In Vitro Cellular & Developmental Biology - Plant (2020) 56:88–97

https://doi.org/10.1007/s11627-019-10015-5

PLANT TISSUE CULTURE

Use of gamma radiation to induce mutations in rice (Oryza sativa L.)

and the selection of lines with tolerance to salinity and drought
Ana Abdelnour-Esquivel 1 & Jason Perez 1 & Miguel Rojas 1 & Walter Vargas 2 & Andres Gatica-Arias 3

Received: 9 October 2018 / Accepted: 26 August 2019 / Published online: 4 February 2020 / Editor: Wagner Campos Otoni
# The Society for In Vitro Biology 2020

Abstract
One of the strategies for genetic improvement of Oryza sativa L. (rice) is the use of radiation-induced mutagenesis to
search for materials suitable for production in marginal conditions, such as drought and salinity. In this present study,
indica rice ‘CR-5272’ embryogenic calluses and seeds were exposed to gamma radiation. Survival and growth of plants
derived from embryogenic calluses and seeds were evaluated in vitro in the presence of selection factors for salinity and
drought (NaCl and sorbitol). Plants regenerated from irradiated calluses and seeds were screened for tolerance to these
conditions and promising lines were identified. Culture of plants regenerated from irradiated calluses (M1) allowed the
selection of 60, 58 and 15 promising lines with tolerance to salinity, drought, and both factors, respectively. Six promising
salt-tolerant lines and four drought-tolerant lines were selected from plants grown from irradiated seeds (M2). A hormetic
response to irradiation was observed in seeds; seeds irradiated at 50 Gy showed increased callus induction and callus
weight. This study allowed advancements in the improvement of rice ‘CR-5272’ through the production of promising lines
that are being evaluated in field studies.

Keywords Climate change . Cobalt-60 radiation-induced mutagenesis . Drought . Embryogenic callus . Salinity

Introduction On a global level, approximately 831 million ha of soil are

affected by salinity and are marginal or unsuitable for agricul-
Oryza sativa L. (rice) is the primary cereal food staple for 3.5 ture and livestock production (Amini et al. 2016). The major-
billion people around the world, providing 20% of the calories ity of the agronomically important crops are susceptible or
in their diets (International Rice Research Institute 2018). only slightly tolerant to salinity. Soil salinization is constantly
Global consumption of rice is projected to reach 763 million increasing due to human activities, such as the increased use
metric tons in 2020 and will increase to 852 million tons by of irrigation in agriculture and the use of poor-quality irriga-
2035 (Khush 2013). Climate change is causing negative ef- tion water (Amini et al. 2016).
fects in agriculture, and rainfall variability has become one of In light of these situations, crop genetic improvement has
the most limiting factors for rice production. Detrimental abi- focused on the search for varieties that are tolerant or resistant
otic stresses generated by rainfall variability, such as salinity to the new conditions brought about by climate change (Reddy
and drought, limit the areas dedicated to irrigated or rain-fed et al. 2017). The search for mutant varieties is one strategy that
rice production (Hedayati et al. 2015). has been used for this purpose. Radiation-induced mutagenesis
is an alternative source for induced genetic variability in rice
and it is of great value in the search for materials that can
* Ana Abdelnour-Esquivel
[email protected] withstand extreme conditions of drought or salinity (Clarke
et al. 2018; Robertson et al. 2018). The Joint Food and
1
Agriculture Organization of the United Nations/International
Centro de Investigación en Biotecnología, Instituto Tecnológico de
Costa Rica, Cartago, Costa Rica
Atomic Energy Agency (FAO/IEAE 2018) Mutant Variety
2
Database has registered the release of more than 3200 mutant
Gamma Irradiation Laboratory, Instituto Tecnológico de Costa Rica,
Cartago, Costa Rica
crop varieties grown throughout the world (Prasanna and Jain
3
2017), including 821 entries for rice (FAO/IEAE 2018).
Escuela de Biología, Universidad de Costa Rica, San José, Costa
Rica
Mutant rice varieties with differential characteristics for
 RADIATION-INDUCED MUTAGENESIS OF RICE 89

