Tissue Culture of a Year-Round Fruiting Variety of Artocarpus

heterophyllus L. in Bangladesh
F.M.S. Azam and M. Rahmatullah Ather-uz-Zaman
Department of Biotechnology and Genetic Engineering Centre for Plant Tissue Culture
University of Development Alternative PROSHIKA, Dhaka
Dhanmondi, Dhaka Bangladesh
Bangladesh

Keywords: jackfruit, clonal propagation, synergistic effect, malnutrition improvement

Abstract
Jackfruit (Artocarpus heterophyllus) is available in Bangladesh between June
to August; however, a variety exists that fruits throughout the year. The edible
portion is considered as a good source of carbohydrates, proteins, vitamins and
minerals. In Bangladesh, malnutrition persists among 35-45% of the population and
child malnutrition rate is more than 50%. Under this circumstance, wide cultivation
of a jackfruit variety that bears fruit throughout the year can play a significant role
towards reducing malnutrition in Bangladesh. Since this particular variety is not
widely available, we have developed a method for its clonal propagation. Healthy
and juvenile shoot tips and nodal segments were collected from field-grown fruit
bearing trees and cultured in Murashige-Skoog (MS) media fortified with different
concentrations and combination of 6-benzyleaminopurine and kinetin following the
sterilization with 0.1% HgCl2. Sprouting and regeneration of micro shoots geared up
when MS was enriched with 3.5 mg/L BAP. Proliferation frequency increased
considerably and multiple shoots were regenerated as a clump of 2-3 shoots in pH
5.8 media within four weeks of inoculation through synergistic effect of 6-
benzyleaminopurine (3.5 mg/L) and kinetin (1.5 mg/L). With the increase of
subculture (up to 10th maximum), frequency of shoot proliferation was enhanced.
Addition of 0.1 mg/L indole-3-acetic acid and 20% coconut water considerably
increased shoot elongation and stimulated growth of the shoots. About 80% rooting
frequency were observed in ½ MS medium with 1.2 mg/L indole-3-butyric acid.
Rooting percentage and their growth were much better in liquid media. After
proper acclimatization, rooted plantlets were transferred to polythene bags
containing garden soil, sand and cowdung (1:1:1). After eight weeks of
transplantation in open-field, more than 80% of the plants had survived. No
morphological variants were observed during the passage of clonal propagation.
This clonal propagation technique could play a significant role in improving
productivity leading to reduced malnutrition.
INTRODUCTION
Jackfruit (Artocarpus heterophyllus L.) belongs to the family Moraceae and enjoys
the crown as national fruit of Bangladesh. The annual production of jackfruit is around
279.5 thousand metric tons per year in Bangladesh. The edible portions are the pulp and
seeds, which are considered as a good source of carbohydrate, proteins, vitamins B1 and
B2 and minerals (Morton, 1966; Burkill, 1997). Jackfruit is one of the cheapest fruit and
referred to as ‘the poor man’s food’ in Bangladesh (Rahman et al., 1995). A family
having five to six members can easily consume one jackfruit at a time and satisfy their
hunger. It is known that one variety exists in Bangladesh which yields fruits throughout
the year, although this variety is yet to have a name or code number. Trees of this variety
have been cultivated at BARI for the past decade.
Propagation of jackfruit plant from seeds is not widely accepted because of high
heterozygosis (Singh, 1986; Samaddar, 1990). Clonal propagation through grafting of
selected genotype is highly desirable but the number of plants produced by these
conventional methods is relatively low (Rowe-Dutton, 1976; Samaddar, 1990). The

