Scientia Horticulturae 79 (1999) 175±183
In vitro propagation of pomegranate (Punica granatum
L. cv. Ganesh) through axillary shoot proliferation from
nodal segments of mature tree
Soumendra K. Naik, Sitakanta Pattnaik, Pradeep K. Chand*
Plant Tissue and Cell Culture Facility, Post-Graduate Department of Botany,
Utkal University, Bhubaneswar, Orissa, India
Accepted 31 July 1998
Abstract
A rapid and efficient procedure is outlined for in vitro clonal propagation of an elite cultivar of
pomegranate (Punica granatum L. cv. Ganesh) using nodal stem segments of a mature tree. Bud
break and axillary shoot proliferation was induced in the nodal segments on Murashige and Skoog
(1962) (MS) medium supplemented with benzyladenine (BA), zeatin riboside (ZR) or thidiazuron
(TDZ), with ZR being most effective. Maximum number of shoots were developed on a medium
containing 2.0 mg lÿ1 ZR. However, the shoots failed to elongate. This problem was overcome by
transferring the shoot clumps to a medium with a lower level of ZR. TDZ was least effective of all
the cytokinins tested. Rooting was induced in 86% of the regenerated shoots in a half-strength MS
medium supplemented with 1.0 mg lÿ1 indole-3-butyric acid (IBA). Upon transfer of the rooted
shoots to an auxin-free half-strength MS medium the primary roots elongated further and
additionally new roots developed. The plantlets were acclimatised and established in soil. # 1999
Elsevier Science B.V. All rights reserved.
Keywords: In vitro propagation; Nodal segment; Punica granatum
Abbreviations: BA, N6-benzyladenine; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; MS,
Murashige and Skoog's (1962) medium; TDZ, thidiazuron; ZR, zeatin riboside
* Corresponding author. Tel.: +91-674-581529; fax: +91-674-409259.
0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 8 ) 0 0 2 1 8 - 0
176
S.K. Naik et al. / Scientia Horticulturae 79 (1999) 175±183
1. Introduction
Pomegranate (Punica granatum L.) is an important tree of the tropical
and subtropical regions of the world which is valued highly for its delicious
edible fruits. In addition, the tree is also cultivated for its pharmaceutical and
ornamental usage. The powdered flower buds are used in bronchitis and the
fruit rind, the decoction of the bark, and the juice of the leaves and young fruits
are used for the treatment of gastrointestinal disorders. Moreover, the fruit rind,
stem and root bark and the leaves are a good source of tannins. The flowers
yield a light red dye which is used for dyeing the cloth. The seeds along with
the fleshy portions are dried and used as a condiment (Anon., 1982).
Conventionally, pomegranate is propagated by stem cuttings including both hard
wood and soft wood cutting, the former being more commonly used. However,
this method is not very efficient as it requires about one year to raise the saplings
(Anon., 1982).
During the past years, micropropagation techniques have been widely used for
the propagation of several plant species. Protocols have also been developed for
in vitro propagation of a number of fruit trees (Bajaj, 1986; Hutchinson and
Zimmerman, 1987). Regeneration of plantlets in vitro through either organogenesis from callus derived from leaf segments (Omura et al., 1987) or anther wall
(Moriguchi et al., 1987), or through embryogenesis from various seedling
explants (Jaidka and Mehra, 1986) have been reported in pomegranate. However,
to date, there is no report on in vitro clonal propagation of this fruit tree directly
using nodal stem segments from mature trees. We report here a rapid and efficient
method for in vitro clonal propagation of an elite cultivar of pomegranate (Punica
granatum L. cv. Ganesh) through axillary shoot proliferation from nodal stem
segments collected from a mature tree.