agronomic traits and consumer-defined quality are recognized for 20 or 30 min each, with constant agitation at 200 rpm. A
(Tai 2007; Yan et al. 2012; Schiocchet et al. 2014). 10 μL volume of TWEEN®-20 (PhytoTechnology
The wide use of mutagenesis for plant genetic improve- Laboratories®, Shawnee Mission, KS) was used per 10 mL
ment can be explained by several factors: the varieties pro- of sanitizing agent. In a laminar flow transfer chamber, seeds
duced are readily accepted; new varieties can be developed in were rinsed, at least, five times with sterile distilled water, then
a shorter time than by conventional breeding methods; and rinsed one time with a 40 or 50% (v/v) biocidal solution of SB
new varieties produced by transgenesis are less favorably ac- ARVI® (0.0412% 2-methyl-4-isothiazolin-3-one (MIT) and
cepted (Masoabi et al. 2018). The production of varieties by 0.1350% 5-chloro-2-methyl-4-isothiazolin-3-one (CIT);
mutagenesis requires the selection of large numbers of indi- Reactivos Químicos Gamma, Laboratorios ARVI S. A,
viduals. Tissue culture is a valuable tool supporting crop im- Cartago, Costa Rica) (Table 1). For induction of embryogenic
provement efforts, as it allows the rapid and massive in vitro callus, 15 sterilized and prepared seeds were placed on 20 mL
screening for variations of interest in irradiated materials. culture medium contained in a 94 × 15 mm Petri plate, as
Using this technique, selection agents for different biotic and described by Bai et al. (2014). The callus induction medium
abiotic stress conditions can be incorporated into the culture consisted of MS (Murashige and Skoog 1962) mineral salts
medium (Maluszynski et al. 2009). Among the most widely and vitamins, supplemented with 2.5 mg L − 1 2,4-
used selection agents are sodium chloride to simulate salinity, dichlorophenoxyacetic acid (2,4-D), 30 g L −1 sucrose,
and polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), 0.3 g L−1 hydrolyzed casein, and 0.5 g L−1 proline (all
and sorbitol to simulate drought. These selection agents re- chemicals previously mentioned were supplied by
duce the water potential to levels similar to those found in PhytoTechnology Laboratories®). The pH was adjusted to
soils under these stress conditions (Bündig et al. 2017). 5.8 with NaOH (EMSURE®, MerckKGaA, Darmstadt,
Environmental stress related to climate change has also Germany) prior to addition of 4 g L − 1 Gelzan®
affected areas of rice production in Costa Rica. Therefore, this (PhytoTechnology Laboratories®) as gelling agent. Medium
current research aimed to produce promising lines with toler- was sterilized by autoclaving at 121.6 kPa and 121°C for
ance to drought and salinity using cobalt-60 gamma irradia- 25 min. Cultures were maintained in darkness at 26 ± 2°C
tion of seeds and embryogenic calluses of the indica rice ‘CR- for 2 wk. After this period, induced calluses were irradiated
5272’ that is widely consumed in Costa Rica, followed by at different gamma rays’ doses or were used to establish the
selection in vitro on medium containing high concentrations sensitivity to salinity and drought.
of sodium chloride and sorbitol. A number of mutant lines
survived the in vitro osmotic and salinity stresses. Radiosensitivity Seeds with a MC of approximately 12% and
Consequently, to the knowledge of the authors, this study embryogenic calluses were placed in an Ob-Servo Ignis irra-
described the production of the first mutagenized rice plants diation system (Institute of Isotopes Co., Ltd. (Izotop),
of ‘CR-5272’ that showed enhanced drought and salinity tol- Budapest, Hungary) with 24 sources of cobalt-60 (type CoS
erance in vitro. 44HH-N; Izotop). Samples were irradiated with a dose rate of
60 Gy min−1. To determine radiosensitivity and the median
lethal dose (LD50), calluses were irradiated at 0, 20, 40, 60, 80,
Materials and methods 100, 200, 300, 400, or 500 Gy and seeds were irradiated at 0,
50, 100, 150, 200, 250, or 300 Gy. The radiosensitivity for
Establishment in vitro Seeds of the indica rice ‘CR-5272’ and seeds was determined based on the capability of seeds to pro-
embryogenic calluses generated from these seeds were used as duce embryogenic calluses on induction medium, the oxida-
experimental material. Seeds were donated by the National tion percentage (necrotic seeds), and fresh and dry weight of
Institute for Agricultural Innovation and Technology produced calluses. Determination of callus induction was
Transfer (INTA San José, Costa Rica). Seed moisture content made following the equation: callus induction = (number of
(MC) was determined to be 10 to 15% with three random seeds producing calluses ÷ number of seeds inoculated) × 100,
samples of 10 g of seeds (fresh weight) that were dried for and seed oxidation = (number of necrotic seeds ÷ number of
48 h at 100°C and weighed again to determine dry weight. seeds inoculated) × 100. Fresh weight was obtained by
Seed moisture content was calculated with the following equa- weighing produced calluses without any remnant of seeds
tion: MC = ((fresh weight − dry weight) ÷ fresh weight) × 100. and the dry weight was determined after drying those same
Prior to surface sterilization and establishment in vitro, the calluses for 48 h at 100°C. In addition, moisture content of
palea and lemma were removed using 100 grit sandpaper. calluses derived from irradiated seeds was calculated with the
Seeds were soaked twice in 3.5% (v/v) sodium hypochlorite equation MC = ((fresh weight of calluses − dry weight of
(diluted from 5.25% commercial solution, Blancox, Bogota, calluses) ÷ fresh weight of calluses) × 100. The radiosensitiv-
Colombia) for 20 min each, or two times in 5.25% (v/v) ity for embryogenic calluses was determined based on the
NaOCl (non-diluted 5.25% commercial solution, Blancox) survival of treated calluses and their ability to produce plants
90 ABDELNOUR-ESQUIVEL ET AL.

