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Fast growing aspens in the development of a plantmicropropagation system based on plant-producedethylene action

Jonas �Ziauka a,b,*, Sigut _e Kuusien _e a,b, Mindaugas �Silininkas b

a Institute of Forestry, Lithuanian Research Center for Agriculture and Forestry, Liepu str. 1, Girionys, LT-53101

Kaunas District, Lithuaniab Joint-stock Company “Euromediena”, S. Daukanto a. 2/10, LT-01122 Vilnius, Lithuania

a r t i c l e i n f o

Article history:

Received 15 June 2012

Received in revised form

20 December 2012

Accepted 9 January 2013

Available online 13 February 2013

Keywords:

Aspen

Hormone

In vitro

Shoot proliferation

Short rotation forestry

Abbreviations: ACC, 1-aminocyclopropane* Corresponding author. Institute of Forestry,

547446.E-mail address: ziauka@gmail.com (J. �Zia

0961-9534/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.biombioe.2013.01.0

a b s t r a c t

Representatives of the genus Populus (poplars), such as Populus tremula L. (European aspen)

and its fast-growing hybrids, are recognized as being among the most suitable tree species

for short rotation coppicing in Northern Europe. Several technologies have been developed

for fast propagation of selected aspen genotypes, including laboratory (in vitro) micro-

propagation, which is usually based on the action of exogenous plant hormones. Seeking to

minimize the use of the latter, the present study was designed to test if the conditions

suitable for increased accumulation of plant-produced gas, including the gaseous plant

hormone ethylene, inside a culture vessel could contribute to commercially desirable

changes in aspen development. Shoot cultures of several European and hybrid (Populus

tremuloides Michx. � P. tremula) aspen genotypes were studied using two different types of

culture vessels: tightly sealed Petri dishes (15 � 54 mm) designed to provide restricted gas

exchange (RGE) conditions, and capped (but not sealed) test tubes (150 � 18 mm) providing

control conditions. Under RGE conditions, not only the positive impact of the ethylene

precursors 1-aminocyclopropane-1-carboxylic-acid (ACC) and ethephon on shoot pro-

liferation was demonstrated but also a several-fold increase, compared to the control

conditions, in the mean shoot number per explant was recorded even on the hormone-free

nutrient medium. Moreover, the shoots developed under RGE conditions were dis-

tinguished by superior rooting ability in the subsequent culture. These results suggest that

a plant micropropagation system based on the action of plant-produced ethylene rather

than of exogenous hormones is possible.

ª 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Fast-growing poplars (Populus) are recognized as a suitable

model for biomass-related scientific studies [1]. In relation to

-1-carboxylic-acid; PGR, pLiepu str. 1, Girionys, LT-

uka).ier Ltd. All rights reserve05

short rotation forestry, a special interest in Northern Europe,

including Scandinavian and Baltic countries, is paid to native

aspen (Populus tremula L.) and its hybrids (particularly with

North American Populus tremuloides Michx.) distinguished

lant growth regulator(s); RGE, restricted gas exchange.53101 Kaunas District, Lithuania. Tel.: þ370 672 04121; fax: þ370 37

d.

b i om a s s a n d b i o e n e r g y 5 3 ( 2 0 1 3 ) 2 0e2 8 21

by their fast growth [2e4]. Several technologies have been

developed for the fast propagation of selected aspen geno-

types, varying from root cuttings [5] to laboratory (in vitro)

micropropagation [6]. In vitro culture provides the opportunity

to obtain the largest number of new shoots during a short

period of time; however, it is usually based on extensive use of

exogenous plant hormones, particularly auxins and cytoki-

nins [7]. This results not only in increased costs but also in

some degree of uncertainty about the nature and persistence

of possible side effects, since exogenously applied hormones

can variously interact with plant-produced hormones.

Therefore, an approach should be developed formore efficient

exploitation of the latter, instead of using the former.

