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Tree Genetics and Molecular Breeding, 2013, Vol.3, No.3, 12
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the
in vitro
raised plantlets more fragile than the
acclimatized plantlets. These histological and bioche-
mical observations in
in vitro
plantlets on comparison
with ex vitro plantlets suggest that some procedural
changes are necessary for acclimatization of these
plants. The successful acclimatization of plantlets may
require the full development of leaves, which may
largely depend on the rooting stage. This means that in
vitro hardening is important and in the present study,
we had attempted to select better-suited plants for
micropropagation. The supplementation of essential
nutrients (½ strength MS plant salt mixture) during
acclimatization resulted in increases survival rate
proving that photoautotrophic phenomenon has
substantial influence on the physiology and develop-
ment in
in vitro
regenerated bael plantlets.
3 Materials and methods
3.1 Explant and growth conditions
Three centimeter long nodal stem segments excised
from mature tree having at least one axillary bud was
used for in vitro regeneration of
Aegle marmelos
Corr.
The nodal explants were washed thoroughly with 2~3
drops of Tween-20 (a liquid detergent) and were
rinsed in running tap water for 30 minutes. Explants
were treated with 5.24 µM carbendazime + 303.5 µM
rifampicin in 100 mL of distilled water and 2~3 drop
of Tween-20 for 1 hour in order to disinfect. To over-
come the problem of
in vitro
oxidative browning, the
explants were given a pre-treatment with an antioxidant
solution comprising ascorbic acid (566 µM), Polyviny
lpyrollidine (250×10
-2
µM) and citric acid (474.8 µM)
for 1 hour. Micropropagation protocol of Pati et al.
(2008a; 2008b) was followed to regenerate plantlets of
Aegle marmelos
Corr.
Eight-week-old rooted plantlets were removed from
agar and thoroughly washed using distilled water and
transferred to culture bottles (450 mL volume) containing
coconut husk. supplemented with ½ strength MS plant
salt mixture (Hi-Media, India). Initially the culture
bottles were incubated in the culture room under at
4000 lux photo intensity maintained at 25 2
℃
tempe-
rature for about 15 days, and then shifted to plant
growth chamber where, illumination was increased to
10 000 lux with high humidity (70%~80%). The
plantlets were then shifted to shade net house (50%
shade) provided with misting. The caps of the bottles
were loosened gradually and the plants were shifted to
pots. Once the plants developed 4~6 leaves, they were
shifted to field during the month of July and August.
3.2 Biochemical Studies
The regenerated plantlets of Aegle marmelos Corr
were analyzed for different biochemical parameters viz.
chlorophyll, reducing sugar, nitrate reductase activity
and protein at various regeneration stages (
in vitro
,
acclimatized, field established and control). 500 mg
leaf samples were collected from each regeneration
stages and used for the biochemical study. The
chlorophyll (a, b and total) was estimated as per the
method described by Arnon (1949). The reducing sugar
was estimated by the method suggested by Ranganna
(1986). Nitrate reductase (NR) activity was measured
by the method of Srivastava (1975). The total soluble
protein was estimated as per the method suggested by
Lowery et al. (1951). For all biochemical studis observ-
ence were measured by DyNA Quant 200 UV-visible
spectrophotometer (Chemito UV-2010, Pvt. Ltd.).
3.3 Histological examination
For histological studies, 0.5 cm
2
leaf, stem and root
samples of were collected from various cultural stages
(8 weeks old
in vitro
and 16 weeks old acclimatized
plants) and fixed into FAA [Formaldehyde (5 mL):
Acetic Acid (5 mL): ethanol (90 mL)]. The fixed
samples were washed thrice with sterile double
distilled water followed by blotted dry on sterile tissue
paper. Then fixed samples were dehydrated through
the series of t-butyl alcohol in ascending order (30%
to 100%). The samples was passed through the
gradated ethanol:xylene mixtures (100% t-butyl
alcohol, 3:1, 1:1, 1:3 and 100% xylene) using automatic
tissue processor electra (Yarco-YSI 104) and embedded
in molten paraffin wax ( melting point 56
℃
) for 8 hrs
in order to completely replace the xylene with paraffin
wax. 15 m thin sections were cut using microtome
(Microm, HM 350S) and stained in 0.1% aqueous
toluedine blue O as described by Jensen (1962). The
slides were observed under stereoscopic microscope
(Leica DFC 320, Japan).
Acknowledgement
We are grateful to Director, CISH, Lucknow for
providing facilities.