Effect of 24-Epibrassinolide on Growth of <i>in Vitro</i> Shoot Tips of Different Yam (<i>Dioscorea</i> Spp.) Species

doi:10.4236/ajps.2013.411280

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American Journal of Plant Sciences, 2013, 4, 2271-2274

Published Online November 2013 (http://www.scirp.org/journal/ajps)

http://dx.doi.org/10.4236/ajps.2013.411280

Open Access AJPS

2271

Effect of 24-Epibrassinolide on Growth of in Vitro Shoot

Tips of Different Yam (Dioscorea

Spp.) Species

Isabelle Engelmann-Sylvestre, Florent Engelmann

IRD, UMR DIADE, Agropolis, Montpellier, France.

Email: florent.en[email protected]

Received September 26th, 2013; revised October 29th, 2013; accepted November 10th, 2013

Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original

work is properly cited.

ABSTRACT

In this work we compared the effect of the growth regulator content of the culture medium on the growth of in vitro

shoot tips of five yam accessions belonging to four yam species (one Dioscorea alata, one D. rotundata, one D. cay-

enensis and two D. trifida). Medium S contained 0.6 µM benzyl adenine, 1.07 µM naphthalene acetic acid and 0.23 µM

gibberellic acid while medium EBR contained 0.23 µM gibberellic acid and 0.1 µM 24-epibrassinolide. After 2 months

of culture, oxidation level was significantly reduced on medium EBR compared to medium S for four of the five acces-

sions tested. By contrast, medium EBR did not have any positive effect on shoot length since length of shoots produced

after 2 months of culture on medium S and EBR were similar, except with accession 3-45T, for which shoot length was

shorter on medium S compared to medium EBR. These results underline the potential of 24-epibrassinolide to reduce

oxidation phenomena during in vitro culture and call for its utilization for regeneration of cryopreserved yam shoot tips,

which is often impeded by oxidation phenomena.

Keywords: Yam; Dioscorea Spp.; 24-Epibrassinolide; Oxidation; Shoot Tip; In Vitro Culture; Cryopreservation

1. Introduction

Cryopreservation (liquid nitrogen, −196˚C) currently is

the only safe and cost-effective option for long-term

conservation of vegetatively propagated plants such as

yam [1]. Indeed, at this temperature, all cell divisions

stop and metabolism is virtually arrested. Explants can

thus be conserved for extended periods (several thousand

years) without modification or alteration, sheltered from

contamination, in a limited volume and with reduced

maintenance.

Cryopreservation protocols have been developed for

Dioscorea alata, D. cayenensis, D. rotundata and D.

floribunda shoot tips using different techniques [2-7]. A

droplet-vitrification protocol jointly established by the

International Institute of Tropical Agriculture (IITA,

Ibadan, Nigeria) and our laboratory has been applied to a

total of 42 D. cayenensis, D. rotundata, D. alata, D. bul-

bifera and D. mangotiana accessions, with an average

recovery of 29% (Gueye et al. unpublished results).

Cryopreservation experiments performed in IRD Mont-

pellier with American yam (D. trifida) shoot tips showed

that, even though positive results were obtained, no re-

producible protocol was yet available for this species

(Engelmann-Sylvestre et al. unpublished results). In all

these reports, the authors mentioned that if high survival

could be consistently achieved, regeneration of whole

plantlets from cryopreserved shoot tips was variable,

depending on the species and the technique used, and

was generally much lower than survival. The occurrence

of severe oxidation phenomena was also consistently re-

ported.

In order to optimize the regrowth rate and pattern of D.

trifida shoot tips, an experiment was performed recently

with one D. trifida accession (N˚ 278) to compare the

effect of the recovery medium used after their cryopre-

servation, which included 6-benzylaminopurine (BAP),

naphthalene acetic acid (NAA) and gibberellic acid

(GA3), with media in which BAP and NAA were re-

placed by 24-epibrassinolide (EBR), zeatine riboside or

meta-topolin [8]. This experiment showed that EBR in-

duced the production of well developed shoots and sig-

nificantly reduced oxidation compared to the other media

tested.

EBR belongs to brassinosteroids (BRs), a class of

Effect of 24-Epibrassinolide on Growth of in Vitro Shoot Tips of Different Yam (Dioscorea Spp.) Species

2272

plant steroid hormones, which possess significant growth-

controlling activity, and are involved in the promotion of

cell elongation, cell division, differentiation, disease re-

sistance, stress tolerance, and senescence throughout the

plant life cycle. BRs have been reported to help modu-

lating the plant antioxidant defence system and thus

scavenging the free radicals and help the plant protecting

itself from oxidative stress [9] and have also been found

to have an activity in vitro. They were reported to in-

crease the rate of cell division and colony formation of

Chinese cabbage mesophyll protoplasts [10] and Petunia

hybrida protoplasts [11]. BRs are also proved to be es-

sential for the differentiation of isolated Zinnia mesophyll

cells into tracheary elements [12] and in the morpho-

genesis of Arabidopsis [13].

