J. Agric. Environ. Sci. Vol. 7 No. 2 (2022) ISSN: 2616-3721 (Online); 2616-3713 (Print)
Publication of College of Agriculture and Environmental Sciences, Bahir Dar University 49
In -vitro evaluation of mung bean (Vigna radiata L. Wilczek) genotypes for drought
tolerance and productivity
Tekle Yoseph*
1
, Firew Mekbib
2
,
Berhanu Amsalu
3
, and Zerihun Tadele
4
1
Southern Agricultural Research Institute, Jinka Agricultural Research Center, Jinka, Ethiopia
2
Haramaya University, School of Plant Sciences, Dire Dawa, Ethiopia
3
International Livestock Research Institute, Addis Ababa, Ethiopia
4
University of Bern, Institute of Plant Sciences, Altenbergrain 21, 3013 Bern, Switzerland
*Corresponding author: tekleyoseph486@gmail.com
Received: November 16, 2022 Accepted: December 14, 2022
Abstract: Drought stress is the most important factor that limits mung bean production and productivity at
large in drought-prone areas of Ethiopia. It is hence necessary to identify and verify drought-tolerant and
productive varieties of major crops grown in drought areas of the country like mung bean. The present study
was conducted to evaluate mung bean genotypes for drought tolerance under in-vitro conditions and to assess
the performance of the in-vitro developed regenerants under greenhouse conditions. The in-vitro experiment
was thus arranged in a factorial experiment using a completely randomized design with three replications.
Three mung bean genotypes, NLLP-MGC-06/G6 (tolerant), VC6368 (46-40-4)/G34 (moderate), and NLLP-
MGC-02/G2 (sensitive) and five polyethylene glycol (PEG) levels (0, 0.5, 1.0, 1.5, and 2.0%) were used. The
analysis of variance exhibited significant differences among the genotypes for all the studied parameters except
the number of roots per shoot. There were significant differences observed among PEG levels for all the studied
parameters. Significant genotypes x PEG interactions were observed for all the studied traits except total roots
per culture and survival percentage. Increasing polyethylene glycol concentration from 0% to 2.0% in the
medium caused a gradual increase in root length from 0.49 cm at 0% PEG to 1.17 cm at 2.0% PEG,
respectively. This revealed an adaptive mechanism to the decreased moisture content in the root zones of plants
and enhanced increased root length to reach deeper water in the soil. Regenerant from the treatment
combinations of G34 (0) exhibited the highest values for the number of primary branches per plant (4.00).
Grain yield for the in-vitro regenerated plants evaluated at greenhouse conditions ranged from 552.52 kg ha
-1
at the treatment combination of G2 (1) to 996.23 kg ha
-1
at the treatment combinations of G6 (0). Most of the
regenerants obtained from NLLP-MGC-06/G6 and VC6368 (46-40-4)/G34 showed the best performance under
the greenhouse for drought-tolerance under the in-vitro condition, suggesting that the accumulated performance
of the tested regenerants under in-vitro conditions was realized under greenhouse conditions. It also indicated
that in-vitro culture is an important tool to identify and verify drought-tolerant genotypes and improve desirable
agronomical traits. Further study is indeed required to understand the mechanism of drought tolerance for in-
vitro-selected somaclones.
Keywords: Drought tolerance, Greenhouse condition, Polyethylene glycol, Somaclonal variation
This work is licensed under a Creative Commons Attribution 4.0 International License
1. Introduction
Drought is a major abiotic stress that adversely
affects plant production in many parts of the world
and had brought a significant yield reduction. Ilker
et al. (2011) suggested that global warming is
noticeable in drought-prone areas and had
significantly affected plant production, thereby
leading to considerable economic and social
problems because of its great importance in human
nutrition. Jaleel et al. (2009) suggested that drought
is a serious problem for crop production and food
security that significantly reduces the turgor
potential of plants. Water stress can result in
reducing crop yield worldwide (Boyer, 1982;
Smirnoff, 1993; Gonzalez et al., 1995). Since yield
is a complex trait and is strongly influenced by the
environment, severe losses can be caused by
drought stress which is common in most arid and
semi‐arid areas. One possible way to ensure the
future food needs for an increasing world
J. Agric. Environ. Sci. Vol. 7 No. 2 (2022) ISSN: 2616-3721 (Online); 2616-3713 (Print)
Publication of College of Agriculture and Environmental Sciences, Bahir Dar University 50
population involves the better use of water through
the development of drought-tolerant crop varieties
that need less water (El‐Shafey et al., 2009;
Mafakheri et al., 2010).
One of the screening techniques based on
physiological traits is the use of various osmotica
to induce stress in plant tissues. Therefore, to
simulate the effect of water stress in vitro, several
researchers have incorporated polyethylene glycol
(PEG) in the culture medium (Handa et al., 1982;
Bhaskaran et al., 1985; Newton et al., 1986;
Newton et al., 1989; Ochatt et al., 1998; Guóth et
al., 2010; Rai et al., 2011; Yang et al., 2012). Sané
et al. (2005) found that the use of PEG under in-
vitro culture allows quick and easy identification of
genotypes tolerant to water stress. Plant cell and
tissue cultures have been implemented as useful
tools to study stress tolerance mechanisms under in
vitro conditions (Bajji et al., 2000). Also, in-vitro-
developed sugar cane regenerants were validated
for agronomical and morphological traits (Gadakh
et al. 2015; Rahman et al., 2016).
