Journal of Cell and Tissue Research Vol.

18(2) 6435-6440 (2018) Original Article

(Available online at www.tcrjournals.com)
ISSN: 0973-0028; E-ISSN: 0974-0910

IN VITRO MASS MULTIPLICATION OF FIG (Ficus carica L.)

THROUGH NODAL SEGMENT EXPLANTS
?
SEN, D. K.¹ AND PATEL, R. M.²

¹Department of Fruit Science, ASPEE College of Horticulture and Forestry, Navsari Agricultural University,
Navsari 396450; ²ASPEE Sakilam Agricultural Biotechnology Institute, Navsari Agricultural University,
Athwa Farm, Surat 395001,Gujarat. E. mail: dipaksen99hort@gmail.com, Cell: 08690091407

Received: May 3, 2018; Accepted: May 24. 2018

Abstract: Fig (ficus carica L.) fruits are used for reduce the risk of prostat, breast and colon
cancer, helps in reducing blood cholesterol. maintain blood pressure and coronary heart attacks..
They contain health benefiting soluble dietary fiber, minerals, vitamins, and pigment antioxidants.
To produce large scale plantation a tissue culture protocol was developed. Maximum
establishment of nodal segment explants observed in Murashige and Skoog (MS) medium
containing 2.0 mg/l 6- benzylaminopurine (BAP). However, MS medium fortified with 1.0 mg/l
BAP + 0.1 mg/l IBA exhibited maximum multiplication rate in second and third sub-cultures.
The maximum frequency of multiple shoots in nodal segment explants (83.33 %) was observed
on treatment MS + 1.0 mg/l BAP + 0.1 mg/ l IBA. In vitro rooting of regenerated shoot developed
in half strength MS medium supplemented with 1.0 mg/l IBA, which produced maximum number
of roots per shoot (9) and length of root (3.0). In vitro grown plantlets having 3.0 cm length of
shoot were transferred to coco peat media under green house, which showed better survival of
plantlets (90.25 %).

Key words: Ficus carica L., Nodal segment explants.

INTRODUCTION deteriorates beyond 390C. The mature tree can

The common fig (Ficus carica L.) is a species of
flowering plant belonging to genus Ficus and family
Moraceae. The fig is believed to be indigenous to
Western Asia and distributed by man throughout the
Mediterranean area. Remnants of figs are found in
excavations of neolithic sites traced to at least 5,000
B.C. The first figs in the New World were planted in
Mexico in 1560. In India only the common fig is
grown.
Fig being a deciduous and sub tropical tree, prefers
areas having arid or semiarid environment, high
summer temperature, plenty of sunshine and
moderate water. Although the plants can survive
temperature as high as 450C, but the fruit quality
withstand at low temperature up to 40C, but good
growth takes place at temperature 150C - 210C . The
size, shape, colour of the skin and pulp quality are
markedly affected by climate. But quality figs are
produced in the region with dry climate especially at
the time of fruit development and maturity. High
humidity coupled with low temperature usually results
in fruit splitting and low fruit quality. Fig is one of the
most salt and drought tolerant crops. It can tolerate
a fairly high level of sulphate or chloride salt. Medium
to heavy, calcareous, well drained, deep (about 1 m )
soil having pH of 7-8 is ideally suitable for cultivation
of fig [1].
Asexual propagation in fig is most commonly
propagated by cuttings to ensure that trees are “true

