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107
Original Research Article
Plant Cell Biotechnology and Molecular Biology 21(37&38):107-120; 2020 ISSN: 0972-2025
MORPHOLOGICAL AND PHYSIOLOGICAL
SCREENING OF ETHYL METHANESULFONATE-
TREATED SUGARCANE CALLI AND PLANTLETS FOR
In vitro MANNITOL-INDUCED OSMOTIC STRESS
TOLERANCE
P. J. DLAMINI, T. SCHULTZ-VILJOEN AND N. R. NTULI
*
Department of Agriculture, University of Zululand, Private Bag X1001, KwaDlangezwa 3886,
South Africa [PJD, TSV].
Department of Botany, University of Zululand, Private Bag X1001, KwaDlangezwa 3886,
South Africa [NRN].
[
*
For Correspondence: E-mail:
NtuliR@unizulu.ac.za]
Article Information
Editor(s):
(1)
Dr. Ahmed Medhat Mohamed Al-Naggar, Professor, Cairo University, Egypt.
Reviewers:
(1)
Romário de Mesquita Pinheiro, Federal University of Pelotas, Brazil.
(2)
Alok Ranjan, Banaras Hindu University, India.
Received: 04 June 2020
Accepted: 10 August 2020
Published: 08 September 2020
_______________________________________________________________________________________________
ABSTRACT
Sugarcane production in South Africa is limited by drought stress and its impact is expected to
increase due to climate change. However, limited research is conducted to develop cultivars that are
suitable for cultivation under water-stressed conditions. This study aimed to select, through
morphological and physiological traits, the ethyl methanesulfonate treatment(s) that can produce
calli and plantlets that are resistant to mannitol-induced osmotic stress. Sugarcane calli were
exposed to ethyl methanesulfonate for 0.5, 1, 2, and 3 hours. To determine optimal selection lethal
doses, calli were cultured on media containing 0, 150, 225 and 300 mM mannitol for further eight
weeks under dark and light conditions. Incubation periods of half an hour and one hour induced
genomic mutations without inhibiting callus growth and plant regeneration abilities. EMS-treated
calli retained whiter, compact and with yellowish friable texture when compared with the control
after the two-weeks recovery period post exposure to osmotic stress. Callus that was exposed for one
hour was able to recover and regenerate plantlets at 225 and 407 mM mannitol stress. However, the
two-hour incubation period and above resulted in stunted and albino plantlets. The LD
50
and LD
90
for selection were calculated as 225 and 407 mM mannitol. The EMS mutagenesis and in vitro
selection for osmotic stress using mannitol can be used successfully to select sugarcane plantlets with
better morphological and physiological responses to water stress in a very short time.
Keywords: Ethyl methanesulfonate mutagenesis; mannitol osmotic stress; sugarcane.
Dlamini et al.
108
INTRODUCTION
Drought is one of the leading causes towards yield
losses in sugarcane (Saccharum spp.) [1]. Its
effects are expected to become widespread due to
high temperatures caused by climate change [2].
Drought imposes osmotic stress that affects
growth, productivity and secondary metabolites in
plants [3]. Mannitol, a six carbon sugar alcohol
[3], is used as an osmotic stress agent to screen for
drought tolerance among plantlets of different
sugarcane cultivars [4]. Ethyl methanesulfonate
(EMS) as a chemical mutagen in combination with
polyethylene glycol as a drought selecting agent,
are used for in vitro callus selection [5]. However,
limited research is available for the combined use
of EMS as a chemical mutagen and mannitol as a
selecting agent for osmotic stress tolerance in
sugarcane.
Sugarcane cultivar NCo376 is identified as the
most drought susceptible cultivar among others
[4]. In tissue cultures, the addition of mannitol to
nutrient solution can affect gene expression,
morphological and physiological characteristics,
and biochemical content in plants [3]. Calli
obtained from drought susceptible Durum wheat
cultivars shrink under high mannitol
concentrations compared with drought tolerant
cultivars [6]. Shrinking of callus under osmotic
stress results because of the cells’ inability to
maintain turgor pressure against the cell walls [7].
High turgor pressure maintains plant form and
facilitates cell expansion and growth [8]. Drought
tolerant plants are characterized by the ability to
maintain cell growth even during water limiting
conditions [9].
