The solicitation of modern biotechnology techniques like tissue culture has
verified to be a powerful tool augmenting forest. Basically, tissue culture
is a technique by which small pieces of a plant regenerate to a complete plant
in a nutritional media under complete aseptic conditions. These explants segregate
and progressively develop either into an unorganised mass of cells called callus
and subsequently discriminate to form plants or directly bestowed rise to shoots
or embryos. Due to the increase in economical importance of leguminous forest
trees impels the application of in vitro micropropagation technique for
multiplication of these trees (Parveen et al., 2010).
During the last few years, many leguminous woody trees have been successfully
in vitro micropropagated using different young as well as mature plant
parts (Borthakur et al., 2012).
Dalbergia sissoo is one of the most influential tree legumes of the
Indian subcontinent and is esteemed mainly for its wood. Dalbergia sissoo
prevalently confessed as sheesham or Indian rosewood (family Fabaceae or
Legumenosae), is a passably fast-growing multipurpose deciduous tree (Thirunavoukkarasu
et al., 2010). It is an important timber-yielding tree. Because of
its great strength, durability and elasticity, the wood is used for making furniture,
cabinets, musical instruments, ornamental veneers and high-quality commercial
plywood (Lal and Singh, 2012). Bulk of this species
is a good source of foreign exchange to the Country. Dalbergia sissoo
is commonly planted as an ornamental, windbreak, shade tree. The wood of D.
sissoo is an excellent source of fuel wood and charcoal due to the presence
of high caloric content. It is also useful in increasing soil fertility through
fixation of atmospheric nitrogen (Thirunavoukkarasu et
D. sissoo also have many medicinal properties. It is used as an aphrodisiac,
abortifacient, expectorant, anthelmintic and antipyretic. It is also useful
in conditions like emesis, ulcers, leucoderma, dysentery, stomach troubles,
skin diseases, blood diseases, syphilis, stomach problems, dysentery, nausea,
nose disorders and expectorant. Different parts such as roots, bark, wood, leaves
and seeds are being used as the remedy in many diseases. D. sissoo
is antidiarrhoeal as it affects bacterial virulence. D. sissoo is
a folk remedy, especially for excoriations, gonorrhoea and skin ailments (Shah
et al., 2010).
Micropropagation of D. sissoo via different plant parts has been
reported for example, nodal segments (Iyer et al.,
2009), axillary buds (Dawara et al., 1984)
derived from mature trees, a cotyledonary node from seven-day axenic seedling
(Lal and Singh, 2012), zygotic embryo (Husaini
et al., 2008) and semi mature cotyledons (Singh
and Chand, 2003). Due to high timber-yielding values and many medicinal
properties, the plant is being over-exploited in recent years. The conventional
method for the propagation of D. sisso through seed is unreliable due
to poor germination and death of the young seedlings under natural environmental
condition. The efficiency of reproduction is also found to be less seed viability
and lack of vegetative propagation methods (Ali et al.,
Hence, the present study has been intended to improve a stable and consistent
procedure of this arrogant plant. That could be used for mass propagation of
this plant species to contest the increasing demand from the timber industry
as well as for the conservation of germplasm.
MATERIALS AND METHODS
Explants preparation: Immature green seeds of Dalbergia sissoo
were collected from mature tree at (6-9 year old) 120 days after flowering.
Seeds were washed under running tap water to remove dust particles for 15 min
and treated with liquid detergent for 5 min and rinsed three times with distilled
water. Further, sterilization treatments were done under a laminar-flow chamber.
The explants were then disinfected with 0.1% (w/v) mercuric chloride for 8 min
under aseptic conditions. After this these explants were then thoroughly washed
3-4 times with sterilized double distilled water to remove the traces of mercuric
chloride now the explants is ready for inoculation on required medium.
Inoculation in culture medium and condition: These sterilized seeds
were inoculated on MS medium without any growth hormone for seed germination.
From 8 to 10 days old in vitro seedling the cotyledonary leaves were
excised and transfer to medium containing 0.5, 1.0, 2.0, 3.0 and 4.0 mg L-1
2, 4-D for callus induction. The pH of the medium was adjusted to 5.75 with
0.1 N NaOH or 0.1 N HCl solution prior to adding 0.8% (w/v) agar. The cultures
were incubated at a temperature of 25±2°C and a photoperiod of 16
h light (intensity of 2000-3000 lux). The cultures were observed after one week.
Embryogenic callus clumps were formed after 2 to 3 weeks. These clumps were
transferred to MS zero media for somatic embryo development.
Subculturing for germination and multiplication: After 3 to 4 week the
initiated somatic embryos was transferred to the medium supplemented with cytokinins
1.0, 3.0 and 5.0 mg L-1 BAP, 1.0, 3.0 and 5.0 mg L-1 Kn
and combination of both 0.5 mg L-1 BAP+0.5 mg L-1 Kn
and 0.5 mg L-1 BAP+1.0 mg L-1 Kn for embryo maturation
and initiation of shoots. The initiated shoot was again transfer for further
multiplication in the medium containing different concentration of Auxin 1.0
mg L-1 IAA, 1.0 mg L-1 IBA and cytokinin+auxin combination
1.0 mg L-1 2,4-D+1.0 mg L-1 BAP, 0.5 mg L-1
NAA+0.5 mg L-1 BAP, 1.0 mg L-1 NAA+1.0 mg L-1
Rooting of shoots and transfer of plantlets to soil: Shoots of 2-3 cm
in height with two or three leaflets derived from cultures were transferred
to MS medium containing 1 mg L-1 NAA+20 g sucrose or IAA or IBA or
200 mg activated charcoal separately for rooting. The rooted shoots were transferred
to MS medium for further elongation of the roots. The rooted plants were washed
with water to remove media then transferred to pots containing autoclaved vermiculite
soil and sand (1:2:1) and covered with polyethylene bags for one week to maintain
high humidity and subsequently exposed to low air humidity for increasing period
and finally polyethylene bags were removed. These hardened plants then transferred
to the greenhouse (Das, 2011; Chinnaraj
and Malimuthu, 2011).