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to type”. Shield or patch budding, or cleft or bark
grafting techniques can also be used to top work an
existing orchard. However, this methods are very
slow, cumbersome, season dependent, plant materials
are not produce on large scale to meet the elite
planting materials.Therefore, it is warranted to search
another alternate method which can help to solve the
problems associated with the conventional method of
propagation to multiply planting material on large scale
rapidly in short time and available around the year.
In vitro culture techniques are becoming increasingly
popular as alternative and feasible means in
vegetative propagation of some commercially
important plants [2,3]. Hence, to meet the demand
of large scale planting material of desirable genotype,
micro propagation technique is only the way to fulfill
the requirements round the year and to produce
sapling materials in large number and rapidly.
Micropropagation is one of the major area in plant
tissue culture which has begun to manifest potential
for mass production of sapling material in short period
of time. This technique has been shown to have
definite and indispensable advantage over the former,
as it ensures extremely rapid rate of multiplication. It
is not season dependent and requires only a limited
quantity of plant tissue as a source of initial explants.
MATERIAL AND METHODS
Preparation and establishment of explants:
Explants were collected from Horticulture nursery,
Navsari Agricultural University, Navsari. Nodal
segment explants from newly emerged shoots
containing one node each were collected from 5-6
years old plants and leaves were removed leaving
the petiole. They were swabbed with cotton and then
dipped in 70 per cent alcohol and washed thoroughly
in running tap water for about 30 minutes to remove
traces of alcohol and dirt. The nodal segment was
pre-treated with bavistin 0.2% (Carbendazim 50 per
cent WP) and streprocyline 0.07 per cent for one
hour. Thereafter, explants were washed with 10 per
cent solution of detergent (Teepol) for 10 minutes.
All traces of detergent were removed by repeated
washing in double distilled water. Further sterilization
procedure was carried out under aseptic condition in
laminar air flow cabinet. The nodal segments was
subjected to surface sterilization using 0.05 to 0.1
per cent HgCl2 solution for 5 minutes and NaOCl @
10 per cent for 10-20 minutes duration. They were
then, thoroughly rinsed at least three times with
autoclaved de-ionized distilled water. The sterilized
nodal segment explants were excised and inoculated
into 250 ml screw caped glass bottle. Initially three
different media MS (Murashige and Scoog, [4], WPM
(Lloyd and McCown [5], and SH (Schenk and
Hildebrandt [6]) were tested for culture establish-
ment. The media was supplemented with 2.0 mg/l
BAP alone and in combination with 2.0 mg/ l BAP +
0.1 mg/l NAA. Sucrose was added at 30.0 g/l and
media was autoclaved at pressure of 15 lb/ inch2 for
20 minutes at approximately 121°C. The pH of the
medium was adjusted at 5.8 prior to autoclaving.
Cultures were incubated for four weeks. Best
medium found in culture establishment was further
used for multiplication.
Shoot multiplication: For multiplication 1.5 to 2.0
cm long raised plantlets from nodal segment explants
were aseptically isolated and transferred to MS
medium supplemented with various concentration of
growth substances BAP (0.5, 1.0 and 2.0 mg/l) alone
and in combination with IBA (0.1, 0.5 and 1.0 mg/l).

Media Composition/Treatment no. Establishment Days taken for Length of Shoot Number of shoots/
(%) establishment (cm) explants
MS +BAP 2.0 mg/l 78.67 7.67 2.1 1.87 Table 1: Effect
(62.5) of different media
SH+BAP 2.0 mg/l 46.33 10.67 0.74 1.1 on establishment
(42.9) of nodal segment
WPM +BAP 2.0 mg/l 71 10.33 1.53 1.3 explants of fig
(57.43) var. Poona Fig
M S +BAP 2.0mg/l +NAA 0.1 mg/l 61.67 9.67 1.72 1.75
Incubation: 4
(51.75)
SH +BAP 2.0 mg/l +NAA 0.1 mg/l 33.67 11.33 0.57 0.75 Weeks Explants:
(35.46) Nodal segment *
WPM+BAP 2.0mg/l +NAA 0.1 mg/l 53.67 10.33 1.25 0.6 Figures in
(47.1) paretheses are arc
S.Em. + 0.58 0.33 0.03 0.03 sin transformed
CD at 5% 1.79 1.02 0.1 0.1
CV% 1.75 5.77 4.33 4.81
value

