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Effect of sucrose, light, and carbon dioxide on plantain micropropagation in

temporary immersion bioreactors

Article  in  In Vitro Cellular & Developmental Biology - Plant · February 2010

DOI: 10.1007/s11627-009-9246-2

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In Vitro Cell.Dev.Biol.—Plant
DOI 10.1007/s11627-009-9246-2
MICROPROPAGATION
Effect of sucrose, light, and carbon dioxide on plantain
micropropagation in temporary immersion bioreactors
Carlos Eduardo Aragón & Maritza Escalona &
Roberto Rodriguez & Maria Jesús Cañal & Iris Capote &
Danilo Pina & Justo González-Olmedo
Received: 10 May 2008 / Accepted: 20 August 2009 / Editor: D. T. Tomes
# The Society for In Vitro Biology 2009
Abstract In vitro physiology and carbon metabolism can be
affected by the sink–source relationship. The effect of
different sucrose concentrations (10, 30, and 50 g L−1), light
intensities (80 and 150 μmol m−2 s−1), and CO2 levels (375
and 1,200 μmol mol−1) were tested during plantain micro-
propagation 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 L−1 sucrose,
80 μmol m−2 s−1 light intensity, and 1,200 μmol mol−1 CO2
concentration. This treatment improved leaf and root devel-
opment, 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. Aragón (*) : M. Escalona : I. Capote : D. Pina :
J. González-Olmedo
Laboratory for Plant Cell and Tissue Culture,
Bioplant Centre, Ciego de Ávila University,
Ciego de Ávila 69450, Cuba
e-mail: eduardo@bioplantas.cu
R. Rodriguez : M. J. Cañal
Department of Biology and Organ of Systems,
University of Oviedo,
Oviedo, Spain
M. J. Cañal
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 hyper-
hydricity (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 μmol mol−1 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 μmol CO2 h−1 per plant for
in vitro cultured banana plants. In addition, they reported
0.0 g L−1 sucrose, 200 μmol m−2 s−1 light intensity, and
1,340 μmol mol−1 CO2 as the best combination of concen-
trations to increase the photosynthetic rate.
 ARAGÓN ET AL.

Culture dry
weight per
On the other hand, light affects the primary carbon

plant (g)

0.13 b
a
a

a
0.25 a
metabolism. Light-induced activation of phosphoenol py-

0.14
0.16
0.10
0.01

0.30

0.01
ruvate carboxylase (PEPC) was described by Dary et al.

*
(2001). The actual paper describes the effects of sucrose,

Culture fresh
light intensity, and CO2 concentration during plantain

weight per
culture in TIB and ex vitro hardening. To our knowledge,

plant (g)

b
a

7.09 a
0.41 c
this information has not been published.

4.47
1.30
0.81
0.21

5.13

0.30
Table 1. Photosynthetic rate and morphological parameters for plantain plants under different mixotrophic treatments at the end of elongation and hardening phases

*
number

2.67 b
a

a
6.17 a
Materials and Methods

Leaf

5.50
4.67
4.00
0.38

6.67

0.37
*

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

Length of
main leaf
propagated in vitro, using the TIB procedure according to

10.50 a
b
b

b
a

2.40 c
Roels et al. (2005). After conventional propagation, five

(cm)

8.17
3.92
3.42
0.46

8.95

0.51
shoots (3.0 cm diameter and 3.0 cm length) were transferred

*
to a 250-mL TIB containing 50 mL culture medium

main leaf
Width of
consisting of Murashige–Skoog basal medium (Murashige

b
b

0.95 b
a

a
3.68 a
and Skoog 1962) supplemented with 30 g L−1 sucrose and

(cm)

2.13
1.27
1.22
0.13

3.85

0.19
100 mg L−1 myo-inositol. The pH was adjusted to 5.8 before

*
autoclaving at 121°C and 118 kPa for 20 min. Cultures were

thickness
maintained at 25°C under a 16:8 h (light/dark) photoperiod

b
b

b
a

0.92 a
0.30 c

Values indicated by different letters are significantly different at the 5% level by Tukey’s multiple range test (n=30)
Stem
with a photosynthetic photon flux (PAR) of 80 μmol m−2 s−1

(cm)