Information on drought stress's effect on the
morphological aspects under in vitro conditions in
mung bean is lacking. Therefore, there is a need to
go to an alternative approach to field experiments
related to moisture stress to induce stress using
polyethylene glycol (PEG) under in-vitro
conditions. Hence, the objectives of this study were
to assess the effect of PEG-induced stress on plant
cells of mung bean genotypes, to select surviving
cell lines under different levels of PEG stress under
in-vitro conditions, and to select suitable
regenerants for drought tolerance.
2. Materials and Methods
2.1. Description of the study area
The study was conducted at the Plant Tissue
Culture Research Laboratory of Areka Agricultural
Research Center, Southern Ethiopia.
2.2. Plant material, treatments, and
experimental design
Three mung bean genotypes with contrasting
drought tolerance including NLLP-MGC-06/G6
(tolerant), VC6368 (46-40-4) /G34 (moderate), and
NLLP-MGC-02/G2 (sensitive) were used for this
experiment. Two genotypes (G34 and G6 ) were
obtained from the Melkassa Agricultural Research
Center, while the third genotype (G2) was a
landrace collected from the southern region of
Ethiopia. The base for selecting these genotypes
was based on the moisture stress response in
drought screening field experiments. The
treatments comprised factorial combinations of
three mung bean genotypes (G34, G6, and G2) and
five polyethylene glycol (PEG
8000
) levels of 0, 0.5,
1, 1.5, and 2% (w/v) adopted from Ferede et al.
(2019). The experiment of the study was laid out in
a completely randomized design in a factorial
arrangement with three replications.
2.3. Culture media and growth conditions
Murashige and Skoog's (1962) medium (MS) was
used as a basal medium with 3% sucrose and
0.75% agar added by melting in a microwave oven.
The pH of all media was adjusted to 5.8 with 0.1 N
NaOH before autoclaving. When the agar became
clear, 50 ml medium was dispensed into culture
tubes and autoclaved at 121°C for 20 minutes
(Ferede et al., 2019).
2.4. Seed sterilization and germination
For sterilization, the seeds were first treated with
70% ethanol for 5 minutes and then washed in 8%
sodium hypochlorite for 30 minutes, followed by
six washes in sterile double distilled water in a
laminar airflow cabinet. The sterilized seeds were
cultured for two weeks under aseptic conditions
containing a semisolid MS medium at 27 °C. After
two weeks, young seedling leaves were excised and
used for callus induction (Ferede et al., 2019).
2.5. Callus induction
Leaf explants (2 cm) were placed on an MS
medium containing 0.75% agar and 3% sucrose for
each treatment. Callus induction was initiated from
the leaf explants placed on MS medium containing
2.4-D (2 mg/l), kinetin (0.2 mg/l), and 1
naphthalene acetic acid (1 mg/l). Different
concentrations of PEG (0, 0.5, 1.0, 1.5, and 2.0%)
were added to the callus induction medium. The
culture tubes were sealed with parafilm and placed
in a growth room at 27 ºC. In all experiments, three
replicates were made, and 10 explants of leaf
segments were placed with one replication
represented by two culture tubes (Ferede et al.,
2019).
2.6. Plant regeneration
After four weeks of incubation, the induced calli
were transferred to culture tubes, sub-cultured
under the same growth conditions, and in the same
MS medium with various concentrations of
J. Agric. Environ. Sci. Vol. 7 No. 2 (2022) ISSN: 2616-3721 (Online); 2616-3713 (Print)
Publication of College of Agriculture and Environmental Sciences, Bahir Dar University 51
PEG
8000
(0, 0.5, 1.0, 1.5, and 2.0%) adopted from
Ferede et al. (2019). The resulting calli were
excised, and transferred, into culture tubes
containing MS medium supplemented with 1.5
mg/l kinetin + 0.2 mg/l NAA + 3% sucrose +
0.75% agar for shoot initiation. By doing this
procedure, the efficiency of the embryogenic calli
was determined and for further regeneration
(shooting and rooting) in the presence of drought
stress, the obtained calli were exposed to PEG
8000
(0, 0.5, 1.0, 1.5, and 2.0%) in the plant regeneration
medium. Rooting was initiated on half-strength
fresh MS medium supplemented with 1.5 mg/l
NAA. The incubation period was two cycles of two
weeks each or two weeks for shooting and two
weeks for rooting (Ferede et al., 2019).
2.7. Acclimatization of regenerated plants
Healthy and well-rooted plantlets were washed to
remove the medium adhered and subjected to
acclimatization, transplanted to the plastic tray
under high humidity by covering the plant with
plastic containing sterilized soils, coco peat, and
compost, and placed under polythene shed with
high humidity (>90% RH) for 3 weeks to harden.
After acclimatization, plantlets were transplanted to
pots under greenhouse conditions, and the survival
percentages were taken four weeks later. Finally,
the plants being survived were assessed for their
agronomic, yield, and yield-related traits (Ferede et
al., 2019).
2.8. Data collection
2.8.1. Callus induction and plant regeneration
Callus induction efficiency (CIE) was assessed as
the number of explants induced callus/ total
number of cultured explants used for each
treatment x 100. Plant regeneration percent (PRP)
was recorded as (number of plantlets/total number
of calli) × 100 after PEG treatment. The total
number of shoots per culture (TSPC) was counted
at the stage of the shoot multiplication when treated
by PEG. Similarly, shoot length (SL) and root
length (RL) were measured using an autoclaved
square paper and a well-sterilized measuring tape
after two weeks of plantlet incubation. The total
number of roots per culture (TRPC) and the
number of roots per shoot (NRPS) were counted at
the stage of the root regeneration medium. Data
were also recorded for rooting percentage as the
percent of rooted shoots (RP) per culture. Survival
percentage (SP) was calculated as the percentage of
surviving plants after four weeks of transfer to pots.
