CO2-enriched atmosphere and supporting material impact the growth, morphophysiology and ultrastructure of in vitro Brazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen] plantlets

ORIGINAL PAPER

CO2-enriched atmosphere and supporting material impactthe growth, morphophysiology and ultrastructure of in vitroBrazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen] plantlets

Cleber Witt Saldanha • Caio Gomide Otoni • Diego Ismael Rocha •

Paulo Cezar Cavatte • Kelly da Silva Coutinho Detmann • Francisco Andre Ossamu Tanaka •

Leonardo Lucas Carnevalli Dias • Fabio Murilo DaMatta • Wagner Campos Otoni

Received: 29 November 2013 / Accepted: 27 February 2014 / Published online: 8 March 2014

� Springer Science+Business Media Dordrecht 2014

Abstract This study aimed to evaluate under photoauto-

trophic conditions the effect of CO2-enriched atmosphere (360

or 1,000 lmol CO2 mol-1 air) combined with two substrate

types (agar or Florialite�) in vitro on plants of Pfaffia glom-

erata, an endangered medicinal species with promising

applications in phytotherapy and phytomedicine. The effects

of the treatments on the growth, stomatal density, Rubisco

activity, carbon isotopic discrimination, metabolite accumu-

lation, photosynthetic pigments and ultrastructural character-

istics were investigated. After a 35-day cultivation period, the

in vitro-growth of P. glomerata nodal segments under the

different treatments resulted in plants with substantial differ-

ences in relation to their growth, photosynthetic pigments,

stomatal density and leaf ultrastructural characteristics. The

enrichment with CO2 coupled with a porous substrate

increased the growth of P. glomerata. The stomatal density in

the abaxial epidermis more than doubled in response to the

high CO2 supply in both supporting types, whereas the Ru-

bisco activity and activation state were both unresponsive to

the treatments. Regardless of the CO2 supply, the plants grown

in agar displayed higher carbon isotope discrimination than

their counterparts grown in Florialite�. We propose that the

long-term photosynthetic performance was improved using

Florialite� as a growth support in combination with a high

CO2 supply. No apparent signs of photosynthetic down-reg-

ulation could be found under elevated CO2 conditions. The

enrichment of in vitro atmospheres with CO2 coupled with a

porous substrate offers new possibilities for improving the

growth and production on a commercial scale of high mor-

phological and physiological quality Pfaffia plants.

Keywords b-Ecdysone � Carbon isotopic discrimination �Photoautotrophic growth � Porous substrate � Secondary

metabolites

Introduction

The application of techniques that allow for in vitro auto-

trophic growth has increased steadily, particularly because

under these conditions, the in vitro physiology resembles ex

vitro physiology, thus ultimately allowing for fast acclima-

tization. In the in vitro propagation of plants, agar has tra-

ditionally been used as the gelling agent, allowing for the

efficient interaction between the explant and nutrients in

addition to serving as a support for plant development

C. W. Saldanha � D. I. Rocha � W. C. Otoni (&)

Laboratorio de Cultura de Tecidos-BIOAGRO, Departamento de

Biologia Vegetal, Universidade Federal de Vicosa, Avenida

Peter Henry Rolfs, Vicosa, MG 36570-900, Brazil

e-mail: [email protected]

C. W. Saldanha

Centro de Pesquisas em Florestas, Fundacao Estadual de

Pesquisa Agropecuaria, BR 287, Acesso VCR 830, km 4,5, Boca

do Monte, Caixa Postal 346, Santa Maria, RS 97001-970, Brazil

C. G. Otoni

Laboratorio de Embalagens (LABEM), Departamento de

Tecnologia de Alimentos, Universidade Federal de Vicosa,

Vicosa, MG 36570-900, Brazil

P. C. Cavatte � Kelly da S. C. Detmann � F. M. DaMatta

Laboratorio de Nutricao e Metabolismo de Plantas,

Departamento de Biologia Vegetal, Universidade Federal de

Vicosa, Vicosa, MG 36570-900, Brazil

F. A. O. Tanaka

NAP/MEPA, Escola Superior de Agricultura ‘‘Luiz de Queiroz’’,

Universidade de Sao Paulo, Piracicaba, SP 13418-900, Brazil

L. L. C. Dias

Universidade Federal de Sao Joao Del Rei, Campus Sete Lagoas,

Rodovia MG 424, km 47, Sete Lagoas, MG 35701-970, Brazil

123

Plant Cell Tiss Organ Cult (2014) 118:87–99

DOI 10.1007/s11240-014-0464-x

(Thorpe et al. 2008). Alternatively, other different porous

substrates have been shown to display potential character-

istics for use as a support for explant growth in in vitro

propagation, namely, vermiculite (Xiao and Kozai 2006;

Cha-um et al. 2011), a compound of vermiculite and cellu-

lose fibers (Florialite�) (Zobayed et al. 1999; Afreen-Zob-

ayed et al. 2000; Xiao and Kozai 2006; Marriot and Sarasan

2010), sugarcane bagasse (Mohan et al. 2004), perlite

(Lucchesini et al. 2006), ‘rockwool’ (Norikane et al. 2010)

and a compound of perlite and vermiculite (Oh et al. 2012).

