Physical Mutagenesis in Cereal Crops

Abstract

The use of physical mutagens to induce heritable genetic variation in crop plants dates back to the beginning of the twentieth century. While X-rays were the first to be used for mutation induction in plants, gamma-rays have been the most widely used physical mutagen. Currently gamma induced mutations represents 60% of the registered mutant varieties in the Mutant Variety Database of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture. Beside gamma and X-rays, other physical mutagens include neutrons, beta particles, alpha particles, protons and ion beam. This chapter introduces the technique of physical mutagenesis with emphasis on gamma-ray and X-ray irradiation of seeds in cereals in the context of inducing genetic variation for resistance to the parasitic weed, Striga. Easy to follow step-by-step protocols are explained including sample preparation, treatment application and post-treatment handling of irradiated seeds. Data collection and graphic illustration are presented to estimate the optimum dose for bulk treatment to determine the radio-sensitivity of cereal crops. The last section briefly explains the development and handling of mutant populations by way of introduction to the rest of this book on mutation breeding in cereals for resistance to Striga.

Introduction

Mutations are sudden heritable changes in the genome, either spontaneous or induced, that enhance genetic diversity allowing evolution and adaptation of organisms to changing environments. The main objective of mutation breeding is to improve well-adapted farmer-preferred varieties through correcting a specific limitation related to productivity, resistance to biotic or abiotic stresses, or aspects of crop quality. Spontaneously occurring mutations are the main drivers of evolution in crop plants. Selection for natural variants with desirable phenotypic impacts on crop quality is the essence of plant breeding. The sum of these mutations accumulated through eons of evolution in the gene pool constitute the genetic diversity of crop species and their wild relatives. The broader this diversity, the greater potential gains in crop adaptability and human guided improvements. Induced mutation has expanded the genetic diversity of crop species and contributed to the development of new and improved crop varieties. Much of the early work in plant mutagenesis was described using X-ray irradiation in maize and barley, and was pioneered by Stadler (1928a, b, 1930). Physical mutagens include gamma and X-rays, neutrons, beta particles, alpha particles, protons and ion beam. Gamma-ray irradiators are the most widely used for the irradiation of plant materials for varietal improvement. The Plant Mutant Variety Database (http://mvd.iaea.org/) of the Joint FAO/IAEA Centre of Nuclear Applications in Food and Agriculture currently holds the records of over 3300 released mutant varieties in a wide range of crop plants of which more than 80% involve the use of ionizing radiation, especially gamma-rays. Tissue penetration depends on the nature of the radiation and this has consequence on the induction of mutations in the DNA. Ionizing electromagnetic radiation such as gamma and X-rays have relatively less biological effectiveness than ionizing atomic particle radiation such as neutrons but have more biological effectiveness than irradiation sources such as UV-light. Relative to X-rays, gamma-rays with their shorter wavelength penetrate tissue deeper. Hard X-rays have deeper tissue penetration than soft X-rays due to their shorter wave length and are hence preferred for use in mutation induction. Changes in DNA caused by gamma-rays include single or multiple nucleotide changes, small insertions and deletions (Yang et al. 2019), larger structural variations due to double-strand breaks and activation of transposable elements (Nielen et al. 2018). Generally, to induce mutations in plants, the planting materials such as seeds and vegetative propagules are exposed to the mutagenic agent. The choice for the appropriate dosage of radiation to be applied depends on the specific plant material, its genetics, and its physiology. By way of introduction, this chapter describes the process of physical mutagenesis with emphasis on gamma and X-ray irradiation, the most widely used mutagens, especially in developing countries. We describe in this introductory chapter all aspects of mutagenesis including the preparation of the seed material, dose optimization, irradiation treatment, post-treatment and handling of the mutant population.

Protocols

Dose optimization. Prior to bulk irradiation, the optimal dose must be determined. The optimum dose of a mutagen is the dose that achieves the optimum mutation frequency with minimal unintended effects. This is usually taken arbitrarily as the dose that causes 50% reduction of vegetative growth of the treated material as compared to the control (GRD₅₀), or the dose that kills 50% of the treated seeds (LD₅₀). In the case of physical mutagenic sources, the optimal dose is determined through the conduct of a radio-sensitivity test (RST), which is a relative measure indicative of the extent of recognizable effects of radiation exposure on the irradiated material. It helps to determine the optimum dose of irradiation required for a specific plant material for a specific outcome.

