ORIGINAL PAPER

Effects of light and ventilation on physiological parametersduring in vitro acclimatization of Gevuina avellana mol

Carolina Alvarez • Patricia Saez • Katia Saez •

Manuel Sanchez-Olate • Darcy Rıos

Received: 18 October 2011 / Accepted: 5 February 2012 / Published online: 22 February 2012

� Springer Science+Business Media B.V. 2012

Abstract The effects of increased photon flux

(100 lmol m-2 s-1), ventilation, and standard in vitro cul-

ture (40 lmol m-2 s-1) with no ventilation were investi-

gated on the physiological and histological characteristics

of microshoots of Gevuina avellana. The increase in pho-

ton flux (light treatment) produced significant improvement

in the fluorescence parameters of photochemical quench-

ing, non-photochemical quenching, electron transport rate

and photochemical efficiency of PSII, compared to the

ventilation and control treatments. Nursery plants showed

similar values compared to the microshoots in the light

treatment, indicating that the plants in the light treatment

developed a management for dissipating excess light.

Moreover, chlorophyll a and b concentrations increased

significantly in both light and ventilation treatments. The

chl a/chl b ratio decreased in the ventilation treatment

compared to the control treatment. Similar results were

found for soluble carbohydrates. Finally, both the photon

flux increase and ventilation had a positive effect on foliar

anatomy, showing a more organized mesophyll and a better

development of the palisade mesophyll compared to the

control treatment. The changes observed in the microshoots

with regards to foliar anatomy and photochemical behavior

were very similar to nursery plants.

Keywords Chlorophyll fluorescence � Gevuina avellana �High irradiance � In vitro acclimatization � Ventilation

Abbreviations

Chl Chlorophyll

ETR Electron transport rate

Fv/Fm Maximum efficiency of PSII

LHC Light harvesting complex

NPQ Non photochemical quenching

PFD Photon flux density

PSII Photosystem II

QA Primary electron acceptor of PSII

qP Photochemical quenching

UPSII Photochemical efficiency of PSII

Introduction

One of the main problems with micro propagated woody

species is the low survival of the micro plants when they are

transferred to ex vitro conditions (Pospısilova et al. 1999;

Xiao et al. 2011). This is mainly due to the physicochemical

conditions inside the culture vessels (Kozai et al. 1997)

associated to a heterotrophic or mixotrophic growth condi-

tions that may generate anomalies (Galzy and Compan

1992). Such anomalies may be deficient physiological and

anatomical characteristics due to low photon flux density

(PFD) commonly used in growth chambers (Seon et al.

2000) and sucrose addition to the growth medium (Arigita

et al. 2002). Both conditions cause in vitro cultivated mi-

croshoots to develop low photosynthetic capacity (Pos-

pısilova et al. 2007), poor management of excess light

through dissipation mechanisms, low activity in the electron

transport chain, and anomalies in the chloroplast

C. Alvarez (&) � P. Saez � M. Sanchez-Olate � D. Rıos

Laboratorio de Cultivo de Tejidos, Facultad de Ciencias

Forestales y Centro de Biotecnologıa, Universidad de

Concepcion, Victoria 631, Casilla 160-C, Concepcion, Chile

e-mail: caalvarez@udec.cl

K. Saez

Departamento de Estadıstica, Facultad de Ciencias Fısicas y

Matemeticas, Universidad de Concepcion, Concepcion, Chile

123

Plant Cell Tiss Organ Cult (2012) 110:93–101

DOI 10.1007/s11240-012-0133-x

ultrastructure (Serret and Trillas 2000). These characteris-

tics cause impairment of the microshoots to withstand ex

vitro conditions including a substantial increase in irradi-

ance (Osorio et al. 2010). Because photosynthesis is one of

the most sensible physiological process to any kind of stress

(Walters 2005), the maximum efficiency of PSII (Fv/Fm)

has been one of the most used physiological parameter to

evaluate plant response to stress (Maxwell and Johnson

2000). According to this, a decrease in Fv/Fm can be

observed through ex vitro acclimation mainly due to severe

increase in irradiance. This behavior has been reported by

Carvalho et al. (2001) in Castanea sativa and Vitis vinifera,

leading to photo inhibition in both species. Also, another

study has informed about a decrease in photochemical effi-

ciency during ex vitro acclimatization (Serret et al. 2001).

