Practice Suggestions for Mutation Breeding

Notes for a Laboratory Manual
Short-Cut-Notes (Quick reading before skipping the introduction)
This Manual is intended as a handy REFERENCE near the laboratory table and not as a textbook in a Class-room.
Therefore the main body, the practical methodology is written for each typical crop with many duplication amongclosely related plant species. For example, for mutagenic treatment of barley, some parts are common with the method
for rice.
General notes on mutagens, structure of a gene and its actions or functions etc are written in the INTRODUNTION part,so that for understanding of genetics of mutation breeding, reading of the introduction is strongly recommended.
However, if you have enough knowledge and want a quick reference, go to the crop species of your interest or to theone closely related to your material.
In mutant plant screening, reproductive manner of your material is important. Generally speaking, the induced mutant
genes are inactivated status, or in other words, they are recessive in nature, so that ONLY HOMOZYGOUS state can
express its mutant character, in seed propagated plants (Mendelian segregation in 3 normal to 1 mutant after self
pollination) and/or vegetatively propagated plants (knock down of dominant gene by somatic mutation in aheterozygotes). Even within seed propagated crops, for self compatible and self incompatible crops, the strategy
should be different. The seeds of barley, wheat and rye look very alike, but genetic ploidy and self compatibility arevery different among these. Barley is a simple diploid, wheat a hexaploid, and rye is self-incompatible.
The major methodologies of crop improvements may reside on cross breeding, where all the chromosomes are mixed
together, then separated into the progenies to be screened for better genotype, with occasional crossing over breaking
the linkage. Mutation Breeding or Transgenic Breeding (so called GM) can modify only a few numbers of genes,
mutation as an inactivation (subtraction), and transformation as an introduction of external gene(s) (addition). While
the former produced more than 2000 cultivars in the world (see IAEA database on internet1 and/or separate file by E.
AMANO2) and widely accepted by consumers. The latter, transgenic breeding, is experiencing difficult phase to be
accepted by the people. As a technology, transformation is a very important methodology of genetics to clarify thefunction of a gene. The difficulty might be due to the use of genes UNFAMILLIER for human being. Some efforts
are being made in Japan to use genes from materials which are familiar to human beings as food materials so thatallergic reaction may not occur for consumers. For the time being, it is still young technology expected to grow.
The basic difference between "mutation" and "transformation" may be in the action of the modified or added gene andits expression. If recessive phenotype or inactivation of a gene is wanted, induced mutation should be the first to be
consulted, but if dominant or actually working gene is wanted, transformation, or cross breeding, to introduce theexternal gene into the crop should be referred.
In some Asian regions, waxy or glutinousness of cereals are highly prized. The waxy character lack a straight chain
starch component, called amylose, by inactivation of a gene for amylose synthetic enzyme. Generally, there is only
one gene in a genome, so that a single mutation can induce waxy mutant gene in a diploid plant, e.g. rice or barley. It
is much easier to induce waxy mutant than transformation (introduction of anti-sense sequence or by gene splitting) or
cross breeding, in the latter, genetic background are completely upset by cross pollination. On the other way, that isreturn form wx to Wx status, it is almost impossible to retrieve normal amylose synthetic function by induced mutation.
If so wanted, only transformation with normal NON-WAXY gene or cross breeding can do the work.

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Introduction
In this section, general genetic information is described. You may skip and go to the crop-wise description if you
know the fundamentals of genetics.