Dose uniformity is highly critical for the mutation induction process, both for conducting the RSTs to estimate the optimum dose and for effective bulk treatment. For RSTs, small samples are treated with a wide range of doses and the effect is measured in the form of a reduction in germination or growth as compared to a control (untreated sample). The parameters measured in the RSTs are used to estimate the lethal doses for 50 and 30% reduction of the germination (LD₅₀, LD₃₀) and growth (GR₅₀, GR₃₀) relative to the control. These estimated values enable the determination of the optimum dose(s) for the bulk mutagenesis treatment. It is very important in RSTs to distinguish effects resulting from the irradiation from differences due to variations in the quality of the seed source such as germinability determined from the control.

Dose uniformity test and safety considerations. Dose uniformity in width and depth of the sample is also taken into consideration for dose optimization. The desired irradiation dose may differ from the actual dose received by the irradiated samples, the latter being defined as the absorbed dose. It is important to accurately determine the absorbed dose after irradiation, especially in X-ray irradiation where the power supply might cause fluctuation in the production of the X-rays. A GAFchromic^® dosimetry system offers a convenient means to accurately quantify the absorbed dose through the use of a radio-chromic film which changes colour depending on the irradiation doses. One or more films placed at different positions near the sample(s) will allow assessment of the absorbed dose and also the dose uniformity. The GAFchromic^® films can be read by a film reader to determine the actual dose applied at the sample level and the uniformity of the treatment across the sample material after each exposure (Fig. 1).

Fig. 1

Films used for the GAFchromic^® dosimetry measurement after physical irradiation showing changes in colour from light green to dark with the dose rates, from 0, 100, 200, 300, 400, 500 Gy

Physical irradiation carries the potential risk of exposure of the operator to ionizing radiation which is mutagenic and carcinogenic. The operation should be handled by trained, authorized personnel in a specialized facility. There is no expected harm from handling of the irradiated seeds and the samples can be handled immediately after the treatment is completed. The treated seeds may be planted immediately or stored in appropriate conditions until the planting time.

Pre-treatment preparation of seed samples. A source of pure and viable seed is critical for a successful mutation breeding programme, once the target variety/genotype has been selected. This may sometimes require seed multiplication. Fresh breeders or foundation seed is preferably used for mutagenesis. Seeds should be multiplied in sufficient isolation to prevent out-crossing and mixing with other seed sources of the same species. Harvested fresh seeds should be separated from chaff, broken and shriveled seeds and disinfected to eliminate microbial contamination if needed.

Seed viability test. Seed viability is tested before proceeding to the irradiation treatment. This can be done by placing a small sample (10–20 seeds) on a moist filter paper in a petri-dish or other appropriate germination test media. For each specific plant species under experimentation, it is important to follow the recommended conditions for seed germination including breaking of dormancy as needed, seed moisture content, imbibition and incubation temperature/light and duration. Germination is scored after 5–7 days (for small grain cereals), or as appropriate, on the germination media. An example for germinating sorghum seeds is given in Chapter “An Agar-Based Method for Determining Mechanisms of Striga Resistance in Sorghum” and for rice in Chapter “Histological Analysis of Striga Infected Plants”. It is recommended to proceed to irradiation treatment only with seeds having a germination percent above 90% (Fig. 2).

Fig. 2

Representative seed viability test of two sorghum cultivars. Germination percentage after 7 days was > 95%

Preparation of seed samples. Once seed viability is determined to be above 90%, the seed can be prepared for RSTs. Determine the number of seeds available for use in RST while ensuring that sufficient amount of seed is available for the bulk irradiation. Ideally 15–20 seeds are packed separately in paper envelopes for each dose treatment for RST (Fig. 3). The number of dose treatments ranges between six and ten depending on the space available for planting and on the range of the dose treatments to be applied. Note that the amount of seeds per treatment and the number of treatments will vary with the crop and the growing media (field, pots, trays, petri-dishes, etc.). At the Plant Breeding and Genetics Laboratory (PBGL), we routinely apply six to seven treatments including an untreated (non-irradiated) sample (control). Treatments are usually replicated two to three times depending on the availability of seeds and planting space.