2.8.2. Growth and yield parameters
The selected regenerants were transferred to the
pots that were labeled based on the genotype name
of the original ex-plant and the PEG level at which
the regenerants were grown.
Plants grown in the greenhouse were evaluated for
different agronomic traits. In this study, a total of
fifteen mung bean regenerants developed from in-
vitro culture were evaluated for morpho-phenologic
traits. The healthy and physiologically matured
regenerants were selected for this study. The
experiment was carried out using a completely
randomized design with three replications at Areka
Agricultural Research Center under greenhouse
conditions in 2020. Data on days to flowering, days
to maturity, peduncle length (cm), plant height
(cm), the number of primary branches per plant,
pod length (cm), the number of pods per cluster,
the number of pods per plant, the number of seeds
per pod, hundred seed weight (g), grain yield per
plant (g), grain yield (kg ha
-1
), biomass yield (kg
ha
-1
), and harvest index were recorded from five
regenerants plants grown in pots.
2.9. Data analysis
Collected data were subjected to analysis of
variance (ANOVA) and the means were separated
using the LSD test at a 0.5% level of probability
using the SAS software version 9.0.
3. Results and Discussion
3.1. Effect of genotypes on callus induction and
plant regeneration
The analysis of variance result showed all the
studied traits were significantly affected by
genotypes except the number of roots per shoot
(Table 1). This shows the existence of inherent
genotypic variability. A similar result was reported
by Tsago et al. (2014) on sorghum and Ferede et
al. (2019) on tef. The highest callus induction
efficiency (16.87%) was noted for the genotype
(G34), while the genotypes G2 and G6 had
relatively lower callus induction efficiency of 15.82
and 15.56%, respectively. In this study, the
observed highest CIE for the genotype G34 might
be due to the genetic makeup of the genotype to
induce good callus as compared to the other two
genotypes. This finding is supported by the
previous reports of (Mekbib et al., 1997; Ferede et
J. Agric. Environ. Sci. Vol. 7 No. 2 (2022) ISSN: 2616-3721 (Online); 2616-3713 (Print)
Publication of College of Agriculture and Environmental Sciences, Bahir Dar University 52
al., 2019) on tef genotypes who suggested that
good callus induced in some of the genotypes was
due attributed to the genetic makeup of the
genotypes.
The highest plant regeneration percent (28.90%)
was noted for genotype G34 while the genotypes
G6 and G2 had relatively similar plant regeneration
percentages of 27.82 and 27.56%, respectively. The
highest rooting percent (73.07%) and the number
of roots per shoot (2.00) were attributed to
genotype G34 (Table 1). This could be attributed to
the high-quality calli obtained from the genotype
(G34), which might be due to the genetic make of
the genotype. This result is supported by the
previous reports of Ferede et al. (2019) on tef
genotypes, Helaly et al. (2013) on wheat, who
reported that callus induction was a critical phase
where the regeneration of plants is highly
dependent on the quality of callus. In contrast, G6
and G2 showed relatively low rooting percentages
and the number of roots per shoot.
3.2. Effect of PEG stress on callus induction and
plant regeneration
The analysis of variance results revealed that all the
studied traits were significantly affected by PEG
levels except total roots per culture and survival
percentage (Table 1), signifying the existence of
differential responses of genotypes to different
levels of PEG. But the total shoots per culture and
survival percentage were not genotype-dependent.
The result showed that as the PEG level increased
the values for most of the studied traits declined
while the number of roots per shoot and root length
increased. The highest mean values of all
parameters except the number of roots per shoot
and root length were observed at 0% PEG which
was reduced at each subsequent higher level of
PEG. On the other hand, the highest number of
roots per shoot and root length of mung bean
regenerants were observed at 2.0% PEG (Table 1).
The highest callus induction efficiency of 22.72%
was observed at 0% PEG and the lowest 10.93%
was observed at 2.0% PEG. The plant regeneration
percentage of 42.11% at 0% PEG was dramatically
decreased to 28.69 % at 0.5%, 25.38% at 1.0%,
23.36% at 1.5%, and reached 20.93% at 2.0% PEG
concentration. The highest rooting percentage of
94.53% was observed at 0% PEG and the lowest
51.69% was observed at 2.0% PEG. On the
contrary, a significant increment of root length was
found at 2.0% (1.17 cm) and 1.5% (1.04 cm) PEG
concentrations respectively, as compared to the
control and the other PEG levels (Table 1). This
reveals an adaptive mechanism to the decreased
moisture content in the root zones of plants that
enhances increased root length to reach deeper
water in the soil. These findings are supported by
the previous reports of Ahmed (2014) on rice and
Ferede et al. (2019) on tef, who found an increase
in root length associated with increasing PEG
concentration and observed similar trends in the
study.
