Role of ethylene and jasmonic acid on rhizome induction and growth in rhubarb ( Rheum rhabarbarum L

ORIGINAL PAPER

Role of ethylene and jasmonic acid on rhizome inductionand growth in rhubarb (Rheum rhabarbarum L.)

Usha P. Rayirath • Rajasekaran R. Lada •

Claude D. Caldwell • Samuel K. Asiedu •

Kevin J. Sibley

Received: 31 May 2010 / Accepted: 30 September 2010 / Published online: 20 October 2010

� Her Majesty the Queen in Right of Canada 2010

Abstract Experiments were conducted to elucidate the

hormonal induction and regulation of rhizome growth in

rhubarb (Rheum rhabarbarum L.). It was found that eth-

ylene is the key regulator of rhizome induction and

development. The role of jasmonic acid (JA) and its

interaction with ethylene in rhizome induction and growth

were also examined. Both ethylene and JA have a signifi-

cant effect on promoting rhizome formation in vitro.

Conversely, the ethylene biosynthesis inhibitor aminoeth-

oxyvinylglycine (AVG) (1.5 lM) inhibited rhizome

induction in multiple-shoot clumps in vitro, and suppressed

the stimulatory effects of exogenously applied ethephon

(1 mg l-1) and JA (10 ng l-1) in promoting mini-rhizome

formation, further confirming the role of endogenous eth-

ylene in the process. In addition, rhizome growth was

significantly enhanced in the presence of both ethylene and

JA (ethephon 1 mg l-1 and JA 10 ng l-1) compared to JA

alone. These results suggest that endogenous ethylene is

the key regulator of rhizome growth in rhubarb and JA

promotes rhizome formation, possibly through inducing

endogenous ethylene synthesis.

Keywords Ethylene � Jasmonic acid � Hormonal

induction � Growth � Mini-rhizome � Rhubarb

Abbreviations

JA Jasmonic acid

ACC 1-Aminocyclopropane-1-carboxylic acid

AVG Aminoethoxyvinylglycine

PGRs Plant growth regulators

Introduction

Rhubarb (Rheum rhabarbarum L.) is a herbaceous cool-

season perennial vegetable having high market potential

for expansion as a commercial vegetable industry in North

America and Europe. The current method of propagation

by rhizome division often results in a very low rate of

propagule production; thus, enhancing rhizome growth is

critical to generate more propagules. The physiological

mechanisms and hormonal relationships that determine the

growth and enlargement of rhizomes in rhubarb are not

known (Rayirath et al. 2009). On the basis of the infor-

mation currently available on the role of plant growth

regulators (PGRs) in promoting vegetative storage organs,

it is expected that ethylene, jasmonic acid (JA), ethylene

gibberellic acid (GA3) and abscisic acid (ABA) can all

modulate the growth and development of these plant

organs (Jasik and de Klerk 2006; Bhatia et al. 1992; Kim

et al. 2005; Zaib-Un-Nissa and Rafiq 1980). Determining

the role(s) of these PGRs in manipulating rhizome growth

in rhubarb will help to identify the key regulators of the

rhizome formation process in rhubarb.

Plant hormones are the key factors that elicit various

physiological changes and modulate plant growth and

development under varying environmental conditions

(Ecker 1995; Etheridge et al. 2005). In general, exogenous

U. P. Rayirath � R. R. Lada (&) � C. D. Caldwell � S. K. Asiedu

Department of Plant and Animal Sciences, Nova Scotia

Agricultural College, 50 Pictou Road, P.O. Box 550,

Truro, NS B2N 5E3, Canada

e-mail: [email protected]

K. J. Sibley

Department of Environmental Sciences, Nova Scotia

Agricultural College, Truro, NS B2N 5E3, Canada

123

Plant Cell Tiss Organ Cult (2011) 105:253–263

DOI 10.1007/s11240-010-9861-y

application of PGRs can positively regulate the formation,

growth and development of various plant organs by acting

as chemical signals that alter source–sink relationships,

long-distance photoassimilate translocation, leaf senes-

cence and other developmental processes (Wardlaw 1990;

Morris 1996). The role of PGRs like JA, GA3, ABA, auxin

and cytokinin in inducing the formation and development

of storage structures like tubers and corms is well dem-

onstrated in several crop species (Bhatia et al. 1992; Jasik

and Mantell 2000; Jasik and de Klerk 2006; Caldiz 1996;

Kim et al. 2005; El-Antably et al. 1967; Zaib-Un-Nissa and

Rafiq 1980; Romanov et al. 2000).

