Plant Cell, Tissue and Organ Culture 66: 67–71, 2001.

© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

67

Research note

Low-cost alternatives for the micropropagation of banana

Andrea Kodym & Francisco Javier Zapata-Arias∗

Plant Breeding Unit, FAO/IAEA Agriculture and Biotechnology Laboratory, International Atomic Energy Agency
Laboratories, A-2444 Seibersdorf, Austria (∗ requests for offprints; Fax: +43-1-2600-28222; E-mail: f.zapata-
arias@iaea.org)

Received 15 March 2000; accepted in revised form 8 February 2001

Key words: in vitro culture, Musa, natural light, starch, sugar, sunlight

Abstract
A 90% resource cost reduction in tissue culture of banana was achieved by replacing tissue culture grade sucrose
and Gelrite in the medium with locally available commercial sugar and a starch/Gelrite mixture and by using sun
light instead of artificial light. The micropropagation of Musa ‘Grande Naine’ by shoot tip culture was used as
model. Thirteen commercial sugars from different countries were tested. Best results were achieved using white
and light brown sugars with low electrical conductivity. Sugars of cane or sugar beet origin were suitable. Starches
of corn or potato could partially substitute for Gelrite and agar. In all experiments, micropropagation rates under
natural light conditions were equal to or higher than under the controlled conditions of a growth room with PPFD
of 65 µmol m−2 s−1 and a 16-h photoperiod. Plants were exposed to average PPFD levels of 58–96 µmol m−2 s−1
and photoperiods ranged from 8–16 hours.

Abbreviations: BA – N-6-benzyladenine; EC – electrical conductivity; EDTA – ethylenediaminetetraacetic acid;

FeEDDHA – ferric ethylendiamine di(o-hydroxyphenylacetate); PPFD – photosynthetic photon flux density;
µS/cm – microSiemens per cm