In vitro cultivated microshoots are also associated with the

low ability of the plantlets to regulate water loss (Apostolo

et al. 2005) due to anatomical abnormalities developed mainly

because of the limited ventilation inside the culture vessels.

This has the effects of producing a poorly developed palisade

mesophyll and excessive intercellular spaces (Wetzstein and

Sommer 1982), stomatal malfunction (Apostolo et al. 2005),

and reduced production of epicuticular waxes (Majada et al.

2001), therefore causing an increase in mortality rates during

transfer to ex vitro conditions. For this reason both PFD and

ventilation management can be essential to overcome the

detrimental effects caused by common in vitro environment.

PFD increase promotes the development of photosynthetic

tissues (Serret and Trillas 2000), increases the photosynthetic

rates (Kozai and Smith 1995), and therefore the growing rates

(Cui et al. 2000). The increase in ventilation inside the culture

vessels promotes the development of normal anatomical

characteristics. Thus, the PFD increase and the application of

ventilated culture vessels could result in the development of

microshoots with desired physiological and anatomical

characteristics similar to those observed in plants that are

produced under nursery or field conditions.

Most of the management of environmental growth con-

ditions has been focused on the ex vitro acclimatization

stage. Even though little attention has been placed in the

management of environmental growth conditions of in vitro

culture, some studies indicate that it may contribute to

lowering stress caused by ex vitro conditions and decreasing

the mortality rates observed during transfer (Huang et al.

2011). Moreover, even less research has been done with the

culture of woody plants. Such is the case of G. avellana,

endemic woody specie from Chile. This woody plant is used

for wood and fruit production (Medel 2000). An important

characteristic of G. avellana is that 50% of the dry weight of

the seed is composed of oils with excellent food and cos-

metic properties, and it is a great source of antioxidants

(Moure et al. 2000). Therefore, the application of propaga-

tion techniques that enhance the continuous, and good

quality homogeneous seed production are needed (Silva

et al. 2012), which can be successful by micro propagation.

Even though propagation protocols are established for this

specie, there are no records of in vitro culture or acclimati-

zation techniques.

Consequently, the objective of our study was to evaluate

the effect of increase PFD and the use of ventilated culture

vessels during the in vitro culture of G. avellana in order to

produce apt explants during the microshoot production

phase. To achieve this we evaluated the parameters asso-

ciated with fluorescence of chlorophyll and physiological

characteristics of carbohydrate contents and photosynthetic

pigments, as well as anatomical characteristics. In addition,

all the previously mentioned parameters measured on in

vitro plants were compared to nursery plants.

Materials and methods

Plant material and growth conditions

Gevuina avellana plantlets were cultivated on MS (Mu-

rashige and Skoog 1962) medium and supplemented with

0.49 lM Indolbutiryc acid (IBA), 0.44 lM bencylamino

purine (BAP) and 0.147 M of sucrose. Microshoots were

sub-cultivated every month. We established two nodal

segments for every culture vessel, where two sub-cultures

were made (S1 and S2) before treatment application.

Microshoots were cultured in growing chambers with a

photoperiod of 16 h of light and 8 h of dark, a temperature

of 25 ± 2�C, a photosynthetic photon flux of 40 lmol m-2

s-1, and a relative humidity of 60%.

In vitro acclimatization treatments

Once the second sub-culture was completed, S2 plants were

divided into three treatment groups, maintaining all the

groups in the same medium (MS supplemented with

0.44 lM of BAP and 0.49 lM of IBA). The control group

was maintained under PFD of 40 lmol photons m-2 s-1 in

an unventilated flask,. The second group was cultivated

under 100 lmol photons m-2 s-1 of PFD, representing the

light treatment. And the third group, the ventilation treat-

ment, was cultivated with ventilated vessels under 40 lmol

photons m-2 s-1 of PFD. The temperature and photoperiod

were constant in all treatments. Thus, the effect of light and

ventilation were independently analyzed over the morpho-

physiological characteristics of G. avellana microshoots.

