Plant Cell Reports (1985) 4:351-354
Plant Cell
A tissue culture technique for rapid clonal propagation and storage
under minimal growth conditions of Musa (Banana and plantain)
Nirmalya Banerjee * and Edmond de Langhe
Laboratorium voor Teeltfysiologie en Tropische Fytotechnie, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92,
B-3030 Leuven, Belgium
Received November 12, 1985 - Communicated by D. von Wettstein
Abstract
A tissue culture technique for rapid clonal propa-
gation and storage under minimal growth conditions
is presented in this paper. Shoot-tip cultures of
M o ~ cultivars (both banana and plantain) are induced
by culturing small excised shoot apices on modified MS
semisolid medium supplemented with various concentra-
tions and combinations of auxins and cytokinins. The
effects of cytokinin concentration in the medium as
well as the genotypic configuration of the cultivars
on the rate of shoot-bud proliferation have been
tested. The established shoot-tip cultures grown on
modified MS semisolid medium supplemented with IAA
(0.18 mg/l) and BA (2.30 mg/l) have been successfully
stored at 15°C with IOOO lux light intensity up to
13-17 months depending on the cultivar. The cultivars
tested in the present investigation seem to vary in
their ability to withstand minimal growth temperature.
Abbreviations
BA - Benzyladenine, IAA - Indoleacetic acid, IBA -
Indolebutyric acid, MS - Murashige and Skoog.
Introduction
The propagation of various cultivars of Mu.6a (both
banana and plantain) by conventional methods has been
described by a number of authors in the past decades
(Aseenco, 1967; De Langhe, 1961 and Hamilton, 1965).
These techniques are laborious and time consuming as
far as the production of a large number of homogeneous
plants is concerned. Moreover, the rate of propagation
of M ~ a plantlets by this method is rather low. In
view of the above facts, there has been an upsurge of
interest in the field of rapid clonal propagation of
plantlets applying meristem culture technique
(Cronauer and Krikorian, 1984; Doreswamy et al., 1983;
Banerjee et al., 1985 and Vuylsteke and De Langhe,
1985). Although there are a few reports available on
clonal multiplication of Musa, further study is needed
to prescribe an optimal technique for rapid clonal
propagation of a large number of divergent genotypes
which would serve as a basic tool in establishing a
gene bank. Parallel to this, emphasis should be laid
on developing a suitable method for medium- and long-
term storage of the meristem cultures. From the
economic point of view, it is undeniably desirable to
minimise the growth rates of the stored cultures in
order to reduce the laborious as well as expensive
subculturing requirements. Moreover, this might have a
Offprint requests to." E. de Langhe
* Present address: Centre of Advance Study in Cell and Chromosome Research, Department of Botany, University of Calcutta, 35 Bally-
gunge Circular Road, Calcutta 700019, India
Reports
© Springer-Verlag1985
beneficial effect on the genetic stability of the
cultures, since mutations generally occur during re-
plication and mitotic phases of the growth cycle. To
date, there are several approaches available for in
wlt~o conservation of germplasm (Henshaw, 1982 and
Henshaw et al., 1980).
The present paper encompasses the aspects of rapid
clonal multiplication of several cultivars of MoAa and
their minimal growth storage at reduced temperature
and low light intensity.
Materials and Methods
Eight triploid cultivars (both banana and plantain)
of different genotype have been used as experimental
material (table I).
Shoot meristems are isolated from the in S ~ u
plants of different cultivars grown in the experimen-
tal glasshouse. The detailed description of the shoot
meristem isolation technique has been given in
(Banerjee et al., 1985).
Isolated shoot-tips are surface sterilized succes-
sively in rectified spirit and in 1.5% calciumhypo-
chlorite solution for 30 seconds and 20 minutes res-
pectively, followed by repeated washing with sterile
distilled water. Finally, shoot meristems of 2-3 mm
size along with 2-3 leaf primordia are excised
aseptically under a stereoscope and immediately placed
onto culture medium. The medium contains MS macro and
modified micro elements (containing no CuSOA.5HgO and
ZnSOL.4H20) supplemented with 3% sucrose, 270 m~/l
glycine, O.I mg/l thiamine HCI, 0.5 mg/l nicotinic
acid and 0.5 mg/l pyridoxine. This medium is further
modified with various concentrations and combinations
of growth regulators to obtain different morphogenetic
responses (e.g. outgrowth of shoots, proliferation of
shoot meristems, induction of roots and maturation of
rooted plantlets). Ascorbic acid (I0 mg/l) is used in
the medium as antioxidant to reduce blackening. The
medium is solidified with 0.45% oxoid agar and the pH
is adjusted to 5.8 prior to autoclaving. Cultures are
incubated at 30°±2°C under constant light of 3000 lux
and 70% relative humidity.
For low temperature storage, established multiple
buds cultured on MS semisolid medium supplemented with
3% sucrose, O.18 mg/l IAA and 2.3 mg/l BA are trans-
ferred to different growth chambers with various low
temperatures and reduced light intensity (IOOO lux).
Survival percentages of the cultures are determined
by transferring a part of the cultures, at 2 months
intervals, to normal growth conditions.
352