Bananas and plantains (Musa spp.) are vegetatively
propagated crops of major importance in tropical and
sub-tropical countries. Micropropagation has become
a routine procedure, but the high costs involved have
prevented laboratories with limited resources from
benefiting from tissue culture technology.
Although alternatives to chemicals of high
purity in media preparation, and to artificially
lit, temperature- and photoperiod-controlled growth
rooms are used in various laboratories, there are few
publications that actually document the utility of such
innovations. Solar illuminated tissue culture rooms are
currently in use in Cuba (Anonymous, 1998). In India,
Ganapathi et al. (1995) reported that commercial grade
sugar could replace analytical grade sucrose, with no
significant change in the frequency of shoot formation
in banana. Refined white and unrefined brown sugars
in the Philippines were effective for coconut embryo
culture (Bonaobra et al., 1994). Inexpensive gelling
agents such as isubgol and sago (Bhattacharyya et al.,
1994), are not readily available. Zimmerman et al.
(1995) used a gelling mixture, consisting of 5% corn
starch and 0.05% Gelrite for the culture of fruit crops.
The aim of this work was to alert emerging laborator-
ies to readily available and inexpensive alternatives for
micropropagation.
In vitro shoot tips of ‘Grande Naine’, a com-
mercially important dessert banana of the Cavendish
subgroup, were bisected longitudinally and cultured in
reusable Magenta boxes (Magenta Corporation, USA)
containing 50 ml MS medium (Murashige and Skoog,
1962) with 20 µM BA, 40 mg l−1 cystein, 1 mg
l−1 thiamine, 4% (w/v) sugar and 0.2% (w/v) Gelrite
(Schweizerhall, Inc.) (Van Duren et al., 1996). As
iron source 37.3 mg l−1 Na2 EDTA and 27.8 mg l−1
FeSO4 .7H2 0 or 96 mg l−1 FeEDDHA were used. The
vessels were covered with cling film.
68
In all experiments, nine explants were cultured in
individual boxes, with 10 replicate boxes per treatment
or treatment combination. After 5 weeks the number
of shoots ≥0.5 cm was counted and the quality of
plantlets (turgescence, shoot size, colour, leaf size)
was evaluated. The micropropagation rate was defined
as the average number of shoots produced per explant.
The data were analysed by ANOVA.
The experiments were initiated in April 1998 and
completed in January 1999. Three locations with dif-
ferent light conditions, a growth room, a sunlit room
and a greenhouse, were used for culture incubation.
Growth room conditions were 25 ◦ C with a pho-
toperiod of 16 h at 65 µmol m−2 s−1 provided by
cool-white fluorescent tubes (Philips TLD 36 W/86).
In a room without air conditioning, the boxes were
placed in front of a window facing west. In the green-
house the cultures were kept on a table which was
covered with plastic netting (4 mm hole size, 55%
light filter) from April to October. Windows were
opened when inside temperatures reached 25 ◦ C and
additional shading curtains on the ceiling closed at
light intensities >900 µmol m−2 s−1 (measured on the
outside of the roof). Air temperature, PPFD and relat-
ive humidity were recorded using Data Hog SDL 2575
(Skye Instruments Ltd., UK) on the culture shelves.
Data were sampled at 30 sec intervals and integrated
over a 30 min period.
During the growth periods (1) April 20 to May 25,
(2) May 25 to June 29 and (3) June 29 to August 3, the
explants were cultured simultaneously in the growth
room, the sunlit room and the greenhouse using dif-
ferent sugars; Sigma sucrose (S5391) and commercial
white sugars from Austria and Cuba. In the 27 treat-
ment combination design (3 sugars × 3 locations × 3
cultivation periods), there were 270 boxes, containing
over 2400 explants.
During November 16 to December 21 the cultures
were grown in the greenhouse and commercial sug-
ars from eleven countries were compared with sucrose
(S5391) from Sigma Chem. Co. (St. Louis, Mo, USA).
The sugars were grouped according to their colour into
5 categories from white (1) to dark brown (5) and were
produced from either sugar beet or cane (Table 1). The
pH and electrical conductivity of 4% (w/v) sugar solu-
tions in distilled water (pH 5.2, EC 1 µS cm−1 at 25
◦ C) were measured before and after autoclaving.
Different gelling agents were tested. Media so-
lidified with household brand corn starch (Knorr
GesmbH, Austria), potato starch (Peru), rice flour
(Thai World Ltd., Thailand), cassava flour (Atco) and
pounded yam (West Africa) were compared with Gel-
rite during the periods of September 17 to October 22
and December 21 to January 25 in the greenhouse.
Starch or flour was mixed with cold water and then
added to the hot medium. After adjustment of pH,
the medium was heated while stirring to dissolve the
gelling agents. Media were decanted into Magenta
containers and autoclaved at 121 ◦ C for 20 min.
Under natural light conditions, results comparat-
ive to artificial light were achieved in plant qual-
ity and quantity, despite wide fluctuations in pho-
toperiod, temperature and light intensity (Table 2).
Sunlit growth rooms eliminate the costs for artificial
lighting, which is US$ 3 per m2 per week in Austria,
not including repairs and spare tubes. Micropropaga-
tion rates were higher in the greenhouse and sunlit
room than in the growth room (Tables 3, 4). Plant
quality was satisfactory in all three locations but under
natural light conditions the plants had larger leaves
and were lighter in colour than those in the growth
room. The availability of iron had an influence on the
colour of plants (Van der Salm et al., 1994). When us-
ing FeEDDHA, a more photostable iron chelate, than
FeEDTA, an increase in leaf colour intensity in all
locations was observed.
There were strong indications of seasonal influ-
ences on the micropropagation rate under natural light
conditions. Highest micropropagation rates were in
the greenhouse during summertime when there were
long photoperiods and abundant sunshine (Figure 1).
During winter with less favourable light conditions
micropropagation rates decreased. The May and June
treatments differed significantly from the Novem-
ber and December treatments (p<0.001). The data
presented in Table 2 also show a significant differ-
ence (p=0.02) between cultivation times. It seemed
that high light intensity favoured plant development
(Marchal et al., 1992, Navarro et al. 1994). On
the other hand, managing light intensity was ex-
tremely important to obtain plants of good quality.
During summer and autumn months the cultures in
the greenhouse were additionally covered with a net-
ting filtering 55% of the light. The problems with loss
of turgescence and browning of leaves (Kodym and
Zapata-Arias, 1999) were thereby overcome. No sig-
nificant interactions among the three factors (location,
cultivation period, sugar) was found.
Commercial sugars are an obvious replacement for
tissue culture grade sucrose at 10% of its price (US$
0.90/kg versus US$ 10/kg, in Austria). Table 1 shows
the characteristics of the tested sugars before autoclav-
 69
Table 1. Effect of different sugars on micropropagation rate (mean value ± S.E.)

