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PHYSIOLOGY
Received: 10 May 2008 / Accepted: 20 August 2009 / Published online: 14 October 2009 / Editor: D. T. Tomes
# The Society for In Vitro Biology 2009
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
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)
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)
10
30
50
Standard error
Significance
Treatments
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
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)
1.33 b
1.82 a
1.08 c
0.11
*
phase (%)
0.74
*
Significance * *
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)
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 analyzed using one-way ANOVA followed by Tukey test
pH 7.4 according to Siegel and Stitt (1990). The catalyzed at 5% level. Nonparametric tests Kruskal–Wallis, Mann–
PEPC reaction was coupled with the L-malate dehydroge- Whitney, and C-Dunnett at 5% level were performed for the
nase (EC 1.1.1.37) (SIGMA) reaction and assayed at 25°C analysis of enzymatic activities and starch determination.
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) Results and Discussion
(Sigma) reaction and assayed at 25°C by monitoring
NADH utilization at 340 nm (Lin et al. 1989). Plants treated with 30 g L−1 sucrose showed the highest
quality when measured at the end of the hardening phase
Starch determination. Leaf and stem material (250 mg) while for in vitro culture in TIB was 10 g L−1 (Table 1). In
collected from in vitro and ex vitro conditions were placed addition, this treatment (30 g L−1) in combination with
immediately in liquid nitrogen and ground with a mortar and 80 μmol m−2 s−1 light intensity and 1,200 μmol mol−1 CO2
pestle. The extract was centrifuged at 10,000×g and 4°C for concentration (LH), improved the percentages of compe-
20 min. The pellet was recovered on KOH 0.2 mol L−1. tent plants (from 80.0% to 91.0%) and survival (from
Later, an enzymatic treatment was applied for total starch 95.71% to 99.80%) at the end of ex vitro hardening
degradation with amyl-glycosidase enzyme (EC 3.2.1.3; (Table 2). The best photosynthetic rate was observed in
Sigma; Thomas et al. 1983). The quantification was referred plants coming from the autotrophy treatment (HH). LH-
to as potato starch control. treated plants showed a favorable photosynthetic rate and a
morphological development presenting the greater foliar
Statistical analysis. All statistical analyses were carried out expansion (width and length), root growth (length and
using SPSS software (version 11.0). The results were 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 93
hardening phase (Table 3). The low transpiration rate concentration during the 3 wk of in vitro phase permitted
(1.08 mmol H2O m−2 s−1) reached at the end of in vitro the development of structures and metabolic capacities for a
phase on LH treatment permitted a better hardening of better subsequent hardening. TIB techniques with CO2
these plantain plants (Table 3). Water status stabilization enrichment allowed the acquisition of high number of
reached through the reduction in transpiration rate in competent plants (91.0%), better plant quality, and better
plantain plants can be because of the imposition of high carbon metabolic rate and survival percentage during ex
CO2 concentration (Pospisilova et al. 2007). The implemen- vitro hardening (99.80%).
tation of 1,500 μmol mol−1 CO2 enhanced the multiplication
rate, the foliar expansion, and the rooting ability of in vitro Acknowledgments This work was supported by funds from the
European Community (INCO project ICA4-CT2001-10063). The
cultured sugarcane (Xiao et al. 2003). author thanks Dr. Jose C. Lorenzo Feijoo and M.D. Fernando Sagarra
On the other hand, PK and PEPC enzymatic activities for the critical comments of the article.
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 References
of the glycolytic pathway (Plaxton 1996). The PEPC
activity was stimulated by the autotrophy treatment with Aguilera D.; Murcia A.; Ruíz C. Effects of carbon dioxide enriched
high light intensity and the CO2 enrichment. Dary et al. irrigation on yield of eggplant (Solanum melongena) production
(2001) observed a similar PEPC activity stimulated by light under green house conditions. Acta Hortic. 559: 155–166; 2001.
