1Department
of Tissue Culture, National Centre for Genetic Resource and Biotechnology
(NACRAB), Moor Plantation, Ibadan, National Biotechnology Research and
Development Agency, (NBRDA) Nigeria.
2Department
of Plant Science and Biotechnology, AdekunleAjasin University, Akungba-Akoko,
Ondo state, Nigeria.
This study investigated the induction of genetic variability in rice
through tissue culture techniques using hydrogen peroxide (H2O2). Two rice
accessions underwent varying soak durations (12 and 24 hours) and
concentrations (0%, 20%, 40%, 60%, 80%, and 100% H2O2). Following soaking,
the seeds were inoculated into Murashige and Skoog (MS) media in a completely randomized design with
three replications under in vitro conditions. Phenotypic traits, including
the number of shoots, shoot length, number of roots, and root length, were analyzed through ANOVA with a significance threshold of
≤ 0.05. Results indicated significant differences among the treated
plantlets and the control group, with specific treatments demonstrating
superior performance in various physical parameters. Notably, the highest
mean number of shoots (2.06) was observed in the control group (12 hours)
for accession NGB00789, followed by 2.00 in the control group (12 hours) for
NGB00792. Conversely, the lowest mean value (0.17) was recorded for NGB00792
soaked with 100% H2O2 for 12 hours. Regarding shoot length, NGB00789 soaked
with 100% H2O2 for 12 hours exhibited the highest mean value (4.44 cm),
while NGB00792 soaked with 60% H2O2 for 24 hours reached a mean value of
4.36 cm. For the number of roots, the highest mean value (6.67) was observed
in NGB00789 under the control condition, followed by 6.22 in NGB00792, also
in the control group. The lowest mean value (0.56) was recorded in NGB00792
soaked with 100% H2O2 for 12 hours. Concerning root length, the highest mean
value (6.01 cm) was achieved in NGB00792 treated with 60% H2O2 for 24 hours,
while NGB00792 soaked with 20% H2O2 for 12 hours reached a mean value of
5.99 cm. The lowest mean value (0.32 cm) was recorded in NGB00789 treated
with 80% H2O2 for 24 hours. In conclusion, this study not only sheds light
on the practical applications of hydrogen peroxide in rice tissue culture
but also underscores the necessity for further research in this domain to
fully harness the potential of mutagenesis as a tool for crop improvement.
Rice
(Oryza sativa L.), a staple crop worldwide,
possesses untapped genetic potential for significantly higher grain yields in
various rice-growing ecosystems. However, realizing this potential remains a challenge,
primarily due to the crop's sensitivity to abiotic stresses, with salinity
being a prominent stressor (Grover et al.,
2000). In Nigeria, rice cultivation, largely led by smallholder farmers
utilizing traditional methods, constitutes over 80% of the national production
(Omoare and Oyediran,
2020). Despite being the top rice producer in Africa, challenges such as
outdated technology, low productivity, and inadequate infrastructure hinder
sustainable production (Afiukwa, 2016; Rosenzweig et al., 2001). Abiotic stresses, including salt
toxicity, drought, and nutrient deficiency, present significant threats to rice
production in Nigeria (Umego et al., 2020). The
impact of drought stress, affecting approximately 23 million hectares of
rain-fed rice globally, is expected to exacerbate with climate change (Serraj et al., 2011; Ahmad et al., 2020).
To mitigate these challenges, it is essential
to develop effective strategies to protect rice production from the adverse
effects of abiotic stresses. A cost-effective approach for enhancing crop
species resilience against production challenges involves employing chemical
mutagenesis. While various chemicals have been utilized for inducing
mutagenesis in crop species, the potential of hydrogen peroxide in this context
remains largely unexplored.
