In Vitro Cell.Dev.Biol.

Plant (2010) 46:8994

DOI 10.1007/s11627-009-9246-2

PHYSIOLOGY

Effect of sucrose, light, and carbon dioxide on plantain

micropropagation in temporary immersion bioreactors
Carlos Eduardo Aragn & Maritza Escalona &
Roberto Rodriguez & Maria Jess Caal & Iris Capote &
Danilo Pina & Justo Gonzlez-Olmedo

Received: 10 May 2008 / Accepted: 20 August 2009 / Published online: 14 October 2009 / Editor: D. T. Tomes
# The Society for In Vitro Biology 2009

Abstract In vitro physiology and carbon metabolism can be

affected by the sinksource relationship. The effect of
different sucrose concentrations (10, 30, and 50 g L1), light
intensities (80 and 150 mol m2 s1), and CO2 levels (375
and 1,200 mol mol1) were tested during plantain micropropagation in temporary immersion bioreactors. Activities of
pyruvate kinase, phosphoenol pyruvate carboxylase, and the
photosynthesis rate were recorded. From the morphological
and practical point of view, the best results were obtained
when plants were cultured with 30 g L1 sucrose,
80 mol m2 s1 light intensity, and 1,200 mol mol1 CO2
concentration. This treatment improved leaf and root development, reduced respiration during in vitro culture, and
increased starch level at the end of the hardening phase. In
addition to that, the number of competent plants was
increased from 80.0% to 91.0% at the end of the in vitro
phase and the survival percentage from 95.71% to 99.80%
during ex vitro hardening.
Keywords Culture vessel headspace . Musa . Nutrition .
Physiology . TIB
C. E. Aragn (*) : M. Escalona : I. Capote : D. Pina :
J. Gonzlez-Olmedo
Laboratory for Plant Cell and Tissue Culture,
Bioplant Centre, Ciego de vila University,
Ciego de vila 69450, Cuba
e-mail: eduardo@bioplantas.cu
R. Rodriguez : M. J. Caal
Department of Biology and Organ of Systems,
University of Oviedo,
Oviedo, Spain
M. J. Caal
e-mail: mjcanal@uniovi.es

Introduction
Plantain (Musa) production is estimated to be a nutritional
source as a key food supplement for Caribbean and African
countries (Lusty et al. 2006). In addition, immunological
research has identified the plantain fruit as a good possible
green vaccine vector (Tripurani et al. 2003). Therefore, the
efficient propagation of plantain plants could be an
alternative to increase the number of selected genotypes.
CEMSA 3/4 AAB is a Cuban cultivar selected by the
improved agricultural yield and growth performance.
Temporary immersion bioreactors (TIB) guarantee a higher
yield of plants (Teisson and Alvard 1995; Etienne and
Berthouly 2002; Escalona et al. 2003; Shu-Han and Yeh
2008). The latter could be a consequence of the better
aeration provoked by the periodic immersion and the
renewal of the headspace, which results in reduced hyperhydricity (Roels et al. 2005; Ziv 2005).
The headspace can also be renewed by CO2 enrichment.
This procedure has shown to affect, in a positive way, some
indicators during conventional in vitro culture. Sha Vallikhan
et al. (2003) reported that a CO2 concentration over
1,450 mol mol1 increased the in vitro multiplication and
rooting rates in Paulownia fortunei. It was also observed a
positive effect of CO2 enrichment on the morphology of
leaves and in the stomata functional ability with the high
number of shoots, number of roots, and elliptical stomata
forms. Nguyen and Kozai (2001) measured photosynthetic
activities between 0.5 and 5.0 mol CO2 h1 per plant for
in vitro cultured banana plants. In addition, they reported
0.0 g L1 sucrose, 200 mol m2 s1 light intensity, and
1,340 mol mol1 CO2 as the best combination of concentrations to increase the photosynthetic rate.

