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.
Received September 26th, 2013; revised October 29th, 2013; accepted November 10th, 2013
Copyright © 2013 Isabelle Engelmann-Sylvestre, Florent Engelmann. This is an open access article distributed under the Creative
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
Open Access AJPS
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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