Plant Cell, Tissue and Organ Culture (2005) 82: 57–66
Springer 2005
Optimization of plantain (Musa AAB) micropropagation by temporary
immersion system
S. Roels1,*, M. Escalona3, I. Cejas3, C. Noceda2, R. Rodriguez2, M.J. Canal2,
J. Sandoval4 & P. Debergh1
1
Department of Plant Production, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653,
9000 Gent, Belgium; 2 Departamento de Biologı´a Organismos y Sistemas, University of Oviedo, Catedrático
Rodrigo Urı´a s/n, 33071 Oviedo, Spain; 3 Laboratory for Plant Cell and Tissue Culture, University of Ciego de
Avila, Carretera Ciego - Moron km 9, 69450 Ciego de Avila, Cuba; 4 Corporacion Bananero Nacional, P.O.
Box 390, 7210 Guapiles, Costa Rica (*requests for offprints; Fax: +32-9-264-6225; E-mail: Sophie.Roels
@UGent.be)
Received 11 June 2004; accepted in revised form 24 November 2004
Key words: in vitro culture, meta-topolin, multiplication rate, Musaceae, temporary immersion
Abstract
The positive and reliable effect of temporary immersion systems on in vitro shoot proliferation was already
proved for different plant genera and it is now presented as an alternative for plantain micropropagation.
Some culture parameters affecting the efficiency of the twin flasks system or temporary immersion bioreactor (Escalona et al., 1999) were investigated. Three different cytokinins (benzyladenine, thidiazuron and
meta-topolin) were added to the culture medium and meta-topolin at a concentration of 4.4 lM was proved
to be the most efficient. Successive subcultures (28 days per subculture) were performed on medium supplemented with meta-topolin, revealing a decrease in multiplication after the 6th subculture. Multiplication
rate was not changed within the ranges of immersion times (4, 12 or 22 min) and frequencies (every 3, 5 or
7 h) tested. The size of the bioreactor (250, 1,000, 5,000 or 10,000 ml) and the volume of medium per
inoculum (10, 20 or 30 ml) were also evaluated and appeared to have an influence on the multiplication. A
proportion of 25–100 ml of headspace per inoculum and 30 ml of medium per inoculum resulted in a
multiplication rate >13 in 28 days.
Abbreviations: BA – N6-benzyladenine; MET – meta-topolin; SP medium – standard proliferation medium; TDZ – thidiazuron; TIB – temporary immersion bioreactor
Introduction
Bananas and plantains (Musa spp.) are among the
most important food crops in the world, with a
production approximating 102 million tons per
year (FAO, 2002); one third is plantain. However,
expansion of plantain production is limited by
shortage of plant material. The transmission of
harmful insects, nematodes, viruses and black
Sigatoka disease by field-grown suckers has
prompted interest in the use of aseptic culture
techniques (in vitro).
High production costs generally limit the
commercial use of in vitro micropropagation.
Using liquid media is considered to be the ideal
solution for automation and reducing production
costs. However, the use of liquid media can be
responsible for other problems such as asphyxia,
hyperhydricity and the need for more complex
equipment (Etienne and Berthouly, 2002). Several
58
methods have been proposed to avoid these
problems, one being the twin flasks system or
temporary immersion bioreactor (TIB), which
allows temporary immersion of the explants (Escalona et al., 1999). Alvard et al. (1993) made a
comparison between five different liquid medium
culture methods and gelled culture medium for
multiplication of the banana ‘Grande Naine’. The
highest multiplication rate (>5 in 20 days) was
observed for explants subjected to temporary
immersion, and the highest accumulation of dry
matter was obtained in aerated liquid medium and
temporary immersion.
In plantain (AAB group) the growth of axillary
buds in vivo is inhibited by a high degree of apical
dominance. Conversely, many well-developed
suckers are often observed on banana (AAA
group). Ortiz and Vuylsteke (1994) have shown
that plantain-banana hybrids manifest a better
sucker development than the plantain parent.