Standard error
Significance

Values indicated by different letters are significantly different at the 5% level by Tukeys multiple range test (n=30)

0.25 a
0.13 b
0.01
*
7.09 a
0.41 c
0.30
*

b
a

6.17 a
2.67 b
0.37
*
10.50 a
2.40 c
0.51
*

b
a

3.68 a
0.95 b
0.19
*
0.92 a
0.30 c
0.04
*

b
b

7.40 a
0.03 c
0.29
*

10

30
50

Environmental Conditions (LL)

Standard error
Significance
Ex vitro

17.25 a
4.58 b
0.87
*

a
a
b

0.14
0.16
0.10
0.01
*
0.30
a
b
c

4.47
1.30
0.81
0.21
*
5.13
a
b
c

5.50
4.67
4.00
0.38
*
6.67
a
b
b

8.17
3.92
3.42
0.46
*
8.95
a
b
b

2.13
1.27
1.22
0.13
*
3.85
a
b
b

0.72
0.50
0.40
0.04
*
0.75
15.33 a
7.25 b
7.67 b
0.92
*
14.48 a
5.26
4.38
0.23
0.21
*
1.07
10
30
50
Environmental Conditions (LL)

a
b
c

Leaf
number
Length of
main leaf
(cm)
Width of
main leaf
(cm)
Stem
thickness
(cm)
Plant height
(cm)

In vitro

Growing conditions during hardening. Plants coming from

TIB and presenting 2.5 cm height and 0.3 cm width and at least
two leaves were defined as competent to face the hardening
phase. Competent plants were transplanted to a sterilized
substrate composed of sugarcane ashes and red soil (1:1, w/w)
distributed in multi-tray containers with 144 holes (52.5 cm

Photosynthetic rate
(mol CO2m2s1)

Plantain shoots (Musa AAB, CEMSA 3/4) were initiated and

propagated in vitro, using the TIB procedure according to
Roels et al. (2005). After conventional propagation, five
shoots (3.0 cm diameter and 3.0 cm length) were transferred
to a 250-mL TIB containing 50 mL culture medium
consisting of MurashigeSkoog basal medium (Murashige
and Skoog 1962) supplemented with 30 g L1 sucrose and
100 mg L1 myo-inositol. The pH was adjusted to 5.8 before
autoclaving at 121C and 118 kPa for 20 min. Cultures were
maintained at 25C under a 16:8 h (light/dark) photoperiod
with a photosynthetic photon flux (PAR) of 80 mol m2 s1
(cool-white fluorescent lamps: Sylvania, Daylight F40T12/D
40 W). Immersions were performed for 4 min every 3 h.
Two different experiments were performed using plantain
plants cultured in TIB. First, different concentrations of
sucrose (10, 30, or 50 g L1) were added to the media culture
to evaluate their effect on the plant physiology and
morphology quality during the elongation phase in TIB.
The best result obtained in this experiment was taken into
consideration to establish the second one. The second
experiment combined different light intensities (80 and
150 mol m2 s1) with different CO2 concentration (375
and 1,200 mol mol1, 98.80% purity) in the headspace of
the culture vessel. The light was provided by different
numbers of fluorescent white lamps (Sylvania, Daylight
F40T12/D 40 W) as light sources, and the intensity was
expressed in PAR. The CO2 enrichment started just 4 h after
the light period as follows: during the first wk of plant
elongation, 6 h; during the second wk, 15 h; and during the
third wk, 24 h per d. The flow was adjusted to
200 mL min1. CO2 was injected to the semi-closed system
formed by the TIB connection described by Escalona et al.
(1999). Samples were collected from the first fully expanded
leaves (main leaf) at days 0 and 21 during in vitro elongation
phase and during the ex vitro hardening.