In plant tissues, auxins and cytokinins, as well as a variety

of stressful environmental factors, are known to increase the

synthesis of ethylene gas, which is also counted among plant

hormones [8,9]. Many authors suggest that ethylene accu-

mulation or signaling should be restricted in order to achieve

better shoot regeneration and growth [10e12], or enhanced

rooting [13,14]; however, some claim a positive role for eth-

ylene in shoot and root development [15,16]. Thus, the present

study was designed to see if the establishment of in vitro

conditions suitable for the increased accumulation of plant-

produced gas inside a culture vessel could induce commer-

cially desirable changes in plant development and, if so, how

these changes are related to the action of ethylene.

2. Materials and methods

2.1. Plant material and growth conditions

The present study involved three Populus genotypes (Table 1)

cloned under laboratory conditions.

At the start of the cloning process, proliferating shoot

cultures were established from 2 to 3 cm long segments (car-

rying at least one vegetative bud) of young aspen twigs that

were collected from the middle part of the crown in early

spring, just before the bud-break. These shoot cultures were

maintained in vitro for several years through successive pas-

sages which were usually done every two months. During

initial phase, the cultures were grown on a solidified (with

8.5 g L�1 phytoagar) Woody Plant Medium (WPM [17]) con-

taining 25 g L�1 sucrose and 0.5 mg L�1 6-benzylaminopurine

(BAP; Duchefa Biochemie, Haarlem, The Netherlands). After

several passages, BAP was excluded from the medium com-

position and, for a period of at least one year, the cultures

were grown on a WPM free of plant growth regulators (PGR).

Table 1 e Data on Populus genotypes and their respective dono

Tree code in the Lithuanian forestseed base catalog; species

Location of thedonor tree

Tree p

Age,

DPL038; P. tremula L. 55�150 N; 23�200 EDPL037; P. tremula L. 55�220 N; 22�140 EDF1001; P. tremuloides Michx. � P. tremula L. 54�520 N; 24�070 E

In the subculture previous to the experiments, cultures

were grown on such a PGR-free medium for a period of

approximately two months (if not stated otherwise). Apical

stem segments carrying two to three buds (including an apical

bud) were used for the experiments. In some of the experi-

ments, nodal stem segments (without apical bud) were also

involved.

The basal WPM without any additional compounds (PGR-

free) was used as control medium in all experiments. In cer-

tain experiments aimed at the study of ethylene’s influence,

the nutrient medium was supplemented with 2-chloroethyl

phosphonic acid (ethephon, an ethylene-releasing com-

pound [18]) or 1-aminocyclopropane-1-carboxylic-acid (ACC, a

natural precursor of ethylene whose conversion to ethylene is

catalyzed by the enzyme ACC oxidase [19]). These chemicals

were obtained from SigmaeAldrich Laborchemikalien GmbH

(Seelze, Germany) and SigmaeAldrich Chemie GmbH (Stein-

heim, Germany), respectively. Also, some of the experiments

involved ethylene signal inhibitor [20] silver nitrate (AgNO3;

Duchefa Biochemie, Haarlem, The Netherlands). ACCwas first

dissolved in 0.5 mL of 1 mmol L�1 NaOH and then diluted with

distilled water to a 50 mL volume, while ethephon and AgNO3

were dissolved in 50mL distilled water (pH value for ethephon

solutionwas setwell below 4.0). All PGR solutionswere filtered

using a 0.22 mm syringe-driven filter prior to adding them (at

the appropriate volume) to the autoclaved nutrient medium,

while the pH value of the medium was adjusted to 4.8 before

autoclaving for 30 min at 121 �C.Glass test tubes and polystyrene Petri dishes were used for

culturing explants. More detailed characteristics of the con-

ditions related to these different culture vessels are given in

Table 2. Here, test tubes are considered to provide control

conditions since they were routinely used while subculturing

Populus explants prior to this study.

In each case, a single explant was provided with 5 mL of

nutrient medium. All cultures were maintained in controlled

environmental conditions under a 16 h photoperiod (white-

light; irradiance 30 mmolm�2 s�1) and a temperature regime of

25 �C/18 �C during day and night conditions.

2.2. Experiments

For testing the impact of ethylene on aspen shoot develop-

ment, the basal nutrient medium for aspen DPL038 explants

was enriched with ethylene precursors ACC and ethephon (at

the concentrations of 1 mmol L�1, 3 mmol L�1, and 5 mmol L�1).