Porous substrates increase the hydraulic conductivity com-

pared to that of the culture medium gelled with agar, favoring

nutrient uptake from the culture medium (Kozai 2010; Oh

et al. 2012). As a consequence, the support material plays an

important role in the in vitro propagation of plants, poten-

tially improving plant vigor and thus the survival rate during

acclimatization (Afreen-Zobayed et al. 1999).

The enrichment of in vitro atmospheres with CO2 has also

been a key factor for improving the growth and production of

high-quality in vitro plants associated with proper acclimati-

zation (Kozai 2010; Xiao et al. 2011). Under photoautotrophic

conditions, CO2 and light are factors that directly affect the

growth and photosynthetic capacity of plants (Kozai 2010;

Norikane et al. 2010). Indeed, a wealth of studies have evi-

denced improved plant growth with increasing CO2 concen-

trations within the inner atmosphere of a culture flask during

in vitro propagation (Kozai 2010; Norikane et al. 2010; Badr

et al. 2011; Cha-um et al. 2011; Xiao et al. 2011). Increased CO2

concentrations are expected to lead to changes in plant physi-

ology, including the reduction (Norikane et al. 2010) or

enhancement (Possell and Hewitt 2009; Yang et al. 2010) of the

activity of ribulose-1,5-bisphosphate carboxylase/oxygenase

(Rubisco), ultrastructural changes (Lucchesini et al. 2006), an

altered metabolic profile (Badr et al. 2011), the accumulation of

secondary metabolites (Zobayed et al. 2003; Mosaleeyanon

et al. 2005; Schonhof et al. 2007; Yang et al. 2010) and modi-

fications of carbon isotope discrimination due to changes in CO2

diffusion (Tazoe et al. 2009). Studies addressing the carbon

isotope discrimination in the in vitro propagation of plants are

scarce in the literature. It has been used in plantlets of Phoenix

dactylifera grown in vitro under conditions of water stress

(Helaly and El-Hosieny 2011) and in Gardenia jasminoides

under different conditions of gas exchange, light and sugar

levels in the culture medium (Serret et al. 1997). However, there

are no reports regarding carbon isotope discrimination and

in vitro propagation in atmospheres enriched with CO2.

Pfaffia glomerata (fafia, ginseng brasileiro), a medicinal

plant that naturally grows in Brazil (Pott and Pott 1994), has

great economic importance due to the production of secondary

metabolites such as b-ecdysone (20E) (Festucci-Buselli

et al. 2008a). Several properties have been associated with

genus Pfaffia, such as anabolic, analgesic, antiinflammatory,

antimutagenic, aphrodisiac, sedative, and muscle tonic

properties (Neto et al. 2005; Fernandes et al. 2005; Festucci-

Buselli et al. 2008a; Mendes 2011). Many patents related to

pharmacological and nutritional properties of genus Pfaffia

have been published (Shibuya et al. 2001; Bernard and Gautier

2005; Olalde 2008; Rangel 2008; Loizou 2009; Higuchi

2011). Because of its economic relevance, the propagation of

P. glomerata plays an essential role in producing raw material

for the pharmaceutical industry (Saldanha et al. 2013). The

in vitro propagation of this species has traditionally been

performed using agar under heterotrophic conditions matched

by axillary bud proliferation (Russowski and Nicoloso 2003;

Maldaner et al. 2006; Skrebsky et al. 2006; Nicoloso et al.

2008; Flores et al. 2010). Currently, studies using agar as a

support for explants have revealed the successful in vitro

propagation of P. glomerata under photomixotrophic and

photoautotrophic conditions with gas exchange with (Salda-

nha et al. 2013) and without (Iarema et al. 2012; Saldanha et al.

2012) CO2 enrichment. Considering all of the above infor-

mation, it is herein hypothesized that the use of alternative

porous substrates and CO2 enrichment could further optimize

the photoautotrophic system.

In this study, we describe a way in which the substrate

type (support for explant) combined with CO2 enrichment

improves the growth and development of the P. glomerata

plants grown in vitro. The growth characteristics, accu-

mulation of metabolites, leaflet gas exchange, Rubisco

activity and ultrastructure changes in Pfaffia leaves are

reported and discussed. The elucidation of such factors is

essential for understanding the behavior of plants in a

photoautotrophic system.

