage suggested that sucros
e might be uptaken by sucrose transporter mainly. In addition, both AGPase an
d α-Amy are higher at this stage suggested that the higher starch content is
the result of higher stasch biosynthetic ability. At the initiation of shoot
regeneration stage, higher expression and enzyme activity of α-Amy, higher So
l-IT, and lower Bound-IT and SS activity were found. These results might expl
ain that the higher glucose content might come from degradation of both starch
and sucrose. In contrast, it showed greatly increase of Sol-IT, Cound-IT, an
d SS at callus induction stage in TN5-M6. It suggested that sucrose uptake fr
om sucrolytic pathway dominantly. At the same time, TN5-M6 showed similar AGP
ase but lower α-Amy activity than TN5. Thestarch accumulation, therefore, mi
ght be caused by lower degradation of starch in TN5-M6. At the initiation of
shoot regeneration stage in TN5-M6, higher Bound-IT and Sol-IT, lower amylase
activity were found. It might suggest that higher glucose content is due to h
igher sucrose uptake by Bound-IT mainly. Because starch content was also decr
eased at the same time, it might have another starch degradation pathway, for
example, SPase, which showed higher SPase activity at this stage in TN5-M6.
According to the observation of immunohistochemistry, it showed the distribut
ions of SPase, β-Amy, and α-Amy are related to starch granules deposition.
In addition, α-Amy is also localized at tracheary elements (TEs).SS1 was more
dominantly localized at TEs and peripheral cells in callus. Besides, SS1 mig
ht be related to starch biosynthesis. On the other hand, SS2 and SS3 were les
s detected and only localized at some specialized tissues and parenchyma. A B
ound-IT-like antibody was used in this thesis. It localized at peripheral cel
ls especially at the side attached culture medium. In addition, it closely re
lated to somatic embryogenesis and organogenesis during shoot regeneration.
When TN5 callus was induced in MSD10 medium containing anthranilic acid andAB
A, it showed slower callus growth, lower cellular water content and osmotic po
tential, and higher starch and soluble sugars contents. After being transferr
ed to regeneration medium, it also showed higher glucose content and decreased
starch and sucrose contents. All the phenomenons were similar to those under
osmotic stress treatment. Higher IAA and ABA levels inhibited all the sucrol
ytic enzyme (Sol-IT, Bound-IT, and SS) and amylase activity. Therefore, the hi
gher soluble sugars and starch contents might be resulted fromsucrose uptaking
by sucrose transporter dominantly and inhibiting starch degradation, respecti
vely. In conclusion, osmotic stress induced some changes of carbohydrate meta
bolism, and was brought about shoot regeneration. Part of those changes were
regulated by changes of endogenous IAA and ABA levels. But there are some pro
cesses induced by osmotic stress might be regulated by other factors during sh
oot regeneration.
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