Results and Discussion fested by the appearance of a clump of numerous fleshy

ESTABLISHMENT OF SHOOT-TIP CULTURE AND PLANTLET
REGENERATION AT STANDARD CULTURE CONDITIONS
The shoot meristem-tips isolated from all possible
parts of in situ plants (e.g. suckers, peepers, dor-
mant buds and pseudostems) give 1OO% survival in cul-
ture (fig. I). The problem of blackening of the cul-
tures can be overcome by a combined effect of the
addition of ascorbic acid and frequent transfer (every
two weeks) of the tissue to fresh medium. It is also
observed that the degree of blackening of the culture
medium is considerably reduced after a few subcultures.
The morphogenetic responses of the explants seem to be
influenced on one hand by the concentration of BA used
in the medium and on the other hand by the genomic
configuration of the cultivars. Thus at low concentra-
tion of BA (0.2 mg/l) only single shoots are regene-
rated in most of the cultivars (fig. 2). These re-
generated shoots can be rooted subsequently by trans-
ferring them onto MS semisolid medium with half
concentration of the maerosalts and 0.2 mg/l IBA
(fig. 3). In most cases root initiation takes place
within 2 weeks. Upon formation of a well ramified root
system (fig. 4) plantlets can be transferred to soil
and raised to maturity. Almost 100% of the rooted
plantlets survive after transplantation to soil.
bulbous structures each of them bearing 2-10 tiny
meristems on their surface (fig. 5). On the contrary,
in the cultivars exhibiting low proliferation rate,
the shoot multiplication occurs by the adventitious
regeneration of small leafy shoots from the originally
explanted shoot meristem-tip (fig. 6). It is also
noticed during the present investigation that the num-
ber of subculturing seems to have an influence on the
proliferation rate. Most of the cultivars revealed
highest proliferation between the 3rd and 6th subcul-
ture (table I). For in vitro storage of germplasm, the
method adopted must maintain a satisfactory level of
genetic stability as well as guaranteeing survival.
Shoot-tip culture thus provides a valid alternative
means of storage of Mu~a germplasm; multiplication is
based on the adventitious as well as non adventitious
production of shoot buds. Furthermore, experience with
a number of horticultural species indicates that the
meristems produced in vitro retain the genetic sta-
bility which is characteristic of in uiuo meristems
(Henshaw et al., 1980).
IN VITRO STORAGE AT REDUCED TEMPERATURE
The principal objective of the minimal growth pro-
cedure is to extend the subculturing interval from the
normal 4-6 weeks to a much longer period (e.g. once in

Table l: Proliferation rates of the Masa cultivars studied in vi~o in different subcultures on modified MS
medium with IAA O.18 mg/l and BA 2.3 mg/l.