Methods of crop improvement
1) Cross breeding
Mix chromosomes then select the best combination of genes
To create a completely new variety, CROSS BREEDING is the major and the most effective methodology. Mutation
or transformation may be good to improve only a few characters leaving most of the other characters specific to thevariety unchanged. On the other hand, cross breeding basically upset the entire genotype of the excellent traits of theparental varieties.
Therefore, if you want to improve only a few characters, most of the other characters remain like the parent, mutation ortransformation may give good results depending on the purpose, recessive trait or dominantly inherited trait. Mutation
may be good for the former and transformation for the latter. Of course, a single cross followed by repeatedback-cross may be used to introduce only a few characters from one parent, although it is laborious and takes long
period of time.
2) Mutation breeding
Subtraction from normal genotype
If recessively inherited character is wanted in otherwise excellent variety, mutation breeding may give good results, likesemi-dwarf lodging resistant rice. Reimei (in Japan) or Calrose 76 (in USA) showed good lodging registance in ricecultivation. Linola (in Australia) might be another excellent case of modification of fatty acid spectrum from drying
oil into edible oil by changing the major element from linolenic acid (18:3) to linoleic acid (18:2).
When a new cultivation regime, like hybrid rice, are considered, temperature or photoperiod depending male sterility,
double pistil, open-hull(but fertile) etc (please be noted that these are all recessive characters and will not expressed in
the F1 hybrids) may be useful, although they may not be appreciated under ordinary cultivation regime.
This method of selecting homozygous mutant plant in M2 may be applicable where self compatible seed propagation
can be expected, however, for crops like most of fruit trees (complicated genotype support the variety traits) like apple,
pear, or seedless crops, banana etc, or many ornamental flowers, e.g. Canna, orchids, Chrysanthemum etc theapplication needs some considerations.
In case of polyploidy crops, mutation work may not give simple and favorable mutant segregation due to the multiple
copies of the related genes. For example, the albino mutant which lost the ability to synthesize chlorophyll are very
good indication of the induced mutation in most of diploid species, but it is very seldom in the mutagenized hexaploid
bread wheat.
3) Transformation (Gene introduction)
Addition of external gene/DNA
In the same way as the mutation work, transformation may give good results in adding the plant by only a few
characters leaving most of other genotype remain unchanged. The introduced external gene should have NORMAL
function in the new environment, both the function of the gene and the new metabolic environment.
In academic studies of a gene, transformation is a fundamental technology to clarify and to prove the gene function.
However, some consumers concern the safety of the output in relation to allergic reaction of human body, which may
occur by the gene products the human being had never exposed to. Some people give their effort on the introductionof rather FAMILLIER gene(s) to the human being so that allergic reaction may not occur by eating the new transgenic
varieties.
Besides the academic values of the technology, there may be some important ideas in this method; 1) to overcome
ploidy difficulty mentioned in mutation work, and 2) introduction of ANTISENSE sequence to overcome multi-copy
gene family of the species. Presently it is still growing methodology, but it will be a powerful tool in future crop
improvement.
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Gene structure

Intron and exon

In describing the activities of a gene, in terms of mutation, its structure is the fundamental. Gene had been considered
to locate on chromosome like a rosary or string of pearls. But recent DNA analyses strongly suggest a structure ofsentences written in only four letters, A, T, G and C and differentiated into exons and introns within a gene. Each gene
generally composed of 2000 to 3000 or more base pairs (or letters). Exons carry genetically meaningful information.
Introns are connecting base sequences which will be finally removed from mRNA by splicing. The spliced exons willbe translated into proteins by function of tRNAs. The protein will get matured to have 3-dimentional structure to be afunctioning enzyme molecule. Following figure3 is an example of an enzyme protein.

This scheme model suggest that if all the letters, or at least spliced exons are in normal condition, it is a normal orfunctioning gene, but if there is a modification in the letter, the gene may lose the function or in other words, the gene
will be get into inactivated condition, the mutated gene. Some ionizing radiations, especially high LET radiations, e.g.
reactor generated neutrons or accelerated ion beams, may introduce deletion of a few bases. Chemical mutagens, e.g.
EMS or dES may introduce substitution of base (or letter). Recent studies presented an explanation on the callus
culture mutation, as activation of retro-transposon, which might be dormant or inactive in ordinary growth or
reproduction conditions. The resulting mutant phenotype may be used as stable genetic resources.
The following figures illustrate example of maize Waxy gene reported in a journal, MGG in 1986 by R. B. Kloesgen et
al4.

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Practice Suggestions for Mutation Breeding