Fig. 3

Photos from left to right, seed counting and packing in paper bags, labelling the bags and assembling bags of the same treatment in one bigger bag labelled with the dose treatment; placing the bags in a desiccator with 60% glycerol for 3–7 days for moisture equilibration to 12–14%

Seeds are labelled for each treatment with the name of the cultivar/genotype, replication number, dose amount, source of irradiation and date of packing (Fig. 3). Seed bags of the same dose treatment are grouped and packed together with the seeds well distributed at the bottom and labelled with the dose and replication. An untreated bag (control) with the same number of seeds is held under the same conditions as the treated ones.

The packed seeds are then placed in a vacuum desiccator with 60% glycerol for moisture equilibration (Fig. 3). The seeds are retained in the desiccator for three to seven days. This equilibrates the seed moisture content of cereals to approximately 12–14%, which is the ideal moisture content for efficient induction of mutation. Water mitigates irradiation through both absorbing radiation and forming free radicals which are directly involved in certain DNA modifications (Spencer-Lopes et al. 2018). Note that critical seed moisture content for mutation induction may vary among plant species. Seed moisture content may be adjusted based on a preliminary test for moisture equilibration in different glycerol concentrations and incubation periods. In addition to the gravimetric method for seed moisture determination using standard oven drying, different types of moisture analyzers are commercially available with varying degrees of accuracy, and both destructive and non-destructive protocols.

Seed irradiation treatment. After moisture equilibration, seeds are removed from the desiccator and transferred to the irradiation facility. Details and description of various irradiation sources and machines used at the PBGL are well described in the third edition of the Manual on Mutation Breeding (Spencer-Lopes et al. 2018). Seed treatment for the widely used gamma-ray and X-ray machines specifically designed for mutagenesis are briefly presented here.

Gamma irradiation. The facility is strictly handled by designated technicians. The operator calculates the time needed for exposure by the assigned treatments based on the current dose rate of the source cell. At PBGL, the ⁶⁰Cobalt cell has a current dose range of 130 Gy per minute. This changes over time as the half-life of ⁶⁰Co is 5.3 years. Gamma-rays are continually emitted by decay of the cobalt isotope to nickel but these are contained within the holding chamber by the radiation absorbing material composing its walls. The operator switches on the machine that lowers the sample compartment into the source chamber, sets the exposure time and places the samples one at a time in the sample compartment. Exposure is relatively rapid and involves remote mechanical lowering of the sample compartment into the ⁶⁰Co source chamber. The irradiation time is calculated by the following equation: Time [min] = (Dose [Gy])/(Dose rate [Gy/min]). Exposure times are typically less than a few minutes. Once the treatment is completed the operator delivers the samples to the requestor.

X-ray irradiation. Like gamma-rays, X-rays are non-particulate electromagnetic radiation emitted as photons but with longer wavelengths than gamma-rays, in the range of 0.01–1 nm. They most commonly cause mutation through breaking DNA strands which are then faultily repaired by cells resulting in genomic deletions (Spencer-Lopes et al. 2018). Because they are much less energetic than gamma-rays, X-rays deliver a much lower dose of radiation, on the order of 2–10 Gy/min. X-ray sources are from special tubes designed to emit radiation upon electrical input. X-ray tubes therefore offer an advantage over gamma-ray generators that rely on certain decaying isotopes in that they can be turned on and off at will through an electrical current. Exposure to reach optimum dose, however, takes much more time than that for gamma-rays. X-ray tubes are also prone to overheating and therefore rely on cooling systems during the long exposure times (up to one hour) required for mutagenic seed irradiation.