Sucrose
(gL1)

Materials and Methods

Table 1. Photosynthetic rate and morphological parameters for plantain plants under different mixotrophic treatments at the end of elongation and hardening phases

On the other hand, light affects the primary carbon

metabolism. Light-induced activation of phosphoenol pyruvate carboxylase (PEPC) was described by Dary et al.
(2001). The actual paper describes the effects of sucrose,
light intensity, and CO2 concentration during plantain
culture in TIB and ex vitro hardening. To our knowledge,
this information has not been published.

Culture fresh
weight per
plant (g)

Culture dry
weight per
plant (g)

ARAGN ET AL.

Treatments

90

0.96
*

Values indicated by different letters are significantly different at 5%

level by StudentNewmanKeuls multiple range test (n=9)

0.03
*
0.31
*
0.35
ns
1.05
*
0.26
*
0.70
*
0.24
*
0.04
*
1.29
ns

0.39 b
0.45 ab
0.48 a
3.49 b
5.26 a
3.98 b
4.69
4.78
4.40
12.39 b
15.05 a
12.33 b
4.33 a
4.21 ab
3.68 b
7.92 b
9.59 a
8.62 b
2.63 b
3.51 a
3.15 b
0.51 b
0.60 ab
0.54 b
14.85
17.49
16.22

0.31 c
2.70 b
4.00
11.44 b
2.27 c
8.22 b
2.55 b
0.66 a
15.64
1.56 ab

Culture dry
weight per
plant (g)
Culture fresh
weight per
plant (g)
Root
number
Length
of roots
(cm)
Leaf
number
Length of
main leaf
(cm)
Width of
main leaf
(cm)
Stem
thickness
(cm)
Plant
height
(cm)

91

Values indicated by different letters are significantly different at the 5% level by Tukeys Multiple Range test (n=30)

0.94
*

c
b
a
b

0.11
*

73.03
94.44
99.80
95.71

b
a
a
b

0.74
*

83.0
90.0
91.0
80.0

1.82 a
1.08 c
1.33 b

High Light High CO2 (HH)

High Light Low CO2 (HL)
Low Light High CO2 (LH)
Low Light Low CO2 (LL)
(Environmental Conditions)
Standard error
Significance

8.93 b
8.40 b
4.96 c

Survival at
hardening
phase (%)

High light low CO2 (HL)

Low light high CO2 (LH)
Low light low CO2 (LL)
(environmental conditions)
Standard error
Significance

Competent
plants (%)

13.60 a

Treatments

High light high CO2 (HH)

Table 2. Percentages of competent plantain plants and the survival at

the end of the hardening phase

Transpiration
(mmol H2Om2s1)

Enzyme extractions and assays. Leaf material (250 mg)

collected from in vitro and ex vitro conditions was placed
immediately in liquid nitrogen and ground with a mortar
and pestle. Enzymes were extracted following the method
described by Geigenberger and Stitt (1991), and the extract
was filtered through cheesecloth (Miracloth) and centrifuged at 15,000g and 4C for 20 min. Protein was
determined according to Bradford (1976) using a commercial kit (Bio-Rad, Hercules, CA). The extracted enzymes,
PEPC (EC 4.1.1.31) and pyruvate kinase (PK; EC
2.7.1.40), were prepared by suspending the powdered plant

Photosynthesis
(mol CO2m2s1)

Physiological parameters. Photosynthetic (micromole CO2

per square meter per second) and transpiration rates
(millimole H2O per square meter per second) were measured
in the first fully expanded leaves. They were sampled 3 h
after the beginning of the photoperiod (1000 h). We used a
Portable CIRAS-2 Photosynthesis System (Europe, PP
Systems, UK). The whole area of the cuvette (PLC6,
2.5 cm2) was covered with the plantain leaf. The carbon
dioxide concentration and the relative humidity of the air
entering the cuvette were 375 mol mol1 and 80%,
respectively, under environmental temperature (2527C).
Before obtaining the experimental data, we measured the
maximum light intensity at which photosynthesis was stable.
This was reached at 600 mol m2 s1. The measurements
were performed on leaves from five plants, with ten repetitions per plant.

