
In Vitro Somatic Embryogenesis in Some Oil Yielding Tropical Tree Species
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dirachta indica were collected from elite trees growing
in dry deciduous forests 50 - 60 days after fruit set. The
fruits were washed with 2% (w/v) detergent solution
(Teepol) for 10 min, further ringed with 70% ethanol for
1 min, surface sterilized with 0.1% (w/v) aqueous solu-
tion of mercuric chloride for 15 min, followed by three 5
min rinses in sterile distilled water. Embryonic axis along
with cotyledons were aseptically cultured on Murashige
and Skoog [14] medium supplemented with various con-
centrations of BA or Kn (0, 0.25, 0.5, 1.0, 1.5, 2.0 mg/l),
NAA or 2,4-D (0, 0.5, 1.0, 1.5, 2.0, 3.0 mg/l) alone or in
combinations for callus induction. The pH of the media
was adjusted to 5.7 using 0.1N NaOH or 0.1N HCl prior
to addition of 0.8% (w/v) agar (Qualigen, India). Rou-
tinely, 20 ml of molten medium was dispensed into 25 ×
150 mm glass tubes (Borosil, India), capped with non-
absorbent cotton plugs wrapped in one layer of cheese-
cloth. The cultures were sterilized at 121˚C and 104 kPa
for 15 mi n.
2.2. Induction of Somatic Embryogenesis
Callus mass (500 ± 20 mg) was transferred to MS me-
dium supplemented with different concentrations of BA,
kinetin and 2,4-D or NAA (0, 0.25, 0.5, 1.0, 1.5, 2.0 , 2.5
and 3.0 mg/l) singly or in combinations for induction of
somatic embryogenesis. L-proline or L-glutamine was
added to the culture medium to enhance the embryogenic
potential. The cultures were incubated under 16h photo-
period with light intensity of 55 µmol·m–2·s–1 provided
by cool, white fluorescent lamps (Phillips, India) at 25˚C
± 2˚C. Subculturing was made every 4 week intervals.
The media were solidified with 0.8% agar-agar. Mor-
phological changes were recorded through visual obser-
vations at 3-week intervals. The embryogenic response
and number of somatic embryos per culture were re-
corded. Each treatment had 20 replicates and the experi-
ment was repeated three times. Data were also recorded
in respect to embryogenic frequency, number of embryos
and frequency of normal embryos per culture.
2.3. Maturation and Germination of Somatic
Embryos
The somatic embryos were transferred to various culture
media with (0.1 - 0.25 mg/l abscisic acid and 2% sucrose)
or without growth regulators for maturation and germi-
nation. The cultures were routinely sub-cultured at
4-week intervals. In another experiment, the cultures
were kept in the dark for 2 weeks which were then trans-
ferred to the light for germination of somatic embryos. In
all the experiments, each treatment consisted of 10 repli-
cations and the experiments were repeated three times.
The somatic embryos were transferred to 1/2 MS me-
dium supplemented with 2% sucrose and abscisic acid
(0.1 - 0.25 mg/l) for germination.
3. Results and Discussion
3.1. Induction of Somatic Embryogenesis
The effects of the different growth regulators on em-
bryogenic callus induction are indicated in Table 1. Fri-
able calli developed from immature zygotic embryos
within 3 - 4 weeks of culture on MS medium supple-
mented with various concentrations of auxins and cyto-
kinins. The maximum proliferation of callu s was noted in
the medium containing BA and NAA or 2,4-D. Kinetin
was not as effective in callus induction as BA. BA with
2,4-D in the medium was observed to be better for callus
proliferation. The proliferated calli were subsequently
sub-cultured on various media for embryogenesis. Em-
bryogenic callus mass developed on MS medium sup-
plemented with 0.5 - 2.0 mg/l BA and 2.0 - 3.0 mg/l
NAA in S. glauca and 0.5 mg/l BA and 1.0 - 2.0 mg/l
2,4-D in case of A. indica.. The medium devoid of
growth regulators did not help in proliferation of em-
bryogenic calli. The maximum rate of embryogenic cal-
lus proliferation was noted on MS medium supplemented
with 1.0 - 1.5 mg/l BA and 2.0 mg/l NAA in Simarouba
glauca and 0.5 - 1.0 mg/l BA and 1.0 - 2.0 mg/l 2,4-D in
Azadirachta indica (Figures 1(a)-(b) and 2(a), Tabl e 1).
Proliferation of friable embryogenic calli in terms of
fresh weight was better in the medium having BA as
compared to Kn. Similar responses were observed when
2,4-D was replaced with NAA. BA at a concentration of
1.0 mg/l along with 2.0 mg/l NAA improved the rate of
embryogenic callus proliferation and improved the pro-
duction of somatic embryos per culture; though NAA
helped in th e proliferation of embryogenic callu s as good
as the 2,4-D, few somatic embryos developed in Sima-
rouba glauca. However, the medium supplemented with
0.5 mg/l BA and 1.5 mg/l 2,4-D showed a higher rate of
proliferation of embryogenic calli in A. indica (Table 2).
Embryogenic callus induction was faster in S. glauca in
the media containing NAA as compared to those having
2,4-D; 2,4-D, however, showed better results in A. indica.
Embryo development in somatic cells was often accom-
panied with cellular stress. Moreover, NAA, the most
frequently used compound for induction of somatic em-
bryogenesis (Figure 2(b)), was known to activate many
stress related genes supporting the hypothesis that somati c
embryogenesis resulted due to extreme stress response of
cultured cells. Proline acted as a potential antioxidant,
which helped in ameliorating the stress [15]. Moon et al
[8] reported that MS medium containing 3% sucrose, 1.0
g/l glutamine along with 2,4-D helped in development of
somatic embryos in Oplopanax elatus. ABA and acti-
ated charcoal appeared to be very important agents for v
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