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Tree Genetics and Molecular Breeding 2014, Vol.4, No.1, 1
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and source of initial tissue/organ which in turn are
related to their endogenous hormonal status (George,
1993). Most of the hardwood species produce
phenolic compounds after wounding (George and
Sherrington, 1984) which is also encountered in
A.
lakoocha
. The phenolic substances on oxidation get
converted into quinines which cause tissue blackening
and inhibit new
in vitro
morphogenetic responses in
plants. Phenolics could be minimized by keeping
explants right from the time of collection to
inoculation in antioxidant solution comprising of
ascorbic acid, citric acid and PVP (Elmore et al., 1990;
Liu and Yang, 2011). Polyvinylpyrollidine (PVP) is a
polyamide which absorbs phenols through hydrogen
bonding and thus prevents their oxidation. It is the
mostly commonly used antioxidant in micro-
propagation of hard wood trees like neem (Gautam et
al. 1993); ascorbic acid was also reported to be potent
antioxidant by Pati et al. (2008) and Abd El-Zaher
(2008) during micropropagation process. In the
present study, theexplants of
A. lakoocha
were given a
pretreatment with PVP (25 µM) for one hour which
was found to be effective in controlling oxidative
browning.
Explants play major role in developing a prototype
system for micropropagation of perennial hard wood
trees. Different types of explants were tried by several
research groups. Earlier, nodal stem segments were
reported by and Pati et al. (2008) and Raj and
Basavaraju (2012) in micropropagation of
A.
mormelos
, an underutiliszed fruit crop;which proved
to be successful because of the presence of protected
axillary buds unaffected by toxicity due to surface
sterilants and regeneration of true to type plantlets.
Optimization of explants was important for successful
micropropagtion technique as this forms the prime
factor for development of an efficient prototype
system. Therefore, preliminary studies on establish-
ment of aseptic cultures with ideal explants were
carried out in
A. lakoocha.
Explants of mature
lakoocha tree exhibit high level of exogenous and
endogenous contamination. Surface sterilization with
0.1% mercuric chloride (HgCl
2
) for 8 min was found
to be most effective for reducing contamination or
infection levels in nodal stem segments in lakoocha.
These results were on par with the earlier reports on
surface sterilization using 0.1% mercuric chloride in
Crataeva nurvala
(Sharma and Pandey, 1996).
Media is another critical factor that positively or
negatively influences the
in vitro
regeneration. In the
present study, MS+BAP 13.33+IAA 2.28+GA
3
0.57
µM was found to be statistically significant in terms of
bud induction, longer axillary shoots and heighest
number of leaves/explant of
in vitro
established
culture. Hamid et al. (2007) reported that jackfruit
explants cultured on MS+BAP 13.33+NAA 0.53µM
produced the highest number of shoots, leaves/explant
and shoot length. However, a combination of BAP
with IAA (MS+BAP 13.33+IAA 1.14 µM) augmented
multiplication of shoots. The synergetic effect of
auxin along with cytokinins on shoot multiplication
and shoot bud induction has been reported by several
workers (Mannan et al, 2006; Abd El-Zaher 2008).
2.2
In vitro
rooting in regenerated shoots
Finally, the microshoots were tested for rooting
efficiency in medium with various auxin levels.
Microshoots incubated on ½ MS IBA 9.85+IAA 1.14+
BAP 0.89 µM+Activated charcoal 500 mg/L signify-
cantly enhanced the root induction (88.16%) and
number (4.33). Roy et al. (1996) obtained successful
rooting of
in vitro
shoots of jackfruit on ½ MS+ IBA
4.9 + NAA 5.37 µM. Most recently, Khan et al. (2010)
have reported higher rooting rate (80%) and (6.9)
numbers of roots per plant were obtained with IBA
14.77 µM in jack fruit.
2.3 Hardening of
in vitro
plantlets
Plantlets grown
in vitro
have been continuously
exposed to controlled conditions for rapid plant
growth and sudden transfer is impossible directly to
pot which causes maximum mortality of
micropropagated plants. A gradual acclimatization to
natural environment is needed to increase survival
rates and hence, an intermediary greenhouse
hardening is done which depends mainly on the carrier
substrates (Hazarika, 2003). Sand+ Soil+ Peat moss as
carrier substrate with ½ strength MS nutrients has
marked effect on the plant height, number of
leaves/explant and overall survival of plants in
lakoocha which was coinciding with the results in