Materials and methods

Plant materials and culture conditions

Leafless, 20-mm-long nodal segments with 2 pre-existing

axillary meristems were taken from in vitro-grown Pfaffia

plants previously maintained under photomixotrophic condi-

tions and inoculated in culture medium consisting of MS salts

(Murashige and Skoog 1962) and vitamins and 100 mg L-1

myo-inositol and 7 g L-1 agar (Merck� KGaA, Darmstadt,

Germany). The culture medium was sterilized by autoclaving

at 121 �C and 0.15 MPa for 15 min, and the pH was adjusted

to 5.7. Powdered MS salts (Sigma Chemical Company, St.

Louis, MO, USA; Cat. no M5519) were added to the medium.

The cultures were stored in sucrose-free culture medium

within a growth room conditioned at 28 ± 2 �C under an

irradiance of 150 lmol m-2 s-1 and photoperiod of 16 h.

Gas exchange between the headspace inside the culture

flasks and the growth room atmosphere was allowed through

two 10-mm-diameter holes in the flask lids, voids which were

covered with hydrophobic polytetrafluoroethylene (PTFE)

88 Plant Cell Tiss Organ Cult (2014) 118:87–99

123

membranes (MilliSeal� Air Vent, Tokyo, Japan) with 0.45-lm

pores. The number of gas exchanges per flask was estimated

according to Fujiwara and Kozai (1995): 0.36 exchanges per

hour for flasks covered with PTFE membranes, while this

value for the hole-free flasks was zero. No subcultures were

performed throughout the 35-day cultivation period.

To obtain either a normal or a CO2-enriched atmosphere,

the culture flasks were placed inside an acrylic chamber

(41 cm wide 9 26 cm high 9 50 cm long) (Saldanha et al.

2013). The CO2-enriched air (a mixture of ambient air with

commercial CO2) was injected into the chamber at a flow rate

of 1 L min-1, and the relative humidity inside the chamber

was maintained at 50 ± 10 %. The CO2 concentration in the

air was 360 or 1,000 lmol mol-1 air, which was adjusted

using an infrared gas analyzer (model S153 CO2 Analyzer,

Qubit System, Kingston, Canada).

The experiment was performed in a completely random-

ized design with 10 replicates, each consisting of two poly-

propylene Magentas� (Sigma Chemical Co., USA) connected

by couplers (Sigma Chemical Co.) to form a 750 mL flask that

contained five nodal segments (experimental unit) and

approximately 100 mL culture medium. The treatments were

arranged in a 22 factorial arrangement consisting of two

environments (360 ± 20 or 1,000 ± 100 lmol CO2 mol-1

air) combined with two types of explant supports [Florialite�

(mixture of vermiculite and cellulose, Nisshinbo Industries,

Japan) or agar (Merck�)], totaling four treatments.

Growth parameters

After 35 days of in vitro-growth, the height of the plantlets was

measured, after which the plantlets were harvested and sepa-

rated into aerial and root portions. Following drying for 3 days

at 60 �C in an oven with forced air circulation, the aerial

(ADM) and root system (RDM) dry masses were computed.

Photosynthetic pigments

Four leaf discs (7 mm in diameter) were taken from the third

pair of fully expanded leaves from the apical meristem

downward and incubated in 5 mL dimethylsulfoxide

(DMSO) for 48 h under darkness at room temperature

(Santos et al. 2008). The absorbances at 665, 645 and 480 nm

(Wellburn 1994) were then determined using a spectropho-

tometer (model Genesys 10UV, Thermo Scientific, USA) in

quartz cuvettes with a 10 mm path length. The calculations

for total chlorophyll (a plus b) and carotenoid contents were

based on the methodology of Wellburn (1994).

Rubisco activity and activation state

Rubisco (EC 4.1.1.39) was extracted according to Gei-

genberger and Stitt (1993). The initial and total activities of

Rubisco were measured using the spectrophotometric,

NADH, enzyme-coupled assay as described by Sharkey

et al. (1991). After adding ribulose-1,5-bisphosphate to the

reaction medium, the NADH oxidation was monitored for

30 min at 340 nm. The results were expressed as nmol g-1

DM s-1. The activation state of Rubisco was calculated as

the ratio of initial to total activity (percent).

Phenolics, sugars and starch

For starch, the total soluble phenolic (TSP) and total sol-

uble sugar (TSS) determinations used approximately

25 mg leaf dry mass to which a mixture of methanol/

chloroform (1:1, v/v) was added (Bligh and Dyer 1959).

The suspension was mixed for 30 min and centrifuged at a

4,0009g for 5 min. Water was incorporated into the

supernatant under agitation, and the mixture was then

centrifuged likewise to separate the phase containing

chloroform from that containing methanol and water.