The machinery housing the X-ray tube varies, with most designed for medical radiography. X-ray machines therefore most commonly use a movable X-ray tube around a stationary target. Some of these can be adapted to irradiate seeds but require extensive testing to position seeds properly and adjust exposure times to achieve optimal dosage. Other machines, like the RS2400^® (RAD Source Technologies Inc., USA) held at the PBGL, are specially designed for mutagenesis and place the target in rotating canisters orbiting a stationary X-ray tube. Seed packets are placed in these canisters, packed in inert parboiled rice and exposed in a preset program at a particular voltage and current for a predetermined time (up to one hour) to achieve the optimum dose. Aluminum shields (0.5 nm) are often used to absorb “soft” X-rays (0.1–1 nm) emitted by the X-ray tube so that only the more mutagenic “hard” X-rays (0.01–0.1 nm) reach the seeds. As with gamma irradiation, seeds are safe to handle after removal from the X-ray machine. The irradiated samples (now called M₁ seeds), together with the untreated control, are transferred to the field, greenhouse or laboratory for planting.

Post-treatment handling of irradiated seeds. The irradiated seeds should be sown as soon as possible after irradiation to minimize post-irradiation damage. If needed, the treated seeds can be stored for a short period of time. For example, in the case of cereals, the seeds may be stored at room temperature for a maximum of four weeks. In the case of longer storage, vacuum packing is used, and storage should be done in the dark and under low-temperature conditions (2–5 °C). These conditions minimize metabolic activity and cellular degradation. Prolonged storage of seed materials after radiation treatment may confound mutagenic effects with ordinary aging.

For planting, the irradiated seeds are transferred to the site of sowing which can be a greenhouse in trays, pots or in any suitable containers, or a laboratory in petri dishes or sandwich blotters, or directly in a field. The choice among these planting media depends on available resources, type and number of treated seeds and mutagenesis goals. In the case of greenhouse planting, the appropriate soil mixture is prepared to ensure optimum drainage, maintain sufficient moisture to the germinating seeds and avoid water logging, e.g., by drilling small holes in the bottom of the plastic trays.

Soil preparation. At the PBGL, local top soil is mixed with commercial sand and peat (Fig. 4) in a 60:25:20 kg ratio. The soil mixture is then distributed and levelled in the plastic sowing trays. An 11.5 × 36.5 × 56.5 cm tray is ideal for a RST involving six doses and 20 seeds per dose in the case of small-grain cereals or similar sized seed.

Fig. 4

Mixing soil components and making a seed bed for radio-sensitivity testing

Assessment of germination and growth rate. Seeds are sown on a well-levelled soil surface after watering the soil slightly to make it uniformly moist. Appropriate multiple-line dibbers are used to ensure uniformity in sowing depth and spacing in order to distribute seeds evenly and to separate samples by dose treatment (usually six furrows). Treatment labels are distributed along the furrows, starting with the control, then incrementally from the lowest to the highest dose treatment, which facilitates visual observation. Envelopes containing the three or two replicates of irradiated seeds are distributed, starting with replicate one, preferably in separate trays. They can be combined if using large trays (Fig. 5). The seeds are covered with a thin layer of dry soil and gently showered with enough water to ensure uniform germination. It is important that watering is optimal. Avoid over-watering or drying to ensure healthy seedling establishment and growth. The plant species and the growing conditions will determine the frequency of irrigation.

Fig. 5

Plastic trays for assessing radiation effects on maize seeds. a Planting furrows for irradiated maize seeds in the order of increasing dose from left to right (0 = control, 75, 150, 300, 450 and 600 Gy). b Even sowing of M₁ seeds before covering with dry soil and then watering

Germination data is collected starting at about 5–7 days after sowing in the case of cereals. Plant height is recorded after two weeks or otherwise, as appropriate, depending on the plant species. It is critical to avoid delays in recording growth-related data as some plant species may recover with time from the damage caused by irradiation.

Data collected on germination or growth rate, e.g., plant height or root length, is used for estimation of the optimum dose. This optimum dose is based on calculations on seedling survival that produce 50 or 30% (LD₅₀, LD₃₀) lethality, or seedling growth reduction based on shoot and/or root growth reduced by 50 or 30% (GR₅₀, GR₃₀) relative to the control (Fig. 6). The radio-sensitivity data based on both seed germination and seedling characteristics/growth are assumed to be highly correlated with the survival rate and fertility of the M₁ population.