Origin Plant source Micropropa- Colour EC pH

gation rate µS cm−1

Cuba brown cane 3.66 ± 0.18 4 137 7.0

no name
Sudan brown cane 3.18 ± 0.18 3 92 6.8
no name
Austria white beet 3.15 ± 0.17 1 6 6.2
Wiener Zucker
Egypt cane 3.14 ± 0.18 2 32 6.5
Middle East Center
Cuba white cane 3.13± 0.18 2 13 5.2
no name
Sigma S5391 cane 3.06 ± 0.17 1 7 6.0
Sigma Chem. USA
El Salvador cane 3.01 ± 0.18 2 56 8.6
Codip
Sri Lanka cane 2.81 ± 0.15 3 35 6.3
Araliya Impex Ltd.
South Africa cane 2.66 ± 0.21 3 58 6.5
marketed by Engelbert. A
Austria brown beet 2.50 ± 0.13 3 1328 5.4
Wiener Zucker
US cane 2.20 ± 0.18 4 440 6.9
Domino Sugar Corp.
UK cane 2.18 ± 0.24 4 452 6.4
Whitworths
Paraguay cane 2.07 ± 0.19 4 1021 7.3
marketed by Engelbert. A
Brazil cane 1.36 ± 0.13∗ 5 1070 7.2
Granovital

Colour coding: 1 white, 2 oyster white, 3 beige, 4 brown, 5 dark brown.

∗ Significantly different from Sigma treatment (ANOVA on Ranks).

Figure 1. Micropropagationrate in the greenhouse. ∗ Bars with the same letter are not significantly different at the 5% level of a Dunn’s Test.
70
Table 2. Environmental conditions during different cultivation periods at three locations (Seibersdorf, Austria)

Location Culturing period Photoperiod Temperature (◦ C) PPFD (µmol m−2 s−1 )

(h, min) Min – Max Mean Daily highs Mean Median of daily highs

Growth chamber all periods 16 21 – 27 25 65 65 65

Sunlit room April 20–May 25 13.56 – 15.36 21 – 32 24 83 – 627 74 270

May 25–June 29 15.36 – 16.04 16 – 33 23 72 – 565 80 350
June 29–Aug. 3 16.04 – 14.58 18 – 30 23 75 - 748 96 195

Greenhouse April 20–May 25 13.56 – 15.36 16 – 36 25 110 – 534 91 220

May 25–June 29 15.36 – 16.04 16 – 41 25 120 – 485 93 230
June 29–Aug. 3 16.04 – 14.58 20 – 40 26 140 – 453 96 245

Sept. 17–Oct. 22 12.31 – 10.29 20 – 33 25 34 – 462 58 145

Nov. 16–Dec. 21∗ 9.12 – 8.19 18 – 31 24 19 – 497 68 146
Dec. 21–Jan. 25∗ 8.19 – 9.07 18 – 27 24 51 – 318 77 185
∗ No netting used.
Outdoor temperatures reached a maximum of 35 ◦ C.