Table 1. Effect of surface-sterilization treatments on in vitro establishment and callus formation of rice seeds (Oryza sativa L. ‘CR-5272’)

NaOCl (3.5% v/v) NaOCl (5.25% v/v) Biocide solution* Fungal contamination (%) Aseptic cultures (%) Callus formation (%)

Twice 20 min 52.2 47.8 39.3

Twice 30 min 25.0 75.0 12.0
Twice 20 min 50% (v/v) 5.6 93.4 42.2
Twice 20 min 40% (v/v) 10.0 90.0 42.3

*Mixture of 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one, (Reactivos Químicos Gamma, Laboratorios ARVI S.A.,
Cartago, Costa Rica)

after their culture on the semisolid regeneration medium. NaCl, 5% (w/v) sorbitol, or a combination of both factors
Survival, regeneration, and oxidation were determined by (150 mM NaCl + 5% (w/v) sorbitol), under the culture condi-
the following equations: survival = (number of calluses show- tions previously described. After 8 wk, survival was evaluated
ing a creamish coloration ÷ total number of irradiated calluses) and plants were classified into four response categories: dead
× 100, regeneration = (number of calluses regenerating plants plantlets, susceptible (percent survival of the line evaluated
÷ total number of irradiated calluses) × 100, and oxidation = less than the non-irradiated control plants), moderately toler-
(number of necrotic calluses ÷ total number of irradiated cal- ant (percent survival greater than in non-irradiated control, but
luses) × 100. The regeneration medium, as described by less than 100%), and tolerant (100% survival of the line eval-
Sudhakar et al. (1998), consisted of MS basal salts, 30 g L−1 uated in presence of the selection factor). Plants from both the
sucrose, 0.5 mg L −1 α-naphthaleneacetic acid (NAA), salt and sorbitol tests were maintained and multiplied on the
3 mg L−1 6-benzylaminopurine (BAP), and 4 g L−1 Gelzan® respective culture medium.
(all reagents supplied by PhytoTechnology Laboratories®). For the selection of promising lines from irradiated seeds,
The pH was adjusted to 5.7 NaOH (EMSURE®, seeds obtained from plants regenerated from seeds irradiated
MerckKGaA) prior to the addition of 4 g L−1 Gelzan® as with 0, 300, 400, or 500 Gy (M2) were surface-sterilized as
gelling agent and autoclaved as before. Radiosensitivity as- described previously and grown in germination medium sup-
says used three repetitions of 50 seeds or 50 calluses. plemented with 0 and 200 mM NaCl, or with 0 and 10% (w/v)
sorbitol, under the conditions described previously. A total of
Sensitivity to selection factors Non-irradiated embryogenic 10,000 seeds were evaluated (1250 seeds per treatment).
calluses were grown on plant regeneration medium supple- Germination was evaluated after 2 wk of culture.
mented with 0, 25, 50, 75, 100, 125, or 150 mM of sodium
chloride (NaCl) to simulate conditions of salinity, or with 0, 5, Data analysis Radiosensitivity assays were analyzed in a
10, 15, or 20% (w/v) sorbitol to simulate drought conditions. completely randomized design by lineal regression. The sen-
Cultures were maintained at 26 ± 2°C with a 16-h photoperiod sitivity to selection factors of calluses and seed data were
and a light intensity of 72 μmol m−2 s−1 provided by 32-W analyzed in one-way analysis of variance (ANOVA) and
fluorescent bulbs (Phillips, Somerset, NJ). Treatments were means were compared by the Tukey HSD (honestly signifi-
repeated 10 times using 15 calluses per repetition. Plant re- cant difference) method. Analyses were performed with a sig-
generation from callus was evaluated after 6 wk of culture. nificance level > 0.05, using the statistical program Minitab
Non-irradiated seeds were surface-sterilized and germinat- Version 17 (Minitab 17 (2010); Minitab Inc., State College,
ed on MS basal medium, supplemented with 30 g L−1 sucrose PA).
and 0, 50, 100, 150, 200, or 250 mM NaCl, or with 0, 5, 10,
15, or 20% (w/v) sorbitol, 4 g L−1 of Gelzan®, and pH adjust-
ed and autoclaved as before (all reagents supplied by Results
PhytoTechnology Laboratories®). Cultures were maintained
in a growth chamber at 26 ± 2°C with a 16-h photoperiod and Establishment in vitro Surface sterilization of rice seeds by
a light intensity of 72 μmol m−2 s−1 provided by 32-W fluo- incubating in 3.5% (v/v) NaOCl for 20 min produced the
rescent bulbs (Phillips). Each treatment was repeated 10 times highest rates of contamination, while incubating two times
using 15 seeds per repetition in a completely randomized de- in 5.25% (v/v) for 30 min each led to more aseptic cultures
sign. Germination and shoot length were evaluated after 2 wk. but the least callus production. On the other hand, incubating
two times in 5.25% (v/v) NaOCl for 20 min each followed by a
Selection of promising lines Plant lines regenerated from 179 final rinse with a 50% or 40% biocidal solution (Table 1)
embryogenic calluses irradiated with 80 Gy (M1) were grown resulted in a lower incidence of contamination and a higher
on regeneration medium supplemented with 0 and 150 mM percentage of aseptic explants and callus formation.
 RADIATION-INDUCED MUTAGENESIS OF RICE 91

