A systematic review on physical mutagens in rice breeding in Southeast Asia

Gamma-rays

Gamma rays are electromagnetic waves with the highest energy and shorter wavelengths that can penetrate deep into biological matter. Analysis of the studies reviewed revealed that gamma rays were used as a source of physical mutagens in 21 out of 28 studies to enhance crop traits. Of these, 13 studies used Cobalt-60, and seven studies used Cesium-137 as the gamma-ray source. The radiation source was not specified in eight of the studies. Cobalt-60, which has a half-life of 5.27 years, is produced by irradiating the stable isotope Cobalt-59 with neutrons in a nuclear reactor (Roberts, 2003). On the other hand, caesium-137 has the longest half-life of 30.1 years, making it the most long-lived isotope (Parsons, 2012). According to Oladosu et al. (2016), gamma rays offer an advantage in terms of accuracy and repeatability as a physical mutagen due to their homogeneous penetration capacity in tissue.

Indonesia has developed the most varieties of rice over the last five (5) years, utilizing 28 varieties of rice, namely Inpago Unsoed 1, Rojolele, Inpari 13, Cirata, Cempo Ireng, Cempo Melik, Melik, Sigupai, Cantek Manis, Rom Mokot, Ramos Mirah, Sanbei, Ciherang, Mentik Wangi, G10 Line, G16 Line, Baas Selem, Inpago Unram-1, Situpatenggang, Batutegi, Sintanur, Sigah, Mira-1, Inpari 10, IR64, Unsyiah-1 Simeulu, Unsyiah-3 Sanberasi, and Unsyiah-5 Sibahak. In contrast to genetic improvement, a physical mutagen is also used in food production for example in Malaysia, a study by Ahmad Zainuri et al. (2020) utilized gamma-rays to enhance the industrial quality of rice flour.

Ion-beam

An ion beam is a charged particle beam that contains an ion. Along with gamma-rays, ion beams have been widely exploited as novel mutagens in mutation breeding programs for the production of new genetic resources in plants, with the increasing number of mutants in the database (Zengliang, 2006; Satoh & Oono, 2019). In Takasaki, Japan, the National Institutes for Quantum and Radiological Science and Technology (QST), has been pursuing fundamental research on the induction of mutation in plants and microorganisms using ion beams accelerated by an azimuthally varying field (AVF) cyclotron at Ion Accelerators for Advanced Radiation Application (TIARA) since the early 1990s. Ion beams have a high linear energy (LET) (about 10–1,000 keV/mm or higher) and cause DNA damage/alterations at high densities, such as clustered damage involving large deletions, translocations, and inversions (Souframanien et al., 2020). One of the most important reasons for the use of ion implantation mutation is that it combines the variables of mass, charge, and energy, and causes damage to biological materials (including genetic components) that primarily leads to the displacement, recombination, and compounding of biological molecules and atoms.

Recent inquire shows there are five (5) studies that utilized ion beams in improving rice traits and consists of four (4) from Thailand and one (1) from Indonesia. In Thailand, the rice varieties KDML-105 and Sangyod were utilized in addition to RD6, whilst, in Indonesia, Pare Ambok, and Pare Lea were used. The seeds were blasted with various ion beams including mixed molecular (N⁺), atomic nitrogen ions (N₂⁺), argon, and carbon under-voltage potential of 30 and 50 kV to an ion fluence of around 1 × 10¹⁶ to 4 ×10¹⁶ ions/cm² (the bombarding time was about 2 min for the fluence of 10¹⁶ ions/cm²). The results showed that in the preliminary test, the optimum conditions to achieve the survival rates of ion bombarded rice seeds from 50% to 70% was the combination of 30 kV and 1,2 and 4 × 10¹⁶ ions/cm² fluences or 50 kV and 1 × 10¹⁶ ions/cm².

Electron beam

In contrast to ion beams, alpha particles, and neutrons, which have a high linear energy transfer (LET), electron beams, gamma-rays, and X-rays are classified as low-LET radiation (Hwang et al., 2014). Low-LET radiation is sparsely ionizing, and multiple exposures may be required to adequately irradiate target tissues. According to Spencer-Lopes et al. (2018), the use of X-rays, gamma-rays, neutrons, and electron beams has proven to be extremely beneficial in medicine, biology, and industry.