The highest number of roots per culture 34.09 was
observed at 0% PEG and the lowest 8.33 was
observed at 2.0% PEG. The mean data of shoot
length revealed that with increasing PEG stress,
shoot length declined in general. The highest shoot
length of 1.41 cm was observed at 0% PEG and the
lowest 0.82 cm was observed at 2.0% PEG. The
highest survival percentage of 90.69% was
observed at 0% PEG and the lowest 55.07% was
observed at 2.0% PEG (Table 1). The reduced
values of regenerants in most mung bean traits at
an increased concentration of PEG might be due to
osmotic stress which prevents water uptake and
might be attributed to the toxic effects of the
increased PEG concentration. Similarly, Haruna et
al. (2019) reported that as the concentration of PEG
increased there was a decrease in callus sizes across
the treatments on wheat genotypes. Likewise,
Tsago et al. (2014) on sorghum reported that there
was a decrease in shoot and root-related traits with
an increase in the concentration of PEG whereas
the mean root number increased with an increasing
level of PEG treatment in each genotype. This
exhibited that as the concentration of PEG
increased; the growth of callus steadily decreased
and vice versa was true. This result has confirmed
the previous reports of Joshi et al. (2011) on rice,
Farshadfar et al. (2012) on wheat, Tsago et al.
(2013) on sorghum, and Ferede et al. (2019) on tef,
who reported that the mean callus induction
efficiency decreased considerably under higher
PEG concentration. The adverse effect of moisture
stress was stronger in higher PEG levels (2.0%
PEG) and about 5.0% of the cultures induced callus
and the induced calli lost their regeneration ability
and further growth was inhibited. Similar results
were reported by (Biswas et al., 2002; Sakthivelu
et al., 2008) ) who stated that the addition of high
PEG-6000 in culture media lowers the water
J. Agric. Environ. Sci. Vol. 7 No. 2 (2022) ISSN: 2616-3721 (Online); 2616-3713 (Print)
Publication of College of Agriculture and Environmental Sciences, Bahir Dar University 53
potential of the medium and adversely affects cell
division leading to reduced further callus growth.
3.3. Effects of Genotype x PEG interaction on
callus induction and plant regeneration
The analysis of variance result depicted that all the
studied traits were significantly affected by the
interaction effects of genotype and PEG levels
(Table 1). The highest number of total shoots per
culture (4.2), total roots per culture (34.36), root
length (1.23 cm), and survival percentage
(93.66%), and the highest shoot length (1.45 cm)
for genotypes (G6) were recorded in the control
treatment. Also, at the PEG concentration of
(0.5%), genotype (G6) showed better plant
regeneration percent and rooting percentage of
30.26 and 94.36%, respectively. The significant
interaction effects were observed due to genotype
by PEG for some of the studied traits, indicating
that the genotypes showed differential
performances across the different PEG
concentrations. This finding confirmed the report
by Leila (2013) on six pearl millet genotypes
exposed to three different (PEG
8000
) levels, and
Tsago et al. (2013) on sixteen sorghum genotypes
exposed to five different (PEG
8000
) levels namely
(0, 0.5, 1.0, 1.5, and 2.0%) who found that
significant differences among genotypes, PEG and
genotype by PEG interactions for shoot length, root
length, shoot number, and root number. A similar
result was reported by Haruna et al. (2019) on
sixteen wheat genotypes exposed to six different
(PEG
6000
) levels namely (0, 5 10, 15, 20, and 2,
5%) and observed that significant differences were
observed among genotypes for the necrotic mass of
the callus. The value of mean shoot length in
control (0.0% PEG) for the genotypes (G34, G6,
and G2) was 1.45, 1.45, and 1.34 cm, respectively
which reduced significantly at each subsequent
level of PEG stress till it reached 0.85, 0.75 and
0.85 cm, respectively at 2.0% PEG concentration.
Generally, inconsistency in regenerants for most of
the studied traits was observed.
J. Agric. Environ. Sci. Vol. 7 No. 2 (2022) ISSN: 2616-3721 (Online); 2616-3713 (Print)
Publication of College of Agriculture and Environmental Sciences, Bahir Dar University 54
Table 1: Callus induction and regeneration of mung bean as influenced by genotypes and PEG levels
Genotype
CIE
(%)
PRP
(%)
TSPC
RP
(%)
TRPC
SL
(cm)
RL
(cm)
SP (%)
G6
15.56b
27.82b
3.04a
72.41b
19.27a
1.05b
0.91a
71.18b
G34
16.88a
28.91a
2.31b
73.07a
18.50b
1.09a
0.82b
76.60a
G2
15.82b