Determination of radiosensitivity The effect of cobalt-60 (Co- and 6B) was significant at concentrations of 10% (w/v)
60) gamma radiation on embryogenic indica rice callus was sorbitol or greater. In general, plants derived from seeds
evaluated. Survival after irradiation with 200 Gy (Fig. 1; black cultured in both selection factors showed similar appear-
diamonds) was approximately 50% (LD50). The percentage of ance to plants derived from seeds cultured in control treat-
plant regeneration from irradiated calluses (Fig. 1; gray ment without NaCl or sorbitol. Differences were described
diamonds) was lower than the percent survival, with an above.
LD50 for regeneration of 60 Gy (Fig. 2B).
When seeds were irradiated with doses higher than 50 Gy, a Selection of promising embryogenic lines Evaluation of lines
marked reduction in callus induction (Fig. 3A) was observed. regenerated from non-irradiated embryogenic calluses in the
A stimulatory radiation effect was observed for callus induc- presence of selection factors showed that NaCl in the culture
tion and biomass accumulation (Fig. 3C, D) at 50 and 250 Gy; medium was more limiting than sorbitol (Table 2). The nega-
a biphasic response was observed for both callus induction tive effect on regeneration was greater in plants grown in the
and biomass accumulation. Calluses derived from irradiated presence of both factors. The M1 lines derived from irradiated
seeds in all doses showed approximately 85% MC, while embryogenic calluses were classified into four response cate-
those derived from non-irradiated seeds showed 39% MC gories (Table 2). As with the non-irradiated controls, suscep-
(data not shown). All doses produced low oxidation rates (be- tibility of the M1 lines was greater in the presence of both
low 20%) on callus derived from treated seeds (Fig. 3B). selection factors. Certain rice lines were identified with toler-
ant and moderately tolerant responses to both selection factors
Sensitivity to selection factors In the evaluation of salt sensi- individually and in combination. The number of tolerant or
tivity of embryogenic calluses (Fig. 4A), no negative effects moderately tolerant lines obtained through irradiation was
on plant regeneration were observed at 0 mM NaCl (99% greater than the number of lines with characteristics of interest
regeneration). Doses of 25, 50, and 75 mM NaCl showed no derived from the non-irradiated control (Table 2). Plants re-
statistical differences. Regeneration was reduced with signifi- generated from calluses cultured in all doses of NaCl and
cant differences from 75 to 150 mM NaCl, with a LD50 of sorbitol showed similar phenotypical appearance to those re-
125 mM NaCl (Fig. 4A). Using sorbitol to simulate drought generated from calluses cultured in control treatment, and no
conditions, plant regeneration was reduced at all concentra- oxidation or necrotic effects were observed in any regenerated
tions evaluated (Fig. 4B), with a LD50 of 5% (w/v) sorbitol. At plants (Fig. 6A, B).
concentrations of 15% (w/v) or greater, no regeneration was Of the M2 seeds evaluated for salt and drought tolerance,
observed (Fig. 4B). six seedlings showed tolerance to 200 mM NaCl and four
The effect of increasing concentrations of NaCl on ‘CR- lines were tolerant to 10% (w/v) sorbitol (Table 3). The six
5272’ seeds was evaluated in vitro (Fig. 5A, C). Decreased plants tolerant to NaCl, along with the four lines derived from
germination and increased oxidation of rice seeds were ob- M2 tolerant to sorbitol and the five lines derived from non-
served at all NaCl concentrations (Fig. 5A). Values were sig- irradiated seeds that tolerated 200 mM salt conditions, were
nificantly different from the control treatment (germination selected for multiplication for further studies (Table 3). All
medium without added salt) at concentrations of 150 mM or salt- and drought-tolerant lines derived from irradiated cal-
greater for both germination and oxidation. The LD50 for luses or irradiated seeds showed essentially the same pheno-
NaCl was 200 mM, based on germination (Fig. 5A) and plant type of ‘CR-5272’ when growing in vitro.
length (Figs. 5C and 6A) data. The effect of sorbitol (drought
factor) on germination (Fig. 5B) and plant length (Figs. 5D
Discussion
100 y = 4E-06x3 - 0.0026x2 + 0.2059x + 90.881
Radiation-induced mutation as a strategy for genetic crop im-
Survival/Regeneration (%)

R² = 0.9412
80 y = -3E-06x3 + 0.0029x2 - 0.8988x + 82.94 provement should begin with the use of high-yielding varie-
R² = 0.906
ties with desirable characteristics but lacking in traits that af-
60
55.0 fect their ability to adapt to specific conditions. Irradiation is
53.3
40 used to produce variation in one or two of the main character-
20
istics influencing adaptation to conditions of interest
(Ahloowalia and Maluszynski 2001). The present study used
0 indica rice ‘CR-5272’, which is preferred by the Costa Rican
0 100 200 300 400 500
Irradiation dose (Gy) population for its grain shape and cooking characteristics.
Figure 1. Effect of gamma radiation (cobalt-60) dose (Gy) on percent
To increase efficiency and reduce the time required to gen-
survival ( ) and percent regeneration ( ) of embryogenic calluses erate varieties, plant breeders combine several techniques,
of rice (Oryza sativa L. ‘CR-5272’) after 8 wk of culture in vitro. such as in vitro culture (cloning) for rapid multiplication and
92 ABDELNOUR-ESQUIVEL ET AL.