Only one study from Thailand, conducted by Promnart et al. (2017), has employed an electron beam to improve the RD31 (Pathum-Thani 31) rice variety. RD31 seeds were exposed to a 440 Gy electron beam before being tested for submergence tolerance. The findings revealed the identification of 317 submergence-tolerant lines (named Sub-1 gene) from a pool of around 3,000 M3 lines.

Neutron

Neutrons have the strongest ionization power among the physical mutagen agents. Neutrons are high-speed nuclear particles with a remarkable penetration power on materials. Neutrons can travel great distances in air and require very thick hydrogen-containing materials (such as concrete or water) to stop them. Fortunately, neutron radiation occurs predominantly within a nuclear reactor, where feet of water provide sufficient protection. The neutron, stable within the perimeters of the atom nucleus, once released will decays with a mean lifetime of approximately 15 min, releasing various kinetic energy. Table 4 shows different neutrons in terms of energy released that are the most used for plant mutation induction.

In Thailand, Malumpong et al. (2019) used a 33 Gy fast neutron (FN) to induce JHN wild type and screen for salt tolerance with 150 mM sodium chloride (NaCl). M1145 lines mutant from JHN was shown to be salinity tolerant in the study, although the authors emphasized that these lines must be tested in the field to confirm the potential for actual salt tolerance.

Several physical mutagens, including gamma-rays, ion beams, and fast neutrons have been explored in this study in terms of their efficient research for rice production enhancement. Gamma rays revealed the most significant mutagens utilized to increase rice plant growth, biochemical characteristics, industrial quality, and abiotic stress. Even though neutrons are potent mutagens, fast neutrons were less popular in rice development techniques due to the significant cost involved.

Plant development

Plant development refers to the processes through which plant structures emerge and mature as plants grow. Any alteration in growth pattern will directly impact maturity and productivity. It was found that the majority of the researchers used a variety of indicators to determine plant development performance. Plant height, number of tillers, number of seed/panicles, 1,000 seed weight, and flowering and harvesting period are among the most frequently used assessment as shown in Table 6. Purwanto et al. (2019) found that irradiated rice seeds with 100 Gy (Cempo Ireng; 134.8 cm and Melik; 137 cm) and 200 Gy (Cempo Melik; 135.6 cm) can produce higher plant height than controls by around 2.3–6.9%. Similarly, Herwibawa, Sakhidin & Dwi Haryanto (2019) discovered that the plant height of Inpago Unsoed 1 (121.75 cm) and Inpari 13 (113.56 cm) increased. However, the treatment was a combination of gamma radiation 100 Gy and immersion in sodium azide (SA) for 2 and 6 h, respectively.

Meanwhile, in an ion beam study (Techarang et al., 2018), plant height M2 mutants from the RD6 rice cultivar ranged from 103 to 135 cm, whereas M4 mutants ranged from 112 to 143 cm. M2 mutants from the Sangyod cultivar have heights ranging from 110 to 179 cm, while M4 mutants have a height ranging from 120 to 130 cm. Both RD6 and Sangyod mutants were shorter than wild cultivars, measuring 152 and 196 cm, respectively. OSRD6-4 and OSRD6-20 were selected from RD6, and OSSY-14, OSSY-17, and OSSY-23 have selected mutants from Sangyod based on the phenotypic variations that could provide higher crop yield including shorter stature, higher tillering capacity, and panicles per plant. Among the selected mutants, OSSY-23 mutant rice produced the most significant results when compared to wild Sangyod rice. According to the OSSY-23 results, the rough seed and brown rice sizes are longer at 2.6 mm × 10.1 mm and 2.2 mm × 7.4 mm, respectively.

In addition to this, Pratiwi et al. (2020) study concentrated on Mentik Wangi (local aromatic rice cultivar) originated from Magelang, Central Java, Indonesia. For the Mentik Wangi M2 mutants exposed to 150, 200, and 250 Gy gamma rays, the results showed that 11 mutant plants (150 Gy), 22 mutant plants (200 Gy), and 24 mutant plants (250 Gy) have short stems and high productivity. Destruction of the cell division process and cell expansion as a result of plant height reduction may be related to biochemical and physiological disruption, fluctuation in ascorbic acid content, and auxin loss in plant cells (Hasan et al., 2020).

