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Tree Genetics and Molecular Breeding, 2013, Vol.3, No.3, 12
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2. Discussion
2.1 Biochemical analysis
During
in vitro
phase, it is generally seen that the
protein production is low because regenerated plants
were photosynthetically inactive therefore protein
biosynthesis requirement remains high (Preece and
Sutter, 1991; Seon et al., 2000). Lower chlorophyll
levels of
in vitro
plants clearly suggest lower or
negligible rate of photosynthesis due to heterotrophic
mode of nutrition that ultimately limits the biosyn-
thesis of protein.
During acclimatization
in vitro
plantlets encounters
severe stress condition. The stress response is exhibited
in the form of culture reduction and production of heat
shock proteins or stress proteins. A decrease in soluble
protein content with increasing temperature has been
observed in other species (Zhou et al., 1995). Therefore
it may be concluded that supplementation of essential
nutrients during acclimatization and photoautotrophic
development of micropropagated plant has increased
survival rate.
In vitro
plants also showed higher levels
of reducing sugars because during micropropagation
carbohydrate reserves are built up in leaves by
manipulating sucrose in shoot multiplication stage prior
to acclimatization. NR is one of the most important
metalloflavoprotein regulatory enzyme associated
with the process of nitrate assimilation and plant
growth which was observed with highest activity in
vitro phase, which significantly decreased during
acclimatization. Since, NR is substrate inducible
enzyme (Haynes, 1964); the presence of high amount
nitrate substrate in tissue culture medium probably
augments the NR activity during
in vitro
phase.
2.2 Histological studies
Histological studies of
in vitro
grown plantlets showed
that leaves had more frequent, smaller elongated
stomata, more irregular shaped epidermal cells and
thin as well as poor developed spongy and palisade
parenchyma then the ex vitro plantlets (Yand and Yeh,
2008). Moreover, the leaf surfaces of micropropagated
plants were more wettable, suggesting less epicuticular
wax deposition on them. These findings suggest that
the possible factors those influencing the hardening of
plants during the last phases of micropropagation
(Gribaudo et al., 2003; Hazel and Harry, 1982). Generally
in vitro
conditions cause rapid growth and shoot
proliferation, thus the plantlet developed shows
certain abnormal histological features like altered leaf
mesophyll, absence of thick cuticle, sunken or functi-
oning stomata and poor water transport (Majada et al.,
1998). Lack of prominent cuticle is a regular feature
of
in vitro
plants and this was proved by the
investigation. This cuticle and epicuticle are primary
centers for containing water permeability (Pospisilova
et al., 2000); this is therefore a major feature leading
to excessive water loss and poor survival rate upon
transpiration. Our findings are in accordance to earlier
reports of Kortemios and Artemios (1997) where in
they have reported that Mg
2+
and sucrose in the
medium influenced the relative size of mesophyll cells,
number of chlorophyll and presence of starch grains.
The addition of sucrose to the substrate media
negatively affected the size of the mesophyll cells
under normal Mg
2+
concentration (35 mg/L), and
positively under high Mg
2+
concentration (105 mg/L).
Sucrose further resulted in an increase in the numbers
of chloroplasts and contained starch grains.
Earlier, Rotondi and Predieri (1996) reported that
highest chlorophyll ‘a’ and ‘b’ contents were recorded
in leaves of pear cultivars viz. Abbe Fetel and Passe
Crassane compact types which also exhibited higher
photosynthetic efficiency. During acclimatization tran-
sitional forms of leaves, petioles, stems and roots
develop that ranged histologically from culture-to
control-type. This trend is analogous to the normal
developmental sequence of organ formation as it
affects the potential for development of successively
formed organs (Danielle et al., 1985). Notable changes
in leaf histology of bael was observed during different
in vitro
stages as well as after transferring plantlets to
soil. The leaf size increased gradually during microp-
ropagation stages. Similar observations were reported
by Brutti et al. (2002) and Apostolo et al. (2005) in
Cynara scolymus
L. leaves. The
in vitro
raised plantlets
shows poorly developed root histology with no cork
cambium, but a single layered epidermis with cortex
around the vascular cylinder and vascular bundles
were very poorly developed (Petrus-vancea, and Sipos,
2010). Non-mucilaginous pith was more prominent,
but the xylem parenchyma and fibers were less, making