2. Materials and methods
2.1. Explant source
A 10-year-old elite tree of Punica granatum L. cv. Ganesh that has been
indexed and maintained in the garden of the Department of Horticulture,
Government of Orissa, Bhubaneswar provided the explants for the present
study. Nodal stem segments (1.0±1.5 cm) collected from that tree were washed
with running tap water for 1 h, treated with an aqueous solution of 5%
liquid detergent Laboline (Qualigens, India) for 3 min and rinsed with distilled
water for 5±6 times. The explants were surface-sterilized with an aqueous
solution of 0.1% HgCl2 for 5 min and rinsed 5±6 times with autoclaved distilled
water.
S.K. Naik et al. / Scientia Horticulturae 79 (1999) 175±183
177
2.2. Culture medium and conditions
Murashige and Skoog (1962) (MS) medium containing 3% sucrose, 100 mg lÿ1
myo-inositol was used. The medium was further supplemented either with 0.5±
3.0 mg lÿ1 zeatin riboside (ZR) or benzyladenine (BA) or 0.01±0.5 mg lÿ1
thidiazuron (TDZ). The pH of the medium was adjusted to 5.8 before gelling with
0.8% agar (BDH, India). Fifteen ml of media were dispensed into 25 ml
Erlenmeyer flasks. Flasks were capped with aluminium foil. All media containing
culture vessels were autoclaved at 104 kPa and 1218C for 15 min. The sterilized
nodal segments (1 explant/flask) were planted vertically in each culture flask.
Each cytokinin treatment consisted of 9 replicate flasks with 1 explant in each.
Cultures were maintained at 25 18C, 60% relative humidity and a particle
(photon) flux density of 35 mE mÿ2 sÿ1 provided by white fluorescent tubes. The
experiment was conducted three times.
2.3. Rooting of the shoots
Shoots with 4±6 expanded leaves (3.5±4.0 cm) developed in vitro were excised
and transferred to half-strength MS medium containing 50 mg lÿ1 myo-inositol,
1.5% sucrose and 0.2% phytagel (Sigma, USA). The medium was further
supplemented with 0.5±2.0 mg lÿ1 indole-3-butyric acid (IBA) or indole-3-acetic
acid (IAA). After 8±10 days the rooted shoots were transferred to an auxin-free
half-strength medium for further elongation of the roots. Each auxin treatment
consisted of seven replicate tubes with one shoot in each and repeated twice.
2.4. Acclimatization and transfer of plants to soil
Well rooted plantlets were removed from the culture medium, the roots were
washed gently with tap water to remove phytagel and then transferred to small plastic
pots (7.5 cm diam) containing autoclaved vermi-compost (Ranjan's Agrotech,
Bhubaneswar). The pots were covered with polyethylene bags to maintain a high
humidity and kept at 25 18C and a particle (photon) flux density of 50 mE mÿ2 sÿ1
provided by white fluorescent tubes for 3 weeks. Plants were then transferred to
larger pots (18 cm diam) containing garden soil (soil : compost, 1 : 1); kept under
shade for another 2 weeks and then transferred to a nursery under full sun.
3. Results
3.1. Bud break and shoot proliferation
The surface-sterilization procedure followed in the present study yielded 95%
of the explants free of apparent microbial contamination. The culture medium
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Table 1
Influence of different cytokinins on bud break and shoot proliferation from nodal segments of
P. granatum
Concentration (mg lÿ1)
Shoot number
Shoot length (cm)
±
±
±
71.0 2.3
93.0 2.5
41.0 2.3
29.0 2.1
1.0 0
2.1 0.1
1.2 0.1
1.0 0
3.2 0.1
3.7 0.2
1.6 0.1
±
ZR
0.5
1.0
2.0
3.0
±
63.0 2.1
85.0 2.1
22.0 2.3
±
2.5 0.2
5.2 0.1
1.2 0.1
±
2.9 0.2a
3.9 0.1a
1.5 0.2a
TDZ
0.01
0.05
0.1
0.5
±
63.0 2.3
52.0 2.5
37.0 2.3
±
1.0 0
1.0 0
1.0 0
±
3.6 0.2b
3.2 0.1b
2.1 0.2b
0.0
BA
0.5
1.0
2.0
3.0
% Bud break
SE) from three independent experiments each with nine replicates.