DPL038 responses to the aforesaid ethylene precursors were

tested both under control and under RGE conditions.

r trees involved in the study.

arameters at the moment of collection of primary explants

years Height, m Stem diameter(at the height of 1.3 m), m

70 33 0.64

70 33 0.66

25 24 0.33

Table 2 e Different culture conditions provided for in vitro experiments.

Conditions Culture vessel Covering/sealing Explant position Number of explantsinside a vessel

Vessel volume for asingle explant

Control Test tubes

(150 � 18 mm)

Plastic caps Vertical 1 w33 mL

Restricted gas

exchange (RGE)

Petri dishes

(15 � 54 mm)

Parafilm

(two layers)

Horizontal 2 w12 mL

b i om a s s an d b i o e n e r g y 5 3 ( 2 0 1 3 ) 2 0e2 822

In an additional experiment aimed at testing the role of

ethylene in the morphogenetic changes induced by RGE con-

ditions, DPL038 explants cultured on basal PGR-free medium

under control conditions were compared to the explants cul-

tured under RGE conditions either on PGR-free medium or on

the media enriched with AgNO3 (20 mmol L�1), either alone or

in combination with ethephon (3 mmol L�1).

The comparison of the responses of aspen explants from

all three different clonal lines (Table 1) to RGE conditions was

made by culturing these explants on PGR-free medium either

under control or under RGE conditions.

In order to make further comparison between the aspen

genotypes DPL038 and DPL037 as well as to evaluate the

influence of the previous subculture’s duration on shoot

development under RGE conditions, shoot cultures of the

aforesaid genotypes were grown for either eight or 18 weeks

under control conditions and then transferred to RGE con-

ditions (either on a PGR-free medium or on one enriched with

ethephon at 3 mmol L-1).

For testing the longer-term impact of RGE conditions,

development of hybrid aspen DF1001 in vitro cultures was

monitored through two subcultures. During the first sub-

culture, apical and nodal DF1001 explantswere cultured either

under control or under RGE conditions, while all the shoots

formed in either type of culture vessels were transferred to

control conditions for the subsequent subculture.

2.3. Data analysis

In all the experiments, each distinct treatment consisted of

three replicates, 16e20 explants per replicate, and these were

organized in a completely randomized design. Experimental

data were collected after eight to ten weeks following culture.

For each particular explant, main shoot length, largest leaf

width, and number of proliferating shoots were recorded. In

the experiment on the development of hybrid aspen DF1001

explants through two subcultures, the number of primary

Table 3 e Shoot development from aspen DPL038 apical stemconditions (in test tubes).

Concentration of a givencompound in the medium,mmol L�1

Shoot length, mm

ACC Ethephon

0 27.3 � 1.6 a 1

1 23.7 � 1.9 a 26.5 � 2.4 a 1

3 18.1 � 1.9 b 24.0 � 2.4 ab

5 12.5 � 1.1 c 27.4 � 1.7 a

Means followed by the same letter are not significantly (P < 0.05) differen

roots per explant was also recorded after each subculture. For

the comparison of the obtained means, a two-tailed Welch’s

t-test intended for use with samples having possibly unequal

variances [21] was performed in Microsoft Excel 2003, calcu-

lating the probability that the means of two different treat-

ments are equal.

3. Results

3.1. Effects of ethylene precursors on aspen developmentunder different culture conditions

The data covering the effects of ethylene precursors ACC and

ethephon on aspen DPL038 apical stem segments under con-

trol conditions are presented in Table 3. The mean shoot

length and leaf width were significantly decreased by

increasing ACC concentrations but not by ethephon. The lat-

ter did not have any impact on shoot development at all. The

mean shoot number remained close tominimal (one shoot per

explant) in all the variants tested.

In contrast, when the same concentrations of ethylene

precursors were tested under RGE conditions (Table 4), the

mean shoot numberwas not only significantly affected by PGR

treatments but also, even on PGR-free medium, exceeded the

corresponding value obtained under control conditions by

several times. Under RGE conditions, shoot proliferation was

increased in a concentration-dependent manner by both ACC

and ethephon. At the concentration of 5 mmol L�1, both of the

aforesaid ethylene precursors increased the mean shoot

number by approximately two times. In contrast, different

ethylene precursors had opposite effects in respect of shoot

elongation, although, similarly as with shoot number, the

mean shoot length under RGE conditions far exceeded the

corresponding value obtained under control conditions.