The TSP concentration was colorimetrically determined

at 725 nm in the methanol/water phase using the Folin-

Ciocalteu (1:1) reagent and tannic acid as standards. The

starch concentration was determined in the pellet resulting

from the methanol/chloroform extraction, to which 3 %

HCl was added to the tubes that contained the extract. The

solution was allowed to rest for 3 h at 100 �C in order to

break down the starch. After centrifuging at 4,0009g for

5 min, the supernatant was collected, and the carbohy-

drates that resulted from the acid hydrolysis, as well as the

soluble sugars that were present in the supernatant of the

methanol/water phase, were quantified at 620 nm using the

anthrone reagent (Fales 1951).

b-Ecdysone

The b-ecdysone (20E) contents in the leaves and the stalk

of in vitro-grown Pfaffia plants was determined using high-

performance liquid chromatography (HPLC) following

previously procedures described (Kamada et al. 2009). The

methanol extracts were analyzed in a Shimadzu HPLC

(model LC-10AI, Tokyo, Japan) equipped with a SPD-

10AI, CBM-10A detector and set up as follows: Bondesil C

18 column (5.0 lm 9 4.6 mm 9 250 mm).

Relative abundance of 13C and 12C from the air and leaf

tissues

For the quantification of the relative abundance of 13C and12C, the air mixtures used for the CO2-enriched

(1,000 lmol CO2 mol-1 air) and not enriched (360 lmol

CO2 mol-1 air) atmospheres were analyzed regarding their

carbon isotopic compositions (CRDS, Isotopic CO2 Ana-

lyzer, Picarro�). For the tissues, a 10-mg (dry matter)

Plant Cell Tiss Organ Cult (2014) 118:87–99 89

123

sample was used to measure the C contents with an ele-

mental analyzer (Carlo Erba, Milan, Italy) and the relative

abundances of 13C and 12C using a mass spectrometer

(ANCA-GSL 20-20, Sercon, Crewe, UK). From these

values, the carbon isotopic composition ratio (d13C) was

determined, after which the carbon isotope discrimination

(D13C) was calculated. Further details regarding this pro-

cedure were previously reported (DaMatta et al. 2003).

Microscopy sample preparation

The fully expanded leaves of Pfaffia plants were sampled after

35 days of in vitro-growth. The leaves were fixed in Kar-

novsky solution (2.5 % glutaraldehyde and 4 % paraformal-

dehyde within sodium cacodylate buffer solution (pH 7.2)

added with 5 mM of calcium chloride) (Karnovsky 1965).

Scanning electron microscopy (SEM)

The fixed samples were dehydrated in an increasing acetone

concentration series, dried to the critical point (CPD 030,

Bal-Tec, Balzers, Germany) and gold-metalized (SCD 050,

Bal-Tec, Balzers, Germany). The analyses were conducted

using a scanning electron microscope (LEO 435-VP, Cam-

bridge, England), and the obtained microphotographs were

used to measure the stomatal density (SD; the number of

stomata per mm2 leaf area) and equatorial stomata size on

both faces of the epidermis. We analyzed five images per

epidermis face per replication (n = 3), totaling 30 images

per treatment. The Fiji platform (Schindelin et al. 2012) was

used for the image analysis.

Transmission electron microscopy (TEM)

The fixed leaf samples were post-fixed with 1 % osmium

tetroxide, dehydrated in an increasing acetone concentra-

tion series, infiltrated and polymerized into Spurr low-

viscosity epoxy resin. The blocks were prepared for ultra-

microtome using a trimmer (EM Trim, Leica Microsystems

Inc., USA). Sections (70-nm thick) were obtained using an

ultramicrotome (Leica UC6, Leica Microsystems Inc.,

USA) and contrasted against uranyl acetate and lead citrate

(Reynolds 1963). The analyses were performed using a

transmission electron microscope (EM900, Zeiss, Ger-

many) fitted with a digital camera at 80 kV.

Statistical analysis

The data were submitted to Bartlett’s test to verify the

homogeneity of the variances. When necessary, the data

were transformed usingffiffiffi

xp

, as was performed for the

height, the aerial and root system and the total dry masses.

The parameters were submitted to analysis of variance

(ANOVA) and Tukey’s test (p \ 0.05). Tables and figures

in this work present untransformed means. All of the sta-

tistical procedures were performed using the SAS software,

version 9.0 (SAS Institute 2003).

Results

The growth of in vitro-grown Pfaffia plants

was enhanced in Florialite� and under elevated CO2

concentrations in the flask environment

The growth performance, as measured via the growth traits of

in vitro-grown P. glomerata plants, was analyzed and was

significantly improved in the plants that were grown in the CO2-

enriched atmosphere (1,000 lmol mol-1) (Table 1; Fig. 1)

compared with their control (360 lmol mol-1) counterparts.

The plant height was unresponsive to the support (agar

or Florialite�), although taller plants were observed under

elevated CO2 concentrations (Table 1). Nonetheless, the

ADM, RDM and TDM were all notably higher in plants

grown in Florialite� with elevated CO2 than in plants

grown in agar with elevated CO2 concentrations (Table 1).

Interestingly, the ADM was 3.2 times higher in plants

grown in Florialite� at elevated CO2 than at normal CO2

supplies.