They suggested that apical dominance in AAB
plantains is controlled by a major recessive gene
(ad), while the dominant allele Ad is probably fixed
in bananas. The Ad gene controls the production
of GA3, which is antagonistic to the inhibition
factor (apical dominance). Presence of the dominant allele resulted in improved suckering behavior of the hybrids as compared with their plantain
parents.
The same phenomenon is observed in vitro.
According to Israeli et al. (1995), proliferation and
multiplication in vitro depend, besides other factors, also on genotype. The micropropagation rate
in the AAA Cavendish sub-group (banana) is 3- to
5-fold for every multiplication cycle, and several
authors mention that the presence of a ‘B’ in the
genome affects multiplication adversely (Wong,
1986; Bhagyalakshmi and Narendra, 1995;
Hirimburegama and Gamage, 1997; Arinaitwe
et al., 2000).
The aim of this study was the optimization of
plantain micropropagation by use of TIB. Several
authors mention shoot proliferation of plantain
using traditional micropropagation on semi-solid
medium. Results on micropropagation rates vary a
lot among cultivars and high concentrations of
cytokinins are often used (Jarret et al., 1985;
Wong, 1986; Bhagyalakshmi and Narendra, 1995;
Hirimburegama and Gamage, 1997; Arinaitwe
et al., 2000). Bhagyalakshmi and Narendra (1995)
also mention the beneficial effect of liquid medium
above agar gelled medium on shoot multiplication
of plantain. No information is available on shoot
proliferation of plantain in temporary immersion
systems. In this work the possibility of micropropagation of plantain in TIB was investigated,
comparing it with traditional micropropagation
on semi-solid medium. In the TIB, the optimal
type and concentration of cytokinin added to the
medium was determined, and used for successive
subculturing of plantain in TIB. Also some other
parameters, inherent to the operation of the TIB
were tested: immersion frequency and time, size of
bioreactor and volume of medium per inoculum.
Methods and materials
Plant material
Establishment phase (21 days)
Sucker shoot tips of plantain CEMSA 3/4 (AAB),
previously tested against Banana Streak Virus
(BSV), were established in vitro on semi-solid
standard proliferation (SP) medium (Table 1),
according to the protocol of INIVIT (National
Institute for Tropical Crops, Agricultural Ministry, Cuba; unpublished information). One shoot
tip was inoculated per 60 ml glass test tube
(diameter 24 mm, length 150 mm, closed with
kap-uts), filled with 20 ml of medium.
Proliferation phase (28 days per subculture)
After the establishment phase, the developing
shoots were subcultured at least three times on the
same medium in 375 ml glass vessels (Meli-jars,
De Proft et al., 1985) before use as starting material
for the experiments. The exact number of preceding
subcultures on semi-solid SP medium is mentioned
for each experiment and depended on the availability of plant material. Because of the slow multiplication ratio of plantain, it was sometimes
necessary to have several subcultures in order to
have enough plant material to start an experiment.
The starting material for the TIB-experiments
consisted of isolated single shoots from which
emerging leaves and roots had been removed; and
divided longitudinally before inoculation in order
to break apical dominance. Only shoots with a
diameter (at leaf base) of at least 3 mm were used,
since smaller shoots could not be divided and
failed to propagate well. Such half shoots, which
59
Table 1. Composition and use of standard proliferation and basic medium as liquid (in TIB) or semi-solid medium (in glass vessels)
Name
Type
Composition
Used for
Standard proliferation medium
Liquid
Experiment 1
Standard proliferation medium
Semi-solid
Basic medium
Liquid
MS salts and vitamins (Murashige and
Skoog, 1962)
3% sucrose
13.3 lM BA
MS salts and vitamins
3% sucrose
13.3 lM BA
2.5 g l)1 Gelrite (Duchefa, The Netherlands)
MS salts and vitamins
3% sucrose
Basic medium
Semi-solid
MS salts and vitamins
3% sucrose
2.5 g l)1 Gelrite
were used for inoculation of the TIB, are hereafter
referred to as ‘inocula’. One multiplication cycle
took 28 days.