Mutagens
Physical agent
Here in this laboratory manual, mainly X-rays and Gamma-rays will be considered. Ion beams and neutrons withvarious energy may be also utilized in specific purposes, but penetration or dosimetry may be beyond the topics
described here, although they are very important elements in irradiation work.
With X-rays, penetration through the materials depends on the high voltage used to operate. In ordinary enveloped
seeds or cuttings, the thickness should be less than a few centimeters, when 100kV to 250kV is used. The sample
thickness may also be important not only for penetration but also in terms of the distance from the radiation source.
The X-ray tube is active only the high voltage is turned ON.
Gamma-rays from 137-Cs (660keV) or 60-Co (1.17MeV+1.33MeV) may penetrate even glass tissue culture flasks.
In case of these Radio Isotope irradiation sources, the gamma-rays are continuously radiated from the source, so that thesafety precautions should be the first priority for the operating personnel.
For botanical staff members, generally dosimetry may be left to the operator/physics staff. Basically, so called
secondary electron equilibrium, is important. The necessary condition is easily achieved by putting 3 to 5 mm acryl
resin-plate or other comparable plastic plate on the irradiated sample.
The irradiation dose should be tested before formal irradiation. In this preliminary exposure experiments, number ofsample per dose-class may not be large, some 20 seeds per dose for example, but the dose range should be extended
from very weak to almost kill all, in 10 classes or so. About 20% reduction dose for the growth (seedling height or114

Practice Suggestions for Mutation Breeding

reduction of the grown (fresh/dry) weight) may be suggested to use (see the example in RICE section). For radiation
biology work, LD50 (50% lethal dose) is a very clear index, but for mutant induction, it is too high to give good
growth/results.
Chemical mutagens
Gamma-rays from 60-Co will be reduced by the HALF-LIFE of five years, which may not be the concerned factor ofmost of biological staff members (except executive and dosimetry staff). However, with chemical mutagens evenbiological staff must handle by themselves in their laboratory. The same half life problem of the agent arises for thechemical mutagen. Compared to 60-Co, the half life of effective elements of most of chemical mutagens in the water
solution is very short, matter of a few hours to several hours. Mutagenic agent, physical or chemical, must be carefully
handled because carcinogenicity is suggested almost all the mutagens. In case of chemical agents, hydrolysis, alkalior acid-disintegration are possible before discarding the wastes, solution or wiped papers etc. The treated materials,
seeds etc, should also be carefully handled.
Ethyl methanesulfonate may be suggested as most effective and easy to handle in storage (refrigeration), use (1% or lesswater solution) and waste disposal after alkali-disintegration. Once there were discussions on the chromosome
breakage ability of EMS, but as far as pure water (distilled and deionized water, and not with any buffering agent) isused, it induces good point mutations including leaky or mis-sense mutations5. Refrigeration (not frozen) storage issuggested for EMS. The cool bial of EMS should not be opened, as the dew drops may hydrolyze and destroy theagent. So, EMS should be stored in a refrigerator in an air-tight container with ample amount of Silica-gel drying
agent (refer to PictureWord file “14EMS”). Take out from refrigerator a few hours before the use, to have temperaturebalancing with the room-temperature. Then, open the bottle. Never try mouth pipetting. If good pipetter is not at
hand, a (plastic) syringe connected to glass pipette by short tubing can a good substitute to measure the agent.
Biological treatment
Remote hybrids often produce aberrant phenotypes in the progeny. Aneuploidy plants（chromosome numbers
deviated from euploidy (diploid, tetrapoid etc.)) will give chromosome aberrant plant which may show aberrant
phenotype. This special phenomena are often used in developing aneuploidy system e.g. in wheat.
Apart from such very special cases, aberrant character which appeared after callus induction, culture and plantregeneration experiment (including anther cultures) are often reported as somaclonal variations. Recent studies
explain the phenomena as activation of otherwise stable RETRO-TRANSPOSON (Hirochika etc6, 7, 8 ). When thecells got special kind of stress like dedifferentiation, e.g. calls induction, the retro-transposon is activated and produce
copies of it as messenger RNA, which is then used in synthesizing DNA sequence(s) ("retro" way!) that will act liketransposon. The period of callus culture increases the number of DNA copies.

In this Figure, Control is ordinary japonica rice
variety, having a stable single band, suggestinghighly stable status under ordinary condition. After
three months of callus culture multiplicated DNA(retro-transposons) had increased significantly. Ninemonths and 16 months gave too many DNAs whichmight be inserted at too many sites inducing too
many gene inactivations or mutations. For further
information, see attached report “Manila2.DOC”
including references by HIROCHIKA.