Table 1 Growth variables of P. glomerata plants in vitro-propagated

under different culture conditions and CO2 concentrations for 35 days

Supporting material CO2 (lmol mol-1) Mean

360 1,000

Height (cm)

Agar 10.12 14.72 12.42a

Florialite� 8.86 16.14 12.50a

Mean 9.49B 15.43A –

Aerial dry mass (g EU-1)

Agar 0.063aB 0.218bA 0.141b

Florialite� 0.095aB 0.303aA 0.199a

Mean 0.079B 0.261A –

Root dry mass (g EU-1)

Agar 0.006aA 0.012bA 0.009b


Mean 0.006B 0.025A –

Total dry mass (g EU-1)

Agar 0.069aB 0.230bA 0.149b


Mean 0.085B 0.279A –

Means followed by the same lower case letter in the column and

capital letter in the rows for each variable are not significantly dif-

ferent by Tukey’s test (p \ 0.05)

EU experimental unit


123

The CO2 enrichment provoked changes in the stomata

size and density on the abaxial epidermis

Irrespective of the growth conditions, the fully expanded P.

glomerata leaves displayed a remarkably lower SD

(p \ 0.05) on the adaxial than on the abaxial epidermis;

such an SD on the adaxial epidermis did not differ

significantly in response to the CO2 treatment in each

support type (Fig. 2; Table 2). Notably, the SD on the

abaxial epidermis was highly responsive to the CO2 treat-

ments, more than doubling in response to the high CO2

supply in both support types (Fig. 2; Table 2). Increases in

the SD on the abaxial epidermis were accompanied by

discrete reductions (11–16 %) in the equatorial dimensions

Fig. 1 Overall aspect on the P.

glomerata in vitro culture.

a Flask sealing detailed. b, d, ePlants grown in agar with 1,000

or 360 lmol mol-1 CO2. c, f, gPlants grown in Florialite� with

1,000 or 360 lmol CO2 mol-1

air. Bar 2 cm


123

(Table 2) of the stomata under elevated CO2 conditions,

regardless of the support type.

The Rubisco activity was unaffected by the growth

conditions

The initial and final activities of Rubisco and its activation

state did not vary significantly (p [ 0.05) in response to the

support type and CO2 treatments (data not shown). In agar-

grown plants, the initial and final activities were 1.65 and

1.95 nmol NAD g-1 FM s-1, respectively (activation state

of 85.3 %), whereas in Florialite�-grown plants the initial

and final Rubisco activities were 1.39 and 1.67 nmol NAD

g-1 FM s-1, respectively (activation state of 83.7 %). In

response to the CO2 treatments, the initial activity ranged

from 1.42 to 1.62 nmol NAD g-1 FM s-1 whereas the final

activity ranged from 1.79 to 1.83 nmol NAD g-1 FM s-1

for the control and enriched CO2 supplies, resulting in

activation states of 80.4 and 88.6 %, respectively.

The carbon isotope discrimination (D13C) was affected

by the substrate type

Due to the large differences in the air isotope composition

ratio in either CO2 atmosphere (-11.36 and -24.12 % for

the control and elevated CO2 treatments, respectively), the

differences in D13C in response to the CO2 supply are not

comparable. Therefore, only the differences in D13C

between the substrate types within each CO2 treatment and

main effects of the substrate types are highlighted.

Fig. 2 Leaf surface of in vitro-cultivated P. glomerata. Scanning

Electron Microscopy. a, b Adaxial epidermis of plants grown under

ambient (360 lmol CO2 mol-1) (a) and high (1,000 lmol CO2

mol-1) CO2 conditions (b) (Bars 100 lm). c–f Abaxial epidermis (c,

d Bars 100 lm; e, f 20 lm)


123

Regardless of the CO2 supply, the plants grown in agar

were better able to discriminate 13CO2 than were their

counterparts grown in Florialite�, thus leading to signifi-

cantly higher D13C values in the former (Table 3).

The concentrations of starch, TSP and TSS varied

as a function of the CO2 concentration and support type

The starch concentration was unresponsive (p [ 0.05) to

the CO2 treatment, ranging from 1.86 to 2.04 % under360

and 1,000 lmol CO2 mol-1, respectively. The in vitro-

grown plants cultivated in agar, however, showed a higher

(p \ 0.05) starch concentration (2.17 %) than that of their

counterparts grown in Florialite� (1.74 %). No significant

interaction (p [ 0.05) between the support type and the

CO2 supply was found for the starch concentration, in

contrast to what was observed for TSS. The highest TSS

concentrations were observed under elevated CO2 condi-

tions regardless of the support type (0.71 and 0.76 % for

360 and 1,000 lmol CO2 mol-1 air, respectively). Under

control CO2 conditions, the plants cultivated in Florialite�

accumulated more leaf TSS (0.80 %) than did their coun-

terparts cultivated in agar (0.62 %).