In addition to its role as a key wounding hormonal signal

(O’Donnell et al. 1996; Leon et al. 2001), ethylene also

participates in regulating a variety of developmental pro-

cesses in plants, including seed germination, lateral bud

stimulation, adventitious rooting, overcoming dormancy

and organ senescence and abscission (Abeles et al. 1992).

Exogenous application of ethylene induces tuberisation in

potatoes and enhances root bulking in carrots (Vreugenhil

and van Dijk 1989; Bhatia et al. 1992; Neuteboom et al.

2002). These results suggest that ethylene could be a pos-

sible rhizome-inducing hormonal signal in rhubarb. How-

ever, the specific role of ethylene in plant morphogenetic

processes such as the induction of vegetative storage

structures has not been reported so far and it has been

suggested that its stimulatory effect on shoot and root

growth depends on the plant species (Dimasi-Theriou et al.

1993).

The plant defence-related signals, JA and its methyl

esters, regulate many physiological and developmental

processes, such as root growth, tuberisation, fruit ripening,

senescence and pollen development (Schaller et al. 2005).

During wound stress, jasmonates activate mechanisms

involved in the healing processes through enhanced cell

division and proliferation (Leon et al. 2001). Jasmonates

are also potent inducers of vegetative storage protein (VSP)

gene expression (Lorenzo and Solano 2005) and play a

significant role in the formation of vegetative storage

organs (Jasik and de Klerk 2006). While the exogenous

application of JA has been reported to induce vegetative

structures like tubers (Koda and Kikuta 1991; Debeljak

et al. 2002; Zhang et al. 2006) and bulbs (Ravnikar et al.

1993; Santos and Salema 2000), it is not known whether

jasmonates induce rhizome growth or whether there is any

interaction between ethylene and JA in this process. Based

on all of this information, it was hypothesised that ethylene

and JA induce and regulate rhizome development in

rhubarb.

To explore the hormonal physiology involved in the

induction and growth of rhizomes in rhubarb, we investi-

gated the role of ethylene and JA in inducing miniature

rhizomes in rhubarb shoots in vitro.

Materials and methods

Plant culture and treatment application

Clonally propagated 7-year-old plants of Rheum rhabarba-

rum L. cv., Sutton were used for obtaining explants to pro-

duce multiple shoots in vitro. Multiple-shoot clumps were

produced in vitro from meristem tips from lateral buds borne

on the rhizome. The buds were surface-sterilised by

immersing in 2% (NaOCl) sodium hypochlorite for 10 min,

followed by four rinses in sterile distilled water. Surface-

sterilised lateral buds were dissected under a microscope to

separate the meristem tips from the outer tissues. These

meristem tips were cultured in sterile Murashige and Skoog

(MS) (1962) basal medium supplemented with 1 mg l-1

each of 6-benzylaminopurine (BAP) and indole-3-butyric

acid (IBA), 20 g l-1 of sucrose and solidified with 8 g l-1

agar (pH 5.5) (Roggemans and Claes 1979; Walkey and

Mathews 1979). A shoot clump refers to the bunch of off-

shoots that arise from a single bud on the rhizome. Seven-

week-old in-vitro-cultured shoot clumps, each consisting of

3–4 miniature shoots, were transferred to MS (1962) basal

medium modified with varying concentrations of PGRs and

30 g l-1 sucrose solidified with 7 g l-1 agar after adjusting

the pH to 5.5. The experiment was conducted in three stages.

In the first stage, selected PGRs, GA3 (10, 20, 40 and

80 mg l-1), ABA (0.1, 1, 10 and 100 lg l-1), JA (10 ng l-1,

1 lg l-1, 10 lg l-1 and 1 mg l-1) and the ethylene-releas-

ing compound, ethephon (2-chloroethylphosphonic acid) (1,

10 and 50 mg l-1), were screened in vitro to investigate

their role in inducing mini-rhizomes in multiple shoots. The

term mini-rhizome as used in this paper refers to a miniature

rhizome produced in vitro from cultured shoot explants. In

order to further confirm the results of the first stage, in the

second stage, the ethylene-releasing compound ethephon (2-

chloroethylphosphonic acid) (1, 10 and 50 mg l-1), the

ethylene precursor ACC (1-aminocyclopropane-1-car-

boxylic acid) (0.5, 1.0, 1.5 mM) and the ethylene biosyn-

thesis inhibitor AVG (aminoethoxyvinylglycine) (0.5, 1.0,

1.5 lM) were used to determine their roles as signals in

inducing mini-rhizomes. While JA and ethephon induced

mini-rhizomes (see the results of the first and second stages),

it was not clear whether JA-induced mini-rhizome formation

is through promoting endogenous ethylene. Also, it was

important to confirm if mini-rhizomes can be induced when

endogenous ethylene synthesis is prevented. To investigate

the interaction between JA and ethylene and the role of

ethylene biosynthesis inhibitor in the process, multiple-

shoot clumps were cultured in MS basal medium modified

with the most effective concentrations of JA, ethephon

(Fig. 1a) and their combination in the presence or absence of

AVG (1.5 lM) with 30 g l-1 sucrose solidified with 7 g l-1

agar after adjusting the pH to 5.5.