The next important parameter was the number of tillers which plays an important role in the determination of grain product because it is related to the number of panicles yielded per area (Purwanto et al., 2019). Local rice varieties viz Cempo Melik (100 Gy), Melik (200 Gy) and Cempo Ireng (300 Gy) irradiated with gamma rays each have an average of 25.3, 23.7, and 24.3 tillers, respectively. However, only Cempo Melik irradiated with 100 and 200 Gy gamma rays, and Melik with 100 Gy gamma-ray has 5–10% more productive tillers than the control (Purwanto et al., 2019). Another study by Kurniawati, Chaniago & Suliansyah (2019), found that exposing Sigah brown rice cultivar to 200 Gy gamma-ray increased the number of tillers by 80%, with 99% of the tillers being productive tillers. Although rice plants exposed to gamma rays exhibited short-statured characteristics, the number of tillers and productive tillers increased surprisingly. In addition to these, the number of stem segments and length of each segment are reduced and the plant becomes shorter, which may result in earlier flowering and harvesting. In a study published by Herwibawa, Sakhidin & Dwi Haryanto (2019), aromatic and non-aromatic rice cultivars showed a 70% or greater increase in the number of tillers when exposed to 100 Gy gamma-ray (Inpago Unsoed 1) and 150 Gy gamma rays (Rojolele, Inpari 13 and Cirata) compared to controls. With the incredible increase in the number of tillers, Inpago Unsoed 1 produces 39%, Impari 13 produces 38%, Rojolele produces 64%, and Cirata produces 69% productive tillers from the total number of tillers. As a result of the promising number of productive tillers, Cirata, Inpago Unsoed 1, and Impari 13 produces 71.58 nos, 71.70 nos, and 76.75 number of tillers, respectively, which is much higher than the control.

Plant development is also widely assessed based on the weight of 1,000 grains of rice yield. In Indonesia, Herwibawa, Sakhidin & Dwi Haryanto (2019), Kurniawati, Chaniago & Suliansyah (2019), and Masruroh et al. (2016) have exposed rice to varying doses of gamma rays and discovered an increase in the total weight of 1,000 grains of rice. In Masruroh et al. (2016), for example, Cempo Ireng which was irradiated with 100 Gy gamma-ray produced 12% higher yield, with 11 days early flowering and harvesting periods than the control. While Ciherang was irradiated with 200 Gy of gamma-ray, there was an increase of 4% in the weight of 1,000 grains, with a record of three (3) days early flowering and harvesting periods compared to control. Another study carried out by Kurniawati, Chaniago & Suliansyah (2019) which employed brown rice (Sigah—M3) as a plant material exposed to 200 Gy gamma rays found that 1,000 grains weight was increased by 9% although the flowering and harvesting periods were delayed by 7 and 2, days respectively, compared to the control. Parallel to this, Herwibawa, Sakhidin & Dwi Haryanto (2019) observed that 1,000 grains weight of aromatic upland (Inpago Unsoed 1), aromatic lowland (Rojolele), non-aromatic upland (Cirata), and non-aromatic lowland (Inpari 13) rice cultivars irradiated with 100 and 150 Gy gamma rays were substantially higher than the control. In Malaysia, Yasmine et al. (2019) discovered a 3–8% increase in the total weight of 1,000 grains among Binadhan-8, NMR-152, and Pongsu Seribu-2 rice varieties. As a result, the appropriate dose of physical mutagens based on the variety of rice can stimulate better rice development from vegetative to the mature phase to produce higher yields.