Hydrogen peroxide functions as a mutagen in
plant breeding experiments, fostering genetic variability for diverse traits in
crops such as cowpea, mung bean, and sesame (Susrama et al., 2022; Ablaku et
al., 2021). It is also implicated in various gene expressions, DNA damage, and
programmed cell death across plant species. In Arabidopsis thaliana, hydrogen
peroxide treatment induced significant changes in gene expression patterns
related to light signaling, nutrient status, and temperature, highlighting its
role in regulating cellular processes. Studies, exemplified by Takáč et al. (2016), reveal that hydrogen peroxide
positively influences flax adventitious root formation by regulating auxin levels, suggesting its potential application for
enhancing flax regeneration capacity. While hydrogen peroxide is extensively
used in various plant studies, specific experiments on its role in inducing
genetic variability in rice are limited, with most rice research concentrating
on its response to oxidative stress and the activation of defense mechanisms.
This study aimed to determine the efficacy of
hydrogen peroxide for inducing genetic variability in rice under tissue culture
conditions. It sets the stage for a comprehensive examination of the mutagenic
effects of hydrogen peroxide and its implications for rice genetic diversity
and agronomic traits.
MATERIALS
AND METHODS
Plant
materials
Seeds
from two rice accessions, namely NGB 00789 and NGB 00792, recognized for their
upland characteristics and higher yield potential, were obtained from the Seed GeneBank Department at the National Centre for Genetic
Resource and Biotechnology (NACGRAB) in Ibadan, Nigeria.
Seed
surface preparation
Healthy
seeds underwent a thorough cleaning process, involving washing with a 0.02%
(v/v) tween-20 solution, followed by rinsing under running tap water. Surface
sterilization was carried out by immersing seeds in 0.15% (v/v) NaOCl2
for 10 minutes, followed by subsequent treatment with 70% (v/v) ethanol for 5
minutes. These steps were performed under aseptic conditions within a laminar
airflow chamber. Sterilized seeds were meticulously rinsed with sterile
distilled water five times and then air-dried on sterile filter sheets within
90 mm Petri dishes.
Inoculation
and culture
Upon
surface sterilization, seeds were introduced onto fresh culture media composed
of Murashige and Skoog (MS)
salts and vitamins (Murashige and Skoog,
1962). These sterilized seeds underwent treatment with varying concentrations
of hydrogen peroxide (H2O2) — 0%, 20%, 40%, 60%, 80%, and 100% — for different
durations: control (untreated), 12 hours, and 24 hours. After treatment, the
seeds were meticulously washed with sterile water before placement on the
prepared MS media. To ensure sterility, the media's pH was adjusted to 5.80
using 0.1 N HCl or NaOH,
followed by autoclaving at 121°C for 15 minutes. Cultures were then maintained
under controlled conditions in a growth room, featuring a temperature of 27 ±
2°C, a photoperiod of 16 hours of light (at an intensity of 10,000 lux), and 8
hours of darkness.
Experimental
design
The
research study was carried out using experimental design a completely
randomized design (CRD) with three replications under in vitro conditions. It
is important to note that the number of seeds or samples per replication was
[insert specific number], and randomization procedures were employed during the
experiment.
Data
collection and analysis
After
two weeks of inoculation, various physical parameters were assessed to monitor
the growth and development of the cultures. Regeneration frequency, denoting
the number of surviving plantlets resulting from H2O2
treatment, was determined. Data obtained were subjected to statistical analysis
using AGRES software, employing analysis of variance (ANOVA) to assess the
impact of H2O2 on mutagenesis induction and regeneration.
RESULTS
AND DISCUSSIONS
The
study aimed to induce genetic variation in rice under tissue culture conditions
using hydrogen peroxide (H2O2) as a chemical mutagen. The impact of H2O2
treatment on desirable phenotypic traits, including the number of shoots, shoot
length, number of roots, and root length, was evaluated. The experiment focused
on the direct regeneration of plants through embryogenesis in Murashige and Skoog (MS) media
treated with H2O2. Analysis of variance (ANOVA) was conducted to assess the
significance of observed variations, with significant levels determined at a
probability threshold of ≤ 0.05 (Table 1).