Figure 2. Regeneration of
embryogenic calluses of rice
(Oryza sativa L. ‘CR-5272’) after
gamma irradiation (cobalt-60)
doses (Gy): 40 (A); 60 (B); 80 (C);
and 500 (D). Evaluations were
made after 8 wk of culture
in vitro. Black bars indicate 2 cm.

a b

c d

selection, molecular methods to screen for specific genotypes, sanitizing agent, due to its efficacy, price, and availability
mutagenesis to increase variation, reverse genetics, double (Smith 2013; Khanam and Chandra 2017; Orlikowska et al.
haploid techniques, and controlled environments to facilitate 2017). The use of other biocidal substances in addition to
and manipulate growth (Ahloowalia and Maluszynski 2001; sodium hypochlorite is recommended when contamination is
Mba 2013). In conventional breeding, screening for mutants persistent. In this present study, the use of the microbial sup-
remains time consuming and expensive, and requires large pressor SB ARVI®, after two 20-min incubation periods in
populations (Schaart et al. 2016). Tissue culture has many sodium hypochlorite, resulted in a greater percentage of asep-
advantages over the conventional plant breeding process, tic material established in vitro (Table 1). The combination of
one of which is the ability to select for desirable characteristics MIT/CIT is a potent wide-spectrum biocide that inhibits the
in a shorter time, due to the ease of modifying culture media development of contaminating bacteria, fungi, and microalgae
through the addition of selection agents. Another advantage of in plant culture medium (Silvetti et al. 2018). This product
tissue culture is the production and growth of plants in con- affects key enzymes in the Krebs cycle and the electron trans-
trolled conditions (Bado et al. 2015). In addition, using this port chain, preventing spore germination and destroying mi-
technique, the number of individuals with the desired pheno- crobial cells (Chemical Laboratory ARVI S.A. 2015).
type can be increased rapidly in a smaller space than that The incubation of shoots and seeds in MIT/CIT as a final
required for field or greenhouse multiplication. step in surface sterilization prior to inoculation in culture me-
This present study was carried out in controlled laboratory dium or for recovery of contaminated materials in vitro has
conditions. Whenever plant material is grown in vitro, initial been reported to improve cultures without affecting explant
surface sterilization of plants is indispensable; bacteria and development (Jiménez et al. 2006; Miyazaki et al. 2010).
fungi are present in the natural plant environment and the However, Compton and Koch (2001) observed negative ef-
culture medium is an ideal environment for the proliferation fects on organogenesis and regeneration of several species
of microorganisms that compete for nutrients (Orlikowska when MIT/CIT was added to culture media. According to their
et al. 2017). Sodium hypochlorite is the most widely used study, this effect is dependent on the concentration used and
 RADIATION-INDUCED MUTAGENESIS OF RICE 93

Calluses induction (%)

Oxidation (%)
Irradiation dose
a Irradiation dose
b

1200
A
1000
Fresh weight (mg)

Dry weight (mg)

800

600
B
400
C
D
200 E
G F

0
0 50 100 150 200 250 300

c Irradiation dose
d
Figure 3. Effect of gamma radiation dose (cobalt-60) on rice seeds Evaluations were made after 4 wk of culture in vitro. Means followed by
(Oryza sativa L. ‘CR-5272’). Induction of embryogenic callus (A); oxi- the same capital letter are not significantly different at P < 0.05. Vertical
dation of tissues (B); fresh weight (mg) (C); and dry weight (mg) (D). bars indicate standard error in (A, B).

the species evaluated. Based on results of the Compton and radiation dose and the response of the variable of interest, such
Koch (2001) study and others reported in the literature, as well as survival, regeneration, fresh weight, dry weight, or tissue
as findings by the present investigation (Table 1), incubation growth (Bado et al. 2015). This relationship is dependent on
of explants in the sanitizing agent is more effective than addi- many conditions, including explant moisture content, geno-
tion of the agent to the culture medium. type of the mother tissue, type of mutagenesis, and source
The first step in using radiation mutagenesis as a technique (in the case of radiation), and should be determined experi-
for genetic improvement is to determine the relation between mentally for each species and tissue type. Determination of the
Regeneration (%)
Regeneration (%)

NaCl (mM) Sorbitol (w/v %)

a b
Figure 4. Effect of selection factor concentration on percent regeneration 4 wk of culture in vitro. Means followed by the same capital letter do not
of non-irradiated rice calluses (Oryza sativa L. ‘CR-5272’). Salinity (mM significantly differ at P < 0.05. Vertical bars indicate standard error.
NaCl) (A) and drought (% (w/v) sorbitol) (B). Evaluations were made after
94 ABDELNOUR-ESQUIVEL ET AL.

Percent

NaCl (mM) Sorbitol (w/v %)

a b
Plant length (cm)

NaCl (mM) Sorbitol (w/v %)

c d
Figure 5. Effect of selection factor concentration on percent germination NaCl) (A, C); drought (% (w/v) sorbitol) (B, D). Plants were evaluated
( ), percent oxidation ( ), and length of plants in centimeters ( ) after 4 wk of culture in vitro. Means followed by the same capital letter do
from seedlings derived from seeds obtained from plants regenerated from not significantly differ at P < 0.05. Vertical bars indicate standard error.
irradiated seeds (M2 seeds) (Oryza sativa L. ‘CR-5272’). Salinity (mM

median lethal dose (LD50) is also essential for the induction of breeding programs and yields sufficient working material for
mutations, since the frequency of negative changes in mutant selection (Bado et al. 2015).
individuals increases with increasing doses of radiation (Bado In the present study, the LD50 for seeds (estimated in
et al. 2015). The LD50 corresponds to the amount of radiation 125 Gy) was based on the percentage of seeds that produced
absorbed at which 50% of the exposed population dies or embryogenic callus (Fig. 3A), and the LD50 for irradiated em-
shows reduced growth. This proportion is considered to be bryogenic callus (Fig. 1) was based on percent survival
the range that favors the appearance of mutations useful for (200 Gy) and plant regeneration (60 Gy), as recommended

Figure 6. Typical lengths of rice

plants (Oryza sativa L. ‘CR-
5272’) derived from seeds
obtained from plants regenerated
from irradiated seeds (M2 seeds)
grown at increasing
concentrations of sodium chloride
(NaCl) (A) and sorbitol (B). Plants
were evaluated after 4 wk of
culture in vitro. White bars
indicate 2.5 cm.