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 Sen and Patel
Table 2: Effect of plant growth regulators on frequency of Subculture was made on the same medium after four
shoot multiplication of various explants of fig var. Poona Fig weeks of culture.
Medium : MS Medium. Incubation : 4 Weeks. * Figure in
paratheses are arc sin transformed value
In vitro rooting and acclimatization: After four
Treatment No. Frequency of multiple weeks of sub-culture individual shoots were
shoots/explants (%)
Nodal segments
transferred into the rooting medium. For rooting stage
MS + BAP 0.5 mg/l 18 MS full and half MS strength with different
(25.1) concentration of IBA alone and in combination with
MS + BAP 1.0 mg/l 30.67 IBA (0.5, 1.0 and 2.0 mg/l) + NAA (0.5 mg/l) were
(33.63)
used. The nutrient medium was gently removed and
MS + BAP 2.0 mg/l 56
(48.45) washed thoroughly in tap water ensuring that all the
MS + BAP 0.5 mg/l + IBA 0.1 mg/l 65.67 adhering agar particles were completely removed
(54.13) without damaging the roots. For acclimatization
MS + BAP 1.0 mg/l + IBA 0.1 mg/l 83.33
different media like vermicompost, soil, coco peat,
65.91
MS + BAP 2.0 mg/l + IBA 0.1 mg/l 48.33
vermicompost : soil. (1:1 v/v) and FYM : soil: sand
(44.04) (1:1:1 v/v) in 300 ml plastic cups were used. In vitro
MS + BAP 0.5 mg/l + IBA 0.5 mg/l 20.33 rooted plants were placed in a culture room at 26 +
(26.8)
2°C. The rooted plantlets were then dipped in 0.05%
MS + BAP 1.0 mg/l + IBA 0.5 mg/l 37
(37.46) bavistin (carbendazim 50% WP) and plantlets in 300
MS + BAP 2.0 mg/l + IBA 0.5 mg/l 43.33 ml plastic cup. They were covered with a plastic cup
(41.17) continuously for 6 to 7 days and kept in an air
MS + BAP 0.5 mg/l + IBA 1.0 mg/l 42 conditioned room. The cover was gradually removed
(40.4)
MS + BAP 1.0 mg/l + IBA 1.0 mg/l 38 after seven days initially for 2 hours followed by 4
(38.06) hours and 8 hours in next four days. The cover was
MS + BAP 2.0 mg/l + IBA 1.0 mg/l 36.67 removed during night and light put-off for next three
(37.27) days. Subsequently the period of keeping the plantlets
S.Em. + 0.25
CD at 5% 0.73
without any cover was gradually increased and after
CV% 0.97 15 days they were brought outside the room in shade.
Within 10 days by gradually exposing them to sun
they were acclimatized to natural environment.
Treatment No. Rooting % Days taken for Length of No. of roots
r oot initiation root (cm) Per shoot
½ MS + IBA 0.5 mg/l 9 19 1.2 1.5
(17.41)
½ MS + IBA 1.0 mg/l 86 9 3 9
(68.06)
½ MS + IBA 2.0 mg/l 51 16 1.2 6
(45.57) Table 3: Effect of IBA,
½ MS + IBA 0.5 mg/l + NAA 0.5 mg/l 11 18 1.57 5 combination of IBA with
(19.36) NAA and strength of media
½ MS + IBA 1.0 mg/l + NAA 0.5 mg/l 59 11 2.2 7 induction of rooting of fig
(50.19)
var. Poona Fig Medium :
½ MS + IBA 2.0 mg/l + NAA 0.5 mg/l 27 14 1.8 5
(31.3) MS Medium, Incubation :
Full MS + IBA 0.5 mg/l 5 20 1.5 2 4 Weeks. * Figure in
(12.88) paratheses are arc sin
Full MS + IBA 1.0 mg/l 39 13 1.9 6 transformed value
(38.64)
Full MS + IBA 2.0 mg/l 23 17 1.67 5
(28.65)
Full MS + IBA 0.5 mg/l + NAA 0.5 mg/l 34 14 1.6 4
(35.67)
Full MS + IBA 1.0 mg/l + NAA 0.5 mg/l 69 10 2.57 8
(56.17)
Full MS + IBA 2.0 mg/l + NAA 0.5 mg/l 21 15 1.4 6
(27.27)
S.Em. + 0.6 0.47 0.06 0.23
CD at 5% 1.76 1.38 0.19 0.68
CV% 3.03 5.36 6.22 7.9

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Treatment no. Survival of Days taken for Length of Shoot(cm)

plantlets (%) establishment of plantlets
Vermicompost 30.5 19.75 5.4
(33.21 Table 4 : Effect of
Soil 50 18 5.6 different potting
(45) mixtures on hardening
Coco peat 90.25 14 6.03
of fig var. Poona Fig *
(72.54)
Vermicompost: Soil. (1:1 v/v) 61 17.25 5.75
Figures in paratheses are
(50.96) arc sine transformed
FYM : Soil: Sand (1:1:1 v/v) 79.5 16 5.9 value
(56.79)
S.Em. + 0.36 0.57 0.08
CD at 5% 1.09 1.71 0.25
CV% 0.72 3.93 1.8

Statistical analysis: Statistical methods were used medium supplemented with 2.0 mg/ l BAP). This
for comparison of treatment means for treatment also showed significantly maximum length
micropropagation. Completely randomized design of shoots (2.10 cm) and number of shoots per explants
(CRD) was used for the experiment. The data were (1.87) followed by MS medium supplemented with
subjected to analysis of variance (ANOVA) and 2.0 mg/l BAP. Earlier workers were used MS and
treatment means were compared [7]. WPM medium and achieved success for
establishment of explants and growth in fig var [8,9]
RESULTS AND DISCUSSION Poona Fig. The multiplication rate of the MS medium
was superior as compared to the WPM and SH
Establishment of explants: The results presented medium.
in Table 1 reveal that medium type influenced
establishment of explants significantly. Maximum Frequency of shoot regeneration and
establishment (78.67 %) was achieved on treatment multiplication: In order to study the multiplication
MS medium supplemented with 2.0 mg/l BAP (Fig. rate, trial was conducted with treatments involving
2) followed by WPM medium supplemented with 2.0 different levels of BAP and IBA. In all, 12 treatments
mg/l BAP. Minimum days taken for establishment of were tested and results are presented in Table 2.
explants (7.67 days) were recorded in treatment MS The sub culturing was done at 4 weeks interval. Each