cultivar/
genome number of shoot-tips/explant

]st subcult 2nd subcult 3rd subcult 4th subcult 5th subcult 6th subcult 7th subcult

mean range mean range mean range mean range mean range mean range mean range

Dwarf
eavendish
(AAA) 7.4 2-I] 7.5 4-10 8.5 4-15 9.4 4-20 12.3 5-20 16.8 5-22 8.5 2-12
Robusta
(AAA) 7.0 2-10 8.5 4-]4 7.8 5-I] 8.0 6-]2 10.7 5-20 9.4 4-15 7.3 3-14
Silk
(AAB) 11.2 6-17 15.0 8-25 13.1 8-22 16.0 11-20 I9.1 12-29 16.7 12-27 11.3 6-27
Prata
(AAB) 3.5 2- 6 6.3 2-11 6.0 2-10 11.7 9-16 8.0 4-11 6.0 2- 9 7.2 3-10
Asamiensa
(AAB) 17.6 8-34 21.6 10-33 23.2 6-40 24.4 ]3-37 30.3 21-41 39.7 11-73 27.8 14-45
Ntanga
(AAB) 24.5 5-39 26.3 4-45 28.3 8-45 26.3 ]9-36 37.3 16-52 21.8 12-43 19.9 ]0-39
Agbagba
(AAB) 16.8 6-35 16.9 8-29 22.6 14-52 19.0 7-47 23.0 8-39 17.6 9-25 19.3 7-23
Bluggoe
(ABB) 8.4 3-25 16.2 8-26 15.0 6-21 20.4 4-49 42.2 27-62 45.9 13-72 39.2 16-76

At higher concentration of BA (2.3 mg/l) in all the
tested cultivars multiplication of shoot-buds is
noticed with the concominant suppression of the shoot
elongation. The morphogenetic responses of the shoot
meristem tips as influenced by genotype and the number
of subcultures are presented in table 1. Different
genotypic combinations exhibit a great variation in
the degree of shoot bud proliferation. In general, a
clear difference in the proliferation rate is observed
between the AAA triploid cultivars and the ones with
one or two B genomes. Thus Asam£e~a, Ntanga, Agbagba
and Bluggoe show fairly high bud proliferation com-
pared to the performance of ~wa~f caven~h, Rob~ta,
Silk and Pro~a. Moreover, in the formerly mentioned
group of cultivars the shoot proliferation is mani-
a year). To date there are several possible approaches
to achieve this goal among which manipulation of the
culture medium and incubation at reduced temperature
and low light intensity are the most accepted as well
as effective ones.
In our laboratory, starting with multiple shoot-bud
cultures (meristem-tip culture) of different cultivars
of Mgt6a, minimal growth storage has been attempted at
various reduced temperatures. Table 2 shows the
results of an experiment where various low tempera-
tures have been tested. Most of the cultivars, cul-
tured on MS semisolid medium supplemented with IAA
(O.18 mg/l) and BA (2.30 mg/l) when incubated at 15°C
remain quite healthy and consequently can be stored up
to 13 to 17 months with relative high survival percen-
 353

tage depending on the genomic configuration of the Table 2: Preliminary results of storage at different
cultivars (table 3). The evaporation of the culture low temperatures of the meristems of eight
medium can be considerably reduced by sealing the cultivars (cvs) of Mu~a.
culture tubes with parafilm, which apparently does
not seem to have any harmful effect. The cultivars duration of incubation
incubation
tested in the present investigation seem to vary in
temperature
their ability to withstand minimal growth temperature. 1.5 months 3 months 12-15 months
Thus the AAB plantains (Asamiensa, Agbagba and Ntanga)
and Bluggoe (ABB genome) are relatively more tolerant most cvs
to reduced temperature than Dwarf eavendish and 5°C turn brown
Pisang nangka (AAA genome) (table 3, fig. 7-8). and die
During the last few years several research papers
have been published on the progress of in vitro sto- most cvs most cvs
rage of temperate plant species using different sto- IO°C stay turn brown
rage techniques. Incubation at reduced temperature healthy and die
has been successfully applied to medium/long-term
storage of various plant species, such as, grapes (12 all cvs all cvs all cvs stay
months - Galzy,1969), strawberry (6 years - Mullin 15°C stay stay healthy with
and Schlegel,1976), apple (12 months - Lundergan and healthy healthy some browning