Genetic variability determines cell ability to adapt
to changing environmental conditions [9]. A well-
developed in vitro culture system for sugarcane
[10] is used concurrently, among other techniques,
with in vitro mutagenesis [5], to develop new
traits. In vitro mutation breeding through chemical
mutagenesis is frequently practiced, where ethyl
methanesulfonate (EMS) is being the most
frequently used mutagen for sugarcane
improvement [11]. Traits that have been improved
in sugarcane using EMS include herbicide
resistance [12,13], fungi resistance [11],
aluminium resistance [14] and drought tolerance
[5]. In addition, in vitro selection reduces the
amount of space, time and labour required for
field selection [4].
The mutagenic effect of EMS depends on the
incubation period, concentration of the mutagen,
as well as growth stage and age of the explant
material [14,15]. Actively dividing cells are
required to incorporate mutations and for that
reason callus tissue is a good substrate for the
treatment [14,16]. Sugarcane has a complex
genome, high chromosome imbalances and a
narrow genetic basis [17]. Therefore, in such
cases, EMS becomes the most viable mutagen of
choice for sugarcane improvement as it results in
random small-scale or point mutations at high
frequencies [13], with low chromosomal
aberration frequencies [18].
Sugarcane cultivar NCo376, which is highly
susceptible to drought, is widely cultivated in
South Africa [19]. There is a lack of research on
the improvement of genetic diversity of this
cultivar using EMS as a mutagen and selecting
under mannitol-induced osmotic stress putative
lines with a potential for drought stress tolerance.
Therefore, this study aimed to screen, through
morphological and physiological traits, ethyl
methanesulfonate treatment(s) that can produce
calli and plantlets from NCo376 cultivar that are
resistant to mannitol-induced osmotic stress.
MATERIALS AND METHODS
Plant Material
Sugarcane cultivar NCo376 collected from the
University of Zululand Orchard Farm Unit
(28°51’S, 31°50’E), was grown in pots in the
greenhouse for six months. Its apical meristems
were cut below the last internode had the leaves
peeled off and the leaf roll surface-sterilized with
70% ethanol.
Initiation of Embryonic Callus
Callus formation was initiated according to
Snyman [20] with some modifications. Leaf roll
was placed in a petri dish containing half-strength
liquid MS medium consisting of 2.2 g/l MS salts
and vitamins [21] and sliced into 30 transverse
Dlamini et al.
109
sections of approximately 3 mm thick each.
Subsequently, 10-disc setts were cultured on
sterile callus induction medium (CIM), which
comprised of 4.4 g/l MS salts and vitamins, 3 mg/l
(300 mg/l stock) 2,4-Dichlorophenoxyacetic acid
(2,4-D), 20 g/l sucrose and 8 g/l agar. The medium
was adjusted to pH 5.8 using HCl or NaOH before
autoclaving. Culture plates were sealed with
parafilm and incubated in a growth chamber
(MRC LE-11) under darkness with the
temperature maintained at 26 ± 1°C for eight
weeks. The calli were sub-cultured on fresh CIM
every two weeks. The 2,4-D stock solution was
prepared by adding 300 mg powdered 2,4-D in 5
ml absolute ethanol, which was gently agitated
until it was completely dissolved and distilled
water was added to make up one litre of stock
solution. This was stored at 4°C and used for six
months.
Mutagenic Treatment of Embryonic Callus
Each embryogenic callus, weighing approximately
0.2 g, was antiseptically immersed in a centrifuge
tube containing 10 ml of half-strength liquid MS
Medium and 16 mM ethyl methanesulfonate
(EMS) as mutagenic agent [12], and incubated for
0, 0.5, 1, 2 and 3 hours. After each incubation
period, EMS-treated calli and controls (not
exposed to EMS) were rinsed three times using
half strength liquid MS medium. Then they were
air dried in empty Petri dishes for five minutes and
cultured again on CIM for a further four weeks in
darkness at 26 ± 1°C. Three Petri dishes were
loaded with 25 calli in each treatment.
The percentage change in callus fresh weight was
determined by recording initial weight before
exposure to EMS and final weight after four
weeks of exposure to EMS. Three culture plates
were used as replicates per treatment duration with
each plate containing 25 calli samples. Percentage
changes in callus fresh weight were determined
using the formula: Fresh weight = 100 × [(final
weight –initial weight) / initial weight].