Establishment in vitro and
subculturing before start
of experiments
Control of experiment 1
Experiments 2, 3 and 4
Elongation phase after
experiment 3
Control of experiment 3
Control of elongation phase
after experiment 3
De Proft et al., 1985). Ten inocula were put in each
type of container.
Greenhouse conditions
Elongation phase (21 days)
Shoots smaller than 3 mm and buds were used for
transfer to the greenhouse, and therefore the
multiplication cycle was followed by an elongation
phase in TIB using basic medium (Table 1).
Media and culture conditions (unless otherwise
stated in experiments) in vitro
Table 1 gives an overview of the composition and
use of the SP medium and basic medium, both for
liquid (TIB) and semi-solid medium.
The pH was always adjusted to 5.7–5.9 before
autoclaving at 112 C, for 30 min. Ten millilitre of
medium were used per inoculum, both in TIB and
semi-solid medium. Cultures were incubated at
25 ± 2 C under cool white fluorescent tubes
(OSRAM 31, 36 W) providing a photosynthetic
active radiation of 40 lmol m)2 s)1 with a photoperiod of 16 h. The concept and operation of the
TIB used in our experiments was based on the
system described by Escalona et al. (1999), using
250 ml Nalgene containers (Nalgene Cat. No.
DS2127-0250). Based on preliminary experiments
(data not presented) inocula were immersed for
4 min every 3 h. For experiments on semi-solid
medium, 375 ml glass vessels were used (Meli-jars,
Shoots obtained after the elongation phase in vitro
were transferred to greenhouse conditions for
acclimatization (60 days). Shoots were planted in
plastic trays, under protective conditions of relative humidity (80%), temperature (28 C) and low
light intensity.
Experiments
We evaluated the efficacy on plantain proliferation
of the following parameters used during the proliferation phase:
(1) Comparison between TIB and semi-solid
medium on SP medium;
(2) Different cytokinins [benzyladenine (BA),
meta-topolin (MET) and thidiazuron (TDZ)] at
different concentrations (0, 1.3, 2.2, 4.4, 13.3
and 22.2 lM);
(3) The effect of seven subsequent subcultures in
TIB;
(4) Other parameters to optimize the culture
conditions in TIB were also tested: immersion
frequency (every 3, 5 or 7 h) and time (4, 12 or
22 min), size of the TIB-container (250, 1,000,
5,000 or 10,000 ml) and volume of medium per
inoculum (10, 20 or 30 ml).
60
Measurements and analysis
At the end of a 28-day proliferation phase, all
shoots (with at least one developed leaf) and buds
(without leaf) were counted and the multiplication
rate [number of shoots and buds at the end of a
subculture/number of shoots inoculated] was calculated. Also morphological characteristics [shoot
height (cm), shoot diameter at leaf base (mm),
number of leaves and roots per shoot, fresh weight
(g)] were evaluated. Each experiment was set up in
three or four repetitions (bioreactors). Morphological characteristics were measured on 30 shoots
(not on buds).
An analysis of variance (ANOVA) was conducted on the data concerning multiplication rate
and shoot morphological parameters using the
statistical program SPSS (SPSS Inc., USA). When
significant differences occurred, means were separated by the Duncan ( p < 0.05) multiple range
test.
number of leaves and roots, whereas shoot height
was significantly higher on semi-solid medium
(5.3 cm) than in TIB (4.5 cm) and shoot diameter
was significantly higher in TIB (5.1 mm) than on
semi-solid medium (3.7 mm). The larger shoot
diameter observed in TIB is an important factor in
the success of multiplication in this culture system
(more detailed in experiment ‘effect of subsequent
subcultures’).
It can be concluded that culturing plantain in
TIB strongly promotes multiplication without
affecting considerably morphological characteristics. According to Etienne and Berthouly (2002),
temporary immersion generally improves plant
material quality, besides the gain in production.