Abiotic stress

Tolerance to abiotic stress has also been used to assess rice mutant performance after irradiation. Abiotic stress is defined as the detrimental effect of non-living factors on plants in a specific environment including drought, floods, salinity, extreme temperature, heavy metals, and other natural causes. Herwibawa, Sakhidin & Dwi Haryanto (2019), Amsal & Ishak-Ishak (2018), Yunita et al. (2020), Meesook, Pongtongkam & Poeaim (2018), Che Mohd Zain et al. (2016), and Kadhimi et al. (2016a), for example, have conducted research to identify drought-resistant rice cultivars. Researchers from Malaysia, Che Mohd Zain et al. (2016) and Kadhimi et al. (2016b) exposed rice seeds (MRQ74 and MR269) to 350 Gy of gamma rays and generated osmotic stress using a varied concentration of Polyethylene glycol (PEG) 6,000 ranging from 0–20%. The results of both types of research revealed that the highest proline concentrations of 29.156 and 40 µMg⁻¹ was noticed at PEG concentration levels of 7% and 20%, respectively, under in vitro condition. Meanwhile, in Thailand, Meesook, Pongtongkam & Poeaim (2018) irradiated riceberry rice seeds with gamma rays at 0, 20, 25, 30, 35, and 40 krad; however, the acceptable survival rate was found to be lower for those above 28 krad, thus, the proline concentrations of leaf were evaluated between 0 to 28 krad. The data showed that proline concentrations were higher in the radiation treatment at 15% PEG (0.175 µMg⁻¹) compared to non-irradiate (0.15 µMg⁻¹) at the same concentration PEG. In Indonesia, rice seeds (Inpago Unsoed 1, Rojolele, Inpari 13, and Cirata) were irradiated with 100 or 150 Gy gamma rays were immersed in sodium azide (SA) for two (2) or six (6) h before being treated with 5% PEG at a pressure of −0.03 MPa. Under the drought stress level of −0.03 MPa, the genetic variants in the M1 generation show diversity, and the best combination of mutagen and cultivar is Inpago Unsoed 1, which was irradiated with gamma-ray 100 Gy and then soaked in SA for 2 h (Herwibawa, Sakhidin & Dwi Haryanto, 2019). Amsal & Ishak-Ishak (2018) exposed the Mira-1 rice variety to 25 and 50 Gy of gamma rays and tested with 20% PEG resulting in two (2) from 20 mutant lines; 28H1 and 30F1 mutant lines showing the best characteristics (number of productive tillers; 12 each, lateral root; 142 and 100, and grain yield per panicle; 80 and 124, respectively) for drought tolerant. Lateral roots can develop on any primary root, including crown roots and embryonic, and are used as a drought tolerance indicator. The ability of the 28H1 and 30F1 rice mutants to absorb water in dry conditions may benefit from having the greatest number of lateral roots. Callus mutation with gamma rays was used by Yunita et al. (2020) in upland rice (Situpatengang and Batutegi) where Situpatengang was irradiated with 24.68 Gy gamma rays and Batutegi was with 22.15 Gy gamma rays. Both varieties were tested with 24.11% and 25.18% PEG, respectively, and both mutant calluses were regenerated on MS +BA 3 mg/L + Zeatin 0.1 mg/L, produced 83 shoots Situpatengang and 73 shoots in Batutegi callus. After acclimatization, the shoots produced 52 mutant lines from Situpatengang rice varieties and 49 mutant lines were produced from Batutegi rice varieties.

Malumpong et al. (2019) used fast neutrons on rice to cope with salt stress during the reproductive stage, and Promnart et al. (2017) used electron beams to explore flood tolerance in Thai rice breeds. A total of 9,000 lines of irradiated M₄ JHN with 33 Gy fast neutron were screened for salt tolerance (150 mM NaCl) and seeded in a field nursery, with M1145 emerging as the salinity tolerant line. Even though the lines were similar under salt stress to Pokkali and FL530 (typical salt-tolerant varieties), the researcher proposed that M1145 should be evaluated in the field to confirm the potential for salt stress. Furthermore, in the flood tolerance study, approximately 3,000 M₃ lines of irradiated electron-ion beam 0.44 KGy were screened for submergence tolerance and 317 tolerant mutant lines were survived and selected for planting for the following season (M₄ and M₅).

A combination of the above study results reported to the impact of irradiation on abiotic stress is expected to be used as a benchmark, particularly in drought, flood, and salinity, in designing the upcoming abiotic resistant/tolerant rice genetic line.