Aragón C.; Escalona M.; Capote I.; Pina D.; Cejas I.; Rodríguez
in tomato plants. The activation of PEPC by high R.; Cañal M.; Sandoval J.; Roels S.; Debergh P.; González-
concentrations of CO2 could be a result of the substrate Olmedo J. Photosynthesis and carbon metabolism in plantain
cooperative induction process. Carbon skeletons formed (Musa AAB) growing in Temporary Immersion Bioreactor (TIB)
through PEPC activity cannot be associated to proteins and ex vitro acclimatization. In Vitro Cell. Dev. Biol. Plant 41:
550–554; 2005.
synthesis because the HH treatment showed the lowest
Aragón C.; Escalona M.; Capote I.; Pina D.; Cejas I.; Rodríguez R.;
concentration of proteins (39.73 mg g FW−1). Maybe, its Noceda C.; Sandoval J.; Roels S.; Debergh P.; González-Olmedo
enzymatic activity can be related to starch synthesis (Dary J. Aclimatización de plantas de plátano “CEMSA ¾ (AAB)”
et al. 2001). The positive effects of carbonic irrigations on provenientes de Biorreactores de Inmersión Temporal. Importan-
cia del almidón en el proceso de aclimatización. Infomusa 15:
the nutrient assimilation and rooting process have been
32–35; 2006.
demonstrated during the in vivo growth of different crops Bradford M. A rapid and sensitive method for the quantification of
(Aguilera et al. 2001; Segura et al. 2001). Oncidium microgram quantities of protein utilizing the principle of protein-
goldiana plants were studied under different CO2 and light dye binding. Anal. Biochem. 86: 248–254; 1976.
Capellades M.; Lemeur L.; Debergh P. Effects of sucrose on starch
environments. The response of primary carbon metabolism
accumulation and rate of photosynthesis in Rosa cultured in vitro.
enzymes was increased by 750 and 1,100 μmol mol−1 of Plant Cell, Tissue Organ Cult. 25: 21–26; 1991.
CO2 (Li et al. 2001). Dary S.; Desjardins Y.; Le V. Sucrose enhances phosphoenolpyruvate
The combination of low light intensity and high CO2 carboxylase activity of in vitro Solanum tuberosum L. under non-
limiting nitrogen conditions. In Vitro Cell. Dev. Biol Plant 37:
concentration (LH) during in vitro culture produced the
130–139; 2001.
highest percentage of competent plants (Table 2). This Escalona M.; Cejas I.; González-Olmedo J.; Capote I.; Roels S.; Cañal
group of plants was compared to the control treatment [low M.; Rodriguez R.; Sandoval J.; Debergh P. The effect of meta-
light intensity-low CO2 concentration (LL)] with regard to topolin on plantain propagation using a Temporary Immersion
Bioreactor. Infomusa 12: 28–30; 2003.
the levels of starch in leaf and stem. Both groups of plants
Escalona M.; Lorenzo J.; González B.; Daquinta M.; Borroto C.;
showed high starch levels in stems at the end of the González J.; Desjardins Y. Pineapple micropropagation in tempo-
elongation phase (Fig. 1). Nevertheless, the CO2 enrich- rary immersion systems. Plant Cell Rep. 18: 743–748; 1999.
ment induced the recovery of starch levels in stems of Etienne E.; Berthouly M. Temporary immersion systems in plant
micropropagation. Plant Cell, Tissue Organ Cult. 69: 215–
plants from LH treatment at the end of the hardening phase.
231; 2002.
The role of starch metabolism during the hardening of Geigenberger P.; Stitt M. A “futile” cycle of sucrose synthesis and
plantain plants propagated in TIB was described by our degradation is involved in regulating partitioning between
team (Aragón et al. 2006). The carbohydrate accumulation sucrose, starch and respiration in cotyledons of germinating
Ricinus communis L. seedlings when phloem transport is
as starch reserves is important for plant survival during
inhibited. Planta 185: 81–90; 1991.
hardening phase (Capellades et al. 1991). Both carbohy- Le V.; Lamaze T.; Champigny M. Effect of light and NO3- on wheat
drates’ (sucrose–starch) reserves in plantain leaf and stem, leaf phosphoenolpyruvate carboxylase activity. Evidence for
respectively, seem to play an important role during covalent modulation of the C3 enzyme. Plant Physiol. 97:
1476–1482; 1991.
hardening phase (Aragón et al. 2005, 2006).