Osmotic Stress Selection Conditions
Calli pieces (15) were cultured in CIM containing
0, 150, 225 and 300 mM mannitol (Sigma-
Aldrich, Johannesburg, South Africa) as an
osmotic agent, for eight weeks in darkness at 26 ±
1°C. Control treatments contained non-mutated
callus (not exposed to EMS) cultured in CIM
containing mannitol (negative control) and
without mannitol (positive control). Calli were
sub-cultured every two weeks. After eight weeks
of exposure to mannitol, calli were sub-cultured
on CIM for two weeks to enable calli to recover.
Calli relative growth rate (RGR) was quantified by
measuring the callus growth (mm) in four
directions where each callus sample was scored
with two lines. The lines were drawn through the
centre of each callus to be measured and each
radial line was labelled R1, R2, R3 and R4. Initial
radial dimensions (mm) were recorded along each
of the four radial lines and the final callus radial
size (mm) was measured after eight weeks of
exposure to the different concentrations of
mannitol. Five replicates were used per treatment.
The sum of the four measurements was
determined and the average was recorded as total
growth of the callus. The RGR was determined
using the formula: RGR = 100 x [(Final callus
radial growth – initial callus radial growth) / initial
callus radial growth]. Callus index of tolerance
(INTOL) was determined using Shekhawat et al.
[22] formula: INTOL = RGR treatment / RGR
control.
After eight weeks of exposure to mannitol-
induced osmotic stress, callus dry weight (DW)
was determined by oven drying the calli at 60℃
for 48 hours. Callus water content (CWC) was
calculated using the formula; CWC = [(FW –
DW) / FW x 100] as described by Errabii et al.
[23], where FW and DW are fresh and dry weights
of the callus, respectively.
The percentage of embryogenic calli was recorded
after two weeks’ recovery period. Whitish and
yellowish calli pieces were selected as potential
embryogenic material; dark brown calli were
considered non-embryogenic/necrotic [24]. The
control percentage of embryonic calli was used to
calculate mannitol lethal dose that inhibits callus
survival by 50% (LD
50
) and 90% (LD
90
) to
determine optimum concentrations for in vitro
selection using GraphPad Prism (v 7.04,
GraphPad, San Diego, CA), using the formula: y =
mx + c. The lethal doses determined were
subsequently used during embryogenic
Dlamini et al.
110
germination to examine plantlet formation abilities
and their morphological characterization.
Embryo Germination and Selection Conditions
To establish plantlets, embryogenic calli were
cultured in embryo germination medium (EGM;
CIM without 2,4-D) supplemented with LD
50
and
LD
90
mannitol. Shoots were initiated in a growth
chamber with cool white fluorescent lights (11120
lux) set at a photoperiod of 16 hours of light and 8
hours of darkness at a temperature of 26 ± 1 °C.
Sub-culturing to fresh media was conducted
biweekly for eight weeks. After eight weeks,
plantlets were transferred to EGM, without
exposure to mannitol, to recover under the same
growth conditions for two weeks. The number of
plantlets generated per callus cluster and the
percentage of stunted and albino plantlets were
recorded.
Statistical Analysis
ANOVA followed by a comparisons test was used
to determine variations across all treatments of
callus exposure to EMS and mannitol. The
interaction between and within treatment groups
was tested using a 5% level of significance
(GraphPad Prism; ver. 7.04, GraphPad Software
Inc., San Diego, CA).
RESULTS
The results are presented in terms of the effects on
calli exposed to ethyl methanesulfonate (EMS) for
different time points, then subsequently to the
effects of mannitol at different concentrations and
finally to plantlet generation after the combined
exposure of calli to EMS and mannitol.
Effect of Ethyl Methanesulfonate on Callus
Ethyl methanesulfonate incubation significantly
reduced the growth percentage of callus fresh
weight after four weeks (Fig. 1). Callus growth
was retarded by exposure to EMS for one hour.
Control calli had the highest relative increase in
fresh weight percentage (13.4%), whereas calli
exposed to EMS for 3 h resulted in the lowest
increase (4.9%).
Fig. 1. Effect of ethyl methanesulfonate incubation time on callus fresh weight after four weeks
Mean values with deferent letter(s) are significantly different at P ≤ 0.05
Dlamini et al.