The leaf TSP concentrations were higher (0.90 %) at

normal than at elevated CO2 levels (0.68 %), independent

of the support type (0.77 and 0.80 % regarding to agar and

Florialite�, respectively). There was no significant inter-

action (p [ 0.05) between the support type and the CO2

supply for the leaf TSP concentrations.

The content of photosynthetic pigments increased

with enhanced CO2 concentrations and in response

to the substrate with higher aeration

The support type (agar or Florialite�) and the CO2 concen-

tration in the environment (360 or 1,000 lmol mol-1) sig-

nificantly (p \ 0.05) affected the concentrations of total

chlorophylls and carotenoids. In vitro-grown Pfaffia plants

under elevated CO2 conditions displayed an increased con-

centration of total chlorophylls (35.2 and 46.2 lg cm-2) and

carotenoids (4.45 and 5.76 lg cm-2), respectively for the

control and elevated CO2 treatments. However, the total

chlorophylls and carotenoids did not differ significantly

(p [ 0.05) in response to the support type under elevated

CO2 conditions (46.2 lg cm-2 on average) (5.76 lg cm-2

on average) In contrast, under control conditions, the Flor-

ialite�-grown plants displayed higher concentrations of total

chlorophylls and carotenoids (41.46 and 5.12 lg cm-2,

respectively) than those of their agar-grown counterparts

(29.0 and 3.77 lg cm-2, respectively).

The CO2 enrichment changed the lamellation pattern

of chloroplasts in Pfaffia leaves

Ultrastructural changes at the chloroplast level were

observed in response to the high CO2 treatment, indepen-

dent of the support type. While under the control CO2

conditions, the chloroplasts displayed a typical oval shape

(Fig. 3a) with thylakoid membranes clearly differentiated

into grana stacks and stroma thylakoids (Fig. 3b), under the

high CO2 conditions, an increased lamellation pattern was

observed (Fig. 3c–f), coupled with a loose organization of

the thylakoid membranes with wavy areas and intense

expansions between them (Fig. 3e, f). The number of grana

stacks was considerably lower under the high than under

the control CO2 conditions.

Table 2 Stomatal density (SD) on the abaxial and adaxial epidermis

and equatorial diameter (ED) of the stomata on the leaves of P.

glomerata plants in vitro-cultivated under different culture conditions

and CO2 concentrations for 35 days


360 1,000

SD (stomata mm-2)

Adaxial surface

Agar 34.4aA 36.1aA 35.2a

Florialite� 30.0aA 28.4bA 29.2b

Mean 32.2A 32.2A –

Abaxial surface

Agar 191.8aB 419.1aA 305.5a


Mean 197.3B 424.3A –

ED (mm)

Agar 0.0250 aA 0.0223 aB 0.0237a

Florialite� 0.0256 aA 0.0215 aB 0.0235a

Mean 0.0253A 0.0219B –

Means followed by the same lower case letter in the column and

capital letter in the rows for each variable are not significantly dif-

ferent by Tukey’s test (p \ 0.05)

Table 3 Carbon isotope discrimination (D13C) in the leaves of P.

glomerata plants in vitro-cultivated under different culture conditions

and CO2 concentrations for 35 days


360 1,000

D (%)

Agar 8.86a 5.37a 7.11a

Florialite� 6.30b 3.59b 4.94b

Means followed by different lower case letters in the column differ

significantly from each other by Tukey’s test (p \ 0.05). Differences

in D13C in response to the CO2 supply are not comparable (see

‘‘Results’’)


123

The b-ecdysone (20E) concentration was enhanced

in plants cultivated in Florialite� under elevated CO2

conditions

The 20E accumulation in both the leaves and stems varied

significantly (p \ 0.05) in response to the CO2 treatment

and support type (Fig. 4) with significant interactions

between these factors. Under the elevated CO2 conditions,

the leaves from plants grown in Florialite� were able to

accumulate more 20E than were those grown in agar,

whereas under the control conditions, the support type did

not affect the 20E accumulation in the leaves (Fig. 4).

Notably, the leaves accumulated 20E to a greater extent

than did the stems (Fig. 4a, b). In the control atmosphere,

the 20E concentrations were similar in either support

type (Fig. 4b), but at high CO2 concentrations, the 20E

Fig. 3 Chloroplast ultrastructure of in vitro-cultivated P. glomerata

plants. Transmission Electron Microscopy. a, b Plants grown in an

atmosphere with ambient CO2 concentrations (360 lmol mol-1)

(Bars 0.5 and 0.1 lm, respectively). c–f Plants grown in a CO2-

enriched atmosphere (1,000 lmol mol-1) (c, d Bars 0.5 lm; d,

e 0.2 lm; f 0.1 lm). Abbreviations: d thylakoid expansion; g grana;

p plastoglobuli; st stroma thylakoids; (arrowhead), ripples in

chloroplast internal membranes


123

concentrations were significantly improved in the stems of

the plants that were cultivated in agar compared to those

that were grown in Florialite�, in sharp contrast to what

occurred in the leaves, as indicated above (Fig. 4).