Fig. 6

Representative sets of photographs from radio-sensitivity testing in sorghum (a and b) and rice (c and d) seedlings two weeks from sowing of the gamma-ray irradiated M₁. Photos a and c are top views of single trays illustrating the trend in growth reduction and germination with increasing dose rate. Photos b and d are single representative seedlings per dose treatment showing the effects on growth after irradiation; from left to right: 0 (control), 75, 150, 300, 450 and 600 Gy

Radio-sensitivity test results following irradiation. Seedling growth or survival parameters measured are used to determine the LD₅₀ or GR₅₀ values for a few different seed crops. Table 1 shows data for plant height collected from three replications of gamma irradiation (⁶⁰Cobat, dose rate 140 Gy/min) of sorghum with doses 0, 75, 150, 300, 450 and 600 Gy. The values in Table 1 are plotted, either as percentage reduction in height or as percentage of the control, against the radiation doses applied.

$$\begin{gathered} \% \;{\text{mean}} = \left[ {\left( {{\text{mean}}\;{\text{of}}\;{\text{treated}}/{\text{mean}}\;{\text{of}}\;{\text{control}}} \right) \times {1}00\% } \right] \hfill \\ \% \;{\text{reduction}} = \left[ {( {{\text{control}}-{\text{treated}}}/{\text{control}}} \right) \times {1}00\% ] \hfill \\ \end{gathered}$$

Table 1 Example of mean sorghum seedling height expressed as a percentage of the control and percentage reduction from the control after gamma irradiation of 20 seeds for each dose replicated three times

The values obtained for the two equations generates two graphs that are symmetrical and opposite in orientation (Fig. 7).

Fig. 7

Plotting of the data in Table 1 to show the effect of irradiation on sorghum seedling height expressed as percentage of non-irradiated (control) and percentage reduction in mean height. Red dots point to dose of GR30 (180 Gy) while green dots point to dose of GR50 (322 Gy)

As the main outcome of the RST, the LD₅₀, GR₅₀, LD₃₀ and GR₃₀ can either be scored directly following the graph or by substituting in the slope equation of the linear trend line (Fig. 8). The doses used for the bulk treatment are usually estimated as LD₅₀ or GR₅₀ plus and minus 20%. Therefore, up to three dosages estimated from the RST can be applied for bulk irradiation, depending on the objective of the mutation breeding programme. In the example shown in Fig. 8 the dose to be applied for the bulk seed can be about 256, 322 and 384 and can be simplified to 260, 320 and 380 Gy if the choice is to apply around the GR50%. However, the same rule applies if the breeder chose lower doses around the GR30.

Fig. 8

Representative examples of photos comparing gamma-ray on two different sorghum varieties three weeks after sowing (top photos) and comparative growth of gamma versus X-ray irradiated rice seedlings (cv. Ashfard), 14 days after sowing (bottom photos)

Relative biological effect of gamma-ray versus X-ray irradiation. Data was compiled from RST experiments on representative varieties of sorghum, rice and maize treated with in-house gamma-ray (⁶⁰Cobalt) and X-ray (RS2400) sources. This data is used to calculate and compare the relative biological effect (RBE) of gamma versus X-ray irradiation (Table 2). The data indicate that there are no significant differences between the two types of irradiation and the RBE ranged between 1.0 and 1.2. Generally, there is slightly greater damage with X-rays, which is most likely due to the different effects of each mutagen on DNA, with X-rays more likely to cause breakages leading to deletions (Fig. 8). As shown in Table 2, there is a very close trend in the RBE and the comparative reduction in germination and growth rates. Different varieties of the same crop may have different sensitivity to irradiation and therefore it is recommended to run a RST before bulk treatment for each variety (Fig. 8).

