Abstract The effects of jasmonic acid (JA) and

methyl jasmonate (MeJA) on potato (Solanum

tuberosum L.) tuberization were studied in

relation to cytokinins using single-node cuttings

(SNCs) in vitro. In three potato cultivars differing

in maturity levels, JA or MeJA (0.0, 2.5, 5.0, 7.5

and 10.0 lM) action was examined with N6-

benzyladenine (BA: 0.0, 22.0 and 44.0 lM) under

optimum tuber-inducing treatment with 80 g L–1

sucrose. Although jasmonates had a stimulatory

role in root growth from SNCs, BA inhibited the

root growth and antagonized the jasmonates

action on root growth promotion. There was a

strong inhibitory effect of BA on stoloniferous

shoot growth, and in combination with jasmo-

nates it could exert a much pronounced inhibitory

effect. Jasmonates did not have any role in tuber

induction in terms of tuber number and source-

sink coordination (harvest index), rather their

effects were counteracted in the presence of

22.0 lM BA in early cultivar. However, they

had a promoting effect on tuber growth after

induction in early cultivar, possibly due to

its lower levels of endogenous gibberellins.

Cytokinins detrimentally affected the tuber

growth, and antagonized the jasmonates action

irrespective of the maturity levels of the culti-

vars. It could, however, increase the tuber dry

matter concomitant with a higher accumulation

of starch. The promoting effect of jasmonates on

tuber dry matter and starch accumulation was

apparent only in early cultivar. There were

significant interactions between cytokinin and

jasmonates for accumulation of reducing and

total sugars in tubers. The results show that

exogenous cytokinins antagonize the jasmonates

action, and the correlated effects of these two

growth hormones interact with the maturing

time of the cultivar during potato tuber forma-

tion in vitro.

Keywords Cytokinins Æ Jasmonic acid Æ Methyl

jasmonate Æ Solanum tuberosum L. Æ Starch ÆTuberization

AbbreviationsBA N6-benzyladenine

JA Jasmonic acid

KA Kufri Ashoka

KBD Kufri Badshah

KS Kufri Sindhuri

MeJA Methyl jasmonate

MS Murashige and Skoog’s medium (1962)

SNC Single-node cutting

D. Sarkar (&) Æ S. K. Pandey Æ S. SharmaDivision of Crop Improvement, Central PotatoResearch Institute, Shimla,171 001 Himachal Pradesh, Indiae-mails: d.sarkar@excite.com; debabrata_s@yahoo.com

Plant Cell Tiss Organ Cult (2006) 87:285–295

DOI 10.1007/s11240-006-9166-3

123

ORIGINAL PAPER

Cytokinins antagonize the jasmonates actionon the regulation of potato (Solanumtuberosum) tuber formation in vitro

Debabrata Sarkar Æ Suman Kumar Pandey ÆSushruti Sharma

Received: 9 April 2006 / Accepted: 12 September 2006 / Published online: 10 November 2006� Springer Science+Business Media B.V. 2006

Introduction

Besides specific environmental cues, such as

short-photoperiod, high-light intensity and low-

nitrogen level, the growth hormones have been

suggested to play a prominent role in the control

of tuberization in potato (Vreugdenhil and

Struik 1989). However, no unequivocal tuberizing

factor has been identified till date, and, therefore,

the recognition of a tuber-inducing signal still

remains an elusive goal (Jackson 1999). Tuberonic

acid (TA) and tuberonic acid glucoside (TAG)

with strong tuber-inducing activities were isolated

from potato leaves (Koda and Okazawa 1988;

Yoshihara et al. 1989). TA was later found to be

structurally related to jasmonic acid (JA) having

similar levels of tuber-inducing activity in vitro

(Koda et al. 1991). JA and its volatile derivative

methyl jasmonate (MeJA) have been detected in a

large number of plant species (Mithofer et al.