An increased shoot vigor, reduced hyperhydricity
and better performance during acclimatization are
some of the advantages of temporary immersion.
Temporary immersion combines the advantages of
semi-solid culture media (gas exchanges) and
liquid media (increased nutrient uptake).
Cytokinin effects on multiplication rate
Results and discussion
Comparison between TIB and semi-solid medium
Figure 1 shows that using liquid SP medium under
TIB-conditions, results in a significantly higher
multiplication rate compared to semi-solid medium. No statistical differences were found for
In the following experiment the basic medium was
supplemented with different cytokinins (BA, MET
or TDZ) at different concentrations (0, 1.3, 2.2,
4.4, 13.3 and 22.2 lM) and the multiplication rate
was evaluated in TIB.
Statistical analysis revealed a significant effect
on the multiplication rate of both type and con-
a
a
5
b
TIB
Semi-solid
4
3
a
b
b
2
1
Multiplication Shoot
rate
height
(cm)
Number of Number of
roots
leaves
Shoot
diameter
(mm)
Figure 1. Multiplication rate and morphological parameters of shoots cultured in TIB or semi-solid using SP medium (preceded by six
subcultures on semi-solid SP medium). One factorial (factor ¼ type of system) analysis of variance ( p < 0.05). Post Hoc Test Duncan:
a, b: significantly different ( p < 0.05).
61
Table 2. Multiplication rate in TIB using basic medium
supplemented with different concentrations and types of
cytokinins (preceded by three or four subcultures on semi-solid
SP medium)
Multiplication rate
Type of cytokinin
Concentration of
cytokinin (lM)
Type of cytokinin
Concentration of cytokinin
Type · concentration
BA
MET
TDZ
0
6.0
8.1
7.5
5.0
b
a
ab
c
1.3
2.2
4.4
13.3
22.2
*
*
ns
6.4 bc
7.2 bc
10.7 a
7.9 b
5.9 bc
Two factorial (factors = type and concentration) analysis of
variance: ns, *: not significant, significant at p < 0.05.
Post Hoc Test Duncan: a, b, c: significantly different
( p < 0.05).
centration of the cytokinin (Table 2). A concentration of 4.4 lM resulted in a significantly higher
multiplication rate (10.7) than all other concentrations. MET resulted in a significantly higher
multiplication rate (8.1) than BA (6.0), but was not
statistically different from TDZ (7.5). The positive
effect of MET compared to BA on in vitro shoot
formation was already reported for some species
(Werbrouck et al., 1996; Wojtania and Gabryszewska, 2001) but not for Musa.
When TDZ was used, a lot of morphologically
aberrant, adventitious buds were formed (Figure 2). These buds look like white fleshy bulbous
structures surrounded by few pale green leaves.
Since we wanted to avoid as much as possible
somaclonal variation, it was decided not to use
TDZ for further multiplication in TIB. Therefore
4.4 lM MET was chosen for the subsequent
experiments.
Effect of subsequent subcultures
In the former experiments, only one cycle was
performed in TIB, preceded by three or more
subcultures on semi-solid SP medium. In the next
experiment, we tested the effect of a number of
successive subcultures of plantain in TIB compared to semi-solid medium, in both cases preceded by three subcultures on semi-solid SP
medium.
Statistical differences in total multiplication
rate were found between TIB and semi-solid
medium in subcultures 4, 5 and 6 (Figure 3, statistical data not shown). During these first subcultures, the multiplication rate was significantly
higher in TIB than on semi-solid medium. In the
subsequent subcultures no more significant differences were observed.
The effect of the number of subcultures was
also evaluated. For semi-solid medium there was
no significant effect of the number of subculture on
multiplication rate, whereas for TIB statistical
differences were found. The multiplication rate in
TIB in subcultures 5 and 6 was significantly higher
than in subcultures 8, 9 and 10. Subcultures 4 and
7 were intermediate between these two groups. A
similar result was obtained by Mendes et al. (1999)
with a decrease of the multiplication rate of Musa
cv. Maçã (AAB) after the fourth subculture.