0.72
0.50
0.40
0.04

0.75

0.04
*

*
(cool-white fluorescent lamps: Sylvania, Daylight F40T12/D
40 W). Immersions were performed for 4 min every 3 h.
Plant height

Two different experiments were performed using plantain

15.33 a

14.48 a
17.25 a
7.25 b
7.67 b

4.58 b
plants cultured in TIB. First, different concentrations of
(cm)

0.92

0.87
sucrose (10, 30, or 50 g L−1) were added to the media culture

*
to evaluate their effect on the plant physiology and
Photosynthetic rate
(μmol CO2m−2s−1)

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
b

b
a

7.40 a
0.03 c
5.26
4.38
0.23
0.21

1.07

0.29
150 μmol m−2 s−1) with different CO2 concentration (375
*

*
and 1,200 μmol mol−1, 98.80% purity) in the headspace of
the culture vessel. The light was provided by different
Sucrose
(gL−1)

numbers of fluorescent white lamps (Sylvania, Daylight

10
30
50

10
30
50

F40T12/D 40 W) as light sources, and the intensity was

expressed in PAR. The CO2 enrichment started just 4 h after
Environmental Conditions (LL)

Environmental Conditions (LL)

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 min−1. 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.

Growing conditions during hardening. Plants coming from

TIB and presenting 2.5 cm height and 0.3 cm width and at least
Standard error

Standard error

two leaves were defined as competent to face the hardening

Significance

Significance
Treatments

phase. Competent plants were transplanted to a sterilized

Ex vitro
In vitro

substrate composed of sugarcane ashes and red soil (1:1, w/w)

distributed in multi-tray containers with 144 holes (52.5 cm
 EFFECT OF SUCROSE, LIGHT, AND CO2 ON PLANTAIN MICROPROPAGATION

Culture dry
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

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

plant (g)

0.45 ab
transferred to a greenhouse (25–27°C; 375 μmol mol−1 CO2,

0.39 b
0.31 c

0.48 a

0.03
2,000 μmol m-2 s−1 as a maximal light intensity). An

*
automatic mist system allowed the relative humidity transition

Culture fresh
from 90% to 80% during hardening phase (21 d): 60 s

weight per
watering every 30 min (1 wk), 30 s watering every 30 min

plant (g)

2.70 b
3.49 b

3.98 b
5.26 a
(1 wk), and 15 s watering every 30 min (1 wk).

0.31
*
Physiological parameters. Photosynthetic (micromole CO2

number
per square meter per second) and transpiration rates

Root

4.00
4.69
4.78
4.40

0.35
ns
(millimole H2O per square meter per second) were measured
in the first fully expanded leaves. They were sampled 3 h

of roots

12.39 b

12.33 b
11.44 b

15.05 a
Length
after the beginning of the photoperiod (1000 h). We used a

(cm)

1.05
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

number

4.21 ab
3.68 b
2.27 c
4.33 a
Leaf

0.26
dioxide concentration and the relative humidity of the air

*
entering the cuvette were 375 μmol mol−1 and 80%,
respectively, under environmental temperature (25–27°C).

Length of
main leaf
Before obtaining the experimental data, we measured the

8.22 b
7.92 b

8.62 b
9.59 a
(cm)

0.70
maximum light intensity at which photosynthesis was stable.

Values indicated by different letters are significantly different at the 5% level by Tukey´s Multiple Range test (n=30)
*
This was reached at 600 μmol m−2 s−1. The measurements
main leaf
were performed on leaves from five plants, with ten rep-
Width of

2.55 b
2.63 b

3.15 b
3.51 a
etitions per plant. (cm)

0.24
*
Enzyme extractions and assays. Leaf material (250 mg)
thickness

collected from in vitro and ex vitro conditions was placed

0.60 ab
0.51 b

0.54 b
0.66 a
Stem

(cm)

0.04
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
height

15.64
14.85
17.49
16.22
Plant

(cm)