2005). They are involved in various morphogenic

events, such as tuberization, tuberous root forma-

tion, bulb formation (Koda 1997) and its dor-

mancy development (Jasik and de Klerk 2006),

senescence, developmental process, wounding,

plant structural determination, thigmomorpho-

genesis (van den Berg and Ewing 1991; Koda

1997), abiotic stress responses and mechanotrans-

duction (Wasternack and Hause 2002). In potato

stolons, shoot cuttings and plantlets cultured in

vitro, JA stimulated tuberization (Koda et al.

1991; Pelacho and Mingo-Castel 1991; Ravnikar

et al. 1992; Pruski et al. 2001, 2002). Even its

endogenous presence was confirmed in roots,

stolons and periderm of newly formed tubers

(Abdala et al. 1996). However, a number of

studies demonstrated that JA was not directly

involved in potato tuberization (Helder et al.

1993; Jackson and Willmitzer 1994).

Now, it is well known that potato tuberization

is regulated by a balance between the levels of

jasmonates and gibberellin (Koda 1997). JA

exerts its effect principally by antagonizing the

effect of GA on microtubule orientation during

potato tuberization (Jackson 1999). It has also

been shown that JA caused a total reversion of

the delaying effect of GA3 on tuberization of

potato shoot cuttings in vitro, and the stimulatory

effect of JA on potato tuberization was antago-

nized in rooted plantlets having a high level of

endogenous GAs (Castro et al. 2000).

Cytokinins have long been suggested to be

involved in potato tuberization (Palmer and Smith

1969), but in contrast to GA and ABA less

attention has been paid to cytokinins. A direct

role of cytokinins in tuberization vis-a-vis tuber

induction has been confirmed by transgenic

approach (Vreugdenhil 2004). For in vitro

tuberization, exogenous cytokinins, especially

N6-benzyladenine (BA), are extensively used to

induce microtubers on a variety of explants

including stolons, shoot cuttings and intact

microplantlets (see review Donnelly et al. 2003).

However, although the presence of cytokinin is

required for tuberization, it is not the stimulus

provided by photoperiodic induction. This essen-

tially shows that cytokinins cannot be the only

factor regulating tuber induction in potato. The

response of a stolon to high-cytokinin levels

depends on the interaction with other hormones,

particularly the levels of GAs (Vreugdenhil and

Struik 1989). It has also been shown that exogenous

cytokinins do not have any role in stolon elonga-

tion and tuber formation in vitro implying that

cytokinins are not the limiting factor for tuber

formation (Xu et al. 1998).

It is not known how the jasmonates (JA and

MeJA) regulate tuber induction and development

in relation to cytokinins. Pelacho and Mingo-

Castel (1991) showed that JA was the stronger

promoter of stolon tuberization in vitro than

kinetin. But the concentration of kinetin tested by

them was about four-times lesser than that

optimally used for cytokinin-induced tuberization

in vitro. Is there any interaction between jasmo-

nates and cytokinins for tuberization in potato? It

has also been proved that BA is more potent than

kinetin for tuberization in vitro. Moreover, in

comparison with JA, the role of MeJA in potato

tuberization is also least investigated to our

knowledge.

In this study, we employed a well-defined in

vitro tuberization system using single-node

cuttings (SNCs) to explore the interaction

between JA/MeJA and BA for tuber formation

in potato. The objectives were (1) to assess the

286 Plant Cell Tiss Organ Cult (2006) 87:285–295

123

comparative efficacy of JA and MeJA for

tuberization under cytokinin-aided inducing and

non-inducing conditions and (2) to examine the

individual and correlated effects of JA/MeJA

and BA on tuber formation in relation to

sucrose-aided optimum tuber-inducing condition.

The study also goes in some way towards

examining the effects JA and MeJA on tuber

formation from SNCs, as compared to that from

stolons and leafy shoot cuttings employed in

other studies.

Materials and methods

Plant material

In potato (Solanum tuberosum L.), tuberization in

vitro is cultivar-specific, essentially modulated by

maturity levels with distinct differences in

response between early and maincrop cultivars

(Naik et al. 1998). Therefore, three tetraploid

(2n = 4x = 48) Indian potato (S. tuberosum

L. subsp. tuberosum) cultivars belonging to dif-

ferent maturity groups, viz., Kufri Ashoka (KA;

early maturing), Kufri Badshah (KBD; medium

maturing) and Kufri Sindhuri (KS; late maturing)

were used in the present study.