111
Differences in the relative growth rate between
control and EMS-treated calli were not significant
when they were not exposed to mannitol, except
for a better growth of callus treated for one hour
than the one treated for three hours (Fig. 2). An
increase in mannitol-induced osmotic stress
resulted in a reduction in the relative growth rates
of both untreated and EMS-treated calli. Stress
induced with 225 and 300 mM mannitol even
resulted in the death (negative relative growth
rate) of control calli, although EMS-treated calli
survived these conditions at different growth
abilities. However, calli exposed to EMS for 1–3
hours, all showed a similar relative growth rate
when subjected to both 150 and 225 mM
mannitol. This resistance to osmotic stress was
also recorded in 2 and 3 hour-treated calli when
further exposed to 300 mM mannitol. However,
callus treated for one hour with EMS grew better
than the control and half-an-hour treated calli,
when exposed to 150 mM mannitol.
Treatment of calli with EMS increased the calli
index of tolerance towards mannitol-induced
osmotic stress, which also increased with the
extension of EMS incubation time (Fig. 3). After
exposure to 150 mM mannitol, the highest
tolerance was in calli treated with EMS for three
hours compared with the control. Calli not treated
with EMS were susceptible (negative values) to
both moderate (225 mM) and high (300 mM)
osmotic stress conditions. Index of tolerance for
calli exposed to EMS for 1–3 hours was
similar for both 150 and 225 mM mannitol-
induced osmotic stress. Again, calli treated with
EMS for three hours maintained the same
tolerance ability even at 300 mM mannitol
exposure.
Water content of both control and EMS-treated
calli was reduced by exposure to mannitol-
induced osmotic stress, compared with the calli
cultured in the media without mannitol (Fig. 4).
However, increase in mannitol concentrations
did not affect the water content of calli treated
with EMS for 1, 2 and 3 hours. Control and 0.5-
hour EMS-treated calli had a gradual decrease in
their water content as the osmotic stress increased.
Fig. 2. Effect of mannitol concentration on callus exposed for different times to ethyl
methanesulfonate on relative growth rate
Mean values with different letter(s) are significantly different at P < 0.05
Fig.
3. Effect of mannitol concentration on callus exposed for different times to ethyl methanesulfon
ate in terms of the index of tolerance
Mean values with different letter(s) are significantly different at
Fig. 4. Water content of calli, exposed to ethyl methanesulfonate for different times, under
mannitol-induced osmotic stress
Mean values with different letter(s) are significantly different at
Dlamini et al.
112
3. Effect of mannitol concentration on callus exposed for different times to ethyl methanesulfon
Mean values with different letter(s) are significantly different at
P < 0.05
Fig. 4. Water content of calli, exposed to ethyl methanesulfonate for different times, under
Mean values with different letter(s) are significantly different at
P < 0.05
Dlamini et al.
3. Effect of mannitol concentration on callus exposed for different times to ethyl methanesulfon
-
Dlamini et al.
113
Embryogenesis of all EMS-treated calli was
similar within each mannitol concentration (Fig.
5). Calli started showing signs of necrosis within
the first week after EMS treatment. The negative
control calli (without mannitol and EMS)
produced the highest amount (93%) of
embryogenic calli (Fig. 5; Fig. 6A). However, this
did not differ from embryonic calli produced by
0.5–2 hour EMS-treated calli that were not
exposed to mannitol, and 0.5-hour EMS-treated
calli exposed to 150 mM mannitol osmotic stress.
Different intensities of mannitol osmotic stress did
not affect embryogenicity of 3-hour EMS-treated
calli, when compared with calli not exposed to
stress. Again, increases in mannitol concentrations
did not affect calli treated with EMS for 1 and 2
hours to produce embryonic calli.
Callus appearing at least 50% brownish were
classified as non-embryogenic (Fig. 6B). After
three weeks of exposure to mannitol, EMS
treatment resulted in early root development in
calli. This was intensified in the 225 mM and 300
mM mannitol treatments (Fig. 6C). Two weeks
after stress relief, EMS treated calli that appeared
to be necrotic due to exposure to water stress
started to develop prolific yellowish callus clusters
(Fig. 6D).
Determination of Mannitol Lethal Doses
Mannitol concentrations lethal doses that inhibited
callus survival by 50% (LD
50
) and 90% (LD
90
), as
determined using calli that were not exposed to
EMS, were LD
50
= 225 mM and LD
90
= 407 mM
(Fig. 7). There was a strong negative correlation
between an increase in mannitol concentrations
and embryonic calli percentage survival (R
2
=
0.92; P = 0.04), meaning that an increase in
mannitol concentration results in a significant
decrease in calli embryogenicity. This trend was
also observed on EMS-treated callus regardless of
the incubation period (Table 1). The determined
lethal doses (LD
50
= 225 mM and LD
90
= 407
mM, respectively) were subsequently used for
callus selection at plantlet germination stage.