Proc. IS on Underutilized Plants 269

Eds.: Jaenicke et al.
Acta Hort. 806, ISHS 2009
application of tissue culture methods for improvement and large-scale propagation of fruit
trees has been well demonstrated (Litz et al., 1985; James, 1988). Successful in vitro
propagation of jackfruit seedlings has been demonstrated (Rahman and Blake, 1988).
Regeneration of plantlets from bud and nodal explants of mature jackfruit trees has been
reported (Jaiswal and Amin, 1990; Roy et al., 1990).
Bangladesh has very high child malnutrition rates. The most recent survey results
revealed that 50.5% children aged <5 years are small for their age and 42.7% children
aged <5 years are underweight for their age. Thus jackfruit, especially the variety which
is available throughout the year, can serve as an efficient means to reduce malnutrition in
Bangladesh.
This research study was undertaken to develop an efficient protocol using tissue
culture technique for the large scale propagation of this year-round fruiting jackfruit
variety by examining the effects of growth regulators and the Murasighe and Skoog (MS)
medium (Murashige and Skoog, 1962).
MATERIALS AND METHODS
This experiment was carried out at the Plant Tissue Culture Laboratory of
PROSHIKA, Bangladesh in 2006 to 2007.
Explant Collection and Sterilization
Fifty to seventy explants were collected for each experiment. These were 10-15
day-old newly-sprouted healthy shoots from 10-12 year-old fruit-bearing trees of the
year-round fruiting variety of jackfruit growing at the Bangladesh Agricultural Research
Institute (BARI). The actual number of explants used in any particular experiment is
detailed in the accompanying tables. After removal of expanded leaves, the shoot apices
and nodes were washed thoroughly under running tap water for 30 min. then treated with
Tween 80 plus Savlon for 10 min. and again rinsed with distilled water. The explants
were surface disinfected with 0.1% HgCl2 (for 5-15 min.) followed by seven times of
rinsing with sterile distilled water in a laminar air flow cabinet. The explants were finally
trimmed, prepared, and cultured on the medium.
Shoot Proliferation Stage
Explants were implanted vertically on MS medium supplemented with different
(0.2, 0.5, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 mg/L) concentrations of 6-
benzylaminopurine (BAP). All media were supplemented with 3% sucrose and 0.8%
Difco Bacto-agar. The pH was adjusted to 5.8 prior to autoclaving the media at 1 kg
pressure per cm2 and 121°C for 20 min. All culture vessels were incubated in a growth
room at 26±2°C with 16 hrs light provided by Philip white fluorescent tube lights with a
photon flux density of 3,000 lux.

Multiple Shooting
In a second experiment, jackfruit explants were cultured on MS medium fortified
with different concentrations and combinations of BAP (3.0, 3.5, 4.0 mg/L) and kinetin
(Kn) from 0.2 to 3.0 mg/L to observe the synergistic effect on multiple shoot formation.

Root Induction
For root induction, micro-shoots were cultured on ½ MS medium enriched with
different concentrations (0.2 to 2.0 mg/L) of indole-3-butyric acid (IBA).

Acclimatization
In vitro derived plantlets were gradually hardened through gradual exposure to
optimum relative humidity and sunlight for seven days and then transferred to a net
house. Plantlets were taken out from the culture vessels/tubes, and then washed carefully
under running tap water to remove any traces of agar. Each plantlet was transplanted to
one of the small poly bags that contained different compositions and combinations of soil

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media to select the best formulation. The soil compositions are shown in Table 1. The
plantlets were immediately covered with perforated polythene bag to prevent desiccation.

Open Field Observation

Acclimatized plants were observed for proper establishment in soil for fifteen days
and field observation was done from January to February 2007 and data were recorded in
one-week intervals.
Data Assemblage
Weekly growth observations were done up to four weeks after establishment, and
experimental data were recorded. The parameters were the following:
i. Shoot length in cm and number of leaves/shoot,
ii. Number of multiple shoot/explant,
iii. Average length of multiple shoots in cm,
iv. Number of root/shoot, and
v. Root length in cm.
Recorded data were analyzed as mean ± SE according to Mian and Miyan (1984).

RESULTS AND DISCUSSION

Surface Sterilization
A survival rate of 82.22% was observed when the excised explants were surface
sterilized with 0.1% HgCl2 solution for 10 min. In previous studies, surface sterilization of
0.2% HgCl2 for 5 min. was done to treated shoot apices and axillary buds of A. chaplasha
Roxb and A. heterophyllus Lamk (Roy and Hadiuzzaman, 1991). In a preliminary
experiment it was observed that lower concentration of 0.1% HgCl2 was suitable for
sterilization, but prolonged treatment (more than 10 min.) manifested in vitro tissue
killing. In another study, 0.1% HgCl2 for 5 min. was used for surface sterilization process
(Amin, 1992a). In the present study, we therefore followed the method of Amin (1992a)
except that surface sterilization with 0.1% HgCl2 was increased from 5 to 10 min. It was
observed that this treatment gave better surface sterilization with good survival rates.