Culture of single-node cuttings in vitro

In vitro culture of these three cultivars were

established from indexed tubers (sprouts), and

they were made virus-free following standard

meristem culture and virus diagnostic procedures

as described earlier (Sarkar et al. 2004). SNCs

from disease-free microplants were cultured in

vitro as described by Sarkar et al. (1997). The

basal medium consisted of MS medium (Murash-

ige and Skoog 1962) supplemented with 20 g L–1

sucrose and solidified with 2 g L–1 gelrite (Sigma,

St. Louis, MI, USA). Five SNCs were cultured

per Magenta� vessel (GA-7; Sigma) containing

50 mL of medium. The cultures were incubated

under a 16 h photoperiod from cool white

fluorescent lights (~50–60 lmol m–2 s–1 PPFD)

at 24 ± 1�C and subcultured on fresh medium at

an interval of 21 days.

In vitro tuberization

For tuber induction in vitro, SNCs with a leaf

subtending the axillary buds from 21-day-old

microplants grown under above conditions were

used as explants. To maintain the explant homo-

geneity in relation to tuber induction and devel-

opment, the SNCs were prepared from middle

nodes of the source microplants. They were

layered horizontally over 30 mL of microtuber

induction medium (Sarkar and Naik 1998) in

90 mm disposable Petri dishes (Greiner Bio-One,

Frickenhausen, Germany). The induction medium

consisted of MS basal medium supplemented with

80 g L–1 sucrose and different concentrations of

BA plus jasmonates (JA and or MeJA) and

solidified with 2 g L–1 gelrite. The presence of

80 g L–1 sucrose in the medium provided an

optimum tuber-inducing conditions in vitro to

counteract the effects of endogenous GAs. Tuber

induction and development were also tested in

hormone-free medium supplemented only with

80 g L–1 sucrose. The pH of the medium was

adjusted to 5.8 before autoclaving at 121�C for

20 min. The Petri dishes were sealed with Parafilm

MTM

(American National Can, Chicago, USA),

and the tuber induction cultures were incubated in

the dark at 20�C (Sarkar and Naik 1998).

Jasmonates and cytokinin bioassays

In the present study, both (±)-JA (Sigma) and

MeJA (Duchefa, Haarlem, The Netherlands)

were supplemented in tuber induction medium

at varying combinations with BA (Sigma). Five

different concentrations of JA or MeJA (0.0, 2.5,

5.0, 7.5 and 10.0 lM) were tested with three

different concentrations of BA (0.0, 22.0 and

44.0 lM). The growth hormones were applied to

the medium by filter sterilization. The standard

concentration of BA for cytokinin-induced in

vitro tuberization is 44.0 lM. Many workers have

confirmed that optimum microtuberization occurs

at this concentration, especially in the absence of

light (Wang and Hu 1982; Wattimena 1983;

Slimmon et al. 1989; Sarkar and Naik 1998).

However, when growth retardants, like CCC is

used, the optimum concentration of BA has been

found to be half of this standard concentration,

Plant Cell Tiss Organ Cult (2006) 87:285–295 287

123

i.e. 22.0 lM (Estrada et al. 1986). Therefore, in

the present study, BA was tested at both 22.0 and

44.0 lM. For each jasmonate treatment, the

experiment was laid out in a factorial (5 · 3 · 3)

completely randomized design (CRD) involving

five concentrations of JA or MeJA, three

concentrations of BA and three cultivars with

five replicate Petri dishes, each containing ten

SNCs.

Data observations

The total duration of in vitro (tuber) induction

cultures was 8 weeks. The in vitro-induced

tubers were harvested, and the observations

were recorded on number of tubers per SNC,

average tuber fresh weight (mg), average stolon

fresh weight (mg), average root fresh weight

(mg) and harvest index (the ratio of tuber yield

to total biomass measured as fresh weight). The

harvested tubers were dried at 70�C for 2 days

for the estimation of percentage tuber dry

matter.