Fig. 5. The effect of mannitol concentration on the survival rate of calli exposed to ethyl methanesul-
fonate for different times
Mean values with different letter(s) are significantly different at P < 0.05
Fig.
6. (A) Embryogenic untreated callus cultured in
untreated callus cultured in 300 mM mannitol; (C)
methanesulfonate mutated calli and
(D) regeneration of yellowish callus clumps after two weeks’
recovery period
Table 1. Regression analysis of
percentage
darkness
Incubation
period (h)
Percentage
survival at LD
50
= 225 mM
Percentage
survival at LD
407 mM
0
0.5
1
2
3
50
46
51
46
53
10
14
23
29
45
Dlamini et al.
114
6. (A) Embryogenic untreated callus cultured in
callus induction medium; (B) necrotic
untreated callus cultured in 300 mM mannitol; (C)
early root development (arrows) on ethyl
(D) regeneration of yellowish callus clumps after two weeks’
percentage
survival on mannitol lethal doses during selection in
Percentage
survival at LD
90
=
407 mM
Regression model Determination
coefficient (R
2
)
Significance
level of the
regression
model (
ŷ = -0.2177*X + 98.66
ŷ = -0.1729*X + 84.76
ŷ = -0.1495*X + 84
ŷ = -0.09194*X + 66.63
ŷ = -0.04079*X + 61.71
0.92
0.77
0.97
0.71
0.47
0.0406
0.1228
0.0176
0.1601
0.3154
Dlamini et al.
(D) regeneration of yellowish callus clumps after two weeks’
survival on mannitol lethal doses during selection in
Significance
level of the
regression
model (
P)
0.0406
0.1228
0.0176
0.1601
0.3154
Dlamini et al.
115
Fig. 7. Linear regression analysis used to determine mannitol concentrations that inhibit survival by
50% (blue circle) and 90% (red circle) (LD
50
and LD
90
, respectively) for cultivar NCo376 using
control calli
Plantlet Generation
When callus was transferred to a growth chamber
with 16:8 h photoperiod for plantlet generation,
calli started greening within the first week, but the
number of plantlets generated per callus clump
were only recorded after eight weeks (Table 2). In
both conditions without mannitol and with 225
mM (LD
50
) concentrations, the differences in the
production of plantlets by control and 1-hour
EMS-treated calli were not significant, but a
significant reduction was recorded in callus treated
for 0.5, 2 and 3 hours with EMS (Table 2). At
LD
90
(407 mM), EMS-treated calli generated
similar numbers of plantlets as the control.
However, calli exposed to EMS for 3 hours did not
develop any plantlets.
Callus treated with EMS produced numerous
stunted plantlets except for 1-hour EMS-treated
callus, when compared with the control (Table 2).
Further, calli exposed to EMS for 2 and 3 hours
produced high numbers of albino plantlets when
compared with the control and other treatments
(Table 2, Fig. 8F).
DISCUSSION
The Effect of Ethyl Methanesulfonate on Callus
Embryogenesis
The decrease in the callus fresh by 8.5% after its
incubation for 3 h in ethyl methanesulfonate
(EMS) solution agrees with the decrease in
sugarcane callus fresh weight after exposure to
EMS as reported by Koch et al. [12] and
Purnamaningsih and Hutami [14]. EMS mutagenic
effect is affected by the incubation period [15].
Long exposure may facilitate absorption of the
mutagen by the cells at an excessive level, and
Table 2. Effect of ethyl methanesulfonate and
generation
Incubation period
(h)
Number of plantlets per callus cluster
Mannitol concentration (mM)
0
Control
0.5
1
2
3
12.47 ± 1.92
a
08.42 ± 1.42
b
13.07 ± 2.88
a
03.53 ± 2.72
c
02.90 ± 2.14
c
6.10 ± 1.64
3.73 ± 1.71
5.75 ± 2.11
1.93 ± 1.39
2.13 ± 1.60
Mean values with different letter(s) within a column are significantly different at
Fig. 8.