In the current investigation, indirect somatic embryogenesis induced from in
vitro raised seedlings by which cotyledonary leaves were used as explant.
The seed germinated within 6-7 days of inoculation on MS medium (without any
growth hormone or zero media).
It was perceived to affect the number of somatic embryos provoked per explant
as well as the number of days taken for induction. The cotyledonary leaves show
the production of callus from the Peripheral surface with in 22-25 days. First
the callus is initiated and multiplied in auxin rich media called Proliferation
Medium (PM). The maximum percentage of callus initiation was reported in the
medium containing 2, 4-D (2.0-3.0 mg L-1) within 3-4 weeks. The fresh
white and fragile callus was obtained shown in Table 1.
The embryogenic callus clumps were transferred from high 2, 4-D containing
medium MS medium supplement with cytokinins for development of embryos embryogenic
clumps developed into embryo when it transferred into medium with zero or low
level of auxin this medium called Embryo Development Medium (EDM).
||Response of multiplication from somatic embryos to various
|| Effect of auxin and cytokinin on multiple shoot formation
The globular glossy white embryos started appearing from the surface of cotyledons
with in 15 days. After sub-culturing which further develop into dark green embryos,
there was also development of leafy structures. The high frequency somatic embryo
development was found to be dependent not only on initial induction phase but
also on medium containing growth hormone. The somatic embryos was significantly
high when it was transferred to the medium containing 1.0, 3.0 and 5.0 mg L-1
BAP and in combination of BAP with kinetin i.e 0.5 mg L-1 BAP+0.5
mg L-1 Kn and 0.5 mg L-1 BAP+1.0 mg L-1 Kn.
From Table 2 it has been revealed that the medium containing
3.0 mg L-1 BAP produces highest level of somatic embryos i.e., 97%
comparison to other media.
For further development of these embryos into complete plantlets, embryos with
well-developed cotyledons were transferred to the growth regulator free medium
with full, half strength MS major salts and other. However, there was no further
development of these embryos into complete plantlets. For maturation and germination
of somatic embryos, these were transferred to MS medium supplemented with Auxin
1.0 mg L-1 IAA, 1.0 mg L-1 IBA and cytokinin+auxin combination
1.0 mg L-1 2,4-D+1.0 mg L-1 BAP, 0.5 mg L-1
NAA+0.5 mg L-1 BAP, 1.0 mg L-1 NAA+1.0 mg L-1
Table 3 depicted that the 47% of multiple shoots were found
in 0.5 mg L-1 NAA+0.5 mg L-1 BAP while callus is formed
in 1.0 mg L-1 2, 4-D+1.0 mg L-1 BAP. When somatic embryos
cultured in MS media supplemented with Auxin 1.0 mg L-1 IAA and 1.0
mg L-1 IBA, 36% somatic embryos germinated.
The age of the explants was found to play an important role in the induction
of indirect somatic embryogenesis. The immature seeds germinate easily compare
to old or mature.
The 2, 4-D is known to play an important role in the induction of embryogenic
cultures. For indirect somatic embryogenesis tissues generally were transferred
from high to low concentration of auxin (2, 4-D), it leads to increase in somatic
embryogenesis. Additionally, it was reported that the higher concentration of
auxin results in the high production of ethanol and ethylene which cause the
repressive effect of auxin on somatic embryogenesis (Singh
et al., 2002; Rastogi et al., 2008).
This study confirmed the production of ethanol and ethylenes by auxin are responsible
to inhibit the somatic embryogenesis.
For further development of embryos into complete plantlets, embryos with well-developed
cotyledons were transferred to the growth regulator free MS medium but there
was no further development of these embryos. From these preliminary studies,
it suggested that preconditioning or further maturation of this somatic embryo,
cytokinin may be necessary for the successful germination into plantlets. The
addition of a cytokinin and auxin to the medium was essential to induce multiple
shoots from the explants (Bari et al., 2008)
after the addition of different concentration of cytokinin and auxin in MS medium
multiple shoots were found. When somatic embryos cultured in MS media supplemented
with auxin, alone somatic embryos were germinated.
The increasing demand of D. sisso in timber industries, the plant is
being over-exploited. In addition, the vegetative propagation methods are less
efficient. So, the plant has been developed through indirect somatic embryogenesis
using MS media supplemented with different auxin (2, 4-D) and cytokinins (BAP,
Kn) and alone. The advantage of somatic embryogenesis in conservation of depleting
flora and rapid and large scale clonal propagation of elite varieties. Somatic
embryogenesis is very useful for genetic transformation and might be helpful
in producing synthetic seeds. The findings of recent investigations have shown
that, it is possible to induce indirect somatic embryogenesis in D. sissoo
and plant regeneration from callus cultures derived from cotyledonary leaves
This study was supported by Grow Tips Biotech Lab, Hazrat Nizamuddin, Bhopal
(M.P.), India with grant number (GTBL/Proj/39/2010).