Under RGE conditions, the maximum mean shoot length was

reached at 3 mmol L�1 ethephon, while the lowest value was

segments on different nutrient media under control

Leaf width, mm Shoot number per explant

ACC Ethephon ACC Ethephon

1.0 � 0.4 a 1.1 � 0.1

0.3 � 0.3 a 10.7 � 0.5 a 1.1 � 0.1 1.1 � 0.1

8.9 � 0.5 b 10.5 � 0.4 a 1.3 � 0.1 1.3 � 0.1

7.6 � 0.3 c 10.4 � 0.4 a 1.2 � 0.1 1.4 � 0.1

t from each other.

Table 4 e Shoot development from aspen DPL038 apical stem segments on different nutrient media under restricted gasexchange conditions (in sealed Petri dishes).

Concentration of a givencompound in the medium,mmol L�1

Shoot length, mm Leaf width, mm Shoot number per explant

ACC Ethephon ACC Ethephon ACC Ethephon

0 64.7 � 4.2 bc 5.1 � 0.3 ab 4.2 � 0.6 c

1 62.7 � 5.6 bc 76.7 � 5.9 ab 5.3 � 0.4 ab 5.5 � 0.3 ab 6.8 � 0.9 ab 5.9 � 0.6 b

3 58.3 � 5.7 c 79.8 � 3.9 a 6.2 � 0.5 a 4.9 � 0.3 b 7.6 � 0.6 ab 6.7 � 0.6 ab

5 39.1 � 3.4 d 69.9 � 4.5 abc 5.9 � 0.4 ab 5.2 � 0.4 ab 8.3 � 0.8 a 8.2 � 0.7 a

Means followed by the same letter are not significantly (P < 0.05) different from each other.

***

***

***

4

6

8

10

12

14

est leaf w

id

th

, m

m

******

***

0

20

40

60

80

100

DPL038 DPL037 DF1001

Main

sh

oo

t len

gth

, m

m

Control Restricted gas exchange

b i om a s s a n d b i o e n e r g y 5 3 ( 2 0 1 3 ) 2 0e2 8 23

recorded at 5 mmol L�1 ACC (in both cases, the differences from

PGR-freemediumwere significant). Meanwhile, the mean leaf

width under RGE conditions was approximately two times

smaller than in control test tubes and remained unaffected by

any of the ethylene precursors.

3.2. Assessment of the role of ethylene in aspen shootproliferation

The results of an additional experiment which involved silver

nitrate (AgNO3) and ethephon treatments indicated that eth-

ylene should be responsible for the vigorous shoot pro-

liferation observed under RGE conditions (Fig. 1). The strong

increase in themean shoot number per explant caused by RGE

conditions was partially reversed by the presence of AgNO3 in

the medium. In turn, the negative effect of AgNO3 on shoot

proliferation was partially overcome by the addition of

ethephon.

3.3. Effect of restricted gas exchange on different aspengenotypes

The experiment whose results are presented in Fig. 2 further

confirmed the ability of RGE conditions to induce radical

changes in aspen shoot development. Aspen explants

c

b

a

a

0

1

2

3

4

5

6

Control RGE RGE +

AgNO3

RGE +

AgNO3 +

Ethephon

Sh

oo

t n

um

ber p

er exp

lan

t

Fig. 1 e Shoot formation on aspen DPL038 explants, grown

either in test tubes (control) or sealed Petri dishes

(restricted gas exchange; RGE). AgNO3 and ethephon

treatments involved 20 mmol LL1 AgNO3 and 3 mmol LL1

ethephon, respectively, added to the nutrient medium

under RGE conditions. Significantly different means

(P < 0.05) are labeled with different letters.