In this study, we compared the regrowth of shoot tips

of a total of five yam accessions including one D.

cayenensis, D. rotundata and D. alata accession and two

D. trifida accessions on medium containing EBR and

GA3, or BAP, NAA and GA3, which are the plant growth

regulators (PGRs) usually added to the regeneration

medium after cryopreservation of yam shoot tips. Our

objective was to observe if the EBR-containing growth

medium which had been successfully employed with D.

trifida accession N˚ 278 [8] was also effective with other

D. trifida accessions and accessions of the other yam

species selected.

2. Materials and Methods

2.1. Plant Material

This study was performed using in vitro shoot cultures of

three yam accessions provided by the International Insti-

tute of Tropical Agriculture (IITA, Ibadan, Nigeria) in-

cluding accessions N˚ 1454 (D. alata), N˚ 2790 (D. cay-

enensis) and N˚ 3675 (D. rotundata) and of two acces-

sions provided by the Institut National de la Recherche

Agronomique (INRA) Guadeloupe, French West Indies

(D. trifida accessions N˚ 278 and 3-45T).

2.2. Methods

Mother-plants were subcultured every 3 - 5 months on

medium containing Murashige and Skoog [14] basal salts

and vitamins, 3% sucrose, 0.2% activated charcoal and

0.7% agar. The pH was adjusted to 5.8 ± 0.1 and the me-

dium was autoclaved for 20 min at 120˚C. Cultures were

maintained at 27˚C ± 1˚C under a 12 h light/12 h dark

photoperiod and a light intensity of 50 µmol·m−2·s−1.

Single node cultures were transferred to yam multipli-

cation medium consisting of MS basal salts and vitamins

[14], 0.476 µM KIN, 0.164 µM L-cysteine, 3% sucrose

and 0.7% agar. After 3 weeks, shoot tips (approx. 1 - 2

mm in length) were excised under the binocular micro-

scope and used for experiments.

Half of the shoot tips were cultured on medium S,

which was used for regeneration of yam shoot tips after

cryopreservation, which consisted of MS mineral salts

and vitamins [14], 0.164 µM L-cysteine, 0.22 mM ade-

nine hemisulfate, 0.6 µM BAP, 1.07 µM NAA, 0.23 µM

GA3 (filter-sterilized), 3% sucrose and 0.7% agar.

The other half of the shoot tips was cultured on me-

dium EBR, consisting of MS mineral salts and vitamins

[14], 0.164 µM L-cysteine, 0.22 mM adenine hemisulfate,

0.23 µM GA3 (filter-sterilized), 0.1 µM 24-epibrassino-

lide (Sigma-Aldrich ref E1641, filter-sterilized), 3% su-

crose and 0.7% agar. Shoot tips were kept in the dark for

1 week, and then transferred to the culture conditions

employed for mother-plants.

2.3. Observations Performed and Statistical

Analysis of Results

The experiment was performed once, with three repli-

cates of 10 shoot tips per experimental condition. After 1

and 2 months of culture on medium S or EBR, the size of

shoot tips (mm) was measured and the oxidation level

was evaluated using a scale from 0 (no oxidation) to 3

(very high oxidation). One-way ANOVA was performed

to compare the growth of shoot tips and oxidation levels

on medium S and EBR. Means were statistically differ-

entiated using Duncan test at a significance level of Po <

0.05.

3. Results

When comparing the growth of shoot tips of the five ac-

cessions studied on the two culture media tested, it ap-

peared that accessions 278 and 2790 had a lower growth

compared to the other three accessions on both S and

EBR medium (Table 1). Studying the effect of the cul-

ture medium on the growth of each individual accession

revealed that shoot length was lower after 1 month on

medium S compared to medium EBR for three acces-

sions (1454, 278 and 3-45T). By contrast, no differences

in shoot length were observed after 2 months of culture

on medium S and EBR, except with accession 3-45T, for

which shoot length was lower on medium S compared to

medium EBR.

When comparing the effect of culture medium on oxi-

dation level, it was observed that accessions reacted dif-

ferently depending on the culture medium and the culture

duration (Table 2). After 1 month on medium S, oxida-

tion was significantly lower in accessions 2790 and 3675,

compared to the other three accessions. After 2 months

on medium S, oxidation remained significantly lower in

accession 2790, and was comparably higher in the four

other accessions. After 1 and 2 months on medium EBR,

oxidation was significantly higher in accession 1454

compared to the other four accessions studied. When

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Effect of 24-Epibrassinolide on Growth of in Vitro Shoot Tips of Different Yam (Dioscorea Spp.) Species

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2273

Table 1. Effect of culture medium and culture duration on shoot length (mm) of five yam accessions. The S medium con-

tained 0.6 µM BAP, 1.07 µM NAA, 0.23 µM GA3 and the EBR medium 0.23 µM GA3 and 0.1 µM 24-epibrassinolide. In lines,

different lower case letters indicate significant differences between accessions (Po ≤ 0.05). In columns, different upper case

letters indicate significant differences between treatments (Po ≤ 0.05).