254 Plant Cell Tiss Organ Cult (2011) 105:253–263

123

The concentrations of the PGRs used in the experiments

were determined based on the previous similar in vitro

studies conducted in similar crop species producing rhi-

zomes and mini-tubers and in rhubarb (Bjornseth 1946) and

also based on initial trial experiments conducted in wide

concentration ranges to determine the optimum concentra-

tion range without damaging the explant tissue, depending

on the active ingredient present in the formulations of these

PGRs (data not provided). The details on the PGR prepa-

rations are given in the Appendix. The cultures were grown

in GA-7 Magenta vessels of size H 77 9 77 9 97 mm

(Sigma-Aldrich, Ontario, Canada) with 50 ml of nutrient

media and maintained at 20 ± 1�C day/night temperature

under a 16-h photoperiod. The light intensity was main-

tained between 100 and 120 lmol m-2 s-1 with cool white

fluorescent lights.

The induction of mini-rhizomes at the base of the clumps

and their growth were monitored in the cultures. After

6 weeks of culture initiation, the plantlets were taken out.

Observations were made on the length of the shoots, shoot

growth, root growth, presence of mini-rhizomes and the

width of the micro-rhizomes produced. For measuring the

shoot growth and root growth, a standardised grade scale

was used (Rayirath 2008). Data on shoot growth and root

growth are not shown. One of the ten replicates in those

treatments which induced mini-rhizomes was sacrificed in

order to take microscopic sections of the mini-rhizomes.

Microscopy

In order to confirm that the growth at the base of in-vitro-

cultured shoot clumps is not a callus tissue but a mini-

Fig. 1 a Mini-rhizome width

(cm) in multiple-shoot clumps.

The multiple-shoot clumps were

incubated in Murashige and

Skoog (MS) basal medium with

various concentrations of plant

growth regulators (PGRs) for

6 weeks. The values represent

the means of ten replicates and

the error bars represent the

standard error (SE) at a = 0.05.

b Left to right: rhizome

induction (initiation) in

ethephon 1 mg l-1 after 1 week

in culture (picture taken from

the bottom of the culture jar);

mini-rhizomes in the explants

cultured with ethephon 1 mg l-1

for 6 weeks; absence of mini-

rhizome growth in control (with

distilled water) explants

Plant Cell Tiss Organ Cult (2011) 105:253–263 255

123

rhizome, microscopic sections of the in-vitro-produced

mini-rhizome and the mother rhizome were compared and

studied. Fresh transverse sections of the in-vitro-produced

mini-rhizomes were made using sterile scalpel blades and

viewed using an Olympus BH-2 compound transmitted

light microscope (Carson Medical and Scientific Co. Ltd.,

Markham, Ontario, Canada) under standard bright-field

conditions and photographed using an Olympus OM-2S

35-mm camera. The sections were not stained since

staining could interfere with the starch which might be

present in the tissues and lead to misinterpretation. Sections

of the in vitro mini-rhizomes were compared with the

transverse sections of the original rhizome obtained from a

live plant grown in the greenhouse. The anatomical struc-

tures of the two different sections were characterised and

compared.

Pot culture and mini-rhizome growth measurements

of in-vitro-cultured rhubarb plantlets

After 6 weeks growth in the culture media with various

PGRs, the shoot clumps which produced mini-rhizomes at

their base and the respective controls were planted in 15 cm

pots filled with the commercial peat-based potting mixture

ProMix BX (Premier Horticulture, Quebec, Canada). The

transplants were kept in a mist chamber for a week to

harden off and were maintained for evaluating mini-rhi-

zome growth further under in vivo conditions in a green-

house at the Nova Scotia Agricultural College (NSAC),

Truro, Nova Scotia, Canada (45� 220 N; 63� 160 W) at 20�/

15�C (day/night) under a 16-h photoperiod. The natural

daylight was supplemented with light from high-pressure

sodium bulbs (400 W). Since one of the replicates from the

in vitro plant outs was used for microscopy study, the

remaining nine replicates were transplanted into the pots.

The plants were watered every third day until the soil was

saturated. After 4 months in pot culture, the plantlets were

gently uprooted and observations were made on the signal

induced mini-rhizome growth and enlargement. The mini-

rhizomes were weighed and the rhizome width and length

were measured. The number of buds produced on the mini-

rhizomes and the number of rhizome branches per plantlet

were also counted. Observations on the root growth, shoot

growth, shoot length and number of leaves were also taken.