Table 3. Micropropagation rates (mean value ± S.E.) in three different locations,

using different white sugars at three cultivation periods

Period Location Sigma Austria Cuba

April 20–May 25 Growth room 2.56 ± 0.20 2.71 ± 0.20 2.78 ± 0.21
Sunlit room 3.24 ± 0.24 3.68 ± 0.20 3.47 ± 0.20
Greenhouse 3.54 ± 0.20 3.45 ± 0.20 3.41 ± 0.20

May 25–June 29 Growth room 2.99 ± 0.20 2.56 ± 0.20 2.36 ± 0.20
Sunlit room 3.60 ± 0.20 3.33 ± 0.20 3.00 ± 0.23
Greenhouse 3.98 ± 0.20 3.69 ± 0.20 3.41 ± 0.20

June 29–Aug. 3 Growth room 2.50 ± 0.20 2.52 ± 0.20 3.40 ± 0.23
Sunlit room 3.61 ± 0.23 3.45 ± 0.21 3.82 ± 0.21
Greenhouse 3.94 ± 0.19 3.82 ± 0.20 4.06 ± 0.20

ing and the associated micropropagation rates. After
autoclaving, the pH of the sugar solutions dropped and
electrical conductivity increased slightly in all sugars.
The most reliable criteria for selecting the sugar were
colour and electrical conductivity, but not pH. There
were no differences observed between purified Sigma
sucrose and white, oyster white and beige sugars (col-
our 1–3) with EC readings of 6 to 137 µS cm−1 .
Brown sugars, which are generally high in minerals
and organic compounds gave high EC readings, ex-
cept for Cuban sugar. Data suggest that dark sugars
(color 4.5) with an EC of 440 µS cm−1 and above lead
to lower micropropagation rates. This may be caused
by inhibitors already present in unpurified sugars or
which formed during autoclaving (Maheshwari et al.,
1980). On all media plant quality was good, although
oxidation was high in explants grown on Brazilian
sugar. In the large scale experiment using Sigma
sucrose and the commercial Austrian and Cuban sugar,
representing sugar beet and cane products with sim-
ilar characteristics, there were no statistical differences
between the micropropagation rates (Tables 3, 4).
Prices for agar (US$ 235/kg) and Gelrite (US$
180/kg) are high compared to starch (US$ 2.30/kg).
Starch at concentrations that supported plant growth
(13% for potato starch and 10% for corn starch) was
difficult to dissolve, pour and clean. To overcome
these problems, corn and potato starch were combined
at 6% with 0.1% agar (Sigma A4675, type E) or at
5% with 0.05% Gelrite. There were no significant dif-

Table 4. Influence of location, sugar and cultivation period on multiplication rates
Location Growth room
Greenhouse
Sunlit room
Cultivation period April 20–May 25
May 25–June 29
June 29–Aug. 3
Sugar Sigma
Cuba, white
Austria, white
ferences between the treatments. In September, the
micropropagation rate for medium with Gelrite was
3.21 (±0.17 S.E.), with potato starch and agar 2.95
(±0.17) and with corn starch and agar 3.11 (±0.21).
In December the micropropagation rate for medium
with Gelrite was 2.77 (±0.17), with potato starch and
Gelrite 2.84 (±0.20) and with corn starch and Gelrite
2.37 (±0.15). Plants on starch media were smaller but
of good quality.
Medium with rice flour did not produce a satisfact-
ory gel, even when combined with agar. Moreover,
plant quality was adversely affected and the medium
turned black. Cassava flour and yam had good gelling
qualities at 14% (w/v) but produced very small shoots.
Success may also vary between different brands and
needs to be determined empirically.
Using natural light and alternative consumables
we were able to reduce the resource costs for micro-
propagation by 90%. Small scale micropropagation
laboratories in tropical and sub-tropical countries can
incorporate low-cost technology for a wide range of
crops.
Acknowledgements
Many thanks to A. Draganitsch for his technical assist-
ance. The authors also acknowledge R. Litz, C. Rigney
and the staff of the Plant Breeding and Genetics Sub-
programme for their critical reading of the manuscript.
We are also grateful to the International Network for
the Improvement of Banana and Plantain (INIBAP),
Musa Germplasm Transit Centre, Leuven, Belgium
for providing in vitro plant material.
71
Mean value ± S.E. Significance
2.70 ± 0.07
3.70 ± 0.07
3.47 ± 0.07
< 0.001
3.21± 0.07
3.22± 0.07
3.45± 0.07
0.02
3.33 ± 0.07
3.30 ± 0.07
3.25 ± 0.07
n.s.
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