27.56b
2.21c
72.34b
18.21b
1.02c
0.94a
72.26b
LSD (5%)
4.34
0.59
0.09
0.35
0.49
0.01
0.04
4.33
Sig. level
***
***
***
***
***
***
***
*
PEG levels
0%
22.72a
42.11a
4.25a
94.53a
34.09a
1.41a
0.49e
90.69a
0.5%
18.69b
28.69b
2.47b
82.54b
22.54b
1.12b
0.83d
81.64b
1.0%
15.38c
25.38c
2.21c
72.07c
16.13c
1.01c
0.92c
74.33c
1.5%
12.69d
23.36d
2.03d
62.21d
12.21d
0.93d
1.04b
65.01d
2.0%
10.93e
20.93e
1.65e
51.69e
8.33e
0.82e
1.17a
55.07e
LSD (5%)
1.03
0.90
0.14
0.53
0.75
0.02
0.06
6.59
Sig. level
***
*
***
***
Ns
***
***
Ns
Genotype
PEG
levels
G34
0%
25.36a
43.53a
2.01d
94.36a
33.51a
1.45a
1.18a
89.85b
G34
0.5%
20.26b
30.26c
3.26b
83.26b
23.26b
1.20c
1.02c
84.60c
G34
1.0%
16.02d
26.02e
2.02d
72.02d
17.02c
1.02e
0.95d
78.69d
G34
1.5%
11.89h
23.89g
2.89c
62.89e
12.89d
0.95f
0.78e
66.23e
G34
2.0%
10.81i
20.81i
2.81c
52.81f
8.81e
0.85h
0.56f
59.84f
G6
0%
21.45b
41.45b
4.20a
94.85a
34.36a
1.45a
1.23a
93.66a
G6
0.5%
17.70d
27.70d
4.18a
82.01c
22.01b
1.03e
1.02c
81.12c
G6
1.0%
14.73e
24.73f
2.04d
72.04d
15.70c
1.00e
0.78e
72.13e
G6
1.5%
12.88g
22.88i
1.77e
61.77e
11.77d
0.89g
0.67f
66.11f
G6
2.0%
11.04i
21.04i
1.06f
51.06g
8.06e
0.75i
0.45g
52.11g
G2
0%
21.34b
41.34b
4.37a
94.39a
34.39a
1.34b
1.10b
88.57b
G2
0.5%
18.12d
28.12d
2.12d
82.35c
22.35b
1.12d
1.07c
79.19c
G2
1.0%
15.40e
25.40f
2.03d
72.17d
15.68c
1.00e
1.05c
72.16e
G2
1.5%
13.30f
23.30h
1.96e
61.96e
11.96d
0.96f
1.04c
62.70f
G2
2.0%
10.94i
20.94i
1.08f
51.19g
8.11e
0.85h
0.46g
53.26g
Sig. level
***
***
***
***
***
***
***
***
LSD (5%)
2.28
1.99
0.31
1.17
1.66
0.06
0.13
14.51
SE±
0.6
0.43
0.01
0.15
0.30
0.0004
0.002
23.03
CV
4.7
2.34
4.11
0.53
2.96
1.96
5.07
6.54
CIE = callus induction efficiency percent, PRP = plant regeneration percent, TSPC = total shoot per culture, RP = rooting
percentage, TRPC = total roots per culture, NRPS = number of roots per shoot, SL = shoot length; RL = root length, SP =
survival percentage; means followed similar letters in column are not statistically difference at p≤0.05
3.4. Evaluation of In-Vitro regenerated plants for
validation under greenhouse conditions
3.4.1. Flowering and vegetative growth
The analysis of variance results revealed that the
regenerants showed highly significant differences
in all the measured flowering and vegetative
growth traits (Table 2). Regenerants of the
treatment combination of genotype G2 x 2% PEG
flowered earlier, which took 33.16 days, while days
to flowering for the regenerant from the treatment
combination of genotype G6x1.0% PEG took
longer time to flower with the value 36.67 days. In
terms of maturity regeneratnts of the treatment
combinations of G2x2.0% (60 days) matured
earlier while those from the treatment combination
of G6x0.5% matured late with a value of 76.00
days.
The in-vitro-developed mung beans having lower
values for days to flowering and days to maturity
were considered drought-tolerant since these
genotypes had ability to escape terminal drought
and could be recommended for drought-prone
areas. Plaza-Wüthrich et al. (2013) reported that
earliness for days to heading and maturity are
important traits on tef for areas with low rainfall to
escape terminal drought, and in high rainfall with
long growing season areas, can be employed in
double-cropping systems.
The highest terminal leaf length (6.86 cm) was
recorded from the regenerant developed from the
J. Agric. Environ. Sci. Vol. 7 No. 2 (2022) ISSN: 2616-3721 (Online); 2616-3713 (Print)
Publication of College of Agriculture and Environmental Sciences, Bahir Dar University 55
treatment combination of G34x0% PEG, while the
least terminal leaf length (3.53 cm) was noted from
the regenerant obtained from the treatment
combination of G34x2.0% PEG. The highest
terminal leaf width of 11.36 and 11.05 (cm) were
recorded from the regenerants from the treatment
combinations of G2x0% PEG and G34x0% PEG,
respectively. The least terminal leaf width (6.36
cm) was obtained from the regenerant developed
from the treatment combinations of G6x2% PEG.
The highest and the least peduncle length of 9.13
cm and 5.43 cm were recorded from the treatment
combinations of G6x0% PEG and G2x2% PEG,
respectively (Table 2). The highest plant height of
44.16 (cm) was recorded from the regenerants from
the treatment combination of G6x0.5% PEG while
the least (38.13 cm) was recorded from G2x0.5%
PEG. The observed variations on vegetative growth
parameters at different treatment combinations of
PEG levels and genotypes might be attributed to
the differential responses of the tested genotypes to
the induced PEG levels.