Data on root and shoot growth are not shown.

Foliar application of ethephon on ex vitro plants

A pot culture experiment was conducted to confirm the

effect of ethephon at 1 mg l-1 [the most effective PGR in

the in vitro studies (Fig. 1)], as a foliar spray on newly

planted rhubarb plant cultures under greenhouse condi-

tions. Uniform size rhizome parts weighing 60 g and

bearing at least two viable buds were planted in large-sized

pots (30 cm diameter). There were ten replicates and a

control without the PGR treatment was also maintained.

The plant cell-tested commercial formulation of ethephon

(Sigma-Aldrich, Ontario, Canada) along with the surfactant

Tween 20 (*0.1%) was applied as a foliar spray using a

hand-pump sprayer, Super Spray GX60 (Green Cross,

Division of CIBA-GEIGY, Canada Ltd.) of capacity

1.25 L. One hundred millilitres of the spray solution was

applied per plant at 8 weeks and 12 weeks after sprout

emergence. The control plants were sprayed with distilled

water containing the same amount of surfactant. The plants

were placed in tall cardboard boxes (cut open at the top) to

prevent drifting of the spray solution to the neighbouring

plants and were sprayed until the leaves were dripping.

After 5 months, the plants were harvested and measure-

ments were made on the rhizome growth.

Experimental design and data analysis

The in vitro and pot culture experiments utilised a com-

pletely randomised design (CRD). All of the experiments

were replicated ten times and were repeated twice. The

general linear models (GLM) procedure (SAS Institute Inc.

1999) was used for the analysis of variance (ANOVA),

with Tukey’s multiple means comparison test being used

for determining differences between the means.

Results

Effect of ethephon and JA on inducing mini-rhizomes

in vitro

Among the PGRs tested, the ethylene-releasing compound,

ethephon, and JA induced mini-rhizomes in in-vitro-cul-

tured rhubarb multiple-shoot clumps. As shown in Fig. 1a,

exogenously applied ethephon and JA induced mini-rhi-

zomes at lower concentrations tested. Within 1 week after

culturing (*5 days), the cultures incubated in the media

with ethephon (ranging from 1 to 50 mg l-1) and JA

(ranging from 10 ng l-1 to 1 mg l-1) exhibited rhizome

induction with a noticeable bulging at the base of the shoot

clumps (Fig. 1b). The width of the induced mini-rhizomes

increased as the length of time in the culture progressed.

Exogenous application of ethephon had a stronger effect in

inducing rhizomes and promoting their growth; rhizome

growth was observed at all concentrations of ethephon

tested. After 6 weeks in culture, the highest rhizome width

of 1.56 cm (Fig. 1b) was achieved in cultures with

1 mg l-1 ethephon. The cultures with 10 ng l-1 and

1 lg l-1 JA were the second best after ethephon 1 mg l-1

in mini-rhizome induction. However, higher concentrations


123

of these growth regulators tested significantly inhibited

mini-rhizome induction and growth. Rhizome induction

was not observed in cultures incubated with all of the tested

concentrations of ABA and GA3. The control plants also

did not produce any mini-rhizomes (Fig. 1b). Good shoot

and root growth was observed in the mini-rhizome-induced

cultures (data not shown). The above data on mini-rhizome

induction and growth indicates a clear involvement of

ethylene and JA as signals in the induction of rhizomes in

rhubarb.

Consistent with previous results, in the second stage of

the in vitro experiments, ethylene (ethephon) promoted

rhizome formation and growth depending on the concen-

tration, with the largest rhizomes produced with 1 mg l-1

ethephon. Like the control cultures (both with DW and

EtOH), the ethylene biosynthesis inhibitor AVG-treated

cultures did not produce mini-rhizomes (Fig. 2a). It could

be possible that AVG inhibited ethylene biosynthesis in

treated plants, but, again, since there was no noticeable

mini-rhizome induction in the control plants, it cannot be

concluded whether that is the reason for the absence of

mini-rhizome formation in AVG-treated cultures. Inter-

estingly, the ethylene precursor, ACC (0.5 and 1.0 mM),

induced mini-rhizomes, but the effect was less significant

compared to that of ethephon (Fig. 2a). The rhizome

induction and growth in the ACC-treated cultures was

noticeable only during the later stages of culturing

(3–4 weeks). AVG also significantly inhibited shoot and

root growth in the cultures (data not shown here). These

results again suggest the possibility that ethylene regulates

rhizome induction and growth in rhubarb.