Table 2 Examples of GR30 and GR50 of some seed crops using a RS2400 X-ray irradiator and a gamma cell irradiator, and the relative biological effect (RBE) of X-ray irradiation over gamma-ray irradiation

Bulk treatment and handling of mutant populations. Initially the mutation breeding project starts with true-breeding highly homozygous seeds for treatment (M0). About 4000–6000 homogenous inbred seeds are prepared, cleaned and packed for irradiation. The proper amount of the M0 seeds depends on the dose, targeted trait and screening protocol. In the first mutant generation (M1), treated seeds are usually grown with spacing to maintain 2–3 tillers. They are protected from outcrossing to ensure self-pollination and each plant is harvested separately. In the second mutant generation (M2), seeds from each M1 plant are planted head-to-row with screening applied, selecting individual mutant plants separately (for qualitative traits). These are planted next to controls (untreated seeds) to verify identity of the mutant and exclude contaminations. In the third mutant generation (M3), seeds of selected mutants are planted for further selection and verification and to propagate selected individuals carrying the mutation. Selection for quantitative trait by head-row may start at M3. Measures are taken to ensure purity of the mutant selections by controlled self- or sib-pollinations. Once the mutant is isolated, the material can be handled in one of the three routes illustrated in Fig. 9. The direct route is through repeated cycles of selfing through M4–M6 generations (Fig. 10) to fix major quantitative genes and eliminate plants with inferior agronomic traits. Seed multiplication occurs at the M6–M7 generations concurrent with preliminary evaluation trials to select the best lines carrying the trait with good agronomic performance. Selections can then be advanced to multi-location trials for the release and registration of the new mutant varieties (Mukhtar Ali Ghanim et al 2018).

Fig. 9

Scheme illustrating the major three options for handling of the identified mutant towards development of mutant varieties. One of the options is to proceed for a few cycles of selection with selfing. A second option involves backcrossing the mutant to the unmutagenized precursor or other elite lines. A third option is the genetic analysis route for gene discovery and marker development

Fig. 10

Schematic drawing showing steps for the direct selection of the targeted mutant trait in M2/M3 and consecutive selfing and selection to better plants with good agronomic characteristics plus the targeted trait and fix them in stable lines in the M4–M6 generations to be advanced for multi-location evaluation for official release and registration

The second route of mutant variety development is though backcrossing to the wildtype (unmutagenized) precursor or other elite lines and cultivars (BC1–BC5) (Fig. 9). Backcrossing can be to the wildtype parent to restore fitness from effects of random mutations in genomic regions outside those controlling the target trait. One or two backcrossing cycles might be sufficient to separate the induced desirable mutant trait from the undesirable background mutations. Improved lines resulting from mutagenesis can then proceed to registration as a new mutant variety.

The third route is to advance the isolated mutants to genetic analysis to understand inheritance of the induced trait and test for allelism if more than one mutant is isolated. Crossing is usually performed between the mutant and wildtype parent or an elite line with contrasting phenotype. Following the segregation in F2 and F3 families will enable inheritance studies. Bulked segregant analysis and comparative analysis of whole genome sequencing of the contrasting bulks can lead to discovery of the mutated genes and further development of molecular markers (see Sect. Conclusion Chapter “Striga as a Constraint to Cereal Production in Sub-Saharan Africa and the Role of Host Plant Resistance” for protocols). Molecular markers for the induced trait will feed subsequent backcrossing to transfer the trait to other varieties (Fig. 9).

There are several technologies which increase efficiency and accelerate the course of these three routes of handling mutant populations enabling fast delivery of the mutant varieties. These are described in Sect. Conclusion of this book.

Conclusion

Simple and easy-to-follow seed irradiation protocols have been developed at the PBGL and are presented here for mutation induction in seed crops with examples of treatment of the targeted cereal crops (sorghum, rice and maize) using both gamma and X-ray as the most widely used physical mutagens. The GR₃₀ and GR₅₀ values for some seed crops (Table 2) are also presented, based on the data collected from radio-sensitivity testing during protocols development. These data may be used as a guide for mutation induction of bulked seed samples. The results presented here may vary depending on the type and characteristics of the irradiator used and the actual experimental conditions. An overview guide is presented for development and handling of mutant populations. The chapter serves as an introduction to the main chapters of the book on screening and efficiency enhancing technologies for development of resistance to Striga in the major cereal crops.