Table 2: Flowering and vegetative growth of mung bean as affected by genotypes and PEG levels
Genotypes
PEG
levels
DTF
(50%)
DTM
(90%)
TLL
(cm)
TLW
(cm)
PDCL
(cm)
PHT
(cm)
BRN
PODL
(cm)
G34
0 %
36.00a
72.66a
6.86a
11.05a
8.75a
40.00b
4.00a
9.16b
G34
0.5%
36.33a
71.66a
6.36a
10.98a
8.23a
41.33a
3.26b
10.66a
G34
1.0%
36.00a
72.66a
4.92c
10.03a
8.74a
42.00a
2.00d
6.66f
G34
1.5%
35.66a
69.66b
4.60d
9.77a
8.57a
41.00a
2.00d
7.53e
G34
2.0%
35.16a
69.66b
3.53e
8.38b
6.60c
39.00b
2.00d
10.98a
G6
0 %
34.33b
70.66a
4.64d
8.60b
9.13a
42.00a
3.00c
11.26a
G6
0.5%
36.33a
76.00a
4.93d
10.25a
7.59b
44.16a
3.00c
10.00a
G6
1.0%
36.76a
74.00a
4.83d
10.76a
7.36b
40.10b
3.00c
10.00a
G6
1.5%
35.66a
70.66a
4.94d
8.36b
8.12a
39.66b
2.00d
9.03c
G6
2.0%
34.33b
69.66b
4.53d
6.36c
5.48d
38.56b
2.00d
7.60e
G2
0 %
35.83a
72.33a
6.60a
11.36a
8.66a
40.00b
3.00c
9.86b
G2
0.5%
36.00a
72.00a
5.00b
9.11a
8.17a
38.13b
2.00d
11.00a
G2
1.0%
36.00a
68.66b
5.17 b
10.22a
8.17a
39.33b
2.00d
8.96d
G2
1.5%
34.16b
68.00b
5.41b
9.17a
6.30c
40.00b
3.00c
10.96a
G2
2.0%
33.16b
60.00c
4.93d
8.96b
5.43d
39.00b
1.00e
5.50g
Sig. level
**
**
***
***
***
***
***
***
SE±
0.89
11.82
0.69
0.28
0.85
1.79
0.01
0.41
CV (%)
2.68
4.87
10.41
9.68
10.87
3.32
2.62
6.94
LSD (5%)
2.85
10.34
1.61
2.78
2.51
4.02
0.19
1.93
DTF = days to flowering, DTM = days to maturity, TLL = terminal leaf length, TLW = terminal leaf width,
PDCL = peduncle length, PHT = plant height, BRN = number of primary branches per plant, PODL = pod
length; means followed similar letters in column are not statistically difference at p≤0.05
3.4.2. Yield related traits
The analysis of variance results depicted that the
regenerants showed highly significant differences
in all the measured yield-related traits (Table 3).
Regenerant from the treatment combinations of
G34x0% PEG exhibited the highest value for the
number of pods per cluster (5) and pods per plant
(19.66). The highest, seeds per pod (11.36), grain
yield per plant (5.22 g), grain yield (996.23 kg ha
-
1
), and harvest index (0.27) were recorded from the
regenerant from the treatment combinations of G6
(0). On the other hand, regenerants obtained from
the treatment combinations of G2 (1) showed poor
performance for pods per cluster (2.66) and pods
per plant (12.00). The highest hundred seed weight
(5.49 g) was recorded from the regenerants
obtained from the treatment combinations of G6
(0), while the least hundred seed weight (3.12 g)
was recorded for the regenerants obtained from the
treatment combinations of G2 (1.5). The highest
biomass yield of 4319.80, 4219.80, and 4219.80
(kg ha
-1
) was recorded for the regenerants obtained
from the treatment combinations of G34 (1.5), G34
(2.0), and G2 (1.5), respectively (Table 3).
The result indicated that an in-vitro culture is an
important tool to screen drought-tolerant genotypes
and improve desirable agronomical traits. In
general, most of the regenerants obtained from G34
and G6 showed the best performance under the
J. Agric. Environ. Sci. Vol. 7 No. 2 (2022) ISSN: 2616-3721 (Online); 2616-3713 (Print)
Publication of College of Agriculture and Environmental Sciences, Bahir Dar University 56
greenhouse and were drought-tolerant under the in-
vitro condition, suggesting that the performance of
the tested regenerants under in vitro conditions was
realized under greenhouse conditions.
Table 3: Yield related traits of mung bean as affected by genotypes and PEG levels
Genotype
PEG
levels
PPC
PPP
SPP
GYPP
(g)
HSW
(g)
GYLD
(kg ha
-1
)
BM
(kg ha
-1
)
HI
G34
0 %
5.00a
19.66a
10.63a
4.11b
4.21c
892.96b
3699.80c
0.24a
G34
0.5%
3.00c
19.00a
8.30d
4.18b
4.05c
596.12d
3819.80b
0.15c
G34
1.0%
3.00c
14.33c
10.46b
4.01b
4.12c
795.21b
3886.50b
0.20b
G34
1.5%
3.66b
14.00c
9.80c
3.97b
4.22c
822.94b
4319.80a
0.19b
G34
2.0%
3.33c
12.66d
7.40d
3.08c
4.22c
555.53e
4219.80a
0.13e
G6
0 %
4.00b
12.66d
11.36a
5.22a
5.49a
996.23a
3686.50c
0.27a
G6
0.5%
3.66b
16.66b
11.10a
3.99b
4.16c
754.74b
4119.80a
0.18c
G6
1.0%
3.00c
15.33b
10.13b
4.14b
4.18c
595.23d
3886.50b
0.15c
G6
1.5%
3.00c
15.33b
10.06b
3.96b
4.12c
729.55b
3953.10b
0.18c
G6
2.0%
3.00c
12.13e
10.16b
3.97b
4.20c
587.16d
3986.50b
0.14d
G2
0 %
3.66b
19.00a
10.20b
5.17a
5.13b
693.94c
3886.50c
0.17c
G2
0.5%
3.33b
18.66a
8.08d
4.20b
4.19c
577.27d
3786.50c
0.15c
G2
1.0%
2.66d
12.00e
10.06b
4.18b
4.08c
552.52e
4019.80b
0.13d
G2
1.5%
3.00c
17.33b
11.06a
3.95b
3.12d
589.57d
4219.80a
0.13d
G2
2.0%
3.00c
15.66b
9.60c
4.07b
4.21c
592.74d
3916.50b
0.15d
Sig. level
***
***
***
***
***
***
**
***
SE±
0.13
2.62
0.14
0.02
0.01
6136.10
29904.00
0.01
CV (%)
10.88
10.37
3.82
3.75
1.56
11.37
4.37
10.10
LSD (5%)
1.09
4.87
1.13
0.46
0.19
235.62
520.15
0.05
PPP = the number of pods per plant, SPP = number of seeds per pod, GYPP = grain yield per plant, HSW =
hundred seed weight, GYLD = grain yield, BM=biomass yield and HI = harvest index; means followed similar
letters in column are not statistically difference at p≤0.05
4. Conclusion
Drought is one of the most liming factors in mung
bean production and productivity. Evaluating mung
bean genotypes in PEG-induced drought conditions
under in-vitro and greenhouse conditions is
important to screen drought-tolerant genotypes.