While the above data indicate that an ethylene and/or

JA-dependant mechanism could exist in the induction of

mini-rhizomes, it is not clear whether JA-induced mini-

rhizome formation is through the promotion of endogenous

ethylene and also whether ethylene biosynthesis inhibitor

acts via inhibiting endogenous ethylene in the rhizome

induction process. To explore the interactions between

ethylene and JA on rhizome formation and further growth,

the ethylene biosynthesis inhibitor AVG (1.5 lM) was

Fig. 2 a Mini-rhizome width in

plantlets grown in ethylene-

releasing compound, ethephon,

precursor 1-

aminocyclopropane-1-

carboxylic acid (ACC) and the

ethylene biosynthesis inhibitor,

aminoethoxyvinylglycine

(AVG). The explants were

incubated in the culture for

6 weeks. The values represent

the means of ten replicates and

the error bars represent the SE

at a = 0.05. b Microscopic

sections showing the structural

similarities observed between

mother rhizomes and in-vitro-

produced signal-induced mini-

rhizomes. The several-layered

epidermis, irregularly arranged

vascular bundles and yellow

bands representing

anthraquinones are visible in

both sections. Scalebars 100 lm


123

used in the culture media, along with the most effective

concentrations of ethephon (1 mg l-1) and JA (10 ng l-1)

and their combinations. After 6 weeks of culturing

explants, consistent with previous results, ethephon

(1 mg l-1) and JA (10 ng l-1) induced mini-rhizomes and

significantly promoted their growth (Fig. 3a). The combi-

nation treatment (JA 10 ng l-1 and ethephon 1 mg l-1)

exhibited better mini-rhizome growth than that of JA alone,

but was significantly less than that of ethephon alone

(Fig. 3a, b). This demonstrated that, in combination with

ethephon, the rhizome growth triggering effect of JA was

further enhanced compared to when applied alone.

As shown in Fig. 3a, c, AVG produced an 88% reduc-

tion in mini-rhizome width in cultures with JA (JA and

AVG), an 90% reduction in cultures with ethylene (ethe-

phon and AVG) and an 89% reduction in cultures with both

JA and ethylene (JA and ethephon and AVG), respectively,

when compared with those without AVG. Considerable

reduction in the mini-rhizome width observed in cultures

treated with AVG 1.5 lM despite the presence of exoge-

nous JA and ethylene further suggests that it is an ethylene-

dependent process. Consistent with the previous results, the

control cultures did not produce mini-rhizomes.

Microscopic studies and structural comparison of in-

vitro-produced mini-rhizomes with the mother rhizome

Structural similarities were observed between the rhizomes

(Fig. 2b). The presence of large amounts of starch made it

difficult to obtain good sections of the mother rhizome. As

shown in Fig. 2b, the epidermal layer was thick, with

several layers of densely packed cells. The vascular bun-

dles in both cases (mini-rhizome and mother rhizome) were

irregularly arranged. They lay scattered and, in between,

there were golden yellow bands/rays, which could possibly

be anthraquinones (Fig. 2b) that are abundant in the rhu-

barb rhizome tissues (Gao et al. 2009). Interestingly, cer-

tain green-coloured structures, which are more likely

chloroplasts, were also observed in some sections. These

structural similarities indicate that the growth at the shoot

bases induced by ethylene and JA are miniature rhizomes.

Growth of induced mini-rhizomes in in vitro plant outs

After 5 months of growth of the plant outs in the pot cul-

ture, they were harvested and measurements were made on

the mini-rhizome growth in vivo. Consistent with the in

vitro results, the mini-rhizomes from the cultures with

ethylene, JA and their combination exhibited the same pace

of growth when compared to the controls (DW and EtOH)

(Fig. 4). As per Table 1, exogenous ethephon (1 mg l-1)

application strongly promoted mini-rhizome growth and

the cultures with the highest width, length, branches and

lateral buds. Rhizome growth was significantly higher in

the cultures with JA (10 ng l-1) compared to the control.

Even though JA–ethephon combination treatment exhibited

considerably higher rhizome growth than JA alone, it was

not superior to those with ethephon alone. Lateral buds

were also significantly promoted by exogenous ethylene

(ethephon) and JA. The PGR-treated cultures exhibited

good shoot and root growth compared to the control in vivo

(data not shown).

Effect of ethephon spray on rhizome growth and lateral

bud production in rhubarb in a pot culture

The foliar application of ethephon (1 mg l-1, the most

effective concentration in vitro) on rhubarb plants at 8 and

12 weeks after shoot emergence significantly enhanced the

number of lateral buds on the rhizome after 4 months in pot

culture. As shown in Table 2, the number of lateral buds

increased two-fold in the treated plants compared with the

control, and there was a significant increase in the rhizome

width, even though the fresh weight did not differ signifi-

cantly between the treated and untreated plants.