It must be mentioned that after each subculture, only shoots with a diameter at leaf base
higher than 3 mm (type 1) were used for inoculation in the next subculture (Figure 4) because
smaller shoots failed to propagate well (data not
presented). Shoots with a smaller diameter and
buds without leaves (Figure 4, type 2 and 3 resp.)
Figure 2. Morphological appearance of shoots in TIB using basic medium supplemented with (a) BA, (b) MET or (c) TDZ (4.4 lM)
(preceded by three or four subcultures on semi-solid SP medium).
Number of shoots per explant per category
62
TIB type 1
TIB type 2+3
9
8
Semi-solid type 1
Semi-solid type 2+3
7
6
5
4
3
2
1
4
5
6
7
8
9
10
Cycle
Figure 3. Distribution of shoots into different types (see Figure 4) after each cycle in TIB and on semi-solid medium using basic
medium supplemented with 4.4 lM MET (preceded by three subcultures on semi-solid SP medium). The total multiplication rate is
formed by the sum of type 1, 2 and 3 shoots per explant.
were used for transfer to greenhouse conditions
after passage through an elongation phase in vitro.
Figure 3 also shows the distribution of the different shoot types after each subculture.
For none of the subcultures statistical differences
in type 1 shoots were found between TIB and semisolid medium, whereas the number of type 2 + 3
shoots in TIB was significantly higher than on semisolid medium in the subcultures 4, 5 and 6. In subsequent subcultures, the number of type 2 + 3
shoots was comparable for TIB and semi-solid
medium. These results indicate that the higher multiplication rate of TIB in subcultures 4, 5 and 6
Figure 4. Shoot types in proliferation after each cycle: (a) type
1: shoot diameter >3 mm; (b) type 2: shoot diameter <3 mm;
and (c) type 3: bud without leaves.
compared to semi-solid medium is mainly due to the
formation of a higher number of type 2 + 3 shoots.
When the effect of the number of subcultures
was evaluated, it was seen that this factor had an
influence on the number of type 1 shoots, both in
TIB and on semi-solid medium. For both systems, the number of type 1 shoots was significantly higher in subcultures 4 and 5 than in
subcultures 7, 8, 9 and 10. Concerning type
2 + 3 shoots, there was only an effect in TIB.
The number of type 2 + 3 shoots in TIB in
subcultures 5 and 6 was significantly higher than
in the other subcultures, and this is reflected in
the total multiplication rate.
Figure 5 shows the cumulative number of shoots
and buds formed after each subculture starting from
one type 1 shoot (which was then divided longitudinally, resulting in 2 inocula) at the beginning of
the 4th subculture. After 10 subcultures 399 shoots
of type 2 + 3 were formed in TIB and 225 on semisolid medium, whereas 41 shoots of type 1 were
formed in TIB and 28 on semi-solid medium.
In fact the total multiplication consists of two
parts. On the one hand, there is the formation of
type 1 shoots, which is superior to one in the first
subcultures. This means that the number of bioreactors can be expanded during the first subcultures, as this type of shoots can be used for
63
TIB type 1
TIB type 2+3
Semi-solid type 1
Semi-solid type 2+3
Number of shoots (cumulative)
400
350
300
250
200
150
100
50
4
5
6
7
8
9
10
Cycle
Figure 5. Cumulative number of shoots and buds formed after each cycle in TIB using basic medium supplemented with 4.4 lM MET
starting from one type 1 shoot at the beginning of the 4th cycle (preceded by three subcultures on semi-solid SP medium).
time, size of the TIB-bioreactor and volume of
medium per inoculum. All experimental conditions
were the same as established before, unless otherwise mentioned.