(A) Untreated callus after two weeks during plantlet formation on embryo growth medium
(EGM); (B) Control plantlets after two weeks on EGM supplemented with 407 mM mannitol (LD
(C) Plantlets after eight weeks on EGM; (D) Callus treated for one hour af
(E) Callus treated for one hour on EGM supplemented with 407 mM
results in
protein denaturation that is followed by
cell death [16,25]. Ethyl
methanesulfonate
mutagenesis has been successfully used to induce
genetic variability [13] and to improve various
traits in sugarcane [11,26,27].
Calli relative growth rate might be an
indication
calli
embryo regeneration potential, where both
0.5 h and 1 h EMS treatments resulted in a rapid
Dlamini et al.
116
Table 2. Effect of ethyl methanesulfonate and
mannitol-induced
osmotic stress on sugarcane plantlet
Number of plantlets per callus cluster
Percentage of phenotypically
abnormal plantlets
Mannitol concentration (mM)
225 407 Stunted Albino
6.10 ± 1.64
a
3.73 ± 1.71
b
5.75 ± 2.11
a
1.93 ± 1.39
c
2.13 ± 1.60
bc
1.80 ± 1.79
ab
2.87 ± 1.06
a
2.27 ± 1.01
a
1.87 ± 1.06
b
No shoots
05.66
b
12.51
a
03.83
b
14.17
a
27.26
a
03.49
b
01.98
b
03.79
b
25.97
a
14.33
a
Mean values with different letter(s) within a column are significantly different at
P < 0.05. Values are mean ± SE, n = 15
(A) Untreated callus after two weeks during plantlet formation on embryo growth medium
(EGM); (B) Control plantlets after two weeks on EGM supplemented with 407 mM mannitol (LD
(C) Plantlets after eight weeks on EGM; (D) Callus treated for one hour after two weeks on EGM;
(E) Callus treated for one hour on EGM supplemented with 407 mM
mannitol; (F) Albino plantlets
protein denaturation that is followed by
methanesulfonate
mutagenesis has been successfully used to induce
genetic variability [13] and to improve various
indication
of
embryo regeneration potential, where both
0.5 h and 1 h EMS treatments resulted in a rapid
increase in relative growth rate as well as a
number of embryos
per callus clump. However, a
reduction in relative growth rates, fewer embryos
per callus clump
, and formation of high numbers
of phenotypically abnormal embryos in calli
exposed to EMS for 2 and 3 h can be attributed to
a decrease in callus proliferation and embryo
initiation with an increase of EMS concentrations
and incubation period [14,28]. Abn
ormal embryo
Dlamini et al.
osmotic stress on sugarcane plantlet
Percentage of phenotypically
(A) Untreated callus after two weeks during plantlet formation on embryo growth medium
(EGM); (B) Control plantlets after two weeks on EGM supplemented with 407 mM mannitol (LD
90
);
ter two weeks on EGM;
mannitol; (F) Albino plantlets
increase in relative growth rate as well as a
high
per callus clump. However, a
reduction in relative growth rates, fewer embryos
, and formation of high numbers
of phenotypically abnormal embryos in calli
exposed to EMS for 2 and 3 h can be attributed to
a decrease in callus proliferation and embryo
initiation with an increase of EMS concentrations
ormal embryo
Dlamini et al.
117
development such as chlorophyll deficiency and
underdeveloped embryos after exposure to EMS
are common in sugarcane [26] and Phaseolus
vulgaris [29].
An incubation of callus in 16 mM EMS for 0.5–1
h was selected in the current study as an optimal
treatment range for inducing useful mutations,
because an optimal concentration and treatment
duration should neither result in significant
inhibition of plantlet regeneration nor in high
numbers of phenotypic abnormalities in plantlets
[30,31]. Similarly, an exposure of callus to 16 mM
EMS for 4 h was optimal for producing herbicide
and drought tolerant mutants [5,12], whereas a
concentration of 40 mM EMS for 2.5 h exposure
of callus is sufficient to produce salt-tolerant lines
[26] in sugarcane. Purnamaningsih and Hutami
[14] recommended treatment with 5–8 mM EMS
concentrations for 0.5–1.5 h incubation duration,
for aluminium tolerance in plantlets. The variation
in optimal concentrations and incubation periods
on the current study and others may be influenced
by the difference in the cultivars used [14].