Chlorophyll fluorescence

Fluorescence signals were measured with a pulse-amplitude

modulated fluorimeter (FMS 2, Hansatech Instruments) in

94 Plant Cell Tiss Organ Cult (2012) 110:93–101

123

the upper microshoot leaves, in both in vitro treatment and

nursery plants. Microshoots were adapted to darkness for

30 min. The different light pulses were applied following

fluorimeter standard protocols described by Van Kooten

and Snel (1990). Minimal fluorescence (F0) was determined

by applying a weak modulated light (0.4 lmol m-2 s-1),

and maximal fluorescence (Fm) was induced by a short

pulse (0.8 s) of saturating light (9,000 lmol m-2 s-1).

After 10 s, an actinic light was turned on for 5 min to obtain

fluorescence parameters during steady-state photosynthesis.

Saturating pulses were applied after a steady-state photo-

synthesis was reached to determine maximal fluorescence

in the light (Fm0) and steady-state fluorescence in the light

(Fs0). Finally, the actinic light was turned off and immedi-

ately a 2 s far-red (FR) pulse was applied to obtain minimal

fluorescence after light-driven steady state (F00). Electron

transport rate (ETR) was calculated according to Genty

et al. (1989) as: ETR = 0.5 9 APSII 9 0.84, where APSII

is the effective quantum yield of PSII and PFD is the inci-

dent photosynthetic photon flux. The 0.5 factor assumes that

the efficiency between the two photosystems is equal and

the light is equally distributed on them, while the 0.84 factor

is the mean value of absorbance for green leaves (Demmig-

Adams et al. 1987). Non-photochemical quenching was

calculated according to Bilger and Bjorkman (1990) as:

NPQ = (Fm - Fm0)/Fm0. Photochemical quenching was

calculated as: qP = (Fm0 - Fs)/(Fm0 - F00). Fluorescence

measurements were performed at PFDs of 1, 90, 193, 279,

348, 564, 786 and 983 lmol m-2 s-1.

Chlorophyll and carotenoid concentrations

Pigment extraction and determination were made using a

spectrophotometer, following the methodology described

by Lichtenthaler and Wellburn (1983). For this, 250 mg of

fresh matter were macerated with 10 mL of 80% acetone;

the process was performed under dark and cold conditions

to avoid pigment degradation.

Soluble carbohydrates and starch determination

Soluble carbohydrates and starch extraction was performed

using 200 mg of dry matter with perchloric acid, following

the methodology described by McCready et al. (1950).

Quantification was obtained with the colorimetric method

using anthrone and glucose (5.55 lM) as a standard

(Steubing et al. 2002).

Foliar anatomy

Foliar sections of microshoots from in vitro culture and

nursery plants were fixed in a FAA solution (formalde-

hyde-acetic acid-alcohol). Then, samples were dehydrated

through increasing series of alcohol and embedded in

paraffin wax. Transversal sections of 20 lm thick were

sliced with a Leica RM2035 microtome and stained with

safranine and fast-green (Ruzin 1999). Photographs were

taken with a camera coupled to an Olympus microscope

and analyzed with Micrometric SE Premium and Image J

softwares. We measured parenchyma, palisade paren-

chyma, spongy parenchyma, and total foliar widths.

Experimental design and statistical analysis

We used a complete random design to evaluate the effect

of the three acclimatization treatments (control, light, and

ventilation) and nursery plants. The sample unit was one

glass vessel with three microshoots in the growing medium

with three replicates each. Data analysis consisted of one-

way ANOVA and differences between treatments were

established with a LSD test (P B 0.05). The fluorescence

curves were analyzed with one-way repeated measured

analysis.

Results

Chlorophyll a fluorescence

Maximum efficiency of PSII (Fv/Fm) did not show sig-

nificant differences between treatments (Table 1) and was

similar to the values found in nursery plants.