cultured on PGR-freemedium under RGE conditions produced

considerably longer shoots with narrower leaves than the

explants grown on the corresponding medium under control

conditions. These differences were observed in all three of the

***

***

*

0

2

4

6

8

10

DPL038 DPL037 DF1001

Sh

oo

t n

um

ber p

er e

xp

lan

t

0

2

DPL038 DPL037 DF1001

Larg

Fig. 2 e Shoot development from the explants of three

Populus sp. genotypes, grown either in test tubes (control)

or sealed Petri dishes (restricted gas exchange). Significant

differences between the samples grown under different

conditions are indicated as follows: *P < 0.05, **P < 0.01,

***P < 0.001.

b i om a s s an d b i o e n e r g y 5 3 ( 2 0 1 3 ) 2 0e2 824

aspen genotypes tested (Fig. 2). RGE conditions also yielded an

increased shoot multiplication rate, with aspen explants

forming between 1.7 (DPL037) and 6.7 (DPL038) times more

shoots than under control conditions.

3.4. Influence of previous subculture’s duration on shootdevelopment under restricted gas exchange

Aspen explants transferred to RGE conditions after a pro-

longed period of time (18 weeks) in the previous subculture

differed in some aspects of shoot development from fresher

(eight-week old) explants (Table 5). In genotype DPL038, the

shoots produced on PGR-free medium by older explants dif-

fered from those produced by fresher explants only in respect

of leaf width (which was larger in the case of older explants).

More significant differences between fresher and older

explants were observed in the case of the medium enriched

with 3 mmol L�1 ethephon. On this medium, fresher explants

produced shorter shoots but the number of shoots produced

was much larger than for older explants. Meanwhile, fresher

DPL037 explants cultured on the medium with ethephon

produced somewhat longer shoots than their older counter-

parts, while the difference in this respect between fresher and

older explants was insignificant on PGR-free medium. The

differences determined by the previous subculture’s duration

weremuchmore significant in respect to leaf width and shoot

number per explant. A prolonged period for the preceding

subculture led to an increase in leaf width and to a decrease in

number of shoots. These differences were significant even on

PGR-free medium and were further increased on the medium

with ethephon. Thus, the pattern of differences between

fresher and older explants in respect of shoot multiplication

was basically the same in the both aspen genotypes: the

ethylene-releasing compound ethephon increased shoot

numbers in fresher but not in older explants. Moreover, it was

observed that, by themean shoot number per explant on PGR-

free medium, fresher DPL038 explants exceeded the corre-

sponding DPL037 explants only by 1.3 times while this dif-

ference reached 2.2 times in the case of older explants.

3.5. Assessment of hybrid aspen development throughtwo subcultures

The experiment conducted on both apical and nodal explants

of hybrid aspen DF1001 and continued through two

Table 5e Shoot development from the apical stem segments ofgas exchange conditions (in sealed Petri dishes).

Nutrient medium Duration ofprevious subculture

(weeks)

Shoot length, m

DPL038 DPL

PGR-free 8 56.8 � 3.5 ab 57.4 �18 48.4 � 4.3 bc 61.6 �

Ethephon, 3 mmol L�1 8 45.3 � 4.4 c 70.4 �18 62.2 � 4.3 a 58.8 �

Means followed by the same letter within a column are not significantly

subcultures showed that developmental abnormalities (such

as narrow leaves) related to culture of explants under RGE

conditions do not lead to impaired plant development in the

subsequent culture. In the first subculture, RGE conditions led

to increased shoot length in apical explants, while the largest

leaf width was decreased in both apical and (even more sig-

nificantly) nodal explants (Fig. 3a). However, when the shoots

developed in the previous stage from differently cultured

explants were compared during subsequent cultures, it was

found that shoots developed from explants which originally

lacked an apical bud and were planted in Petri dishes for the

first subculture were longer than the shoots produced by

explants of the same type but cultured only under control

conditions (Fig. 3b). Meanwhile, no differences in respect of

leaf width were observed between the explants which

encountered different environmental conditions during the

previous subculture.

In respect of organogenesis during the first subculture, RGE

conditions led to increased shoot numbers in both apical and

nodal explants and to decreased root numbers in nodal

explants (Fig. 4a). Meanwhile, when uniform (control) con-

ditions were provided for the second subculture, the explants

previously cultured under RGE conditions were distinguished

by significantly enhanced root formation (Fig. 4b).