Discussion

The present study demonstrates that increased CO2 con-

centrations within the in vitro environment led to the

increased growth (and vigor) of Pfaffia plants (Table 1).

Most importantly, this study is the first to report significant

interactions between CO2 enrichment and the support type

and the in vitro-growth, ultrastructure, photosynthetic gas

exchange and contents of carbohydrates and metabolites

using P. glomerata as a case study. We contend that P.

glomerata is suited for growth under elevated CO2 condi-

tions; therefore, the technology of CO2 enrichment, cou-

pled with an adequate growth support, may be promising

for the proper in vitro propagation of this species.

Photosynthetic performance is expected to increase

with elevated CO2 concentrations in a support

type-dependent manner

Under the present experimental conditions, the improved

growth under the high CO2 supply must obviously be

coupled with increased photosynthetic rates considering

that 90–95 % of plant dry mass is derived from photo-

synthetically fixed carbon (Kruger and Volin, 2006). When

all of the external factors are equal, the magnitude of the

photosynthetic rate is chiefly governed by the CO2 entry

into the leaf though the stomata, which is driven by the

stomatal conductance and by the CO2 fixation capacity,

which is largely but not exclusively associated with the

Rubisco activity and/or activation state (DaMatta et al.

2010). With an increasing substrate (CO2) availability, a

higher carboxylation and a lower oxygenation rate (lower

photorespiration rates) of Rubisco are expected (Ainsworth

and Rogers 2007; Bader et al. 2010; Kirschbaum 2011),

thus leading to increased photosynthetic rates. Further-

more, we propose that the stomatal conductance should

have increased in this study, given that the SD increased

remarkably, while the stomatal size (indirectly evaluated

by the equatorial dimensions) decreased only slightly under

high CO2 conditions. This result is unexpected because the

stomatal conductance (and SD) is consistently (but not

universally) decreased under elevated CO2 conditions

(Ainsworth and Long 2005; Leakey et al. 2009). In any

case, the proposed increases in stomatal conductance

should have allowed higher rates of diffusion of CO2 from

the atmosphere to the chloroplasts, thus contributing fur-

ther to improve the photosynthetic rates and ultimately

increasing the biomass production under elevated CO2

conditions.

The Florialite� porosity is estimated in 89 % of its own

volume, whereas in agar matrix the porosity should be near

zero after gelling, though affecting the in vitro-growth of

plants (Afreen-Zobayed et al. 1999). Here, we propose that

the expected positive effects of an elevated CO2 supply on

photosynthesis strongly depend on the support type. When

comparing the plants grown under elevated CO2 conditions

in agar or Florialite�, the differences in the total biomass

accumulation (and photosynthesis) were unlikely to have

been associated with stomatal behavior or the Rubisco-

mediated CO2 fixation capacity (given that the changes in

the SD and Rubisco were minimal, if any). In contrast,

those differences should be accounted for by long-term

higher photosynthetic rates in the Florialite�-grown plants

than in their counterparts that were grown using agar as a

support. Compelling evidence for this assumption is pro-

vided by the carbon isotope discrimination pattern, which

depends on either the photosynthetic rates or stomatal

conductance or both and expresses the magnitude of gas

exchange over time instead of as a discrete measurement

(Farquhar et al. 1989). Given that we have no reason to

Fig. 4 b-Ecdysone (20E) concentration in the leaves and stems of P.

glomerata plants in vitro-cultivated under different growth conditions

and CO2 concentrations for 35 days. a Leaf. b Stem. Means followed

by the same lowercase letter do not differ significantly between CO2

treatments within the same substrate type. Means followed by the

same uppercase letter do not differ significantly between substrate

types within each CO2 treatment (Tukey’s test, p \ 0.05)


123

claim differences in the stomatal conductance in response

to the support type, the significantly lower D13C in the

Florialite�-grown plants (regardless of the CO2 supply)

should therefore imply a higher long-term photosynthetic

capacity in these plants. This result may largely explain the

observed improved biomass of these plants, as noted under

elevated CO2 conditions.

The CO2 enrichment did not provoke any apparent

down-regulation of the photosynthetic process

The degree of down-regulation (acclimation) of photo-

synthesis in response to CO2 enrichment is usually more

evident under conditions that restrict plant growth (Arp

1991; Ronchi et al. 2006); such a down-regulation could

largely decrease the stimulatory effect of elevated CO2

concentrations on plant growth (Woodward 2002). Here,

we have compelling evidence to support the fact that no

apparent photosynthetic acclimation took place under high

CO2 conditions. First, the Florialite�-grown plants that

accumulate more biomass also displayed lower D13C val-

ues, indicating higher long-term photosynthetic rates.