Shoot Proliferation
Highest frequency (92.30%) of sprouting and adventitious shoot proliferation was
observed when implanted on MS medium supplemented with BAP at 3.5 mg/L (Figs. 1
and 2) and the highest length of shoot was 3.81 cm (Table 2). The higher concentrations
of BAP produced multiple shoots but excessive amount (BAP 4.5 mg/L) had deteriorating
effect on shoot proliferation resulting in curled leaves. BAP-induced shoot proliferation
from the shoot apices has also been reported in Artocarpus heterophyllus (Amin and
Jaiswal, 1993). However the shoot proliferation rate as observed in the present study was
much higher than that reported earlier for jackfruit (about 80% bud explants produced
deep-green, mossy inflorescence at the axils of basal nodes) (Amin, 1992a). A
combination of BAP and NAA resulted in proliferation of shoots up to 5.2±0.17 cm in A.
heterophyllus (Roy and Hadiuzzaman, 1991). Superiority of BAP over other cytokinins
has been recorded in other tree species (Zaman et al., 1993; Pattnaik and Chand, 1997).

Multiple Shoot Formation

After the shoot proliferation phase, BAP at 3.5 mg/L was used for multiple shoot
proliferation along with Kn at different concentrations (0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0
mg/L) to observe any synergistic effect (Table 1). Highest percentage (58.06%) of
multiple shoot proliferation was observed at Kn (2.0 mg/L) + BAP (3.5 mg/L) having 2
shoots/explant with 1.54 cm shoot length. However, with a decrease of Kn (1.5 mg/L), the
percentage of multiple shoot proliferation decreased (47.50%) but the number of multiple
shoot (3 shoots/explant) increased (Fig. 3). Addition of IAA 0.1 mg/L and 20% coconut
water showed green and expanded leaves. In Tectona grandis (Gupta et al., 1980) both

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BAP and Kn were observed to be essential. The superiority of BAP over other cytokines
for multiple shoot formation in hardwoods was also reported (Vieitez and Vieitez, 1980).

Root Induction
All concentrations of IBA induced the roots, but maximum response (80%) and
healthy roots (6.17 roots/shoot and length 3.2 cm) were noticed when shootlets were
cultured on ½ MS enriched with IBA 1.2 mg/L (Table 3 and Fig. 4). Any increase of
concentration of IBA (>1.5 mg/L) induced callus formation at the base of the shoots and
showed leaf necrosis. Omission of agar from rooting medium showed same response.
IBA 2.0 mg/L showed about 68% rooting, and 83% rooting efficiency has been recorded
in jackfruit with IBA and Į-naphthalene acetic acid (NAA), 2 mg/L each (Amin, 1992b).
Healthy roots were induced in A. heterophyllus with 1.5 mg/L IBA (Roy and
Hadiuzzaman, 1991). In jackfruit, 90% rooting was achieved on a media consisting of ½
MS with IBA and IAA, 1.0 mg/L each (Islam et al., 1993). It was observed that 1-2 mg/L
each of IBA and NAA induced maximum rooting (Jaiswal and Amin, 1990) while the
present investigation showed better response by only using IBA.
Hardening and Acclimatization
After hardening, the in vitro raised plantlets were transplanted in potted media and
were kept in a net house. After 2-3 weeks of transplantation, highest survival rate (80%)
was achieved in planting media consisting of garden soil, sand, and cowdung (1:1:1)
(Table 1). On the other hand, Islam et al., (1993) observed about 85% survival of jackfruit
plantlets in the field. However, Amin et al., (1993) reported only 50% survival rate after
transplantation.
Field Observation
After 6-8 weeks, the in vitro propagated plantlets resembled the general
morphological characteristics of the mother plant but showed no detectable variation in
growth on the basis of soil types (Table 1). Highest length (5.50 cm) and highest number
of leaves (4.60) were observed from soil formula 3 (Fig. 5).

CONCLUSION
With this experiment, a protocol for the micropropagation of a unique variety of
A. heterophyllus which fruits throughout the year was established from shoot tip and
nodal explants. It may be noted that all previous studies were conducted with varieties of
this species which fruits for only two to three months in any given year. In our
knowledge, this is the first successful description of micropropagation of this unique
variety. Successful micropropagation of this variety can lead to widespread cultivation
and for obtaining this fruit throughout the year, which in turn can lead to a cheap source
of nutritious food available to the general population and specifically to the nutritionally
impoverished population of Bangladesh and other countries where this variety can be
cultivated.
Literature Cited
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from explants of Jackfruit trees. Plant Tissue Cult. 2:27-30.
Amin, M.N. 1992b. In vitro rooting of Jackfruit (Artocarpus heterophyllus) microcuttings
derived from mature trees. Plant Tissue Cult. 2:129-133.
Amin, M.N. and Jaiswal, V.S. 1993. In vitro response of apical bud explants from mature
tree of Jackfruit (Artocarpus heterophyllus). Plant Cell Tiss. and Org. Cult. 33:59-65.
Burkill, H.M. 1997. The useful plants of west tropical Africa. Vol. 4, 2nd Ed. Royal
Botanic Gardens, Kew, UK.
Gupta, P.K., Nadgir, A.L., Mascarenhas, A.F. and Jagannathan, V. 1980. Tissue culture
of forest tree: Clonal propagation of mature trees of Tectona grandis L. (Teak) by
tissue culture. Plant Sci. Let. 17:259-268.