A preliminary experiment showed a significant
decrease of the multiplication rate and of morphological characteristics when the cultures were
immersed every 1 h, this was not the case for 3 and
5 h. Therefore, a second experiment was set up in
order to compare immersion frequencies of every
3, 5 and 7 h (Table 3), as well as experiments to
compare different durations of immersion (Table 4), sizes of bioreactor (Table 5) and volume of
medium per inoculum (Table 6).
Although immersion frequency and time are
considered to be important parameters for the
efficiency of the TIB system, they did not have an
effect on the multiplication rate of plantain within
micropropagation. On the other hand, there is the
formation of type 2 + 3 shoots, used for transfer
to greenhouse conditions. Since the number of
type 1 shoots decreases in both systems after
subculture 5, but the number of subcultures only
affects total multiplication rate in TIB, it can be
concluded that the decrease in number of type 1
shoots is not the main cause for the decrease in
multiplication; the main cause is the decrease of
the number of type 2 + 3 shoots. It can be concluded that the decrease in type 2 + 3 shoots
corresponds to a reduction of the multiplication
potential of the type 1 shoots.
Other parameters
Other parameters related to the operation of the
TIB were also tested: immersion frequency and
Table 3. Multiplication rate and morphological characteristics of shoots treated with different immersion frequencies in TIB using
basic medium supplemented with 4.4 lM MET (preceded by three or four subcultures on semi-solid SP medium)
Immersion frequency
Multiplication
rate
Shoot height
(cm)
Number of
leaves
Number of
roots
Shoot diameter
(mm)
Fresh mass (g)
Every 3 h
Every 5 h
Every 7 h
10.3
10.1
9.1
3.6 b
3.7 b
4.3 a
3.5
3.6
3.6
1.1
1.8
1.8
5.0
5.0
5.3
0.57
0.63
0.70
One factorial (factor = immersion frequency) analysis of variance ( p < 0.05).
Post hoc test Duncan: a, b: significantly different ( p < 0.05).
64
Table 4. Multiplication rate and morphological characteristics of shoots treated with different immersion times in TIB using basic
medium supplemented with 4.4 lM MET (preceded by 11 subcultures on semi-solid SP medium)
Immersion time
(min)
Multiplication rate
Shoot height
(cm)
Number of leaves
Number of roots
Shoot diameter
(mm)
4
12
22
4.6
4.0
5.9
5.2 a
4.8 a
2.4 b
2.5 a
2.7 a
2.2 b
0.8
1.0
0.9
4.0
4.8
4.1
One factorial (factor = immersion time) analysis of variance ( p < 0.05).
Post hoc test Duncan: a, b: significantly different ( p < 0.05).
Table 5. Multiplication rate and morphological characteristics of shoots produced in different sized TIB using basic medium
supplemented with 4.4 lM MET (preceded by three or four subcultures on semi-solid SP medium)
Size of TIB
(ml)
Headspace per Multiplication Shoot height
inoculum (ml) rate
(cm)
Number of
leaves
Number of
roots
Shoot diameter Fresh mass
(mm)
(g)
250
1,000
5,000
10,000
25
100
500
1000
4.2
4.1
3.3
3.2
3.4
2.8
0.8
1.4
6.4
5.6
5.3
4.8
13.5 a
14.1 a
9.2 b
6.4 b
4.4
3.9
3.1
3.6
a
a
c
b
a
a
b
b
a
a
b
b
a
b
c
bc
1.19
1.15
0.52
0.56
a
a
b
b
One factorial (factor = size) analysis of variance ( p < 0.05).
Post hoc test Duncan: a, b, c: significantly different ( p < 0.05).
Table 6. Multiplication rate and morphological characteristics of shoots treated with different volumes of medium per inoculum in
TIB using basic medium supplemented with 4.4 lM MET (preceded by three or four subcultures on semi-solid SP medium)
Volume of medium per inoculum (ml)
Multiplication rate
Shoot height (cm)
Number of leaves
Number of roots
10
20
30
11.9 b
11.7 b
13.8 a
4.0 b
3.1 c
5.0 a
3.1 b
3.2 b
3.7 a
2.8 ab
2.2 b
3.2 a
One factorial (factor = medium volume) analysis of variance ( p < 0.05).