4. Discussion

4.1. Impact of restricted gas exchange on plantdevelopment and ethylene action

In vitro culture intended for rapid production of genetically

identical plant material is normally carried out in closed

vessels, thus protecting the aseptic culture from infection and

preventing desiccation of the plant and nutrientmedium. This

restricts gas exchange between the atmosphere inside the

vessel with that external to it, making growth and develop-

ment of plants or explants dependent not only on the com-

position of the nutrient medium but also on the composition

of the gaseous atmosphere. The physical properties of the

culture vessel and how it is sealed are among the most

important factors that affect the accumulation of gases [22].

Thus, in the present research, small-volume, tightly sealed

Petri dishes served as vessels designed to restrict gas

exchange in comparison with standard glass test tubes. Use of

two aspen genotypes (DPL038 andDPL037) under restricted

m Leaf width, mm Shoot number per explant

037 DPL038 DPL037 DPL038 DPL037

4.8 b 5.4 � 0.4 b 6.4 � 0.4 b 5.8 � 0.7 b 4.4 � 0.6 b

4.4 ab 6.6 � 0.3 a 8.9 � 0.5 a 5.3 � 1.1 b 2.4 � 0.4 c

3.3 a 5.0 � 0.3 b 5.4 � 0.3 b 10.1 � 1.0 a 6.5 � 0.7 a

2.3 b 5.7 � 0.3 b 9.5 � 0.4 a 5.6 � 0.9 b 2.5 � 0.2 c

(P < 0.05) different from each other.

***

***

0

2

4

6

8

10

12

14

Apical Nodal

Explant type

La

rg

es

t le

af w

id

th

, m

m

0

2

4

6

8

10

12

14

Apical Nodal

Explant type in previous

subculture

La

rg

es

t le

af w

id

th

, m

m

**

0

10

20

30

40

50

60

Ma

in

s

ho

ot le

ng

th

, m

m

Control

Restricted gas

exchange

a

***

0

10

20

30

40

50

60

Ma

in

s

ho

ot le

ng

th

, m

m

Control

Restricted (previously)

gas exchange

b

Fig. 3 e Shoot growth parameters in the in vitro culture of hybrid aspen (DF1001) through two subcultures. The results for

both the first (a) and second (b) subcultures reflect explant-type and culture vessel differences of the first subculture.

Significant differences between the samples grown (during the first subculture) under control and restricted gas exchange

conditions are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

b i om a s s a n d b i o e n e r g y 5 3 ( 2 0 1 3 ) 2 0e2 8 25

the former led to significant morphogenetic changes, includ-

ing increased number of shoots produced by a single explant,

which was the most important outcome of aspen culture

under RGE conditions from a commercial point of view.

Since shootmultiplication in plants is usually related to the

activity of certain hormones, particular attention was given in

this case to the gaseous plant hormone ethylene. Although, in

standard protocols for plant micropropagation, various cyto-

kinins (and not ethylene gas or ethylene-releasing com-

pounds, such as ethephon) are usually applied to induce shoot

multiplication [23e25], there are nonetheless reports about

the positive role of ethylene in cytokinin-inducible organo-

genetic processes, such as axillary shoot proliferation [9,26].

However, accumulation of ethylene inside culture vessels is

not appreciated unambiguously. Many authors recommend

that ethylene accumulation in culture vessels should be

restricted by ventilation, adsorption, or ethylene synthesis

inhibition in order to avoid the impairment of plant develop-

ment by ethylene [11,27,28]. Nonetheless, the results of the

present study suggest that ethylene may have a decisive role

in the positive outcome of aspen shoot culture under restric-

ted gas exchange, since the ability of both ACC and ethephon

to enhance shoot proliferation on aspen explants in small-

volume Petri dishes was demonstrated. Moreover, shoot

proliferation was impaired by reported [20] ethylene signal

inhibitor AgNO3.