Second, no indication of decreased stomatal conductance

could be deduced. Third, the acclimation of key photo-

synthetic traits, such as the Rubisco activity and activation

state, was not observed. Fourth, despite the higher TSS

under the elevated CO2 conditions (likely a result of higher

photosynthetic rates), there was no accumulation of starch,

as commonly observed when the photosynthetic process is

down-regulated (Arp 1991; Ainsworth and Long 2005).

Indeed, the Florialite�-grown plants displayed the lowest

starch concentration, suggesting an elevated sink strength.

This result is consistent with the expected higher photo-

synthetic rates (Arp 1991) of these plants. Fifth, the con-

centration of photosynthetic pigments was up-regulated.

Given that chlorophylls and nitrogen are strongly related to

each other, it is tempting to speculate that the nitrogen

concentration was at least uncompromised under elevated

CO2 conditions, in contrast with a wealth of reports that

demonstrate decreased nitrogen levels under these condi-

tions (DaMatta et al. 2010; and references therein). Finally,

the increased lamellation pattern of the chloroplast internal

membrane system supports our claims that photosynthesis

was stimulated under high CO2 conditions. Such an

increase may represent an improved surface area, leading

to a greater light energy capture (consistent with the higher

pigment concentration under elevated CO2 conditions) by

the photosystems localized in the membrane system

(Redondo-Gomez et al. 2010), which is vital to fuel the

augmented CO2 assimilation under high CO2 conditions.

Similar observations were previously reported in the leaves

of Platanus orientalis (Velikova et al. 2009) and Solanum

tuberosum (Sun et al. 2011) cultivated under elevated CO2

conditions and may represent adaptive changes at the cel-

lular level in response to the environmental conditions

(Mostowska 1997; Velikova et al. 2009).

Further consequences of long-term CO2 enrichment

on secondary metabolites

Conflicting information has been reported on the effects of

CO2 enrichment on the pools of secondary metabolites,

ranging from increased (Castells et al. 2002; Mosaleeyanon

et al. 2005; Schonhof et al. 2007), unaltered (Castells et al.

2002; Zobayed and Saxena 2004) or decreased (Coley et al.

2002; Zobayed et al. 2003) concentrations. These varying

responses may be species/genotype-dependent (Castells

et al., 2002), in addition to being affected by the stage and

kinetics of growth (Neumann et al. 2009). Here, we showed

that the TSP concentration was down-regulated by the high

CO2 supply, apparently suggesting an inverse relationship

between the biomass accumulation and phenolic contents

in P. glomerata.

In contrast with the TSP, the concentration of 20E

increased in response to the CO2 enrichment. P. glomerata

plants display varying patterns of 20E accumulation within

their organs, with the highest accumulation under ex vitro

conditions (normal CO2) in their roots (Festucci-Buselli

et al. 2008b). The 20E concentrations in the leaves in this

current study are similar to those that were found in P.

glomerata roots by Festucci-Buselli et al. (2008b) under ex

vitro growth for 120 days. The increased 20E in the leaves

from Pfaffia plants (Fig. 4) in response to Florialite� and a

CO2-enriched atmosphere may be related to the higher

aeration that the Florialite� provides compared to agar,

favoring the primary and secondary metabolisms. During

the growth of cellular suspensions of Morinda citrifolia, for

example, the increase in the accumulation of secondary

metabolites is regulated by the culture medium aeration

(Ahmed et al. 2008).

The effect of different supports and CO2 concentrations

on the growth of in vitro-grown P. glomerata plants is

reported in the present study. We clearly demonstrate that

the plants displayed improved growth coupled with an

improved photosynthetic performance when cultivated

under an increased CO2 supply and in Florialite�. Indeed,

no signs of photosynthetic down-regulation could be

observed. We also show that the plants with a higher bio-

mass also produced higher amounts of 20E. This study

highlights that a photoautotrophic system under CO2

enrichment may be attractive for the achievement of

autotrophy by CO2, thus potentially being useful for the

large-scale commercial production of Pfaffia seedlings or

even for producing Pfaffia biomass containing high levels

of b-ecdysone.


123

Acknowledgments The authors thank the National Council for

Scientific and Technological Development (CNPq) [MCT/CNPq

480675/2009-0; PQ 303201/2010-10 to WCO], the Minas Gerais

State Research Foundation (FAPEMIG) [CAG-APQ-01036-09], and a

CAPES (PNPD) for financial support. CWS was a recipient of a

scholarship from CAPES (PNPD). The Microscopy and Microanal-

ysis Center (NMM) of the Federal University of Vicosa, and NAP-

MEPA (USP/ESALQ, Piracicaba) are also acknowledged.

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Documents

CO2-enriched atmosphere and supporting material impact the growth, morphophysiology and ultrastructure of in vitro Brazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen] plantlets