Post hoc test Duncan: a, b, c: significantly different ( p < 0.05).
the ranges that were tested (Table 3 and 4).
Immersion frequency only affected shoot height,
resulting in significantly larger shoots when immersed every 7 h. A longer duration of the
immersion (22 min) resulted in significantly smaller shoots and a lower number of leaves, whereas
no statistical differences were found between 4 and
12 min.
A significantly higher multiplication rate was
achieved when the volume of the container was
250 or 1000 ml, compared to 5000 and 10,000 ml.
Since the number of inocula was the same in each
container (10), this result gives an indication about
the proportion headspace/inoculum. A proportion
of 25–100 ml headspace per inoculum appeared to
be the best (Table 5). This result is important for
further upscaling, because it indicates that it is
recommended to use 100–400 inocula in the
10,000 ml-container in order to obtain an ideal
proportion headspace/inoculum.
It could be seen that the higher volume of
medium (30 ml per inoculum) resulted in a significantly higher multiplication rate for plantain
(Table 6). Shoot height and number of leaves were
significantly higher for 30 ml then for 10 or 20 ml.
The volume of liquid medium per inoculum is also
one of the important factors controlling the morphogenic response and shoot quality in TIB
systems. Lorenzo et al. (1998) determined that
50 ml of medium per inoculum was optimal for
65
Sacharum spp., while it was 200 ml for pineapple
(Escalona et al., 1999). For plantain, the highest
volume of medium (30 ml) that was tested resulted
in better multiplication. Further investigations are
necessary in order to determine the optimal volume of medium, which might be higher than
30 ml.
Rica (CORBANA) with plants produced in TIB or
on semi-solid medium using 4.4 lM MET or BA.
The effect of the system (TIB or semi-solid medium) and the cytokinin (BA or MET) on survival,
growth, flowering and harvest time, fruit production and somaclonal variation will be evaluated.
The efficiency of the TIB and the possibility of the
use of MET in plantain micropropagation need to
be evaluated in view of these results.
Conclusion
The positive effect of temporary immersion on in
vitro shoot proliferation was already proved for
banana (Alvard et al., 1993), pineapple (Escalona
et al., 1999), sugarcane (Lorenzo et al., 1998) and
calabash tree (Murch et al., 2004) and is now
demonstrated for plantain. MET at a concentration of 4.4 lM appeared to be the better choice
for optimization of the multiplication of plantain
in TIB. Immersion time and frequency did not
have an effect on the multiplication of plantain
within the ranges that were tested. However, since
these parameters are considered to be of great
importance in the efficiency of the TIB (Etienne
and Berthouly, 2002) an extension of these
experiments could result in further optimization
of the multiplication protocol. It was proved that
the size of the bioreactor and the volume of
medium per inoculum affected the multiplication
of plantain in TIB. A proportion of 25–100 ml of
headspace per inoculum and 30 ml of medium
per inoculum resulted in the best multiplication
rate, however further investigations still can
reveal a more optimal volume of medium per
inoculum.
Subsequent subcultures of plantain were performed in TIB and it was seen that in the first
subcultures the total multiplication rate in TIB
was higher than on semi-solid medium, but
decreased after some subcultures and became
equal to semi-solid medium. The total multiplication rate was divided into two parts: type 1
shoots used for further multiplication in vitro and
type 2 + 3 shoots used for transfer to greenhouse
conditions. The higher multiplication rate in TIB
was caused by a higher number type 2 + 3
shoots in TIB, which could not be reached on
semi-solid medium.
At this moment field experiments are going on
at the National Corporation of Bananas in Costa
Acknowledgement
This work was supported by funds from the
European Community (INCO project ICA4-CT2001-10063).
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