Discussion

The absence of mini-rhizomes (Figs. 2a and 3c) in AVG-

treated plant cultures and AVG’s antagonistic effect on

mini-rhizome induction and growth in cultures treated with

exogenous ethephon and JA strongly indicate that ethylene

is a key regulator and/or inducer and growth enhancer of

rhizomes in Rheum rhabarbarum plantlets cultured in

vitro. Interestingly, the ethylene precursor, ACC, was also

effective in inducing mini-rhizomes but to a lesser extent

than ethephon. It appears as if, in the treated cultures, AVG

inhibited the formation of ethylene precursor ACC) from

S-adenosylmethionine (SAM) by inhibiting the rate-limit-

ing enzyme ACC synthase (Yang and Hoffman 1984),

limiting endogenous ethylene and inhibiting the rhizome

induction process. Even though JA triggers promoter

activities of ACC synthase genes (Tang et al. 2008),

enhancing ethylene biosynthesis, here, in the cultures with

JA and AVG, AVG might have counteracted JA’s effect by

antagonistically inhibiting endogenous ethylene.

Several previous studies have demonstrated that

exogenous ethylene could induce endogenous ethylene

through the upregulation of ethylene biosynthesis genes

(Alonso et al. 2003; Van Zhong and Burns 2003).

Investigating the role of endogenous ethylene in cotton

fibre cell elongation, Shi et al. (2006) have shown that

exogenously applied ethylene induce the expression of

ACO genes (1-Aminocyclopropane-1-Carboxylic Acid

Oxidase) involved in the oxidation of ACC to ethylene


123

(Yang and Hoffman 1984), thereby, inducing endogenous

ethylene synthesis, while the cotton ovules treated with

ethylene biosynthesis inhibitor AVG suppressed fibre cell

elongation through inhibiting endogenous ethylene. The

regulation of the expression of ACO genes, thus, has

functional significance (Prescott and John 1996) and it

Fig. 3 a Mini-rhizome width in

plantlets grown in JA, ethephon

and their most effective

combinations in the presence

and absence of AVG. The

explants were incubated in the

culture for 6 weeks. The valuesrepresent the means of ten

replicates and the error barsrepresent the SE at a = 0.05.

b Plants after 6 weeks growth in

1 mg l-1 ethephon and

10 ng l-1 JA combination and

1 mg l-1 ethephon (top), and

the mini-rhizome growth in

1 mg l-1 ethephon and 10 ng

l-1 JA combination and 1 mg

l-1 ethephon (bottom). The

explants were cultured in MS

basal medium with PGRs for

6 weeks (pictures taken from

the bottom of the culture jars).

c Left to right: mini-rhizome

growth inhibited in explants

incubated with 10 ng l-1 JA and

1.5 lM AVG combination;

1 mg l-1 ethephon and 1.5 lM

AVG combination; 1 mg l-1

ethephon, 10 ng l-1 JA and

1.5 lM AVG combination

treatment


123

has also been proved that at least two ACO genes in

Arabidopsis thaliana are ethylene-inducible (Alonso et al.

2003; Van Zhong and Burns 2003). Hence, in this study,

exogenous ethylene (ethephon) appears to induce mini-

rhizomes in treated cultures through enhanced ethylene

biosynthesis, which is inhibited by AVG, resulting in the

suppression of mini-rhizome growth in the combination

cultures. However, the present investigation, being purely

physiological, cannot address the molecular mechanisms

which result in these differential responses to the biosyn-

thetic inhibitors. The results presented in Fig. 1a suggest

that GA3 and ABA did not have any significant role in the

process as supported by previous studies (Obasi and Atanu

2004; Zheng et al. 2005). Even though ABA induces tuber

formation and growth in potato by counteracting the effect

of GA3 (Xu et al. 1998), scientific evidence on its role in

inducing rhizome growth in vegetatively propagated

perennial crops is not available thus far.

The comparison of microscopic sections of in-vitro-

generated mini-rhizomes and mother rhizomes of a normal

plant grown in the greenhouse clearly revealed some

structural similarities (Fig. 2b). These structural similari-

ties strongly support that the growth obtained at the base of

the explants treated with effective concentrations of ethe-

phon and JA is a miniature rhizome. A rhizome is a stem

modification that is a common storage organ in vegeta-

tively propagated crops (Hartmann et al. 2002). In some

herbaceous perennials which over-winter (like rhubarb),

rhizomes serve as carbon reserves for survival (Moore et al.