Treatments

length, 29.5 cm width, and 4.0 cm height). Then, they were

transferred to a greenhouse (2527C; 375 mol mol1 CO2,
2,000 mol m-2 s1 as a maximal light intensity). An
automatic mist system allowed the relative humidity transition
from 90% to 80% during hardening phase (21 d): 60 s
watering every 30 min (1 wk), 30 s watering every 30 min
(1 wk), and 15 s watering every 30 min (1 wk).

Table 3. Photosynthetic rate, transpiration rate, and morphological parameters determined in plantain plants under different mixotrophic treatments at the end of elongation and hardening phases

EFFECT OF SUCROSE, LIGHT, AND CO2 ON PLANTAIN MICROPROPAGATION

92

ARAGN ET AL.

Table 4. Enzymatic activities (PK and PEPC) and proteins concentration determined in plantain plants under different mixotrophic treatments at
the end of elongation phase in TIB
Treatments

PK (Ug FW1)

PEPC (Ug FW1)

Proteins concentration (mgg FW1)

High light high CO2 (HH)

High light low CO2 (HL)
Low light high CO2 (LH)
Low light low CO2 (LL) (environmental conditions)
Standard error
Significance

69.14
71.61
33.95
48.15
4.22
*

16.97
13.89
10.80
10.64
1.10
*

39.73
48.52
57.45
78.96
4.74
*

a
a
c
b

a
b
c
c

c
bc
b
a

One unit corresponds to 1 mol of substrate transformed per h. Values indicated by different letters are significantly different at the 5% level by
StudentNewmanKeuls multiple range test (n=9)

material in 1 mL of 50 mmol L1 HEPESKOH buffer at

pH 7.4 according to Siegel and Stitt (1990). The catalyzed
PEPC reaction was coupled with the L-malate dehydrogenase (EC 1.1.1.37) (SIGMA) reaction and assayed at 25C
by monitoring NADH utilization at 340 nm (Le et al. 1991)
using a Pharmacia Spectrophotometer. The PK reaction was
coupled with the L-lactate dehydrogenase (EC 1.1.1.27)
(Sigma) reaction and assayed at 25C by monitoring
NADH utilization at 340 nm (Lin et al. 1989).
Starch determination. Leaf and stem material (250 mg)
collected from in vitro and ex vitro conditions were placed
immediately in liquid nitrogen and ground with a mortar and
pestle. The extract was centrifuged at 10,000g and 4C for
20 min. The pellet was recovered on KOH 0.2 mol L1.
Later, an enzymatic treatment was applied for total starch
degradation with amyl-glycosidase enzyme (EC 3.2.1.3;
Sigma; Thomas et al. 1983). The quantification was referred
to as potato starch control.
Statistical analysis. All statistical analyses were carried out
using SPSS software (version 11.0). The results were

Plants treated with 30 g L1 sucrose showed the highest

quality when measured at the end of the hardening phase
while for in vitro culture in TIB was 10 g L1 (Table 1). In
addition, this treatment (30 g L1) in combination with
80 mol m2 s1 light intensity and 1,200 mol mol1 CO2
concentration (LH), improved the percentages of competent plants (from 80.0% to 91.0%) and survival (from
95.71% to 99.80%) at the end of ex vitro hardening
(Table 2). The best photosynthetic rate was observed in
plants coming from the autotrophy treatment (HH). LHtreated plants showed a favorable photosynthetic rate and a
morphological development presenting the greater foliar
expansion (width and length), root growth (length and
number), and values of fresh and dry weight at the end of

150

100
b

c
BE

b
b

200

Starch Concentration
(mg,g FW-1)

Starch Concentration
(mg,g FW-1)

Results and Discussion

200

50

analyzed using one-way ANOVA followed by Tukey test

at 5% level. Nonparametric tests KruskalWallis, Mann
Whitney, and C-Dunnett at 5% level were performed for the
analysis of enzymatic activities and starch determination.

a
150

100

c
c

50

14

21

t (days)
Leaf

d
0

c
cd

e
BE

14

21

t (days)
Stem

Figure 1. Starch concentration for the treatment of low light

intensitylow carbon dioxide concentration (LL; environmental conditions) (A) and low light intensity-high carbon dioxide concentration
(LH) (B). Measures at the beginning of elongation phase (BE) and

Leaf

Stem

during the hardening phase (021 d). Values indicated by different

letters are significantly different at 5% level by StudentNewman
Keuls multiple range test (n=9).