Biochemical properties

Radiation may also alter the gene that regulates biochemical compounds in plants. Anthocyanin, the bioactive substance with antioxidant properties in rice that may give anti-inflammatory and anti-cancer benefits, is one of the primary target qualities of the next generation of genetically modified crops particularly in black rice (Chen & Lin, 2013; Ciulu, de la Cadiz-Gurrea & Segura-Carretero, 2018). According to Yamuangmorn & Prom-U-thai (2021), anthocyanin can reduce cell damage when plants are exposed to biotic and abiotic stress, and anthocyanin’s contribution to plant structure has been identified as protecting against free radicals during physiological metabolism. Generally, the subspecies indica and japonica were reported to have total anthocyanin contents ranging from 32.40 to 50.30 mg/100 g, and 121.30 to 155.90 mg/100 g, respectively (Pedro, Granato & Rosso, 2016). However, according to Masruroh et al. (2016), Cempo Ireng rice exposed to 500 Gy of gamma-ray resulted in a 52.174% increase in anthocyanin levels compared to controls (18.623%). A similar study conducted by Purwanto et al. (2019) discovered the highest concentration of anthocyanin in Cempo Ireng rice irradiated at 200 Gy of gamma-ray with 155.946 mg/100 g and Cempo Melik with 142.492 mg/100 g (300 Gy of gamma-ray). In contrast, Melik rice had lower anthocyanin contents with 137.523 mg/100 g (100 Gy of gamma-ray) compared to control 146.323 mg/100 g as the radiation exposure increased (Purwanto et al., 2019). This suggests that radiation has a strong effect on important biochemical contents of rice and may contribute to enhancing the property at the right dosage.

Nutritional quality

Higher nutritional value food is constantly required for human health. Agricultural practices (soil preparation, sowing, manuring, irrigation, etc.), post-harvest handling (threshing, drying, cleaning, milling, grading, storage, and packaging), cultivar varieties and manipulations, followed by selection through breeding and genetic mechanisms, all have an impact on the nutrient content of rice. It is necessary to carefully manage the entire process to avoid nutritional losses including in the selection of grains for seeds to be used in the following cropping. The current study provides an update on the effect of physical mutagens on the rice’s nutritional properties.

The protein content is one of the nutrients that represents the nutritional quality of the rice yield. According to Amagliani et al. (2017), the protein content of rice grain varies and can be classified as brown rice, white or milled rice, and rice bran. The protein content of each type of rice grain ranged from 7.1% to 8.3% (brown rice), 6.3% to 7.1% (milled rice), and 11% to 15% (rice bran). Current reviews have found that Masruroh et al. (2016) have investigated the effects of five (5) levels of gamma radiation (100, 200, 300, 400, and 500 Gy) on two (2) rice cultivars to improve rice nutritional quality of Ciherang and Cempo Ireng. As a result, the protein content of Ciherang rice exposed to 400 Gy gamma rays increased by 43%, with a protein content of 8.98% compared to the control, which was 6.26%. In comparison to the control (7.73%), Cempo Ireng exposed to 500 Gy gamma-ray increased by 30%, with a protein content of 10.09%. Another study by Techarang et al. (2018) on protein content in brown rice (M2 generation) from RD6 mutants exposed to ion beams (4 × 10¹⁶ ions/cm² (50 kV)) found that OSRD6-4 and OSRD6-20 are 9.1/100 and 8.7/100 g, respectively, and are higher than the wild RD6 (8.3/100 g). Similarly, OSSY-23 (8.9/100 g) and OSSY-17 (9.3/100 g) were mutants selected from Sangyod rice mutants in M2 generation irradiated with an ion beam (4 × 10¹⁶ ions/cm² (50 kV)) also showed high protein content compared to wild Sangyod (8.6/100 g). The findings demonstrated that when physical agents are used in the induced mutation breeding technique, rice characteristics have a high possibility and chance of improving rice quality.