1998). Anatomically, rhizomes mostly lack a proper stele

pattern and form shoot and root buds from the nodes

(Raven et al. 2005; Hartmann et al. 2002). The presence of

structures, which are more likely chloroplasts (seen as

green-coloured structures), further confirms that it is not a

Fig. 4 Mini-rhizome weight (g) of in vitro plantlets treated with

1 mg l-1 ethephon, 10 ng l-1 JA and their combination (1 mg l-1

ethephon, 10 ng l-1 JA) after 4 months in pot culture. The valuesrepresent the means of nine replicates and the error bars represent the

SE at a = 0.05

Table 1 Growth of in-vitro-induced mini-rhizomes after transplantation to pots

Treatments Rhizome

width (cm)

Rhizome

length (cm)

Rhizome

weight (g)

No. of

rhizome branches

No. of buds

Ethephon 1 mg l-1 6.6a 6.9a 53.5a 5.1a 19.1a

JA 10 ng l-1 4.8b 5.0b 38.2b 3.4b 10.2c

JA 1 lg l-1 5.5b 4.9b 36.2b 3.8b 7.4d

Ethephon 1 mg l-1 and JA 10 ng l-1 5.4b 5.4b 49.4a 3.1b 11.7b

Control DW 2.7c 4.8bc 11.1c 1.4c 3.2e

Control EtOH 1.6c 2.8c 6.9c 0.8c 2.4e

The in-vitro-produced rhubarb plantlets with the induced mini-rhizomes were grown under controlled conditions in the greenhouse for 4 months

and then uprooted and observations were made

EtOH ethanol, DW distilled watera–e Means with the same letters indicate no significant difference based on Tukey’s test at a = 0.05; n = 9

Table 2 Foliar treatment with ethephon on plants in pot culture

Treatments Rhizome width (cm) Rhizome weight (g) No. of buds

Ethephon 1 mg l-1 8.6a 117.6a 22.2a

Control 7.5b 110.9a 11.6b

Measurements were made on the rhizome growth of pot-grown plants 5 months after foliar treatment with ethephona, b Means with the same letters indicate no significant difference based on Tukey’s test at a = 0.05; n = 10


123

root structure and the presence of vascular structures also

supports that it is not a mere callus growth but a well-

differentiated plant organ.

To further support the results of the in vitro studies,

exogenously applied ethephon in the form of foliar spray

on in situ rhubarb plants during active vegetative growth (8

and 12 weeks after shoot emergence) significantly

enhanced the number of potential buds on the rhizome.

Being a gaseous hormone, ethylene is not translocated

through the vascular system. Ethylene is neither actively

transported nor degraded (Bleecker and Kende 2000). The

induction of ethylene synthesis by signals such as auxin or

wounding usually occurs through the activation of ACC

synthase through increased gene expression (Bleecker and

Kende 2000). However, the exact molecular mechanism of

ethephon- and JA-induced rhizome growth is yet to be

uncovered. Here, exogenous ethylene appeared to trigger

adventitious bud development in the sprayed plants, pos-

sibly through breaking lateral bud dormancy. Previous

reports indicate that ethylene (and ethylene-releasing

compounds) could positively regulate developmental pro-

cesses like adventitious root formation (Liu et al. 1990),

trigger adventitious bud development releasing lateral bud

dormancy (Reid 1987), enhance cambial activity and

induce radial swelling of storage organs (Reid 1987; Ne-

uteboom et al. 2002).

Our study clearly indicates the specific role of ethylene

in the induction, growth and development of rhizomes in

rhubarb. Although ethylene has a pleiotropic role in various

processes of plant growth and development, specific roles

of this hormonal signal in plant morphogenesis, such as the

formation and development of vegetative storage organs or

vegetative reproductive structures, has not been reported so

far.

In the present study, JA at lower concentrations

(10 ng l-1 and 1 lg l-1) significantly induced mini-rhi-

zomes in rhubarb plantlets (Fig. 1a). However, the rhizome-

inducing effect of JA was suppressed in the presence of

ethylene synthesis inhibitor, AVG, and, at the same time,

further enhanced by ethylene (ethephon) when they were

combined and applied exogenously in the growing media.

This implies that, while JA could promote rhizome induc-

tion, it requires the presence of endogenous ethylene as

AVG inhibited JA-induced rhizome growth. Also, it is

possible that, since JA induces ethylene synthesis (Tang

et al. 2008), the JA-induced mini-rhizome synthesis may be,

perhaps, due to JA-induced ethylene. Though synergistic

interactions between ethylene and JA have been proposed to

contribute to diverse plant responses to abiotic and biotic

stresses (Zhu et al. 2006; Feys and Parker 2000; Glazebrook

2001; Lorenzo et al. 2003), the relationships between JA and

ethylene in regulating developmental processes, other than

plant defence responses, has not been studied intensively.