Sugars and starch determination

Sugars and starch in freshly harvested tubers

were estimated as described by Sharma et al.

(2004). Total soluble sugars, reducing sugars

and starch were expressed as mg glucose

equivalents per g microtuber fresh weight. For

each treatment, there were three independent

replications for the estimation of soluble sugars

and starch.

Statistical analyses

Prior to statistical analyses, the data on average

stolon fresh weight (mg), average root fresh

weight and tuber dry matter (%) were trans-

formed into square roots. For each jasmonate

(JA or MeJA) treatment, the three-way analy-

ses of variance (ANOVAs) were calculated

using the standard procedure, and the

means were separated by Least Significant

Difference (LSD) test. The term significant has

been used to indicate differences for which

p £ 0.05.

Results

All tested factors, viz., cultivar, JA/MeJA and BA

significantly affected tuber induction and devel-

opment from SNCs. The main effects of BA were

highly significant for tuber induction, growth and

biochemical parameters. JA had a significant

interaction with BA for number of tubers per

SNC, tuber weight, stolon fresh weight, root fresh

weight and tuber dry matter (%). For MeJA, the

significant interactions with BA were apparent

only for root fresh weight, harvest index and

tuber dry matter (%). Since three-way interac-

tions involving the cultivar, jasmonates and BA

were significant for most of the characters, only

the means and mean comparisons for the three-

way interactions are presented.

Effects of jasmonates on root

and stoloniferous shoot growth

JA significantly stimulated root growth, measured

as fresh weight, from SNCs. Independent of the

cultivars, the stimulatory effect of JA on root

growth was apparent up to 5.0 lM. Averaged

over the cultivars, about three-times increase in

root growth occurred at 5.0 lM JA (75.0 mg FW/

SNC) as compared to control 80 g L–1 sucrose

medium (25.5 mg FW/SNC). When the medium

contained 22.0–44.0 lM BA, rooting was com-

pletely inhibited, and JA even at higher concen-

trations (‡5.0 lM) could not repress this BA-

induced inhibitory effect. MeJA also had a

stimulatory effect on root growth, but its effect

was not so much pronounced (40.0 mg FW/SNC

at 7.5 lM MeJA averaged over the cultivars) and

uniform over the cultivars. Like JA, it also failed

to repress the inhibitory effect of BA on root

growth, especially in medium (KBD) and late

(KS) cultivars.

JA significantly inhibited stoloniferous shoot

(SS) growth, measured as fresh weight, even at

2.5 lM in early (KA) and late (KS) cultivars. The

addition of BA to the induction medium resulted

in a considerable decline in SS growth (2.4–6.8-

fold reduction as compared to the control). When

the BA-medium contained 2.5 lM JA, there was

a further decline in SS growth in all the cultivars.

The inhibitory effect of MeJA on SS growth was

288 Plant Cell Tiss Organ Cult (2006) 87:285–295

123

observed at higher concentrations (7.5–10.0 lM)

only in early cultivar (KA). In general, MeJA did

not have any effect on BA-induced inhibition of

SS growth, except in early cultivar (KA) where it

could significantly promote the SS growth at

5.0 lM.

Effects of jasmonates on tuber induction

and development

In cytokinin-aided in vitro tuberization under

optimum tuber-inducing treatment with 80 g L–1

sucrose, generally a single tuber is induced per

SNC. Therefore, it is not surprising that there was

not much variation in tuber number per SNC in

the present experiments. Both JA and MeJA did

not have any effect on tuber number; however,

there was a significant JA · BA interaction for

tuber induction (Fig. 1). JA at 2.5–5.0 lM was

found to inhibit tuber induction when the medium

contained 22.0 lM BA, but this JA-induced

inhibitory effect on tuber induction did not occur

in the presence of 44.0 lM BA. The inhibitory

effect of MeJA on tuber induction in the presence

of 22.0 lM BA was observed only in early cv. KA

(data not shown).

