Molecular Plant Breeding, 2016, Vol.7, No.15, 1-12
10
division property, quickly heals phloem and stops
exudation. For this purpose IAA was added as an
auxin in every micropropagation medium in addition
to BAP. Moreover this problem was overcome by
subsequent subculturing of explants that indicated
browning. Two-three subcultures were enough to
lessen the exudation enough, so it could not affect the
shoot formation.
Induction and multiplication of
in vitro
shoots of
almost all woody tree species is easier if the explants
are obtained from developing plantlets, generally said
to be at juvenile stage and they give superior results
than mature explants (George, 1993). The harder the
explant cutting became, the longer the sterilization
time required and yet harder to control contamination.
The bacteria survive longer on hard explants and grow
faster under favorable conditions. Soft cuttings grew
faster and illustrated less contamination upon
initiation, which is why young juvenile explant
cuttings with fresh green buds were selected for
micropropagation.
Micropropagation is done via different concentrations
of various growth regulators, either alone or in
combination. Inclusion of auxins and cytokinins in the
micropropagation medium greatly influences the shoot
formation and development. Generally response and
the quality of
in vitro
cultures are highly influenced by
the concentrations and type of nutrients present in the
culture media (Niedz and Evens, 2007). Outcome of
different micro & macronutrients on growth and
number of
in vitro
culture and micropropagation was
investigated by many researchers (Cohen, 1995; Rout
et al.,
1998; Thirunavoukkarasu and Debata, 2002).
In
vitro
shoot induction in most of the plant species is
done using various cytokinins, among which BAP is
reported to be the most responsive and efficient
(Arumugam and Rao, 1996; Behera
et al.,
2008;
Nayak
et al.,
2007; Pradhan
et al.,
1998; Purohit and
Dave, 1996). Carelli and Echeverigary, (2002) showed
that BAP produces more shoots as compared to
Kinetin and 2iP in micropropagation systems. BAP
has also been reported the superlative growth
regulator for shoot development and multiplication in
Shisham by several researchers. Bari
et al.,
(2008)
reported an average 2.2 shoots form callus of Shisham,
via combination of 1.5 mg/L BAP + 0.5 mg/L IAA in
MS media. Current study also indicated that the
addition of BAP, among all cytokinins, proved to be
substantially useful for
in vitro
shoot formation from
juvenile buds of Shisham. We obtained a maximum
2.78 number of shoots using MS medium
supplemented with 6.6 µM BAP + 1.71 µM IAA.
However deformity and abnormal shoot induction was
reported as a result of high BAP concentration by
some researchers (Carelli and Echeverrigary, 2002).
3 Materials and Methods
3.1 Explants to be used
Material for Shisham micropropagation was collected
from the territory of University of Agriculture
Faisalabad. Axillary and epicormic shoots were used
as explants in this study. Cuttings of 1.5-2.5 cm, each
containing at least one nodal bud were used. To the
best of our information, Shisham trees that were used
as source for getting explants (axillary and epicormic
shoots), were wild genotypes to the Faisalabad area,
and thus the potential value as clonal material was
unidentified.
3.2 Explant sterilization
Explants were thoroughly washed and treated with
tween- 20 (7drops/100ml) followed by the treatment
with 0.7% Chloroxylenol solution. These explants
were dipped in 70% ethanol for 10 seconds and then
treated with 0.1 % Mercuric Chloride (HgCl
2
) solution
for 5 minutes. All these operations were done under
high axenic conditions in laminar air flow cabinet.
Ultra pure water was used for the preparation of all
these solutions to be used in sterilization of explants.
3.3 Media preparation and culture conditions
Micropropagation media were prepared by using
growth regulators of varying concentrations in
addition to MS Salts and sucrose in common. For
micropropagation eight media combination with
varying levels of IAA and BAP were studied (Table 3).
For root induction, half MS media with and without
IAA and IBA were used (Table 4). Gellan gum powder
was used as solidifying agent and pH was adjusted to
5.8 using NaOH and HCl. Micropropagation media
were poured into test tubes that were wrapped by
using polypropylene sheets and then autoclaved at
121°C, 15
psi
for 20 minutes. Culturing of explants on
micropropagation media were done under aseptic
conditions in laminar air flow hood. These cultures
were incubated and maintained in controlled
conditions at 25 ± 2°C for 16/8 hrs light/dark period.
