Molecular Plant Breeding, 2016, Vol.7, No.15, 1-12
2
dozen fungal diseases are reported in IndoPak. But in
last few years this tree has been badly affected by a
disease called; Shisham decline or dieback, that was
announced as an epidemic for Shisham all over the
Pakistan in 1998 (Naz, 2002; Bajwa et al., 2003).
Die-back disease of Shisham was first time observed
and reported in Nepal. This disease has now been
reached epidemic proportion in Bangladesh and other
countries of South Asia. This disease is characterized
by wilting and subsequent loss of side branches,
leading to so called stagheadness.
To combat with these problems, tissue culture and
biotechnological approaches have been employing for
many decades. Micropropagation of woody trees has
been attempted many times but still is not emerged as
routine. In
D. sissoo
, during the last couple of years,
several attempts have been done to develop efficient
micropropagation. Impact of various explants and
media types on micropropagation have been studied
which resulted in small success but a breakthrough is
still to come which will make
D. sissoo
micropropagation a routine work. Therefore in this
study,
an effective and source independent
micropropagation protocol was established that would
be a milestone for all in vitro studies in
D. sissoo
.
1 Results
Tissue culture has key role in providing rapid and
healthy plant multiplication techniques that are crucial
for plant species facing certain biotic and abiotic
stresses in nature. Various diseases, especially dieback,
are badly affecting our natural Shisham resources;
hence establishment of an efficient micropropagation
protocol is necessary. This study addresses the
development of source independent micropropagation
protocol for
D. sissoo
. For this purpose two explant
sources i.e. Axillary and epicormic shoots, were
selected and micropropagation response was evaluated
on different media combinations.
1.1 Micropropagation Response of Shisham
(
Dalbergia sissoo
)
For establishing micropropagation in
D. sissoo
eight
different
micropropagation media (MPM)
combinations supplemented with varying con-
centrations of BAP and IAA were studied. The data
was collected in the form of number of shoots
obtained per explant and ANOVA table was
constructed.
The analysis of variance table (Table 1) depicts the
considerable significant variation among media
combinations. MPMS 5 gave maximum number of
2.78 shoots per explant (Figure 1) followed by MPMS
7 and MPMS 2 on which 2.72 and 2.68 average shoots
per explant were obtained respectively. The other five
combinations showed a comparatively low response
for shoot induction. Among which MPMS 3 showed
the lowest response with 2.15 mean numbers of shoots
per explant (Table 2 and Figure 1).
Both explant
sources responded well
for
micropropagation, epicormic shoots giving 2.53
shoots per explant and Axillary shoots giving 2.46
shoots per explant. On these micropropagation media,
single shoots as well as bunch of shoots/multiple
shoots were observed (Fig 2, Fig 3, Fig 4, & Fig5).
But the media to explant interaction represented
non-significant
results,
which means the
micropropagation response does not depend upon the
explant source (Response of different micro-
propagation media is shown in Fig6, Fig 7, Fig 8, Fig
9, Fig 10, Fig 11, Fig 12 & Fig 13). A common thing
that had been seen was the formation of calli from
explants at media-explant junction. The callus
formation was seen to be increasing with the increase
in BAP concentration, so MPMS 4 & MPMS 8 giving
maximum callus. Different shoot development stages
are shown in Fig 14 & Fig 15.
1.2 Root Induction from Micropropagated Shoots
of
D. sissoo
For root induction three rooting media were studied.
Well developed micropropagated shoots were shifted
to root induction media. Root initiation from few
micropropagated shoots were started after 20-25 days
of shifting on root induction media (Fig 16 & Fig 17).
Of these, one rooting medium; RM2 (Rooting medium
2) was already reported by Thirunavoukkarasu et al.,
2010 and we used it as control. In Shisham root
induction of in vitro regenerated shoots is very poor
and in our study we attempted this challenge but very
little success was achieved in case of rooting.