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Islam, M.S., Sen, J., Alam, N. and Roy, S.K. 1993. Propagation of jackfruit (Artocarpus
heterophyllus) through zygotic embryo culture in vitro. Short Communications, Plant
Tissue Cult. 3:51-55.
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Intl. Cong. Plant Tissue and Cell Culture, Amsterdam, The Netherlands.
James, D.J. 1988. Cell and tissue culture technology for the genetic manipulation of
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Intercept. Dorset, England.
Litz, R.E., Moore, G.A. and Srinivasan, C. 1985. In vitro system for propagation and
improvement of tropical fruits and palms. Hort. Soc. 7:157-198.
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and utilization. Proc. Fla. State Hort. Soc. 78:336-344.
Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays
with tobacco tissue cultures. Physiologia Plantarum 15:473-497.
Pattnaik, S.K. and Chand, P.K. 1997. Rapid clonal propagation of three mulberries,
Morus cathayana Hemsl, M. Ihou Koiz and M. serrata Roxb. through in vitro culture
of apical shoot buds and nodal explants from mature trees. Plant Cell Rep. 16:503-
508.
Rahman, A.K.M.M., Huq, E., Mian, A.J. and Chesson, A. 1995. Microscopic and
chemical changes occurring during the ripening of two forms jackfruit (Artocarpus
heterophyllus L). Food Chem. 52:405-410.
Rahman, M.A. and Blake, J. 1988. Factors affecting in vitro proliferation and rooting of
shoots of jackfruit (Artocarpus heterophyllus Lam.). Plant Cell Tissue and Organ
Culture 13:179-187.
Rowe-Dutton, P. 1976. Artocarpus heterophyllus- jackfruit. p.269-290. In: R.J. Garner
and S.A. Chaudhuri (eds.), The propagation of tropical fruit trees. FAO, CAB,
London.
Roy, S.K., Rahman, S.L. and Majumder, R. 1990. In vitro propagation of Jackfruit
(Artocarpus heterophyllus Lam.). J. Hort. Sci. 65:355-358.
Roy, S.K. and Hadiuzzaman, S. 1991. Micropropagation of two species of Artocarpus
through in vitro culture. Bangladesh J. Bot. 20:27-32.
Samaddar, H.N. 1990. Jackfruit. p.638-649. In: T.K. Bose and S.K. Mitra (eds.), Fruits:
tropical and subtropical. Naya Prokash, Calcutta.
Singh, A. 1986. Fruit physiology and production, 2nd revised edition. Kalyani Publishers,
New Delhi-Ludhiana.
Vieitez, A.M. and Vieitez, M.L. 1980. Culture of chestnut shoots from buds in vitro.
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Tables

Table 1. Growth observation of plantlets in poly bags.

Experimental soil No. of plantlet Ave. length No. of Ave. time Growth
formula transplanted/ of plant leaves/plant required vigor
survival rate (cm) (days) (visual remark)
(%)
Garden soil 12/58.33 4.80±0.084 4.14±0.260 60 +1
Garden soil + Sand (1:1) 10/70.00 4.90±0.179 4.20±0.184 60 +
Garden soil + Sand + 15/80.00 5.50±0.071 4.66±0.188 60 +++
Cowdung (1:1:1)
Garden soil + Sand + 12/66.66 5.20±0.073 4.30±0.163 60 ++
poultry liter (1:1:1)
Garden soil + Organic 13/53.84 5.10±0.069 4.15±0.132 60 ++
fertilizer (1:1)
Garden soil 10/40.00 3.10±0.054 3.40±0.250 60 +
(hot water treated)
Garden soil 12/33.33 2.80±0.103 3.75±0.250 60 –
(0.01% formalin treated)
1
– = Poor, + = good, ++ = better, +++ = best.

Table 2. Hormonal effects on in vitro shoot regeneration and multiple shoot proliferation
on MS medium of A. heterophyllus.