As indicated by the results of the present study, restricted

gas exchange, besides increased shoot proliferation, should be

also responsible for thedevelopmentof abnormally long shoots

with narrow leaves. Although ethylene gas is more frequently

reported as a hormone that inhibits plant shoot growth [8,29],

some authors suggest that the specific effect of ethylene on

shoot elongation depends on the particular interaction

between ethylene and growth hormone gibberellin, and this

interaction, in turn, depends on the particular environmental

conditions, such as relative humidity [30]. The studies con-

ducted on some flooding-resistant plant species (e.g., Rumex

palustris) reveal that strong enhancement of shoot growth

characteristics for these plantswhen in submerged conditions,

although directly regulated by gibberellin, is triggered by

accumulated ethylene gas produced by the plant and trapped

inside due to the low rate of underwater diffusion; this leads to

a subsequent increase in gibberellin levels [31e33]. This model

might contribute to an explanation of the increased shoot

elongation in Petri dishes, since, according to Jackson [34], both

the sealing of explants inside small-volume culture vessels and

submergence result in the similar restriction of gas exchange

between the internal and external environments.

****

0

1

2

3

4

5

6

Apical Nodal

Explant type in

previous subculture

Ro

ot n

um

ber p

er exp

lan

t

0

1

2

3

Sh

oo

t n

um

ber p

er exp

lan

t

Control

Restricted (previously)

gas exchange

***

0

1

2

3

4

5

6

Apical Nodal

Explant type

Ro

ot n

um

ber p

er exp

lan

t

* ***

0

1

2

3S

ho

ot n

um

ber p

er exp

lan

t

Control

Restricted gas

exchange

a b

Fig. 4 e Organogenesis parameters in the in vitro culture of hybrid aspen (DF1001) through two subcultures. The results for

both the first (a) and second (b) subcultures reflect explant-type and culture vessel differences of the first subculture.

Significant differences between the samples grown (during the first subculture) under control and restricted gas exchange

conditions are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

b i om a s s an d b i o e n e r g y 5 3 ( 2 0 1 3 ) 2 0e2 826

It is not completely clear whether there is a direct con-

nection between enhanced shoot elongation and adventitious

shoot formation observed under RGE conditions. Although the

previous research on aspen shoot cultures [35] revealed a

positive effect of gibberellins on both shoot elongation and

shoot proliferation, the results of the present study suggest

that the ability to promote shoot proliferation is quite a spe-

cific feature of ethylene. This is indicated by the similarities

between ethephon and ACC effects in respect of shoot pro-

liferation, in contrast to the other estimated parameters of

shoot development and, particularly, to shoot length. The

induction of shoot proliferation under restricted gas exchange

conditions could be also related to the ability of ethylene to

cause defects in apical dominance, which is a reasonably well

reported aspect of ethylene action. For instance, in Petunia,

ethylene interrupts apical dominance by decreasing the ratio

of auxin to cytokinin [36]. Sanyal and Bangerth [37], working

with apple seedlings and mature trees, confirmed the stim-

ulating effect of horizontal orientation of shoots on ethylene

production and subsequent interruption of polar auxin

transport which, in turn, is known to contribute largely to

apical dominance by preventing the outgrowth of axillary

buds [38]. Moreover, Wan et al. [39] demonstrated that polar

transport of auxin in the xylem parenchyma of aspen is an

important inhibitor of root suckering and adventitious shoot

formation. Since, during the present study, the culture of

aspen explants under RGE conditions involved not only the

use of small-volume Petri dishes but also the horizontal ori-

entation of explants in such vessels (vertical growth was

largely restricted by the small height of Petri dishes), ethylene-

induced shoot proliferation could have resulted not only from

restricted gas exchange but also from continuous mechanical

stress (caused by the covering of a vessel) and, subsequently,

increased ethylene production.