1st sub culturing 2nd sub culturing 3rd sub culturing 4th sub culturing
4
Number of Shoots per Culture

3.5
3
2.5
2
1.5
1
0.5
0
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12
Fig. 1 Treatment

Fig.1 : Effect of serial sub culturing on multiplication of nodal segment explants of fig var. Poona fig M1 – W + BAP
0.5 mg/l,M2 – W + BAP 1.0 mg/l, M3 – W + BAP 2.0 mg/l,M4 – W + BAP 0.5 mg/l + IBA 0.1 mg/l, M5 – W + BAP 1.0 mg/l
+ IBA 0.1 mg/l, M6 – W + BAP 2.0 mg/l + IBA 0.1 mg/l, M7 – W + BAP 0.5 mg/l + IBA 0.5 mg/l, M8 – W + BAP 1.0 mg/
l + IBA 0.5 mg/l, M9 – W + BAP 2.0 mg/l + IBA 0.5 mg/l, M10 – W + BAP 2.0 mg/l + IBA 1.0 mg/l, M11– W + BAP 2.0
mg/l + IBA 1.5 mg/l, M12– W + BAP 2.0 mg/l + IBA 2.0 mg/l

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 Sen and Patel

Fig. 2 Fig. 3

Fig. 4 Fig. 5
Fig. 2: Establishment of explants on MS medium + 2.0 mg/l BAP. Fig. 3: Shoot multiplication on MS medium + 1.0 mg/l BAP
+ 0.1 mg/l IBA. Fig. 4: In vitro rooting on half MS + 1.0 mg/l IBA. Fig. 5: Hardening of plantlet in potting mixture of coco
peat.

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 J. Cell Tissue Research
shoots of cultures were cut into nodal segments (2.0
cm size) and were used for multiplication. The
treatment MS fortified with 1.0 mg/l BAP + 0.1 mg/
l IBA was found significantly superior for producing
high frequency of multiple shoots (83.33%) in culture
from nodal segment explants (Fig. 3). The dominant
buds of vegetative apex are stimulated to grow and
elongated into presence of cytokinin and also produce
new axes. For the shoot proliferation BAP was found
more effective when used in combination with auxin
in pomegranate [10]. Similarly, Subramani et al. [11]
also reported high frequency of shoot multiplication
from nodal segment and shoot tip of noni.
Effects of serial sub culturing on shoot
multiplication: The result for shoot multiplication
with serial sub culturing of nodal segment explants is
shown in (Fig. 1). Regeneration of multiple shoots at
the rate of 6 folds and highest length of shoots were
observed in treatment MS + 1.0 mg/l BAP + 0.1 mg/
l IBA) up to second subcultures then, declined. The
declined trend has also been reported in papaya [12]
and grape [13].
Effect of IBA, NAA and strength of the medium
on rooting of in vitro shoots: Results on in vitro
rooting response to different level of IBA and
combination of IBA with NAA supplemented in half
and full strength of MS medium is presented in Table
3. The maximum rooting (86.00 %), minimum days
taken for root induction (9.0 days), number of roots/
shoot (9.0) and length of rooted shoot (3.0 cm) was
found in treatment half MS + 1.0 mg/l IBA (Fig. 4)
followed by Full MS + 1.0 mg/l IBA + 0.5 mg/l NAA.
Response of rooting was reduced either with increased
or decreased level of IBA. Reduction in rooting
response at higher concentration of auxin was
observed in pear [14] and black plum [15]. Similar
findings were reported in papaya [16,17].
Effect of different potting mixtures on survival
of in vitro raised plantlets: The maximum per
cent survival of plantlets (90.25%) was reported in
coco peat (Table 4). The potting mixtures used in the
present investigation helped in giving better grip for
the roots and ample aeration. Coco peat was found
as the best hardening medium, which gave maximum
survival per cent of in vitro raised plantlets of fig var.
Poona Fig (Fig. 5). These results are consistent with
earlier finding of Subramani et al. [11] in noni.
6440
CONCLUSION
The present investigation on in vitro mass
multiplication of fig through nodal segment explants
clearly demonstrated its potentiality for rapid clonal
propagation. It appeared that using the present
protocol of in vitro propagation, large number of
plantlets can be produced in a year starting from single
nodal segment explants.
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