For tuber growth measured as fresh weight, the

promoting effect of JA was observed only in early

cultivar, but not in medium and late ones (Figs. 1,

2a). However, at higher concentrations, it not

only caused a reduction in tuber growth, but also

induced morphological changes in tuber shape

(Fig. 2b). In general, BA inhibited tuber growth.

Irrespective of the cultivars, an inhibition of tuber

growth occurred when JA was supplemented in

the medium with BA, although its effect was not

uniform over the BA levels (Figs. 1, 2c, d). The

trend of MeJA effect on tuber growth was similar

to that of JA effect; at 2.5 lM it had a promoting

effect on tuber growth only in early cultivar when

the nutrient medium contained only 80 g L–1

sucrose (Fig. 1). There was no significant Me-

JA · BA interaction, and in combination with

BA it depressed the tuber growth further only in

early cultivar (KA).

Harvest index is a function of total biomass

(source) and tuber yield (sink) in vitro. In the

present study, BA was found to significant

increase the harvest indices in all the cultivars

(Fig. 1). Both JA and MeJA did not have any

positive effect on harvest index. In combination

with BA, JA did not show any synergistic effect

on harvest index in relation to what obtained

when BA was supplemented alone in the med-

ium. In medium (KBD) and late (KS) cultivars,

there was a significant decline in harvest index

with increasing concentrations of MeJA (Fig. 1).

Effects of jasmonates on tuber quality

Tuber dry matter was between 15 and 17% when

the tubers were induced on medium containing

only 80 g L–1 sucrose (data not shown). BA

caused a significant increase in tuber dry matter

(19.2–22.9%) in medium (KBD) and late (KS)

cultivars. When JA was supplemented at 2.5 lM,

there was a significant increment in tuber dry

matter (20.5%) only in early cultivar (KA). Like

JA, MeJA at 2.5 lM also had a promoting effect

on tuber dry matter (18.1% as compared to

16.2% in the control) in early cultivar. In general,

MeJA in combination with BA significantly

increased the tuber dry matter in all the cultivars,

although this promoting effect was more conspic-

uous in medium (20.9–25.3%) and late cultivars

(22.8–25.5%). In contrast, the combined effect of

JA and BA on tuber dry matter was not so much

prominent as that of MeJA and BA.

There were significant interactions between

jasmonates and BA for reducing sugars, total

sugars and starch in tubers induced on SNCs in

vitro. In general, BA was found to have a

promoting effect on reducing and total sugar

levels in tubers (Figs. 3, 4). JA significantly

increased the levels of reducing and total sugars

in all the cultivars at 2.5–5.0 lM when the

medium contained only 80 g L–1 sucrose

(Fig. 4). In the presence of BA, JA could exert

a further promoting effect on reducing and total

sugar contents. No significant promoting effect of

MeJA on reducing and total sugar contents could

be detected in the present study (Fig. 4).

When the tubers were induced on medium

containing only 80 g L–1 sucrose, the tuber starch

content was in the range of 80–120 mg g–1 FW

(Figs. 3, 4). There was a significant increase in

starch content when the medium contained BA.

On medium containing 2.5–5.0 lM JA there was

Plant Cell Tiss Organ Cult (2006) 87:285–295 289

123

a significant increase in starch content in all

the cultivars, as compared to that obtained at

80 g L–1 sucrose-medium. In the presence of

22.0 lM BA, JA increased the starch levels

further, and increasing BA level did not have

any further promoting effect on starch levels. In

contrast, MeJA could increase the starch

content only in early cultivar (KA). However,

Jasmonic acid (µM)

0.0 2.5 5.0 7.5 10.0

Har

vest

inde

x

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Tub

er fr

esh

wei

ght (

mg)

0

50

100

150

200

250

300

0.0 2.5 5.0 7.5 10.0

Har

vest

inde

x

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0.0 2.5 5.0 7.5 10.0

Methyl jasmonate (µM)