0 125 150 200 250 0 5 10 15 20

NaCl (mM) Sorbitol (w/v %)
a b
 RADIATION-INDUCED MUTAGENESIS OF RICE 95

Table 2. Selection of promising plant lines of rice (Oryza sativa L. ‘CR-5272’) with tolerance to salinity (NaCl) and drought (sorbitol) regenerated
from embryogenic calluses irradiated with 80 Gy (M1)

Selection factor Survival of Irradiated M1 lines*

non-irradiated lines**
Dead plantlets Susceptible Moderately tolerant Tolerant Gain***

5% (w/v) sorbitol 97 31 43 45 60 8
150 mM NaCl 58 56 8 57 58 57
150 mM NaCl + 5% (w/v) sorbitol 24 129 17 18 15 9

A total of 179 lines were evaluated for each selection factor and compared to non-irradiated controls
*Number of irradiated lines surviving in vitro culture in presence of selection factors
**Number of non-irradiated lines surviving in vitro culture in presence of selection factors
***Increase in the number of moderately tolerant and tolerant lines with respect to non-irradiated lines

by Mba (2013). According to Hase et al. (2018), radiosensi- irradiated calluses) and M2 plants (from germinated irradiated
tivity is directly related to the moisture content of the plant M1 seeds) were evaluated for their response to conditions of
tissue. In the current study, the response in seeds was higher salinity and drought, in order to identify promising tolerant
compared to regeneration in irradiated embryogenic calluses, lines. Salinity is a severe abiotic stress generated by the pres-
which is a more hydrated tissue. ence of soluble salts, primarily NaCl, that accumulate in the
Although irradiated seeds tended to show a decrease in soil profile naturally or by irrigation (Reddy et al. 2017). The
callus induction (Fig. 3A) and callus weight (Fig. 3C, D), in majority of crops do not tolerate salt stress, but the range of
the current study, a hormetic effect was observed at 50 Gy. susceptibility is genotype-dependent (Hase et al. 2018).
Hormesis is a physiological effect that occurs at low doses of a It has been shown that non-penetrating polymers, such as
toxic agent, which cannot be predicted by the effect caused at PEG and sugars in the culture medium, reduce the water po-
high doses of the agent (Baldwin and Grantham 2015). Some tential in a similar manner to drought in soil (Bündig et al.
biological systems respond positively or are stimulated by 2017). Several research groups have used sorbitol under
exposure to low doses of radiation, depending on tissue sen- in vitro conditions to evaluate drought tolerance in numerous
sitivity. In the present study, this effect was seen only in the crops. In the present study, survival tended to decrease with
response of irradiated seeds and not in irradiated callus tissue. increasing selective agent concentrations (Figs. 4 and 5), as
This increase in water content was observed in various tissues has been shown in previous works (Marssaro et al. 2017;
when irradiated (Al-Enezi and Al-Khayri 2012). Razavizadeh and Adabavazeh 2017).
After genetic variability has been generated, tools for rec- The effects of salinity and drought are related, considering
ognizing variants with differential characteristics are needed. the management systems used for dryland and irrigated rice
For in vitro screening, selection agents can be incorporated into (Hedayati et al. 2015). Production areas that depend on irriga-
culture media to simulate different conditions of abiotic stress. tion can be expected to have increasing soil salinity, whereas
This method furnishes the required rapid and efficient means areas of rainfall-dependent production are likely to experience
for early selection of genotypes of interest (Maluszynski et al. yield reductions and reduced acreage, due to climate change
2009). In this present study, M1 plants (regenerated from (Ahloowalia and Maluszynski 2001). The results of this

Table 3. Selection of promising

plant lines of rice (Oryza sativa L. Dose of radiation Selection factors for M2 germination
‘CR-5272’) based on radiation of M1 seeds (Gy)
dose and number of germinated 200 mM NaCl* Sorbitol 10% (w/v)*
M2 seeds tolerant to salinity and
drought Germinated Oxidized NR** Germinated Oxidized NR**

M0 5 5 1240 0 0 1250
300 2 15 1233 1 0 1249
400 0 0 1250 0 0 1250
500 4 17 1229 3 5 1242

n = 1250 seeds per treatment

M0, non-irradiated seeds (0 Gy); * in number of seeds; NR**, non-responsive seeds; M1, plants derived from
seeds irradiated at 0, 300, 400, or 500 Gy; M2, seeds obtained from plants regenerated from M1 irradiated seeds
96 ABDELNOUR-ESQUIVEL ET AL.