Mannitol In vitro Screening
Mannitol lethal doses of LD
50
and LD
90
calculated
as 224 and 407 mM mannitol, respectively, used in
the current research to select for osmotic stress
tolerance cell lines of sugarcane, were lower than
those reported at plantlet level under in vitro
conditions [4]. Plant tolerance to osmotic stress
can be determined by applying the selecting agents
such as sorbitol, polyethylene glycol and mannitol
to the culture media [32]. During in vitro culture,
establishment of an optimum concentration of
dehydration agent that affects cells, tissues, organs
and whole plants is important as it can decrease
the chances of selecting false positive tolerant
lines [33]. In vitro culture selection is a cost-
effective alternative approach to select for stress-
tolerant cell lines after in vitro mutagenesis [5].
Screening for drought stress tolerance under field
conditions is complicated due to the effect of
genotype by environmental interaction and high
cost due to the lengthy breeding period [34,35].
The decrease in callus relative growth rate, index
of tolerance and embryogenicity with an increase
of mannitol concentration during selection under
darkness reported in the current work, agrees with
other studies [6,23,36]. Mild and severe osmotic
stress resulted in the shrinking of the control callus
as demonstrated by negative relative growth rate
and index of tolerance, probably because the
NCo376 cultivar used in the current study is
susceptible to drought stress [4]. Calli obtained
from drought susceptible cultivars shrink under
high mannitol concentrations compared with that
from drought tolerant cultivars [6]. Shrinking of
callus under osmotic stress results because of the
cells’ inability to maintain turgor pressure against
their cell walls [7]. High turgor pressure maintains
plant form and facilitates cell expansion and
growth [8]. Reduction of relative growth rate in
the current research is potentially a survival
strategy for a long-term osmotic stress, because
osmotic stress tolerant cells reduce their growth
rate by reducing the number of mitotic and
differentiating cells to maintain the growth cycle
[8], resulting in a decrease in biochemical
activities that require water and thus ensuring the
long-term survival of the plant cells [9,37].
Regeneration of prolific yellowish callus clumps
(cell recovery) post osmotic stress recorded in the
current research was also observed in other studies
that used callus culture [6]. Both EMS treatment
and mannitol stress appeared to enhance early root
formation during selection under darkness.
Abnormal callus characterized by early root
development was previously reported on EMS
mutated calli under stress conditions that include
herbicide [12] and fungal infection [11]. Early
development of roots is a consequence of both the
chemical mutagens and stress treatment. The calli
that showed early root development was unable to
develop into proper plantlets (roots and shoots)
probably because they shifted towards indirect
organogenesis, which is undesired [12].
The highest plantlet formation was recorded in the
control treatment, followed by callus exposed
LD
50
and LD
90
. A decrease in plantlet formation
with an increase of osmotic stress in growth media
observed in the current research was also reported
on studies that used polyethylene glycol or
mannitol as an osmoticum [5,6]. In LD
90
callus
treated for one hour in EMS produced the highest
number of plantlets per callus clump and lowest
number of abnormal shoots when compared with
Dlamini et al.
118
the callus treated for two and three hours. These
findings suggest that treatment of callus for one
hour may have resulted in the successful induction
of osmotic tolerance.
CONCLUSION
Ethyl methanesulfonate-treated (mutated) calli had
higher relative growth rate, index of tolerance and
recovery ability under osmotic stress. Treatment of
callus for one hour was optimal for inducing
useful mutations without inhibiting callus
embryogenicity nor increasing phenotypically
abnormal plantlets. The use of lethal doses (LD
50
and LD
90
) as the stress thresholds, permits rapid,
less-resourced and less labour-intensive selection
of callus lines that are resistant to osmotic stress.
Therefore, the establishment of plantlets under
mannitol stress can be used to select for potential
drought tolerant mutant lines. Future studies will
characterize drought tolerant lines under field
conditions based on their relative water content,
photosynthetic rate, membrane damages, and
general agronomic traits’ performance when
compared with wild type. In additions, genes that
may have been affected by ethyl methanesulfonate
mutagenesis and type of mutation will be
identified to assess the observed enhanced
drought-tolerant traits on the mutant.
ACKNOWLEDGEMENTS
The authors would like to thanks the laboratory
staff members in the Departments of Agriculture,
Botany, Chemistry, as well as Biochemistry and
Microbiology, University of Zululand for their
assistance on this project. Dr SJ Snyman from
South African Sugarcane Research Institute and
Ms M Timothy from the Dube Tradeport,
AgricLab are greatly appreciated for their
invaluable contribution towards this research.
COMPETING INTERESTS
Authors have declared that no competing interests
exist.
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