LSD0.05

LSD0.05

Kufri Ashoka Kufri Badshah Kufri Sindhuri

Tub

ers

per

expl

ant

0.0

0.5

1.0

1.5

LSD0.05

Tub

er fr

esh

wei

ght (

mg)

0

50

100

150

200

250

300

LSD0.05

0.0 2.5 5.0 7.5 10.0 0.0 2.5 5.0 7.5 10.0

LSD0.5

0.0 2.5 5.0 7.5 10.0

0.0 µM BA22.0 µM BA44.0 µM BA

Fig. 1 Effects of jasmonic acid and methyl jasmonate ontuber induction and development from single-node cut-tings in relation to BA in three potato cultivars differing in

maturity levels. Means are separated, according to LeastSignificant Difference (LSD) test at p £ 0.05

290 Plant Cell Tiss Organ Cult (2006) 87:285–295

123

MeJA at 5.0–10.0 lM could significantly in-

crease the starch levels in the presence of

22.0 lM BA, irrespective of the cultivars

(Fig. 4).

Discussion

Although several reports have been published

concerning the effect of JA on potato tuberiza-

tion (Koda et al. 1991; Pelacho and Mingo-Castel

1991; Ravnikar et al. 1992; Helder et al. 1993;

Jackson and Willmitzer 1994; Castro et al. 2000;

Pruski et al. 2001, 2002), our study is the first one

to our knowledge to analyze the efficacy of

jasmonates (JA and MeJA) for potato tuber

induction and development in relation to exoge-

nous cytokinins. Our results clearly showed that

jasmonates did not affect tuber induction. Neither

did they have any favourable effect on in vitro

source-sink coordination. The inhibitory effects

of jasmonates on tuber induction in the presence

of 22.0 lM BA, especially in early cultivar is

difficult to explain, but may perhaps be due to an

altered balance of ethylene/gibberellin ratio

(Vreugdenhil and Struik 1989). However, the

promoting effects of both JA and MeJA on tuber

growth only in early cultivar suggested that their

effects are maturity-specific. A favourable effect

of JA on tuber growth after induction was also

reported by Pelacho and Mingo-Castel (1991).

Koda and Kikuta (2001) observed that JA-

induced in vitro tuberization in potato was

dependent on the maturing time of the cultivar;

the later the maturing time of the cultivar, the

lower the response. The relative higher JA-

responsiveness of early cultivar is assumed to be

due to the lower levels of endogenous GAs (Koda

1997; Koda and Kikuta 2001), which are known to

antagonize the JA action (Jackson 1999; Castro

et al. 2000). Since relatively high GA levels

reduce or stop tuber growth, it seems highly

Fig. 2 Development of tubers from single-node cuttingsof potato cv. Kufri Ashoka in vitro on nutrient mediasupplemented with sucrose and various combinations ofJA plus BA after 8 weeks of culture. (a) Eighty g L–1

sucrose (bottom) and 80 g L–1 sucrose plus 2.5 lM JA(top). (b) Eighty g L–1 sucrose plus 10.0 lM JA. (c) Eighty

g L–1 sucrose plus 22.0 lM BA and a series of JAconcentrations: 2.5, 5.0, 7.5 and 10.0 lM JA from left toright. (d) Eighty g L–1 sucrose plus 44.0 lM BA and aseries of JA concentrations: 2.5, 5.0, 7.5 and 10.0 lM JAfrom left to right

Plant Cell Tiss Organ Cult (2006) 87:285–295 291

123

plausible that jasmonates could only exert their

favourable effect on tuber growth in early culti-

vars, because their endogenous GA levels are

lower than that of late cultivars. Cultivar-specific

JA response in potato tuberization was also

reported by Pruski et al. (2001).

We unambiguously proved that exogenous

cytokinins did not have any stimulatory role in

tuber growth after induction. Although several

studies have reported the favourable effect of

cytokinins, particularly BA, on tuberization in

potato (Hussey and Stacey 1984; Donnelley et al.