This technique is crucial because the results of the
in-vitro were reproduced or realized in the
greenhouse. It also indicated that an in-vitro culture
is an important tool to develop drought-tolerant
genotypes and improve desirable agronomical traits
under greenhouse conditions for further field
verification. Therefore, some regenerants
performed better under the greenhouse conditions
were became drought-tolerant under the in-vitro
condition. In general, most of the regenerants
showed the best performance under the greenhouse
and were drought-tolerant under the in-vitro
condition, suggesting that the performance of the
tested regenerants under in vitro conditions was
realized under greenhouse conditions. This
suggests the accumulated performance of the tested
regenerants under in-vitro conditions was realized
under greenhouse conditions. Further study is
indeed required to understand the mechanism of
drought tolerance for the in-vitro selected
somaclones and to put the recommendation on a
strong basis.
Conflict of interest
The authors declare that there is no conflict
ofinterest in publishing the manuscript in this
journal.
Acknowledgment
The authors extend their gratitude to the Southern
Agricultural Research Institute (SARI) for the
financial support of this research. The authors also
recognize Jinka Agricultural Research Center
(JARC) for its administrative facilitation during the
implementation of this research.
References
Ahmed, M.E. (2014). Assessment of Seedling
Growth under Osmotic Stress and
Morphological Characterization of Deep Water
Rice (Oryza sativa L.) Genotypes. MSc Thesis.
Bangladesh Agricultural University.
J. Agric. Environ. Sci. Vol. 7 No. 2 (2022) ISSN: 2616-3721 (Online); 2616-3713 (Print)
Journal of the College of Agriculture & Environmental Sciences, Bahir Dar University 57
Bajji, M.S., Lutts, S. and Kinet, J.M. (2000).
Physiological Changes After Exposure to
Recovery from Polyethylene Glycol Induced
Water Deficit in Callus Culture Issued from
Durum Wheat (Triticum durum) Cultivars
Differing in Drought Resistance. Journal of
Plant Physiology, 156: 75-83.
Bhaskaran, S., Smith, R.H. and Newton, R.J.
(1985). Physiological Changes in Cultured
Sorghum Cells in Response to Induced Water
Stress I. Free Proline. Plant Physiology, 79:
266-269.
Biswas, J., Chowdhury, B., Bhattacharya, A. and
Mandal, A.B. (2002). In vitro Screening for
Increased Drought Tolerance in Rice. In Vitro
Cellular & Developmental Biology-Plant, 38:
525–530.
Boyer, J.S. (1982). Plant productivity and
environment. Science, 218: 443–448.
El‐ Shafey, N.M., Raifa, A.H., Mahmoud, M.A.G.
and El Sheihy, O. (2009). Pre-exposure to
Gamma Rays Alleviates the Harmful Effect of
Drought on the Embryo-Derived Rice Calli.
Australian Journal of Crop Science, 3(5): 268-
277.
Ferede, B., Mekbib, F., Assefa, K., Chanyalew, S.,
Abraha, E. and Tadele, Z. 2019. In vitro
Evaluation of Tef [Eragrostis tef (Zucc)
Trotter] Genotypes for Drought Tolerance.
Ethiopian Journal of Agricultural Sciences,
29(3): 73-88.
Gadakh, S.S., Patel, D.U. and Patil, A.B. (2015).
Evaluation of Sugarcane (saccharum spp.
complex) Mutants for Yield, Yield
Contributing Traits and Quality Parameters.
International Journal of Advanced Biological
Research, 5(3): 220-228.
Gonzalez, E.M., Cordon, A.J., James, C.L. and
Arrese-Igor, C. (1995). The Role of Sucrose
Synthase in The Response of Soybean Nodules
to Drought. Journal of Experimental Botany,
46(10): 1515–1523.
Guóth, A., Benyo, D., Csiszar, J., Galle, A.,
Horvath, F., Cseuz, L., Erdei, L. and Tari, I.
(2010). Relationship Between Osmotic Stress-
Induced Abscisic Acid Accumulation, Biomass
Production and Plant Growth in Drought-
Tolerant and -Sensitive Wheat Cultivars. Acta
Physiologiae Plantarum, 32: 719–727.
Handa, A.K., Bressan, R.A., Handa, S. and
Hasegawa, P.M. (1982). Characteristics of
Cultured Tomato Cells after Prolonged
Exposure to Medium Containing Polyethylene
Glycol. Plant Physiology, 69: 514-521.
Haruna, M.K. Aguoru, C.U., Iheukwumere, C.C.,
Ogbonna, C.I.C. and Salisu, I.D. (2019). In
Vitro Callus Induction Potentials of Wheat
Genotypes Using Mature Embryo As Ex-Plant
Source Under Different Levels of Polyethylene
Glycol (PEG). Journal of Biological Sciences
and Bioconservation, 11(2): 34-56.