Both photochemical PSII efficiency (UPSII) and elec-

tron transport rate (ETR) (Fig. 1a, b) were significantly

different among treatments. Control and ventilation treat-

ments showed that ETR did not exceed 22 lmol m-2 s-1.

Due to the higher PFD, the light treatment showed a sig-

nificant increase in ETR, achieving a maximum value of

45.4 lmol m-2 s-1. The decrease in UPSII was not as

drastic compared to the values observed in the control and

ventilation treatments that had the same light intensity.

UPSII and ETR from control and ventilation treatments

presented values significantly lower than those observed in

nursery plants. However, the light treated microshoots

showed similar values to nursery plants.

Table 1 Effects of light and ventilation treatments on maximum PSII

efficiency (Fv/Fm) of Gevuina avellana leaflet grown in vitro

Treatments Fv/Fm

Control 0.795 ± 0.0208a*

Light 0.775 ± 0.0251a

Ventilation 0.759 ± 0.0380a

Nursery 0.779 ± 0.0244a

* Means in each column followed by different lowercase letters are

significantly different (P B 0.05) according to the LSD test

Plant Cell Tiss Organ Cult (2012) 110:93–101 95

123

Regarding the fluorescence quenching parameters,

photochemical quenching (qP) and non-photochemical

quenching (NPQ) (Fig. 1c, d) differed between in vitro

treatments. qP decreased with the increasing PFD in all

treatments. The highest qP value was found in the light

treatment at 90 lmol m-2 s-1. Ventilation and control

treatments at the same PFD presented the lowest values of

qP, this trend was maintained until 786 lmol m-2 s-1. The

light treatment also had significantly higher values of NPQ

compared to the control and ventilation treatments. The

maximum NPQ in the light treatment was observed at

983 lmol m-2 s-1, whereas in the control and ventilation

treatments the NPQ value was found at 1 lmol m-2 s-1.

Nursery plants presented the highest values for all param-

eters measured (Fig. 1) compared to in vitro treatments.

Pigment content

Concentrations of chlorophyll a and b were significantly

affected by in vitro treatments (Fig. 2a). Chlorophyll a

increased with PFD application and ventilation in relation

to the control treatment and nursery plants. Concentrations

of chlorophyll b had the greatest increment in those mi-

croshoots in the ventilated vessels, reaching values similar

to those of nursery plants. Thus, chl a/chl b (Fig. 2b) ratio

was lower in nursery plants than in vitro microshoots from

control and light treatment, the ventilated microshoots

showed lower chl a/chl b ration than control treatment, and

similar values than nursery plants. Thus, chl a/chl b ratio

(Fig. 2b) was lower in nursery plants compared to in vitro

microshoots. However, the ventilation treated microshoots

were significantly different than the control treatment, but

did not differ significantly compared to the nursery plants.

Soluble carbohydrates and starch concentration

Soluble carbohydrates were significantly different among

in vitro acclimation treatments (Fig. 3). In the light treat-

ment the increase in PFD caused a gain in soluble carbo-

hydrates in relation to control and nursery plants. The

ventilation treatment showed no significant differences

compared to the control and light treatments. Nursery

plants showed higher starch concentration compared to in

vitro microshoots. In vitro treatments did no show signifi-

cant difference in starch concentration among them.

Nursery plants also showed higher starch concentration

compared to soluble carbohydrate content. An opposite

trend was observed on in vitro treatment, where there was

lower starch concentration compared to the soluble car-

bohydrate content.

Fig. 1 Effective quantum yield of PSII a electron transport rate, b photochemical quenching (c) and non-photochemical quenching d on

Gevuina avellana Mol. microshoots cultured in vitro in response to increased light intensities

96 Plant Cell Tiss Organ Cult (2012) 110:93–101

123

Foliar anatomy

Nursery plants showed a mesophyll, spongy parenchyma

and palisade parenchyma significantly thicker than in vitro

microshoots (Fig. 4). The leaves of nursery plants devel-

oped a greater spongy and palisade parenchyma, with

elongated cells in the palisade parenchyma. When com-

paring in vitro microshoots with the control treatment we

observed many air spaces across the leaf and no differen-

tiation between palisade and spongy parenchyma. We

observed a more differentiated parenchyma with less air

spaces (Fig. 5) when comparing light and ventilation

treatment. However, the light treated microshoots pre-

sented an anatomy that is more similar to nursery plants.