4.2. Practical implications of plant-produced ethylene inaspen micropropagation

In order to increase the economic effect of aspen breeding

for woody biomass production, selection of the most pro-

ductive aspen hybrids, genotypes, and clonal lines should be

accompanied by effective and convenient methods for veg-

etative propagation. Two main alternatives are usually sug-

gested in this context: either aspen propagation from root

cuttings [5,40] or micropropagation through in vitro culture

[6,7]. The latter option is more suitable for continuous plant

b i om a s s a n d b i o e n e r g y 5 3 ( 2 0 1 3 ) 2 0e2 8 27

multiplication on an industrial scale since it is more effective

in terms of the number of newly produced plants [6] and less

dependent on natural environmental conditions (which can

be favorable or unfavorable for root growth and root suck-

ering [41], therefore having a significant influence on the

success of plant propagation from root cuttings). On the

other hand, in vitro propagation is inseparable from the rel-

atively high costs necessitated by the specific technical

equipment required, as well as from the use of exogenous

plant hormones which are added to the nutrient medium

designed for aspen shoot multiplication. Since different

hormones are applied for two different purposes (cytokinins

for shoot multiplication and auxins for rooting), researchers

usually aim to determine a proper balance between these

hormones in the nutrient medium at different stages of

in vitro propagation [7]. However, an earlier study by Gon-

zalez et al. [16] shows that both shoot proliferation and root

formation during aspen in vitro culture depend on ethylene

action. Although the aforementioned work was conducted

using a standard shoot multiplication medium enriched with

both cytokinin (higher concentration) and auxin, it indicates

that positive results in aspen micropropagation can be ach-

ieved through enhanced ethylene action. Building on this

premise, the ability of RGE conditions to increase shoot

proliferation even on the medium free of any exogenous

hormones or their precursors was demonstrated during the

present study.

In order to support the exploitation of plant-produced

ethylene as a commercially attractive alternative to the use

of exogenous hormones, two main conditions must be ful-

filled. These include, firstly, a high rate of shootmultiplication

and, secondly, having a sufficiently short period between the

subcultures purported for shoot proliferation under RGE con-

ditions. The results of the present study indicated that these

two conditions are fully compatible. Fresher explants, as

compared to those planted after a prolonged previous sub-

culture, were more able to produce an increased number of

adventitious shoots under RGE conditions and, particularly,

under ethephon treatment, thus indicating that theymight be

more sensitive to ethylene than older explants. Meanwhile,

the data from the comparison of DPL038 and DPL037 explants

suggest that the differences between the rates of shoot pro-

liferation in different aspen clonal lines can be partially over-

come by the use of appropriate (for instance, fresher) explants.

In regard to the possibility to apply RGE conditions for shoot

multiplication in various aspen clonal lines, the case of the

genotype DF1001 should be noted. Although the donor tree

DF1001 (hybrid aspen) differed from the European aspen donor

trees DPL038 and DPL037 both by origin and by age, the ten-

dency to increasedshootproliferationby itsexplantsunderRGE

conditions was essentially similar. This suggests a potentially

wide application of the discussed approach which was found

helpful both for shoot multiplication and for subsequent root-

ing, since RGE conditions resulted not only in increased shoot

proliferation but also in increased root formation during the

subsequent culture. Moreover, certain morphological abnor-

malities (suchasnarrowleaves)observedunderRGEconditions

were not retained for the subsequent culture.

The overall results of the present study point to the fact

that the use of exogenous hormones can be replaced by

more ecological means, aiming at increased ethylene syn-

thesis and accumulation. For instance, the use of small-

volume unventilated vessels would be recommended for

aspen shoot multiplication on PGR-free nutrient medium.

Since ethylene production in plant tissues is known to be

enhanced by various abiotic stress factors [42], it seems

promising to study how shoot multiplication rates on PGR-

free medium could be further increased by some other

simple means of manipulation in regard to the tissue cul-

ture environment. Accordingly, the positive results

obtained so far in aspen cultures point to the potential

usefulness of equivalent studies on other fast-growing tree

species that are, either as well as poplars or as an alter-

native [43], recommended for the development of short

rotation forestry.

5. Conclusions

The results of this study indicate that in vitro conditions spe-

cifically designed for increased gas accumulation in a culture

environment lead to increased aspen shoot multiplication,

and therefore suggest the possibility of developing an efficient

plant micropropagation system based on the action of plant-

produced ethylene rather than on the use of exogenous

hormones.

Acknowledgments

The paper includes research findings which have been

obtained through the long-term research programme “Sus-

tainable Forestry and Global Changes” implemented by the

Lithuanian Research Center for Agriculture and Forestry.

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