1.29
was filtered through cheesecloth (Miracloth) and centri-
ns
fuged at 15,000×g and 4°C for 20 min. Protein was
determined according to Bradford (1976) using a commer-
(mmol H2Om−2s−1)

cial kit (Bio-Rad, Hercules, CA). The extracted enzymes,

Transpiration

PEPC (EC 4.1.1.31) and pyruvate kinase (PK; EC

2.7.1.40), were prepared by suspending the powdered plant
1.56 ab

1.33 b
1.82 a
1.08 c

0.11
*

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

(µmol CO2m−2s−1)

the end of the hardening phase

Photosynthesis

Treatments Competent Survival at

plants (%) hardening
13.60 a
8.93 b
8.40 b
4.96 c

phase (%)
0.74
*

High Light High CO2 (HH) 83.0 b 73.03 c

(environmental conditions)

High Light Low CO2 (HL) 90.0 a 94.44 b

High light high CO2 (HH)

Low light high CO2 (LH)

High light low CO2 (HL)

Low light low CO2 (LL)

Low Light High CO2 (LH) 91.0 a 99.80 a

Low Light Low CO2 (LL) 80.0 b 95.71 b
(Environmental Conditions)
Standard error 0.94 0.96
Standard error
Significance
Treatments

Significance * *

Values indicated by different letters are significantly different at 5%

level by Student–Newman–Keuls’ multiple range test (n=9)
 ARAGÓN 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 FW−1) PEPC (Ug FW−1) Proteins concentration (mgg FW−1)

High light high CO2 (HH) 69.14 a 16.97 a 39.73 c

High light low CO2 (HL) 71.61 a 13.89 b 48.52 bc
Low light high CO2 (LH) 33.95 c 10.80 c 57.45 b
Low light low CO2 (LL) (environmental conditions) 48.15 b 10.64 c 78.96 a
Standard error 4.22 1.10 4.74
Significance * * *

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

material in 1 mL of 50 mmol L−1 HEPES–KOH buffer at
pH 7.4 according to Siegel and Stitt (1990). The catalyzed
PEPC reaction was coupled with the L-malate dehydroge-
nase (EC 1.1.1.37) (SIGMA) reaction and assayed at 25°C
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 25°C 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 10,000×g and 4°C for
20 min. The pellet was recovered on KOH 0.2 mol L−1.
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
analyzed using one-way ANOVA followed by Tukey test
at 5% level. Nonparametric tests Kruskal–Wallis, Mann–
Whitney, and C-Dunnett at 5% level were performed for the
analysis of enzymatic activities and starch determination.
Results and Discussion
Plants treated with 30 g L−1 sucrose showed the highest
quality when measured at the end of the hardening phase
while for in vitro culture in TIB was 10 g L−1 (Table 1). In
addition, this treatment (30 g L−1) in combination with
80 μmol m−2 s−1 light intensity and 1,200 μmol mol−1 CO2
concentration (LH), improved the percentages of compe-
tent 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). LH-
treated 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

200 A 200 B
a a
Starch Concentration
Starch Concentration

150 150
(mg,g FW-1)
(mg,g FW-1)

100 100
c
c
b b b c
50 b b 50 cd
b d d
b b d

0 c 0 e
BE 0 7 14 21 BE 0 7 14 21
t (days) t (days)
Leaf Stem Leaf Stem

Figure 1. Starch concentration for the treatment of low light during the hardening phase (0–21 d). Values indicated by different
intensity–low carbon dioxide concentration (LL; environmental con- letters are significantly different at 5% level by Student–Newman–
ditions) (A) and low light intensity-high carbon dioxide concentration Keuls multiple range test (n=9).
(LH) (B). Measures at the beginning of elongation phase (BE) and
 EFFECT OF SUCROSE, LIGHT, AND CO2 ON PLANTAIN MICROPROPAGATION
hardening phase (Table 3). The low transpiration rate
(1.08 mmol H2O m−2 s−1) 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 implemen-
tation of 1,500 μmol mol−1 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 FW−1). 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 μmol mol−1 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 enrich-
ment 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 (Aragón et al. 2006). The carbohydrate accumulation
as starch reserves is important for plant survival during
hardening phase (Capellades et al. 1991). Both carbohy-
drates’ (sucrose–starch) reserves in plantain leaf and stem,
respectively, seem to play an important role during
hardening phase (Aragón et al. 2005, 2006).
The combination of a high sucrose level (30 g L−1),
80 μmol m−2 s−1 light intensity and 1,200 μmol mol−1 CO2
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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