Li C.; Sun W.; Hew C. Up-regulation of sucrose metabolizing
The combination of a high sucrose level (30 g L−1), enzymes in Oncidium goldiana grown under elevated carbon
80 μmol m−2 s−1 light intensity and 1,200 μmol mol−1 CO2 dioxide. Physiol. Plant. 1: 15; 2001.
94 ARAGÓN ET AL.
Lin M.; Turpin D.; Plaxton W. Pyruvate kinase isoenzymes from mixotrophic and photoautotrophic conditions. Biol. Plant 46:
green alga Selenastrum minutum. Purification and physical and 161–166; 2003.
immunological characterization. Arch. Biochem. Biophys 269: Shu-Han Y.; Yeh D.-M. In vitro leaf anatomy, ex vitro photosyn-
219–227; 1989. thetic behaviors and growth of Calathea orbifolia (Linden)
Lusty C.; Akyeampong E.; Davey M.; Ngoh G.; Markham R. A staple Kennedy plants obtained from semi-solid medium and tempo-
food with nutritious appeal. Infomusa 15: 32–35; 2006. rary immersion systems. Plant Cell, Tissue Organ Cult. 93:
Murashige T.; Skoog F. A revised medium for rapid growth and bioassays 201–207; 2008.
with tobacco tissue cultures. Physiol. Plant. 15: 473–497; 1962. Siegel G.; Stitt M. Partial purification of two forms of spinach leaf
Nguyen Q.; Kozai T. Growth of in vitro banana (Musa spp.) shoots sucrose-phosphate synthase which differ in their kinetic proper-
under photomixotrophic and photoautotrophic conditions. In ties. Plant Sci. 66: 205–210; 1990.
Vitro Cell. Dev. Biol Plant 37: 824–829; 2001. Teisson C.; Alvard D. A new concept of plant in vitro cultivation
Plaxton W. The organization and regulation plant glycolysis. Annu liquid medium: temporary immersion. In: Terzi M. Celia R.
Rev Plant Physiol Plant Mol Biol. 47: 185–214; 1996. Falavigna A. (eds) Current issues in plant molecular and cellular
Pospisilova J.; Synkova D.; Haisel D.; Semoradova S. Acclimatization biology. Kluwer, Dordrecht, pp 105–110; 1995.
of plantlets to ex vitro conditions: effect of air humidity, Thomas W.; Rufty J.; Steven C. Changes in starch formation and
irradiance, CO2 concentration and abscisic acid (a review). Acta activities of sucrose phosphate synthase and cytoplasmic
Hort 748: 29–38; 2007. fructose-1-6-bisphosphatase in response to source-sink altera-
Roels S.; Escalona M.; Cejas I.; Noceda C.; Rodríguez R.; Cañal M.; tions. Plant Physiol. 72: 474–480; 1983.
Sandoval J.; Debergh P. Optimization of plantain (Musa AAB) Tripurani S.; Reddy N.; Sambasiva K. Green revolution vaccines,
micropropagation by temporary immersion system. Plant Cell, edible vaccines. Afr. J. Biotech. 2: 679–683; 2003.
Tissue Organ Cult. 82: 57–66; 2005. Xiao Y.; Lok Y.; Kozai T. Photoautotrophic growth of sugarcane
Segura M.; Parra J.; Lorenzo P.; Sánchez-Guerrero M.; Medrano E. plantlets in vitro as affected by photosynthetic photon flux and
The effect of CO2 enrichment on cucumber growth under vessel air exchanges. In Vitro Cell. Dev. Biol. Plant 39: 186–
greenhouse conditions. Acta Hort 559: 45–49; 2001. 192; 2003.
Sha Vallikhan P.; Kozai T.; Nguyen Q.; Kubota C.; Dhawan A. Ziv M. Simple bioreactors for mass propagation of plants. Plant Cell,
Growth and water relations of Paulownia fortunei under photo- Tissue Organ Cult. 81: 277–285; 2005.