Data (X
a
Shoot length was recorded following transfer to a medium containing 1.0 mglÿ1 ZR.
b
Shoot length was recorded following transfer to a medium containing 0.5 mglÿ1 BA.
turned brown within 3 days of inoculation of the explants. The browning
secretion was first noticed at the cut ends of the explants which gradually spread
into the surrounding medium. Shoot development was completely inhibited and
the explants died within a week. The browning problem was more intense with
explants collected from the older shoots than those from the younger shoots. This
problem was overcome by transferring the nodal segments to a fresh medium of
the same composition at 24 h interval. The browning secretion was stopped after
two consecutive transfers and the explants exhibited normal shoot development.
Shoot development could not be induced in the nodal stem segments on a
growth regulator-free MS medium. Addition of a cytokinin to the medium was
essential to induce bud break and multiple shoot formation in the explants
(Table 1). MS medium containing BA at an optimum concentration of 1.0 mg lÿ1
induced bud break in 93% of the explants in 12±15 days (Fig. 1). Each explant
developed 2.1 shoots averaging 3.7 cm length in 30 days (Table 1). On the other
hand 85% of the explants exhibited bud break within 10±12 days on a medium
containing 2.0 mg lÿ1 ZR and from each explant an average of 5.2 shoots were
developed in 22±25 days (Fig. 2). These shoots elongated within 4±6 days upon
transfer to a medium containing a lower concentration of ZR (1.0 mg lÿ1) and
S.K. Naik et al. / Scientia Horticulturae 79 (1999) 175±183
179
Fig. 1. Shoot development from a nodal segment of P.granatum after 15 days of culture on
MS 1.0 mglÿ1 BA.
Fig. 2. Development of multiple shoots from a nodal segment of P. granatum on MS
2.0 mglÿ1 ZR after 25 days of culture.
attained an average height of 3.9 cm. Concentrations of BA or ZR higher than the
optimum level had an inhibitory effect on shoot development (Table 1).
In another experiment TDZ at 0.01±0.5 mg lÿ1 was used to induce shoot
development from the nodal explants (Table 1). The optimum concentration of
TDZ, at which maximum number of explants exhibited bud break, was
0.05 mg lÿ1. The frequency of bud break was 63% and from each explant a
single shoot was developed in 15±18 days. However, the shoot failed to elongate.
This problem was overcome by transferring the shoot to a medium containing
0.5 mg lÿ1 BA. Within 3±4 days of transfer the shoot elongated and attained an
average height of 3.6 cm in 10 days. The frequency of shoot development was
reduced markedly at higher concentration of TDZ.
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Table 2
Influence of different auxins on rooting of the in vitro formed shoots of P. granatum
Auxin (mg lÿ1)
% rooting
Root numbera
1/2 MS basal
±
±
IAA
0.5
1.0
2.0
±
43.0 1.2
28.0 1.5
±
3.1 0.2
1.8 0.2
IBA
0.5
1.0
2.0
38.0 1.3
86.0 1.5
42.0 1.3
3.9 0.1
6.8 0.1
2.5 0.2
SE) from three independent experiments each with seven replicates.
Data (X
Root number was recorded following transfer to an auxin-free half-strength MS medium
a
3.2. Rooting of shoots
Rooting could not be induced in the excised shoots in an auxin-free halfstrength MS medium even after 30 days. Of the two different auxins tested, IBA
was more effective (Table 2). Half-strength MS medium containing IBA at
1.0 mg lÿ1 induced rooting in 86% of the shoots in 8±10 days and from each
shoot 3±4 roots were developed (Fig. 3(a)). The rooted shoots were then
transferred to an auxin-free half-strength MS medium in which the primary roots
Fig. 3. (a) Root development from an excised shoot of P. granatum on half-strength
MS 1.0 mg lÿ1 IBA after 10 days of culture. (b) A well-rooted shoot of P. granatum on an
auxin-free half-strength MS medium at day 7 of culture.