Table 3: Survival percentages ){ of different cultivars at 15°C and 1000 lux light.

period of Dwarf Pisang Silk Asamiensa Agbagha Ntanga Bluggoe

incubation cavendish nangka
at 15°C (AAA) (AAA) (AAB) (AAB) (AAB) (AAB) (ABB)

Months survival % survival % survival % survival % survival % survival % survival %

I 100 100 ]00 |00 100 100 100

3 100 100 100 100 100 100 ]00
5 100 100 83.3 100 I00 100 100
7 100 83.3 83.3 100 100 91.6 100
9 66.6 66.6 83.3 9].6 83.3 91.6 100
11 50 66.6 50 91.6 83.3 91.6 ]00
13 50 50 25 83.3 66.6 83.3 ]00
15 25 33.4 25 66.6 50 83.3 91.6
17 0 -- 0 43.4 50 50 91.6

The survival percentages were calculated on the basis of 12 tubes containing cold stored explants of different
cultivars which were withdrawn every two months from the cold room and incubated in normal culture conditions
for testing their ability to survive.

Janick, 1979), potato (Westcott,1981), Lolium muf~-

florum ( D a l e , 1 9 8 0 ) , Me~icago sativa, Trifolium repen~
and T. pra~ens (Cheyne and Dale,]980; Bhojwani,1981).
Although reduced temperatures in the range 5-I0°C
have been found suitable for in vf2Ao storage of
meristem cultures of several temperate species, me-
ristem cultures of more tropical species like sweet
potato (Ipomoea b~a~a~) and related Ipomoea spp.
show considerable chilling damage at these tempera-
tures (Alan,]979), but satisfactory reduction in the
growth rate is achieved at ]5-18°C. These results
are in agreement with our findings where meristem
cultures of different genotypes of ~usa suffer from mmmmmmiR
chilling damage during incubation at 5-10°C. However,
this low temperature sensitivity is not necessarily
typical of all the tropical species, as meristem
culture of Colocasia esculenta (Staristsky et ai.~1985) iiiili
i!iiiiii!i
and Xanthosoma s a g i ~ f o l i u m (Acheampong, 1982) could
be stored at 9-I0°C for 3 years and I year respecti-
vely. Figure 1~3 Different stages of plant r e g e n e r a t i o n
through in v/tro meristem culture in Musa { b~nana
and pla~ta/n ). 1. Excised shoot apex after I week
of culture. 2. Regeneration of single shoot from
excised shoot apex of'Dwarf cavendish'. 3. Rooting of
regenerated shoots.
354

Figure 4. Further development of the rooted plantlets before transplantation to soil. Figure 5. Proliferation of
shoot buds in 'Bluggoe' at IAA 0.18 mg/l and BA 2.3 mg/l. Figure 6. Proliferation of leafy shoots in 'Dwarf
cavendish' at IAA 0.[8 mg/l and BA 2.3 mg/l.
Figures 7-8. Shoot tip cultures of 'Dwarf cavendish' and 'Bluggoe' after ] year of storage at 15°C and 1000 lux
light intensity. 7. Dwarf cavendish. 8. Bluggoe.

Acknowledgement De Langhe E (1960) Bulletin d'information de I'INEAC

The financial support from International Board of
Plant Genetic Resources (IBPGR) which enabled the
first author to carryout this investigation is grate-
fully acknowledged. Thanks are also due to Dr. F.
Dumortier and Ir. J. Schoofs for their valuable
suggestions during the preparation of the manuscript.
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