The exogenous application of JA rapidly enhances ethylene

emission in tomato and supports the indication that they act

synergistically in the expression of proteinase inhibitors

(PIN) genes in response to wound stress (O’Donnell et al.

1996). Ethylene is found to act downstream of JA in the

wound signal transduction pathway (O’Donnell et al. 1996).

Similarly, methyl jasmonate acts synergistically with ACC

in enhancing ethylene production and promoting gum for-

mation in tulips through stimulated ACC oxidase activity

(Saniewski et al. 2003). The interactive role of these hor-

monal signals in developmental processes such as root hair

formation has been clearly demonstrated in recent studies

(Zhu et al. 2006). Studies on the promoter activities of

Arabidopsis ACC synthase genes have recently shown that

exogenous JA could enhance the activity of Arabidopsis

ACC synthase (the rate-limiting step in ethylene biosyn-

thesis) promoter AtACS4 in the wild-type genotype (Tang

et al. 2008). The above explanations indicated that ethylene

is the key player in the process of rhizome induction and

development and JA might act through ethylene induction in

the process. All of these results support a significant role of

the PGRs ethylene and JA in the induction and growth of

rhizomes in Rheum rhabarbarum.

Taken together, the findings of this study strongly suggest

that ethylene is the rhizome induction signal in rhubarb. The

significant antagonistic effect of AVG on rhizome growth

even in the presence of exogenous ethylene and JA further

confirms this and suggests that JA’s role in the process is

possibly through stimulating endogenous ethylene levels

(Zhu et al. 2006; Tang et al. 2008). Further experiments

focusing on the interaction of ethylene and JA utilising

respective biosynthesis and action inhibitors would certainly

disclose the cross-talk between the two hormones in the

regulation of rhizome induction and growth in rhubarb. The

findings of such investigations would improve our under-

standing of how these two hormones regulate growth and

development processes in different crop species. However,

unlike in model plant species like Arabidopsis, in the

absence of hormonal signalling mutants of this plant, it is

very challenging to unveil the hormonal signalling pathway

involved in this developmental process.

Acknowledgements Financial support from AgriFocus 2000—

Technology Development Program (Nova Scotia Department of

Agriculture and Fisheries) and Knol Farms Ltd., Nova Scotia, Can-

ada, to Dr. Lada is gratefully acknowledged.

Appendix: details of the plant growth regulator

application

Plant-cell-tested commercial formulations of growth regu-

lators from Sigma-Aldrich, Canada, were used. Stock

solutions of GA3 (1,000 mg l-1), ABA (100 mg l-1) and


123

JA (1,000 mg l-1) were prepared by dissolving them first

in four drops of 95% ethanol and diluted with distilled

water to the final stock concentrations. ACC, AVG and

ethephon (Sigma-Aldrich, Canada) were dissolved in dis-

tilled water to make the stock solutions of concentrations

1,000 mg l-1, 50 mg l-1 and 1,000 mg l-1, respectively.

ABA and GA3 were autoclaved with other media compo-

nents, whereas all other growth regulators were filter-ster-

ilised and added to the autoclaved culture media after

cooling to 45–50�C. An ethanol control (with four drops of

95% ethanol) and a distilled water control were maintained

in all three sets of experiments, since most of the plant

growth regulators (PGRs) were dissolved in ethanol before

making up the stock solution.

The concentrations of each PGR used in each stage of

the experiments are given below.

Treatments in stage I

10 ng l-1, 1 lg l-1, 10 lg l-1, 1 mg l-1 JA

0.1 lg l-1, 1 lg l-1, 10 lg l-1, 100 lg l-1 ABA

10 mg l-1, 20 mg l-1, 40 mg l-1, 80 mg l-1 GA3

1 mg l-1, 10 mg l-1, 50 mg l-1 ethephon

Control DW—control with distilled water (DW)

Control EtOH—control with ethanol (EtOH)

Treatments in stage II

1 mg l-1, 10 mg l-1, 50 mg l-1 ethephon

0.5 mM, 1.0 mM, 1.5 mM ACC

0.5 lM, 1.0 lM, 1.5 lM AVG



Treatments in stage III

1 mg l-1, 10 mg l-1 ethephon

10 ng l-1, 1 lg l-1 JA

1 mg l-1 ethephon and 1.5 lM AVG

10 ng l-1 JA and 1.5 lM AVG

10 ng l-1 JA and 1 mg l-1 ethephon

10 ng l-1 JA, 1 mg l-1 ethephon and 1.5 lM AVG



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Role of ethylene and jasmonic acid on rhizome induction and growth in rhubarb ( Rheum rhabarbarum L

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