S.K. Naik et al. / Scientia Horticulturae 79 (1999) 175±183
181
Fig. 4. Regenerated plants of P. granatum in soil.
elongated within 5±7 days and a few more new roots were developed (Fig. 3(b)).
In 21 days the number of roots per shoot was 6.8 (Table 2). The rooting
percentage was markedly reduced at higher concentration of IBA.
3.3. Establishment of plants in soil
The percentage survival of the plantlets was 68% after transfer to vermicompost. Eighty percent of the plants transferred to soil survived (Fig. 4). The
regenerated plants did not show any detectable variation in morphology when
compared with the donor plant.
4. Discussion
Establishment of in vitro cultures of woody plants is greatly affected by the
browning of the medium and necrosis of explants. Browning is generally
considered to result from the oxidation of phenolic substances released from the
cut ends of the explants by polyphenol oxidases or peroxidases. The methods
commonly employed to overcome the harmful effect of browning include the use
of adsorbing agents such as activated charcoal or polyvinylpolypyrrolidone,
inclusion of antioxidants in the medium or soaking the explants in antioxidant
solution, transfer of explants to fresh medium at frequent intervals or sealing the
cut ends of the explants with paraffin wax (Broome and Zimmerman, 1978;
Weatherhead et al., 1978; Lloyd and McCown, 1980; Gupta et al., 1980; Amin
and Jaiswal, 1988; Bhat and Chandel, 1991). In the present study the browning
problem was overcome by repeatedly transferring the explants to fresh medium of
the same composition.
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S.K. Naik et al. / Scientia Horticulturae 79 (1999) 175±183
In P. granatum, highest number of shoots per explant was recorded only on a
medium containing ZR. This was unlike that reported for several other fruit trees
including guava (Amin and Jaiswal, 1988) and mulberry (Pattnaik et al., 1996) in
which BA was most effective among the cytokinins in inducing bud break and
shoot proliferation in the explants.
Thidiazuron is now being widely used for micropropagation of several woody
plants because of its tremendous ability to enhance axillary shoot proliferation
(Huetteman and Preece, 1993). In the present study, however, it was less effective
than the adenine type cytokinins such as BA or ZR. Shoots developed on a TDZcontaining medium failed to elongate. The inhibition of shoot elongation by TDZ
may be consistent with its high cytokinin activity (Huetteman and Preece, 1993).
This has been reported in several fruit tree species including Malus spp. (van
Nieuwkerk et al., 1986) and Populus (Russel and McCown, 1986). To overcome
this problem, transfer of shoot cultures to a medium often lacking TDZ or with a
different growth regulator has been suggested. The use of a primary and
secondary media has been successful in Populus (Russel and McCown, 1986). In
P. granatum, shoots developed on TDZ medium elongated following transfer to
medium containing BA.
In the present study of the two auxins tested for rooting (IAA and IBA), the
latter was more effective. IBA was reported to have favoured root induction in
several tree species including Artocarpus heterophyllus (Rahman and Blake,
1988) and Morus nigra (Yadav et al., 1990).
The in vitro propagation of pomegranate via axillary shoot proliferation from
nodal segments producing rooted plantlets in 12 weeks as described in this paper
is a rapid method as compared to the conventional method of using hard wood
cutting. Additionally, ours is a direct and more efficient method in ensuring
trueness-to-type than other in vitro procedures reported earlier for pomegranate
through callus-mediated organogenesis/embryogenesis (Jaidka and Mehra, 1986;
Omura et al., 1987; Moriguchi et al., 1987).
Acknowledgements
We thank the Department of Horticulture, Government of Orissa, Bhubaneswar, for permitting us to use the experimental plant material.
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