present study should provide useful findings for rice breeding Chemical Laboratory ARVI S. A (2015) Supresor Microbiológico S.B.
ARVI: Inhibidor/controlador de contaminación para cultivos in vitro,
programs focused on the search for elite materials with poten-
Ficha técnica, Online document http://www.arvicr.com/productos/
tial for adaptation to conditions of drought and salinity. fichas-tecnicas/solucion-biocidaft.pdf. Accessed on January 15,
2019
Clarke D, Lázár AN, Saleh AFM, Jahiruddin M (2018) Prospects for
Conclusions agriculture under climate change and soil salinisation. In: Nicholls
RJ, Hutton CW, Adger WN, Hanson SE, Rahman M, Salehin M
(eds) Ecosystem services for well-being in deltas. Palgrave
In this current study, the LD50 for gamma irradiation was de- Macmillan, Cham, pp 447–467
termined for seeds and calluses of indica rice ‘CR-5272’, and Compton ME, Koch JM (2001) Influence of plant preservative mixture
sensitivity to salinity and drought were determined in vitro. (PPM) TM on adventitious organogenesis in melon, petunia, and
tobacco. In Vitro Cell Dev Biol–Plant 37:259–261. https://doi.org/
Lines with tolerance to these conditions in vitro were selected 10.1079/IVP2000144
by adding sodium chloride and sorbitol, respectively, to the Food and Agriculture Organization of the United Nations/International
culture medium. The benefits of using the techniques of gam- Atomic Energy Agency (FAO/IAEA) (2018) Mutant Variety
ma irradiation together with plant tissue culture selection were Database. http://mvdiaeaorg/ Cited 5 October 2018
evident to identify tolerant lines, as a first step in a program of Hase Y, Satoh K, Kitamura S, Oono Y (2018) Physiological status of
plant tissue affects the frequency and types of mutations induced
genetic improvement of this rice variety, which is widely con- by carbon-ion irradiation in Arabidopsis. Sci Rep 8:1394–1404.
sumed because of its preferred culinary characteristics. The https://doi.org/10.1038/s41598-018-19278-1
selected lines will be further evaluated under field conditions. Hedayati P, Monfard HH, Isa NM, Hwang DJ, Zain CRCM, Uddin MI,
Zainal Z (2015) Construction and analysis of an Oryza sativa (cv.
Funding information The authors gratefully recognize the financial sup- MR219) salinity-related cDNA library. Acta Physiol Plant 37:91.
port of the Consejo Nacional de Rectores (CONARE) of Costa Rica (VI- https://doi.org/10.1007/s11738-015-1837-4
252-2015) and the Vice Presidencies for Research and Extension of the International Rice Research Institute (2018) World rice statistics online
Instituto Tecnologico de Costa Rica and the Universidad de Costa Rica. query facility. http://ricestatirriorg:8080/wrsv3/entrypointhtm Cited
9 October 2018
Jiménez VM, Castillo J, Tavares E, Guevara E, Montiel M (2006) In vitro
Compliance with ethical standards propagation of the neotropical giant bamboo, Guadua angustifolia
Kunth, through axillary shoot proliferation. Plant Cell Tissue Organ
Conflict of interest The authors declare that they have no conflict of Cult 86:389–395. https://doi.org/10.1007/s11240-006-9120-4
interest. Khanam B, Chandra R (2017) Optimization of surface sterilization pro-
cess of selected dye-yielding plants for isolation of bacterial endo-
phytes. In: Mukhopadhyay K, Sachan A, Kumar M (eds)
Applications of biotechnology for sustainable development.
References Springer, Singapore, pp 45–50. https://doi.org/10.1007/978-981-
10-5538-6_7
Ahloowalia BS, Maluszynski M (2001) Induced mutations—a new par- Khush GS (2013) Strategies for increasing the yield potential of cereals:
adigm in plant breeding. Euphytica 118:167–173. https://doi.org/10. case of rice as an example. Plant Breed 132:433–436. https://doi.
1023/A:100416232 org/10.1111/pbr.1991
Al-Enezi NA, Al-Khayri JM (2012) Effect of X-irradiation on proline Maluszynski M, Szarejko I, Bhatia CR, Nichterlein K, Lagoda PJ (2009)
accumulation, growth, and water content of date palm (Phoenix Methodologies for generating variability Part 4, Mutation tech-
dactylifera L.) seedlings. J Biol Sci 12:146–153. https://doi.org/10. niques. In: Ceccarelli S, Guimaraes EP, Weltzien E (eds) Plant
3923/jbs.2012.146.153 breeders and farmer participation. FAO, Rome, Italy, pp 159–194
Amini S, Ghadiri H, Chen C, Marschner P (2016) Salt-affected soils, ISBN 978–92–5-106382-8
reclamation, carbon dynamics, and biochar: a review. J Soils Marssaro AL, Morais-Lino LS, Cruz JL, Ledo CA, Santos-Serejo JA
Sediments 16:939–953. https://doi.org/10.1007/s11368-015-1293-1
(2017) Simulation of in vitro water deficit for selecting drought-
Bado S, Forster BP, Nielen S, Ghanim A, Lagoda PJ, Till BJ, Laimer M tolerant banana genotypes. Pesq Agropec Bras 52:1301–1304.
(2015) Plant mutation breeding: current progress and future assess- https://doi.org/10.1590/s0100-204x2017001200021
ment. In: Goldman IL, Michler CH, Ortiz R (eds) Plant breeding
Masoabi M, Lloyd J, Kossmann J, van der Vyver C (2018) Ethyl
reviews. Willey-Blackwell, Hoboken, NJ, pp 23–88 ISSN: 0730-
methanesulfonate mutagenesis and in vitro polyethylene glycol se-
2207
lection for drought tolerance in sugarcane (Saccharum spp.). Sugar
Bai C, Rivera SM, Medina V, Alves R, Vilaprinyo E, Sorribas A, Zhu C Tech 20:50–59. https://doi.org/10.1007/s12355-017-0524-8
(2014) An in vitro system for the rapid functional characterization of
Mba C (2013) Induced mutation unleashes the potentials of plant genetic
genes involved in carotenoid biosynthesis and accumulation. Plant J
resources for food and agriculture. Agronomy 3:200–231. https://
77:464–475. https://doi.org/10.1111/tpj.12384
doi.org/10.3390/agronomy3010200
Baldwin J, Grantham V (2015) Radiation hormesis: historical and current
Minitab 17 (2010) Statistical Software by Minitab Inc. Retrieved from
perspectives. J Nucl Med 43:242–246. https://doi.org/10.2967/jnmt.
http://www.minitab.com/es-mx/products/minitab/ on December 13,
115.166074
2018
Bündig C, Vu TH, Meise P, Seddig S, Schum A, Winkelmann T (2017)
Miyazaki J, Tan BH, Errington SG (2010) Eradication of endophytic
Variability in osmotic stress tolerance of starch potato genotypes
bacteria via treatment for axillary buds of Petunia hybrida using
(Solanum tuberosum L.) as revealed by an in vitro screening: role
Plant Preservative Mixture (PPM™). Plant Cell Tissue Organ Cult
of proline, osmotic adjustment and drought response in pot trials. J
102:365–372. https://doi.org/10.1007/s11240-010-9741-5
Agron Crop Sci 203:206–218. https://doi.org/10.1111/jac.12186
 RADIATION-INDUCED MUTAGENESIS OF RICE 97