MS + No. of Percentage Ave. no. Ave. length Ave. length of Ave. leaves/
hormone explants of explants of multiple of shoot multiple shoot shoot
(mg/L) inoculated responded shoot (cm) (cm) (Mean±SE)
(%) (Mean±SE) (Mean±SE)
BAP
0.2 39 0.00 - 0.00 - 0.00
0.5 47 8.51 - 0.15±0.035 - 2.75±0.025
0.8 58 15.51 - 0.54±0.013 - 3.00±0.160
1.0 41 26.82 - 0.74±0.011 - 3.09±0.160
1.5 57 36.84 - 1.12±0.004 - 3.38±0.200
2.0 43 53.48 - 1.56±0.017 - 3.69±0.250
2.5 48 70.83 - 2.22±0.048 - 4.00±0.280
3.0 55 80.00 - 2.75±0.009 - 4.25±0.220
3.5 52 92.30 - 3.81±0.013 - 4.41±0.200
4.0 61 60.65 - 2.30±0.004 - 4.13±0.220
4.5 45 37.77 - 1.22±0.005 - 3.76±0.010
BAP + Kn
3.0 + 0.2 40 00.00 0.00 - 0.00 -
3.0 + 0.5 37 00.00 0.00 - 0.00 -
3.0 + 1.0 48 00.00 0.00 - 0.00 -
3.0 + 1.5 52 40.38 1±0 - 1.5±0.045 -
3.0 + 2.0 46 56.52 1±0 - 1.8±0.060 -
3.0 + 2.5 45 15.55 1±0 - 1.3±0.044 -
3.0 + 3.0 50 06.00 1±0 - 1.3±0.050 -
3.5 + 0.2 38 00.00 0.00 - 0.00 -

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Table 2. Continued.

MS + No. of Percentage Ave. no. Ave. length Ave. length of Ave. leaves/
hormone explants of explants of multiple of shoot multiple shoot shoot
(mg/L) inoculated responded shoot (cm) (cm) (Mean±SE)
(%) (Mean±SE) (Mean±SE)
3.5 + 0.5 25 12.00 1±0 - 1.43±0.005 -
3.5 + 1.0 36 25.00 2±0.166 - 1.62±0.005 -
3.5 + 1.5 40 47.50 3±0.108 - 1.78±0.007 -
3.5 + 2.0 31 58.06 2±0.140 - 1.54±0.016 -
3.5 + 2.5 28 39.28 1±0 - 1.38±0.005 -
3.5 + 3.0 35 14.28 1±0 - 1.30±0.054 -
4.0 + 0.2 25 00.00 0.00 - 00.00 -
4.0 + 0.5 30 06.66 1±0 - 01.44±0.014 -
4.0 + 1.0 32 18.75 1±0 - 01.52±0.004 -
4.0 + 1.5 28 42.85 2±0.117 - 01.68±0.010 -
4.0 + 2.0 35 45.71 1±0 - 01.46±0.006 -
4.0 + 2.5 27 00.00 0.00 - 00.00 -
4.0 + 3.0 30 00.00 0.00 - 00.00 -

Table 3. Effect of IBA for root induction in vitro raised shootlets.

½ MS + different No. of No. of Rate of Ave. Ave. Callus

concentration explant explant root roots/shoot length formation
of IBA inoculated responded induction of root
(mg/L) (%) (cm)
0.2 36 8 22.22 3.75±0.163 0.8±0.026 –1
0.5 40 13 32.50 4.20±0.155 1.8±0.053 –
0.8 42 24 57.14 4.60±0.187 2.2±0.010 –
1.0 38 29 76.31 5.24±0.191 2.8±0.048 –
1.2 35 28 80.00 6.17±0.176 3.2±0.051 –
1.5 45 28 62.22 4.50±0.154 2.6±0.055 +
1.8 30 14 46.66 3.64±0.132 1.5±0.062 ++
2.0 39 13 33.33 2.61±0.140 0.5±0.052 +++
1
– = no callus, + = low callus, ++ = moderate callus, +++ = high callus.

Figures

Fig. 1. Regenerated micro-shoot on MS medium fortified with BAP (3.5 mg/L) after 2
weeks of culture.

275
Fig. 2. Shoot proliferation after 8th subculture in the same medium.

Fig. 3. Clusters of profuse branching of proliferated shoots on MS + BAP (3.5 mg/L) +

Kn (1.5 mg/L).

Fig. 4. Adventitious rooting on MS medium enriched with IBA (1.2 mg/L).

Fig. 5. Eight weeks old plants established in a mixture of garden soil, sand, and cowdung
(1:1:1).

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