Discussion

In vitro traditional culture promotes the development of

deficient anatomical and physiological characteristics.

These plants are not able to endure the stress when they are

transferred to ex vitro conditions, resulting in high mor-

tality rates. However, this can be reversed by managing

environmental conditions during in vitro culture.

When in vitro plants are transferred to ex vitro condi-

tions, photosynthesis is highly affected due to an increase

in PFD (Walters 2005). In vitro cultivated microshoots

showed high values for the maximum photochemical effi-

ciency of PSII (Fv/Fm) indicating a lack of excess absor-

bed energy. This also implies that an increase in PFD is

enough to influence the other fluorescence parameters

(ETR, APSII, qP and NPQ) without producing harmful

effects on the photosynthetic apparatus. Regarding the

photoprotective mechanisms, Demmig-Adams and Adams

(1992) have indicated that it is achieved by dissipating the

excess energy that could cause damage through photo-

chemical use of energy (qP) and thermal dissipation

(NPQ). In the case of G. avellana, we observed an increase

of NPQ in the light treatment indicating that these micro-

shoots developed a mechanism to manage the excessive

energy through thermal dissipation. These results are

Fig. 2 Pigment content of

Gevuina avellana micro shoots

under different in vitro

acclimation treatments (control,

light and ventilation treatments)

and nursery plants. Chlorophyll

concentration a and b and

carotenoids (a) and Chl a/ b

ratios (b). Different lowercaseletters are significantly different

(P B 0.05) according to the

LSD test

Fig. 3 Carbohydrates and starch concentration of Gevuina avellana

leaflets grown under different acclimations in in vitro treatments

(control, light and ventilation treatments). Different lowercase lettersare significantly different between treatments (P B 0.05) according to

the LSD test

Fig. 4 Foliar anatomy (total width, and mesophyll, spongy meso-

phyll and palisade mesophyll widths) of Gevuina avellana leaflets

grown under different in vitro acclimation treatments and foliar

anatomy of nursery plants. Different lowercase letters are signifi-

cantly different between treatments (P B 0.05) according to the LSD

test

Plant Cell Tiss Organ Cult (2012) 110:93–101 97

123

similar to the results observed in nursery plants. The

increase in NPQ prevents over-reduction of the electron

transport chain, increasing the protection against photo-

damage, a characteristic that is also found in studies of

other plant species (Demmig-Adams and Adams 1995;

Park et al. 1996; Semoradova et al. 2002). In the same way,

the increase of qP in the light treatment indicates the

development in photosynthetic ability, because these high

values are related to the presence of QA in the oxidized

state (Schreiber et al. 1986), which is consistent with high

ETR and APSII values found in the microshoots with

increased PFD. On the other hand, control and ventilated

microshoots showed significantly lower values in qP and

NPQ than those observed in the light treatment and nursery

plants. The lack of a photo-protection mechanism in both

control and ventilated treatments may lead to the formation

of reactive oxygen species once microshoots are trans-

ferred to ex vitro conditions (Muller et al. 2001), and these

responses are likely related to the low PFD at which these

microshoots were developing during in vitro culture (Saez

et al. 2012a). These results also indicate that quenching

capacity can be altered through in vitro acclimatization

(Fracheboud and Leipner 2003).

Moreover, the augment in APSII and ETR of micro-

shoots in the light treatment indicates an increase in pho-

tosynthetic rates and finally in reserves, which allows to

conclude that plants cultivated under higher PFD are con-

tributing to growth through photosynthesis (Fila et al.

