Abstract:
Propagation of Red sandalwood either through seeds or vegetative means is not given
satisfactory results. Tissue culture has proved to be a promising technique for conservation
and large scale multiplication of several woody species. Therefore the overall objective of this
research was to develop a feasible in vitro micro propagation protocol for red sandalwood
through tissue culture, which would be a reliable mass propagation method.
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Shoot tips and semi hardwood cuttings were surface sterilized using different concentrations
of clorox with different exposure time durations followed by 70% ethanol with 2 minutes
exposure time. Surface sterilized shoots were transferred to Murashige and Skoog (MS) and
Me Cowns Woody Plant (WPM)) media with or without 0.1 % activated charcoal. Browning,
survivals, number of new leaves produced and rate of forming new shoot buds were
evaluated. Ex vitro derived shoot tips and semi hard wood cuttings, shoot tips excised from
twenty- day- old in vitro germinated seedlings, cotyledonary nodal segments, mesocotyls and
twenty- day- old in vitro derived seedlings were evaluated to identify best proliferation
medium and best Benzyl Amino Purine (BAP) and Napthalene Acetic Acid (NAA)
concentrations by evaluating number of shoots, leaves, branches and shoot height.
Proliferated shoots were excised for in vitro as well as ex vitro rooting. Three Indol Butric
Acid (IBA) concentrations with 12 to 24 hours shaking periods were applied as the pulse
treatment and subsequently transferred to 0.1 and 1 mg/1 IBA containing liquid and solid half
strength MS media. Number of roots produced, root length and browning of shoots were
evaluated. In vitro rooted plantlets were transferred to pots containing sand: coir dust, cow
dung and top soil in different ratios and placed under laboratory conditions and then
transferred to plant house and maintained under high humidity. Finally plantlets were
transferred to poly bags containing cow dung: top soil: sand (1:1:2). Survival rates, time taken to appear new shoot branches, number of new leaves formed and height increment of the
plants were evaluated. Experiments were designed according to Complete Randomized
Design and all parametric data were analyzed using SAS statistical software. Mean
separations were carried out using Least Significant Difference test (LSD). Non-parametric
data were analyzed using chi square test. Each treatment was replicated twenty times.
Results revealed that 15% Clorox with 10 minutes exposure time followed by 70% ethanol for
two minutes exposure time was the best treatment combination for surface sterilization of
shoot tips by getting 80% aseptic cultures, with 80% survival. WPM medium incorporated
with 0.1% activated charcoal is the best establishment medium where shoot tips showed 95%
survival, without browning and producing highest number of new leaves and shoot buds. B5
medium with 2 mg/1 BAP and 0.4 mg/1 NAA gave maximum number of shoots and shoot
branches from cotyledonary nodal segments (4.95, 4.20), mesocotyl segments (4.00, 3.75), in
vitro derived shoot tips excised from 20- day- old seedlings (3.00, 2.5), shoot tips (2.95, 3.15)
and semi hardwood cuttings (0.00, 3.45) detached from one- year- old plantlets maintained
under plant house conditions and in vitro germinated seedlings (3.25, 3.5) respectively during
four weeks time period. Explant, mesocotyls gave the highest mean shoot height (4.55 cm)
and ex vitro derived shoot tips produced the highest mean number of leaves per shoot (4.00)
in B5 medium with 2 mg/1 BAP and 0.4 mg/1 NAA, after four weeks. Adventitious roots were
formed directly on 2 cm length stem cuttings when they were exposed to pulse treatment for
twelve hours. After pulse treatment, stem cuttings were transferred to half strength solid MS
medium containing 0.1 mg/1 IBA to form roots.
In vitro rooted plantlets were successfully acclimatized in potting media containing sand: coir
dust 1: 1 ratio. It is recommended to place plantlets in a humid chamber during the first six
weeks time for better survival. Before repotting or field establishment, plants have to be
. maintained under plant house conditions for another four weeks or more.