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Tree Genetics and Molecular Breeding, 2013, Vol.3, No.3, 12
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rapid growth shoot proliferation and plantlet developed,
resulted certain abnormal characteristics such as
altered leaf morphology and mesophyll structure, poor
photosynthesis, sunken and malfunctioning stomata
(Ziv, et al., 1986; Hazarika, 1996). In order to devise
an efficient acclimatization procedure, it was imperative
to investigate some of the crucial biochemical (total
chlorophyll, reducing sugar, NR activity and protein)
and histological changes during various micropropagation
stages, which contribute towards growth and develop-
ment of plants.
1 Result and Analysis
1.1 Biochemical analysis of regenerated plants
The results revealed that a significant variation in
biochemical parameters which were observed among
the various culture stages. The chlorophyll (a, b and
total) synthesis was recorded lower (0.022, 0.020 and
0.042 mg/g) during
in vitro
phase, while higher
chlorophyll levels (0.093 mg/g) was recorded during
acclimatization. The
in vitro
plants showed lower level
(3.22 mg/g) of reducing sugars than ex vitro grown
plants. Highest NR activity (1.35×10
-2
NO
2
/h/g fresh
weight) was observed during
in vitro
phase, which
decreased significantly during acclimatization. The
protein levels remain low during acclimatization
(0.048 µg/g) as compared to control plant (Table 1).
1.2 Histological study of leaf
Histology of acclimatized plant leaf (Figure 1B;
Figure 1D) showed single layered epidermis with thick
cuticle and well developed double layered palisade
mesophyll. The lower side had spongy parenchyma
with air spaces. Stomata were present in lower epidermis.
The
in vitro
leaf histology showed single layered
epidermis with almost no cuticle (Figure 1A; Figure 1C)
on both adaxial and abaxial sides. The palisade
parenchyma was single layered and poorly developed
but the spongy parenchyma was prominent and well
developed and the vascular tissue was observed to be
poorly developed. It also showed few and open
stomatal apparatus with fully turgid guard cells.
1.3 Histological study of stem
Both the
in vitro
stem (Figure 1E) and acclimatized
stem (Figure 1F) had a polystelic structures. Vascular
bundles were arranged in a ring and they were
conjoint collateral and open. In the acclimatized stem
cork cambium was well developed which was lacking
in the
in vitro
stem. Both the
in vitro
and acclimatized
stems had well developed parenchymatous pith but the
pith was mucilaginous in the case of acclimatized stem.
The acclimatized stem showed distinct secondary growth
with well-developed wood and large woody vessels,
while the
in vitro
stem showed primary medullary rays.
The epidermis was uniseriate in both the cases, but it
was covered with cuticle in the acclimatized stem,
while no cuticle was present in the
in vitro
stem. The
acclimatized stem showed distinct endoderm which
was absent in the
in vitro
stem. The cortex was
parenchymatous in the
in vitro
stem while it was
collenchymatous in case of acclimatized Stem.
Table 1 Biochemical analysis of nodal stem segment-regenerated plants of
Aegle marmelos
. Different letters within column indicate
statistically different values according to Tukey’ test (
p
=0.05)
Treatment
Biochemical attributes
Chlororophyll
Reducing sugar (mg/g) Nitrate reductase
assay (NO
2
/h/g FW)
Protein
(µg/g)
'a'
'b'
'a+b' (mg/g)
In vitro
0.021d
0.019c
0.040b
3.117c
1.323a
0.046c
Acclimatized
0.047c
0.044b
0.090b
4.727c
1.090b
0.027d
Field grown
0.060b
0.055b
0.078b
7.210b
0.670c
0.061b
Mother/control
0.125a
0.097a
0.222a
9.267a
0.320d
0.069a
SE(m) ±
0.004
0.005
0.017
0.344
0.063
0.002
CD (0.05)
0.013
0.016
0.056
1.138
0.209
0.007
Note: Source: Pati et al., 2008b