2003), ours is the first one to demonstrate that

exogenous cytokinins do not promote, rather

inhibit tuber growth even at concentrations opti-

mum for in vitro tuberization in the dark. This is

in agreement with Pelacho and Mingo-Castel

(1991) who reported that JA-induced tuber fresh

weight was about 6.4-times the kinetin-induced

tuber weight. By the way, both JA (Pruski et al.

2001) and cytokinins (Palmer and Smith 1969)

have been proved to be most beneficial when

tuberization is occurred in the dark. This clearly

precludes any possibility of our results having

been influenced by contrasting requirements of

both these growth regulators for tuberization in

vitro. Our results further show that cytokinins

antagonize the jasmonate-effect on tuber growth

after induction, although this antagonizing effect

is more conspicuous with JA than MeJA irre-

spective of the cultivars. Dermastia et al. (1994)

showed that JA at a low concentration (1.0 lM)

caused an increase in the ratio of active to

inactive cytokinins in potato plantlets in vitro,

but there was no change in the total concentra-

tions of endogenous cytokinins. This report was

the basis of the possibility that one of the roles

that jasmonates play in tuberization might be

0.0 2.5 5.0 7.5 10.0

Red

ucin

g su

gars

(mg/

g fr

esh

wei

ght)

0

5

10

15

20

0.0 2.5 5.0 7.5 10.0 0.0 2.5 5.0 7.5 10.0

0.0 2.5 5.0 7.5 10.0

Tot

al s

ugar

s(m

g/ g

fres

h w

eigh

t)

0

10

20

30

40

50

0.0 2.5 5.0 7.5 10.0 0.0 2.5 5.0 7.5 10.0

0.0 2.5 5.0 7.5 10.0

Sta

rch

(mg/

g fr

esh

wei

ght)

0

50

100

150

200

250

0.0 2.5 5.0 7.5 10.0 0.0 2.5 5.0 7.5 10.0

0.0 µM BA22.0 µM BA44.0 µM BA

Jasmonic acid (µM)

LSD0.05

LSD0.05

LSD0.05

Kufri Ashoka Kufri Badshah Kufri Sindhuri

Fig. 3 The accumulation of reducing sugars, total sugarsand starch in tubers grown in vitro from single-nodecuttings under various concentrations of JA in relation

BA. Means are separated, according to Least SignificantDifference (LSD) test at p £ 0.05

292 Plant Cell Tiss Organ Cult (2006) 87:285–295

123

through an increase in the level of active cytoki-

nins (Koda 1997). However, in a later study, it

was demonstrated that the cytokinin content of in

vitro-cultured potato plantlets exposed to 1.0 lM

JA was about half that of the control plantlets

(Dermastia et al. 1996). It seems highly likely that

this may be the reason why exogenous application

of cytokinins resulted in a reduction in tuber

growth during jasmonate-induced tuberization in

potato. There appears to be no information about

the endogenous levels of cytokinins in stolons or

SNCs in vitro during jasmonate-induced tuber-

ization in potato under contrasting conditions of

tuber-inducing and tuber-noninducing stimuli.

Future studies in this direction would provide

further insight into this aspect.

Cytokinin was proved to be more potent than

jasmonates in regulating the source-sink coordi-

nation during tuberization in vitro. We assume

that its positive effect on source-sink interactions

is attained in association with other endogenous

tuberization signals. This was further supported

by the observation that cytokinin increased the

tuber dry matter irrespective of cultivars. Sea-

brook et al. (2004) have suggested that the

accumulation of dry matter is controlled by

the developing tuber (sink), whereas the increase

in water (tuber fresh weight) is controlled

by the leaves (source) during tuberization in

vitro. It indicates that there exists a dynamic

equilibrium between the leaf of a single-node

cutting and the developing tuber, suggesting that

dry matter and water accumulation are under the

control of two separate genetic mechanisms

(Seabrook et al. 2004). This could be the reason

why cytokinin despite having a negative effect

on tuber fresh weight had a favourable influence

over tuber dry matter vis-a-vis source-sink

0.0 2.5 5.0 7.5 10.0

Red

ucin

g su

gars

(mg/

g fr

esh

wei

ght)