EFFECT OF SUCROSE, LIGHT, AND CO2 ON PLANTAIN MICROPROPAGATION

hardening phase (Table 3). The low transpiration rate

(1.08 mmol H2O m2 s1) reached at the end of in vitro
phase on LH treatment permitted a better hardening of
these plantain plants (Table 3). Water status stabilization
reached through the reduction in transpiration rate in
plantain plants can be because of the imposition of high
CO2 concentration (Pospisilova et al. 2007). The implementation of 1,500 mol mol1 CO2 enhanced the multiplication
rate, the foliar expansion, and the rooting ability of in vitro
cultured sugarcane (Xiao et al. 2003).
On the other hand, PK and PEPC enzymatic activities
increased under HH conditions (Table 4). The PK activity
was stimulated by the high light intensity. This activation is
closely related to the sucrose assimilation and the activation
of the glycolytic pathway (Plaxton 1996). The PEPC
activity was stimulated by the autotrophy treatment with
high light intensity and the CO2 enrichment. Dary et al.
(2001) observed a similar PEPC activity stimulated by light
in tomato plants. The activation of PEPC by high
concentrations of CO2 could be a result of the substrate
cooperative induction process. Carbon skeletons formed
through PEPC activity cannot be associated to proteins
synthesis because the HH treatment showed the lowest
concentration of proteins (39.73 mg g FW1). Maybe, its
enzymatic activity can be related to starch synthesis (Dary
et al. 2001). The positive effects of carbonic irrigations on
the nutrient assimilation and rooting process have been
demonstrated during the in vivo growth of different crops
(Aguilera et al. 2001; Segura et al. 2001). Oncidium
goldiana plants were studied under different CO2 and light
environments. The response of primary carbon metabolism
enzymes was increased by 750 and 1,100 mol mol1 of
CO2 (Li et al. 2001).
The combination of low light intensity and high CO2
concentration (LH) during in vitro culture produced the
highest percentage of competent plants (Table 2). This
group of plants was compared to the control treatment [low
light intensity-low CO2 concentration (LL)] with regard to
the levels of starch in leaf and stem. Both groups of plants
showed high starch levels in stems at the end of the
elongation phase (Fig. 1). Nevertheless, the CO2 enrichment induced the recovery of starch levels in stems of
plants from LH treatment at the end of the hardening phase.
The role of starch metabolism during the hardening of
plantain plants propagated in TIB was described by our
team (Aragn et al. 2006). The carbohydrate accumulation
as starch reserves is important for plant survival during
hardening phase (Capellades et al. 1991). Both carbohydrates (sucrosestarch) reserves in plantain leaf and stem,
respectively, seem to play an important role during
hardening phase (Aragn et al. 2005, 2006).
The combination of a high sucrose level (30 g L1),
80 mol m2 s1 light intensity and 1,200 mol mol1 CO2

93

concentration during the 3 wk of in vitro phase permitted

the development of structures and metabolic capacities for a
better subsequent hardening. TIB techniques with CO2
enrichment allowed the acquisition of high number of
competent plants (91.0%), better plant quality, and better
carbon metabolic rate and survival percentage during ex
vitro hardening (99.80%).
Acknowledgments This work was supported by funds from the
European Community (INCO project ICA4-CT2001-10063). The
author thanks Dr. Jose C. Lorenzo Feijoo and M.D. Fernando Sagarra
for the critical comments of the article.

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