Effect
of H2O2 treatment on phenotypic traits
A
notable decrease in all assessed traits was observed with increasing duration
of mutagenic treatments, both for the 12-hour and 24-hour treatments with
full-strength MS media (Table 2). Significant differences (p ≤ 0.05) were
identified among the treated plantlets and the control group. Certain
treatments performed better in terms of physical parameters. The number of
shoots showed the highest mean value (2.06) in the control group (12 hours) for
rice accession NGB00789, followed by a mean value of 2.00 in the control group
(12 hours) for NGB00792. In contrast, the lowest mean value (0.17) was recorded
for NGB00792 soaked with 100% H2O2 for 12 hours. For
shoot length, the highest mean value (4.44 cm) was achieved in NGB00789 soaked
with 100% H2O2 for 12 hours, while NGB00792 soaked with
60% H2O2 for 24 hours reached a mean value of 4.36 cm.
Regarding the number of roots, the highest mean value (6.67) was observed in
NGB00789 without H2O2 treatment (control), followed by a
mean value of 6.22 in NGB00792, also in the control group. The lowest mean
value (0.56) was recorded in NGB00792 soaked with 100% H2O2
for 12 hours. The highest mean value (6.01 cm) for root length was achieved in
NGB00792 treated with 60% H2O2 for 24 hours, while a mean
value of 5.99 cm was observed in NGB00792 soaked with 20% H2O2
for 12 hours. The lowest mean value (0.32 cm) was recorded in NGB00789 treated
with 80% H2O2 for 24 hours.
Table 1: Analysis of variance of H2O2-induced
phenotypic traits in rice under tissue culture conditions
Source of variation
DF
SHTN
SHTL
RTN
RTL
Treatment
5
0.018*
0.024*
0.039*
0.041*
Hr
1
1ns
0.063ns
0.161ns
0.234ns
Treatment × Hr
5
0.021*
0.027*
0.072ns
0.078ns
Error
24
Probability of
significant level when p ≤ 0.05
NS: Number of shoots;
SL: Shoot length; NR: Number of roots; RL: Root length
Table
2: Mean and standard deviation of H2O2-induced phenotypic traits in rice under
tissue culture conditions
Treatment
Number of shoots
Shoot length (cm)
Number of roots
Root length (cm)
H202
20% (12hr) NGB 00789
1.33
± 0.97bc
3.28
± 2.56abc
4.72
± 3.71bcd
3.21
± 2.44def
H202
40% (12hr)
1.11
± 0.90cde
3.07
± 2.54bc
4.06±3.54cdef
4.17±3.05bcd
H202 60% (12hr)
0.00±0.00h
0.00±0.00h
0.00±0.00j
0.00±0.00i
H202
80% (12hr)
0.67±0.97defg
1.54±2.25def
2.50±3.84efgh
1.79±2.61fgh
H202100%
(12hr)
1.72±0.57ab
4.44±2.07a
5.17±2.09abcd
3.38±1.59de
CONTROL
(12h)
2.06±0.54a
3.38±1.27abc
6.67±3.41a
5.62±1.62ab
H202
20% (24hr)
1.06±0.94cdef
1.31±1.14efg
2.28±2.22fghi
1.19±1.06ghi
H202
40% (24hr)
1.89±0.32a
3.54±1.45abc
5.67±1.61abc
4.43±1.38bcd
H202
60% (24hr)
1.67±1.28ab
2.34±1.77cde
4.28±3.18cde
3.83±3.12bcd
H202
80% (24hr)
0.33±0.49gh
0.70±1.02fgh
0.67±0.97hij
0.32±0.49hi
H202100% (24hr)
0.00±0.00h
0.00±0.00h
0.00±0.00j
0.00±0.00i
CONTROL
(24hr)
1.83±0.38ab
2.28±0.24cde
3.67±1.81def
3.11±1.43def
H202
20% (12hr) NGB 00792
1.83±0.38ab
3.24±1.29abc
5.17±2.43abcd
5.81±2.10a
H202
40% (12hr)
0.67±0.97defg
1.44±1.67efgh
1.11±1.67ghij
2.21±3.11fgh
H202
60% (12hr)
1.17±0.99cd
2.55±2.33cd
2.78±2.51efg
3.09±2.75def
H202
80% (12hr)
0.61±0.91efg
1.33±2.15efg
1.39±2.19ghij
1.93±3.01efg
H202100%
(12hr)
0.17±0.38gh
0.08±0.19h
0.56±1.29ij
0.68±1.57ghi
CONTROL
(12h)
2.00±0.00a
2.96±1.59bc
6.22±2.69ab
5.53±2.03ab
H202
20% (24hr)
1.78±0.65ab
2.73±1.39bc
5.44±2.66abcd
5.99±2.24a
H202
40% (24hr)
1.72±0.67ab
3.84±1.75ab
4.94±2.53abcd
4.63±2.19abcd
H202
60% (24hr)
1.89±0.32a
4.36±1.96a
5.56±2.97abcd
6.01±2.18a
H202
80% (24hr)
0.56±0.92fg
1.13±1.87efgh
1.83±3.19ghij
0.99±1.66ghi
H202100%
(24hr)
0.22±0.55gh
0.14±0.35gh
0.67±1.64hij
0.69±1.59ghi
CONTROL
(24hr)
1.94±0.23a
3.27±1.56abc
5.39±2.30abcd
4.97±2.07abc
DISCUSSION
Mutation
induction has been recorded for a long period. Nevertheless, many improvements
have been made to increase the mutation frequency from modern technology. Rice
is an important cereal crop species for economic values and value addition
chain globally, and since it presents a small genome and displays synteny with other crops, many achievements in rice
structural and functional genomics have been expanded to other crop species.