Amylose content was the next characteristic that represents nutritional quality in rice. The amylose content of the milled rice is a major determinant of rice texture, specifically rice hardness, and this contributes to amylose content playing an important part in decision making in rice breeding programs. Amylose content in rice is divided into five (5) categories: waxy (0–2%), very low (3–9%), low (10–19%) intermediate (20–25%), and high (>25%) (Cuevas et al., 2016). Techarang et al. (2018) discovered two (2) RD6 rice mutants (M2 generation), OSRD6-4 and OSRD6-20, that showed beneficial changes in amylose content after being exposed to an ion beam (4 × 10¹⁶ ions/cm² (50 kV)). The amylose content of the M2 generation of RD6 rice which is OSRD6-4 (16.4/100 g) was higher than that of the RD6 (wild type) with amylose content of 7.0/100 g and OSRD6-20 (6.8/100 g). Meanwhile, M2 generation from Sangyod rice revealed no significant difference in amylose content between rice mutants OSSY-14 (19.0/100 g), OSSY-17 (15.5/100 g), and OSSY-23 (16.9/100 g) compared to wild Sangyod (17.7/100 g) when exposed to ion beam (4 × 10¹⁶ ions/cm² (50 kV)). This implies that wild rice Sangyod, Sangyod mutants (OSSY-14, OSSY-17, and OSSY-23), and OSRD6-4 belong to the low amylose content group, whereas wild RD6 and RD6 mutants (OSRD6-20) belong to the group very low amylose content. When cooked, rice with a very low amylose content becomes softer, moister, and stickier in texture than rice with low amylose content. Furthermore, according to Prom-u-thai & Rerkasem (2020), grain with a higher amylose content absorbs more water, resulting in a higher volume expansion ratio, such as the volume of cooked rice compared to its precooked volume. Based on the findings, it is possible to conclude that using the optimum physical mutagens such as gamma rays and ion beams may result in a wide range of morphological changes, including amylose content (glutinous to non-glutinous) and protein content.

Industrial quality

There is currently no common definition of “industrial rice quality,” and there is even less agreement on how it should be quantified. Rice quality was difficult to define precisely because “rice quality” is subjective and context-specific compared to agronomic traits which include the ability of rice to boost production and/or alleviate abiotic or biotic stresses in crops (Custodio et al., 2019). Consumer perceptions of rice quality differ between areas, countries, cities, and levels of urbanization. According to Jaafar, Lalp & Mohamed@Naba (2018), consumer attitudes, and extrinsic and intrinsic attributes were factors that influence consumers’ purchase intention toward food products in Malaysia. In this study, rice quality characteristics such as grain shape, size, color, cleanliness, softness, and aroma can be categorized as intrinsic elements, whereas extrinsic elements include price, labeling, packaging, and branding. The intrinsic quality elements could also apply to rice-based products such as rice noodles.

In Ahmad Zainuri et al. (2020), gamma irradiation (4, 6, and 8 KGy) on flat rice noodles was found to cause changes in physicochemical properties such as texture, color, and cooking quality. Increasing the gamma-ray dosage, decreased the pH value, lightness (dark to white), cooking yield (%) (weight of noodle after cooked/weight of uncooked noodle), and hardness of the flat rice noodle. However, this increases cooking loss (noodle particles leaked into the cooking water), breaking length and color changes. Parallel to this, it was found that 4 KGy of gamma-ray lowers microbial growth and 8 KGy helps extends up to ten (10) days of shelf life of flat rice noodles at room temperature. As a consequence, gamma rays not only promote growth and yield, respond to abiotic stress, and improve the biochemical properties and nutritional values, but it also improves a product’s industrial quality and potentially extend storage duration.

Semsang et al. (2018) employed low energy N-ion beams in addition to gamma rays to improve the industrial quality of rice. The introduction of a low-energy ion beam as a source of irradiation comes with characteristics such as low damage rate, high mutation rate, and broad mutation spectrum. Bearing this in mind, Semsang et al. (2018) conducted a study focused on seed quality losses that occur throughout the growing season, harvesting, and storage as the primary goals by using ion beam mutation in Thailand. The results showed that HyKOS21 (a secondary mutant of KDML 105) derived from the Thai jasmine rice variety, KDML 105 (as a wild type), and BKOS6 (primary mutant of KDML 10) have favorable traits such as short stature, photoperiod insensitivity, and high yield potential. Not just that, HyKOS21 lacks the expression of the LOX-1 and LOX-2 genes, indicating that it increases the life span of seeds. This is evidenced by the fact that the germination percentage of seeds stored in natural conditions can reach up to 90% on average after 18 months of storage, compared to less than 50% for wild type.