Murashige T, Skoog F (1962) A revised medium for rapid growth and bio Schiocchet MA, Noldin JA, Raimondi JV, Neto AT, Marschalek R,
assays with tobacco tissue cultures. Physiol Plant 15:473–497. Wickert E, Eberhardt DS (2014) SCS118 Marques—new rice culti-
https://doi.org/10.1111/j.1399-3054.1962.tb08052.x var obtained through induced mutation. Crop Breed Appl Biotechnol
Orlikowska T, Nowak K, Reed B (2017) Bacteria in the plant tissue 14:68–70. https://doi.org/10.1590/S1984-70332014000100012
culture environment. Plant Cell Tissue Organ Cult 128:487–508. Silvetti T, Merlini L, Brasca M, Galante YM (2018) Aerogel from chemo-
https://doi.org/10.1007/s11240-016-1144-9 enzymatically oxidized fenugreek gum: an innovative delivery sys-
Prasanna S, Jain SM (2017) Mutant resources and mutagenomics in crop tem of isothiazolinones biocides. Appl Microbiol Biotechnol 102:
plants. Emir J Food Agric 29:651–657. https://doi.org/10.9755/ejfa. 2683–2692. https://doi.org/10.1007/s00253-018-8804-0
2017.v29.i9.86 Smith RH (2013) Plant tissue culture: techniques and experiments, vol 3.
Razavizadeh R, Adabavazeh F (2017) Effects of sorbitol on essential oil Academic Press. San Diego, California, USA, pp 45–52. https://doi.
of Carum copticum L. under in vitro culture. Rom Biotechnol Lett org/10.1016/B978-0-12-415920-4.00004-9
22:12281–12289 https://www.rombio.eu/vol22nr1/16_Raazavideh. Sudhakar D, Bong BB, Tinjuangjun P, Maqbool SB, Valdez M, Jefferson
pdf. Accessed 21 Nov 2018 R, Christou P (1998) An efficient rice transformation system utiliz-
Reddy IN, Kim BK, Yoon IS, Kim KH, Kwon TR (2017) Salt tolerance in ing mature seed-derived explants and a portable, inexpensive parti-
rice: focus on mechanisms and approaches. Rice Sci 24:123–144. cle bombardment device. Transgenic Res 7:289–294. https://doi.
https://doi.org/10.1016/j.rsci.2016.09.004 org/10.1023/A:1008870012568
Robertson AD, Zhang Y, Sherrod LA, Rosenzweig ST, Ma L, Ahuja L, Tai TH (2007) Induced mutations in rice (Oryza sativa L.). Isr J Plant Sci
Schipanski ME (2018) Climate change impacts on yields and soil 55:137–145. https://doi.org/10.1560/IJPS.55.2.137
carbon in row crop dryland agriculture. J Environ Qual 47:684–694.
Yan W, Hu B, Zhang Q, Jia L, Jackson A, Pan X, Huang B, Yan Z, Deren
https://doi.org/10.2134/jeq2017.08.0309
C (2012) Short and erect rice (ser) mutant from ‘Khao Dawk Mali
Schaart JG, van de Wiel CC, Lotz LA, Smulders MJ (2016) Opportunities
105’ improves plant architecture. Plant Breed 131:282–285. https://
for products of new plant breeding techniques. Trends Plant Sci 21:
doi.org/10.1111/j.1439-0523.2011.01943.x
438–449. https://doi.org/10.1016/j.tplants.2015.11.006