05-06-2013, 03:22 PM
In vitro Regeneration of Sandal (Santalum album L.) from Leaves
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Abstract:
The first successful induction of adventitious shoot buds on Santalum album L. leaves is reported. De novo shoots were
induced directly on leaves without any callusing stage. A leaf length of 0.5-1.5 cm only showed bud inducing potential. Bud formation
occurred on both MS and WPM basal media, although, liquid media were more responsive. Among the plant growth regulators, BAP
at low concentrations (0.44 and 2.22 µM) was effective in this organogenetic process but exogenous auxin application failed to illicit
a similar morphogenetic response. Epiphyllous shoot formation was more pronounced on leaf lamina in which the dorsal and ventral
leaf surfaces were equally highly regenerative; the response, however, varied in different parts of the leaf.
Introduction
Santalum album L. is an important tree species
cultivated in a wide range of areas because of its many
applications. In vitro and particularly somatic
embryogenesis, technology has been used for quite some
time in sandal for the regeneration of plants (Lakshmi
Sita et al., 1980; Bapat et al., 1985; Bapat et al., 1990;
Mujib et al., 1997; Surajit et al., 1998). There are no
published reports on shoot bud formation directly from in
vitro cultured leaves for sandal.
Histology
For histological analysis, leaves were fixed in
formalin:acetic acid:alcohol (FAA, 1:1:18). Then they
were sequentially dehydrated in ethyl alcohol and tertiary
butyl alcohol (TBA) series. Following adequate paraffin
infiltrations, paraffin blocks were made and sections were
cut with a rotary microtome (Reichert Ultracut) at 8-10
µm, stained with a 1% (w/v) aqueous solution of crystal
violet and examined under light microscope for shoot bud
development.
Results
In the media supplemented with kinetin, the leaf
segments remained green for long periods (3-4 months)
with no shoot bud formation. With the addition of 2,i-p a
similar response was observed. Shoot bud production
was exclusively obtained in BAP supplemented media
(Figure 1a). Of the levels evaluated 0.44-2.22 µM BAP
was effective for shoot bud formation. Frequency of
shoot bud formation was, however, relatively low, i.e at
2.22 µM, 13.95% of the cultured leaf explants showed
direct shoot bud formation in solid medium. In addition
to the formation of buds, BAP also occasionally induced
small, slow growing creamy white calli. Kinetin and 2,i-p
also produced little callus from the leaf surfaces.
However, callusing potential was poor (2.32-17.14%), in
all the tested cytokinins callus was only induced in solid
media at lower concentrations.