Helaly, M.N., Mohamed, Z.A. Fouda, R.A. and
Arafa, A.A. (2013). In vitro Studies on Bread
Wheat Genotypes for Drought Tolerance
Using Polyethylene Glycol. Journal of Plant
Production, 4(4): 605-620.
Ilker, E., Tatar, O., Tonk, F.A. and Tosun M.
(2011). Determination of Tolerance Level of
Some Wheat Genotypes to Post-Anthesis
Drought. Turkish Journal of Field Crop, 16(1):
59-63.
Jaleel, C.A., Manivannan, P., Wahid, A., Farooq,
M., Somasundaram, R. and Paneerselvam, R.
(2009). Drought Stress In Plants: A Review on
Morphological Characteristics and Pigments
Composition. International Journal of
Agriculture and Biology, 11: 100-105.
Leila, R. (2013). Response of Tunisian
Autochthonous Pearl Millet (Pennisetum
glaucum (L.) R. Br.) to Drought Stress Induced
by Polyethylene Glycol (PEG) 6000.
International Research Journal of Genetic
Engineering, 1(4): 051-053.
Mafakheri, A., Siosemardeh, A., Bahramnejad, B.,
Struik, P.C. and Sohrabi Y. (2010). Effect of
Drought Stress on Yield, Proline and
Chlorophyll Contents in Three Chickpea
Cultivars. Australian Journal of Crop Science,
4(8): 580‐ 585.
Mekbib, F., Mantel, S.H. and Buchanan-
Wollastone, V. (1997). Callus induction and In
vitro regeneration of tef (Eragrostis tef (Zucc.)
J. Agric. Environ. Sci. Vol. 7 No. 2 (2022) ISSN: 2616-3721 (Online); 2616-3713 (Print)
Journal of the College of Agriculture & Environmental Sciences, Bahir Dar University 58
Trotter) from leaf. Journal of Plant
Physiology, 151: 368-372.
Murashige, T. and Skoog, F.A. (1962). A revised
Medium for Rapid Growth and Bioassays with
Tobacco Tissue Cultures. Physiology of Plants,
15: 473-497.
Newton, R.J., Bhaskaran, S.,Puryear, J.D. and
Smith, R.H. (1986). Physiological Changes in
Cultured Sorghum Cells in Reponse to Induced
Water Stress II. Soluble Carbohydrates and
Organic Acids. Plant physiology, 81: 626-629.
Ochatt, S.J., Marconi, P.L., Radice, S., Arnozis,
P.A. and Caso, O.H. (1998). In vitro Recurrent
Selection of Potato: Production and
Characterization of Salt Tolerant Cell Lines
and Plants. Plant Cell Tissue Organ Culture,
55:1.
Plaza, S., Cannarozzi, G.M. and Tadele, Z. (2013).
Genetic and phenotypic diversity in selected
genotypes of tef [Eragrostis tef (Zucc.)]
Trotter. African Journal of Agricultural
Research, 8(12): 1041-1049.
Rahman, M.M., Ivy, N.A., Mian, M.K., Rasul,
M.G. and Hossain, M.M. (2016). Performance
of Sugarcane Somaclones Under Field
Condition. International Journal of Plant
Biology & Research, 4(2): 1056.
Rai, M.K., Kaliaa, R.K., Singh, R., Gangola, M.P.
and Dhawan, A.K. (2011). Developing Stress
Tolerant Plants Through In Vitro Selection—
An overview of the Recent Progress.
Environmental and experimental botany,
71(1): 89–98.
Sakthivelu, G., Devi, M.K.A., Giridhar, P.,
Rajasekaran, T., Ravishankar, G.A., Nedev, T.,
and Kosturkova, G. (2008). Drought Induced
Alterations in Growth, Osmotic Potential and
In Vitro Regeneration of Soybean Cultivars.
General and Applied Plant Physiology, Special
Issue, 34(1-2): 103-112.
Sané D., Ould, K.M. and Diouf, D. (2005). Growth
and Development of Date Palm (Phoenix
dactylefera L.) Seeding Under Drought and
Salinity Stresses. African Journal of
Biotechnology, 4(9): 966-972.
Smirnoff, N. (1993). The Role of Active Oxygen in
the Response of Plants to Water Deficit, and
Desiccation. New Phytologist, 125: 27–58.
Tsago, Y., Andargie, M. and Takele, A. (2013). In
Vitro Screening for Drought Tolerance in
Different Sorghum[(Sorghum bicolor (L.)
Moench] Varieties. Journal of Stress
Physiology and Biochemistry, 9(3): 72-83.
Tsago, Y., Andargie, M. and Takele, A. (2014). .In
vitro Selection of Sorghum [Sorghum bicolor
(L.) Moench] for Polyethylene Glycol (PEG)
Induced Drought Stress. Plant Science Today,
1(2): 62-68.
Verma, D., Ansari, M.W., Agrawal, G.K., Rakwal,
R., Shukla, A., and Tuteja, N. (2013). In vitro
selection and field responses of somaclonal
variant plants of rice cv PR113 for drought
tolerance. Plant signaling & behavior, 8(4):
e23519.
Yang, L., Li, Y. and Shen, H. (2012). Somatic
Embryogenesis and Plant Regeneration from
Immature Zygotic Embryo Cultures of
Mountain Ash (Sorbus pohuashanensis). Plant
Cell Tissue Organ Culture, 109: 547–556.