1998). This is consistent with the increase in soluble car-

bohydrates concentration in the light treatment, which

agrees with Amancio et al. (1999) showing that a higher

PFD (90 lmol m-2 s-1) increases carbohydrates concen-

tration in V. vinifera micro plants, indicating that autotro-

phy acquisition is promoted by light. Similar results have

been shown by Ticha et al. (1999) and Kadlecek et al.

(2001) in Nicotiana tabacum, and Fuentes et al. (2005) in

Cocos nucıfera. On the other hand, the increase in carbo-

hydrates observed in the ventilation treatment could be

explained by the increase in CO2 inside the culture vessels

(Serret et al. 1997; Cournac et al. 2001; Badr et al. 2011).

This has been demonstrated by Lian et al. (2002), indi-

cating that Limonium ‘‘Misty Blue’’ micro cuttings that

grow under photomixotrophic condition (30 g L-1 sucrose

and CO2) have higher concentrations of starch, reducing

and non-reducing sugars.

Although chlorophyll content is not directly related to

the photosynthetic capacity (Fujiwara et al. 1992), it is a

good indicator of the photosynthetic apparatus status. Thus,

the raise in PFD caused an increase in chlorophyll a content

compared to the control treatment, indicating a better

antennae organization (Saez et al. 2012b), which is

reflected by an improvement in the fluorescence parameters

Fig. 5 Cross-section of Gevuina avellana leaves of control (a), light (b) and ventilation treatments (c) and for nursery plants (d). Bars represent

100 lm in a, b and c and 50 lm in d

98 Plant Cell Tiss Organ Cult (2012) 110:93–101

123

previously described. On the other hand, a high chl a/chl b

ratio observed in in vitro microshoots is in agreement with

a less developed photosynthetic apparatus and low photo-

synthetic activity compared to nursery plants (Fig. 2)

(Serret et al. 1997). The results observed in the ventilation

treatment agree with those of Chanemougasoundharam

et al. (2004) for Solanum tuberosum, where ventilation

caused a significant increase in the chlorophyll content.

This response can be related to a decrease in ethylene

concentration inside the vessels. Hazarika (2006) reported

that the accumulation of this gas causes the degradation of

photosynthetic pigments, and enhances the formation of

hyperhydric microshoots (Gaspar 1986). The hyperhyd-

ricity characteristics, such as lack of mesophyll differen-

tiation and big intercellular spaces (Kevers et al. 2004),

were observed in the leaves of the control treatment

(Fig. 5a), this has been reported in several in vitro cultured

species such as Helianthus annuus (Fauguel et al. 2008),

Cynara scolymus (Debergh et al. 1981), Dianthus cario-

phyllus (Ziv et al. 1983), Castanea sativa (Vieitez et al.

1985) and Pityopsis ruthii (Wadl et al. 2011), among oth-

ers. Therefore, both increase in light intensity and venti-

lation promote a better development of palisade

parenchyma, better differentiation between the palisade

and spongy parenchyma, and a lower number of intercel-

lular spaces. The effect of an increase in PFD is supported

by Goncalves et al. (2000) in C. sativa, where microshoots

presented thicker leaves and a more organized parenchyma.

The improvement of histological parameters in the light

and ventilation treatments is beneficial, because the ana-

tomical abnormalities observed in the control treatment

result in dehydration during transfer to ex vitro conditions

(Apostolo and Llorente 2000), which is the main cause of

mortality in microshoots.

Finally, both light and ventilation treatments improved

the physiological characteristics of G. avellana micro-

shoots that were produced in vitro. However, the in vitro

light treatment was more successful in improving photo-

chemical efficiency, developing mechanisms for excess

light dissipation and improving morphological character-

istics compared to the ventilation treatment. It is also

important to highlight that these characteristics were

accomplished during the in vitro process and can help the

plants endure the stress of ex vitro transfer. In addition, it is

important to evaluate the effects of both treatments on this

specie and the different stages of in vitro culture in future

research, and if the changes acquired during the in vitro

treatment application affect the behavior during the transfer

to ex vitro conditions.

Acknowledgments We thank Dr. Leon Bravo of the University of

Concepcion for providing the pulse-amplitude modulated fluorimeter

FMS2.

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