0

5

10

15

20

0.0 2.5 5.0 7.5 10.0 0.0 2.5 5.0 7.5 10.0

0.0 2.5 5.0 7.5 10.0

Tot

al s

ugar

s(m

g/ g

fres

h w

eigh

t)

0

10

20

30

40

50

0.0 2.5 5.0 7.5 10.0 0.0 2.5 5.0 7.5 10.0

0.0 2.5 5.0 7.5 10.0

Sta

rch

(mg/

g fr

esh

wei

ght)

0

50

100

150

200

250

0.0 2.5 5.0 7.5 10.0 0.0 2.5 5.0 7.5 10.0

0.0 µM BA22.0 µM BA44.0 µM BA

Methyl jasmonate (µM)

LSD0.05

LSD0.05

LSD0.05

Kufri Ashoka Kufri Badshah Kufri Sindhuri

Fig. 4 The accumulation of reducing sugars, total sugarsand starch in tubers grown in vitro from single-nodecuttings under various concentrations of MeJA in relation

BA. Means are separated, according to Least SignificantDifference (LSD) test at p £ 0.05

Plant Cell Tiss Organ Cult (2006) 87:285–295 293

123

coordination. Cytokinin was also found to in-

crease the starch accumulation in tubers. As

starch is the major component of the dry matter

content of potato tubers accounting for about

70% of the total solids, this may explain the

occurrence of high-dry matter in cytokinin-

induced tubers in vitro. The beneficial effect of

jasmonates on tuber dry matter was apparent only

in early cultivar in which they had also stimula-

tory influence over tuber growth. Incidentally,

the promoting effects of jasmonates on starch

accumulation in tubers at relatively low concen-

trations were also evident in early cultivar. This

again substantiates the beneficial role of jasmo-

nates in tuber growth after induction depending

on the maturing time of the cultivars through a

regulation of endogenous GA levels. Our results

on starch accumulation in relation to jasmonates

and cytokinins based on in vitro bioassays can

well generalize their effects on potato tuberiza-

tion in vivo since starch synthesis and composi-

tion in tubers grown in vitro have been shown to

be identical to field-grown tubers (Veramendi

et al. 1999).

We also observed that both cytokinin and JA

had a promoting effect on the accumulation of

reducing and total sugars in tubers. The biochem-

ical machinery for starch synthesis and break-

down is present during all stages of tuber

development, and is coordinately up- or down-

regulated (Vreugdenhil 2004). Therefore, it is not

surprising as to why these two growth hormones

resulted in a high levels of sugars concomitant

with increased starch accumulation in tubers

induced in vitro. However, cytokinin, JA and

cultivars interacted among themselves to regulate

the levels of reducing sugars, total sugars and

starch in tubers. This invariably indicates that

these two growth hormones affect endogenous

sugar and starch levels of developed tubers

depending on the maturing time of the cultivars.

However, it must be noted that our study

describes the partitioning of sugars and starch in

tubers after they have developed fully vis-a-vis

matured in vitro, and therefore, might be its

result, not the cause. A time-course estimation of

the partitioning of sugars and starch during

various stages of tuber development from single-

node cuttings under varying treatments of exog-

enous cytokinins and jasmonates will be needed

to examine the specific roles of these two growth

hormones in tuber formation in vitro. Reducing

sugar levels can be an estimate of the maturity of

tubers grown in vitro (Xiong et al. 2002; Sharma

et al. 2004). Since both BA and JA caused an

increase in reducing sugars in tubers, their yet

another role in tuberization can be attributed to

tuber maturity, possibly through an endogenous

regulation of GA: ABA balance. However, fur-

ther investigation is needed to study the effect of

jasmonates in relation to cytokinins on tuber

dormancy release.

Acknowledgements Comments and suggestions on themanuscript from the Editor and two anonymous reviewersare gratefully acknowledged.

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