Shoot length (mm)

Medium Culture duration Accession N˚

1454 278 2790 3-45 T 3675

S 1 month 5.27 ± 2.83 a/B 2.97 ± 2.31 b/B 1.89 ± 1.15 b/A 1.86 ± 1.22 b/B 4.65 ± 2.56 a/A

2 months 6.73 ± 2.56 bc/A 11.60 ± 9.77 a/A 3.47 ± 2.55 d/A 3.97 ± 2.81 cd/B 8.15 ± 3.76 b/A

EBR 1 month 6.67 ± 2.22 a/A 6.57 ± 5.10 a/A 1.55 ± 0.69 b/A 3.13 ± 2.90 b/A 4.95 ± 2.72 a/A

2 months 7.93 ± 3.03 b/A 13.96 ± 4.14 a/A 5.15 ± 3.98 c/A 6.70 ± 3.70 bc/A 7.65 ± 3.56 b/A

Table 2. Effect of culture medium and culture duration on oxidation level (0 to 3) of five yam accessions. The S medium con-

tained 0.6 µM BAP, 1.07 µM NAA, 0.23 µM GA3 and the EBR medium 0.23 µM GA3 and 0.1 µM 24-epibrassinolide. In lines,

different lower case letters indicate significant differences between accessions (Po ≤ 0.05). In columns, different upper case

letters indicate significant differences between treatments (Po ≤ 0.05).

Oxidation level

Medium Culture duration Accession N˚

1454 278 2790 3-45 T 3675

S 1 month 2.07 ± 0.94 a/A 2.00 ± 0.87 a/A 0.32 ± 0.48 b/A 2.28 ± 1.07 a/A 0.50 ± 0.89 b/A

2 months 2.43 ± 0.73 ab/A 1.93 ± 0.83 b/A 1.11 ± 0.94 c/A 2.48 ± 0.83 a/A 2.15 ± 1.18 ab/A

EBR 1 months 0.97 ± 0.67 a/B 0.20 ± 0.41 b/B 0.10 ± 0.31 b/A 0.13 ± 0.35 b/B 0.00 ± 0.00 b/B

2 months 1.43 ± 0.77 a/B 0.54 ± 0.74 bc/B 0.60 ± 0.60 bc/A 0.73 ± 0.52 b/B 0.25 ± 0.55c/B

studying the effect of the culture medium on the oxida-

tion level measured on each individual accession, it was

observed that the EBR medium induced significantly

lower oxidation at 1 and 2 months in all accessions stud-

ied except in accession 2790, for which no significant

differences between experimental conditions were noted.

The positive effect of EBR on oxidation level after 2

months of culture on medium EBR compared to medium

S is illustrated on Figure 1 with accessions N˚ 3675 (D.

rotundata) and 1454 (D. alata).

4. Discussion

Our experiments showed that culture for 2 months of

shoot tips of five yam accessions, representing four

different yam species, on medium EBR, which contained

24-epibrassinolide, had a positive effect on oxidation

level compared to culture on medium S, which contained

BAP and NAA. Indeed, oxidation level was significantly

reduced on medium EBR for four of the five accessions

tested. This result confirms our previous observations

performed on D. trifida accessions N˚ 278 [8] and under-

lines the potential of 24-epibrassinolide to reduce oxida-

tion phenomena during in vitro culture.

Figure 1. Effect culture for 2 months of shoot tips of yam

accessions N˚ 3675 and 1454 on S medium (0.6 µM BAP,

1.07 µM NAA, 0.23 µM GA3) or EBR medium (0.23 µM

GA3 and 0.1 µM 24-epibrassinolide) on oxidation level (0 -

3). Bars represent 10 mm.

By contrast, even though medium EBR induced higher

shoot growth after 1 month compared to medium S, this

positive effect was no more visible after 2 months of

culture since shoots grown on medium S and EBR had

similar lengths, except with accession 3-45T, for which

shoot length was shorter on medium S compared to

medium EBR.

Effect of 24-Epibrassinolide on Growth of in Vitro Shoot Tips of Different Yam (Dioscorea Spp.) Species

2274

It has been demonstrated in numerous species that

modifying the PGR composition of the recovery medium

can have a dramatic effect on regrowth of cryopreserved

plant material [1].

Notably, alteration of post-cryopreservation culture

media with PGRs and their application at various stages

of growth recovery was crucial for regeneration of shoot

tips and formation of in vitro plantlets in D. alata and D.

bulbifera [5,7]. A recent report showed that the addition

of vitamin E, vitamin C or both vitamins together, which

have antioxidant properties, at different steps of the pro-

tocol (pre- and/or post-cryopreservation) had a positive

effect on regrowth of cryopreserved blackberry shoot tips

[15].

In view of the potential of the PGR 24-epibrassinolide

in reducing oxidation, which has been shown in this

work, experiments will be performed to test its effect on

regrowth of cryopreserved yam shoot tips using the five

accessions employed in this study.

5. Acknowledgements

This work has received financial support from ARCAD,

a flagship programme of Agropolis Fondation (Montpel-

lier, France) (I. Engelmann-Sylvestre). The assistance of

Giuseppe Barraco for performing the statistical analysis

of results is acknowledged.

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