Rice was already objective from the initial findings of mutation induction of
recent research work, which includes more than the 9th century of
research. Much has been gained agronomically and
conservatively through the increase in rice variability. Chemical mutagenesis
offers several advantages, including ease of handling, cost-effectiveness, and
specificity compared to physical mutagenesis methods. However, it is crucial to
exercise caution due to the potentially carcinogenic nature of chemical
mutagens. Research in crop mutagenesis has demonstrated the superiority of
chemical mutagens over ionizing radiations, as they induce milder genetic
effects and fewer chromosome breaks. Rapoport (1966)
made significant contributions to the field by introducing the concept of
"microgenetics." This concept elucidates
gene structure, function, mutagen mode of action, mutation origin, and fixation
in progeny.
This review summarized the data related to
the role of hydrogen peroxide that we have published during the past 15 years
using rice (cultivar TN1) as a model plant. It is clear that, in rice plants,
hydrogen peroxide not only acts as a toxic compound but also as a signaling
molecule associated with basal salt media. In considering the relationships
between hydrogen peroxide production and basal salt media and response to
mutation, we would also remember that these stresses almost occur in the field
together with other abiotic stress conditions. Such combinations could include
phenotypic and temperature factors. How these different stress combinations
affect hydrogen peroxide production and basal salt media is a subject of active
research that should be taken into reward. Chloroplasts are one of rice cells'
most significant sources of hydrogen peroxide. Extensive insight mutagens play
an important role in cereal crops especially rice by using hydrogen peroxide in
signaling seems to be necessary. However, it would be a
great admire and demand to notify all the changes in gene expression regulated
by phenotypic expression or abiotic stresses in rice using transcriptomic analyses. The combined use of mutagenic
treatment with in vitro culture was applied to play the role of alteration
in genetic variations. This was delineated by best growth value and green plant
regeneration frequency of irradiated Hydrogen peroxide-adapted affected
chlorophyll content of the plant and some formed callus induction as compared
with non -non-mutagenized control shown in (Tables 1 and 2). Some results were recorded
by other authors (Bhagwat and Duncan, 1998; Cheema, et al., 2002; Lee, et
al., 2003). It was believed that minimal stress or proper stress on the
callus by irradiation might not induce irreversible genotypic changes but could
stimulate chlorophyll pigmentation reduction or callus formation and plant
regeneration. Furthermore, the haploid induction technique can nowadays be
efficiently combined with several other plant biotechnological techniques,
enabling several novel breeding achievements, such as hybrid breeding, improved
mutation breeding, reverse breeding, and genetic transformation.
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Cite
this Article:Hassan, KO; Ajayi, AT; Anukwu, BC; Ajongbolo, FB; Lawyer, EF; Ilesanmi,
AO; Afolayan, AO (2024). Induce genetic variability
in rice (Oryzasativa L.)
under Tissue Culture condition. Greener
Journal of Agricultural Sciences, 14(3): 153-157.