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For mutagenic treatment, only a few months may be suggested. Too long callus culture may produce only albino
regenerated plants perhaps due to too much transposons produced. Further studies suggest that the mutant thus
induced, and the causing retro-transposons are stable under ordinary field agriculture. One possible difference fromthe radiation induced mutation, in which complete randomness can be expected, may be in the preference or distortionby the biological system. For example, in some mutants, e.g. rolled leaf in rice, all the members of the family often
show the same mutant character. If it were dominant mutation, there must be some normal plants segregated to 3
dominant mutants to 1 normal. The reason of such abnormal segregation still remains to be studied. Otherwise,mutants thus developed and detected will be used as stable mutant line. Please be noted that other “transposon”
mutants may revert to original non-mutant phenotype relatively frequently.
The regenerated plant may be in R0 generation as the researchers in this field call it. R0 is comparable to M1 ofirradiated or chemically treated materials, that is heterozygous generation regarding the mutated gene(s).
It should be noted that “generation designation is different in M (mutagenized) and R (regenerated).
Mutation is caused by insertion of the (retro-) transposon into existing gene(s). Perhaps complete inactivation of theinserted gene may be expected by insertion of the large sequence of transposon. Screening of mutant should be madein the R1, i.e. M2 comparable generation.

Dominant / recessive
This is to repeat the description of when and where the technique should be applied to improve crop plants.
Cross breeding: Effective for all STABLE genes
This is a fundamental methodology to improve crop plants. Recent tissue culture techniques may be useful in remotehybrid making through embryo rescuing. In some cases, cell fusion may be used to produce somatic hybrid throughprotoplast. The basic pollination hybrid is to make chromosome mixture, and then screening of favorable genotype(phenotype) is the fundamental procedure to create new varieties.
Mutation: Effective only for recessive characters
When the gene inactivation or its phenotype (recessively inherited character) is required, mutation induction will be asimple, low cost and easy way to get good results. However, where gene exist in multicopies, like polyploid plants,
other methodology must be consulted also.
Transformation: Effective only for dominant genes
When fully active gene or a new and active gene is wanted this transformation method may give good results. Some
consumers concern the safety of the newly introduced materials (protein as a gene product), but the technique itself isvery powerful tools in academic research work on the gene. Some people are trying to introduce genes wellaccustomed to us, human being, for long time in certain food materials. For example, only moving one gene from one
bean species to other bean species may not give serious allergic reaction for human, but it was effective to reduce insect
damage in azuki bean.
Anti-sense DNA sequence introduction may have another very interesting power, in some expression SUPERDOMINANT inheritance expressing the RECESSIVE character. This may be a very good study item for the future
molecular work.

Extra-information
Size of chimerism (mutant sector)
Besides the gene genetics, structure of plant tissues and reproductive characteristics needs special attention in mutant
induction and screening. When a plant/seed is mutagenized, e.g. in rice seed/embryo, perhaps mutation will occur
116Practice Suggestions for Mutation Breeding

singular in a nucleus of a cell, in other words, the mutant gene will be hetero-zygously present. After self pollinationof such heterozygotes, Mendelian segregation will produce homozygous recessive mutant plants in about 25% of theprogeny family. However, suppose a cucumber or maize, seeds of which had been treated by effective mutagen.
The embryo in the treated seed had already in a multi-cellularcondition. One cell may have a mutated nucleus, but the next or theneighboring cells may not. Such heterogeneous cell population
form chimerism in the M1 plant. Then, it may highly be possible
the male pollen of a maize formed on the top of the plant mightcome from different chimeric mutant tissues from the lower female
flower/cobs of the same plant. If such occurs, self pollination
within the same plant is not really the self pollination in the case ofrice, i.e. within the heterozygous tissues, to have Mendelian
segregation. It may sound strange, but it may be visualized if you use a leaf color heterozygotes as the treatment
material, e.g. Yg2/yg2 in maize. If you have many M1 plants you may see chimerism like the Photos (maize maletassel albino mutant tissue) shown here.
In vegetatively propagated plants, visualization may be difficult, but the genetics and idea are the same. In these cases,
non-chimeric or so-called solid mutant tissue/sport may be available after a few vegetative generations, in M1V3,
M1V4 or later
Brushing Up a Mutant
Induced mutant line very often contains "unfavorable simultaneous mutant gene(s)", which very often degrades themutant quality to be a practical variety. Too hasten evaluation of the mutant may also evaluating the MINUS factorsof these extra mutant genes. Unfavorable simultaneously induced mutant gene(s) may be cleaned up by backcrossing
and re-segregation of the intended mutant phenotype again. Very often, a single backcross to the original line may begood enough to recover the original seed fertility, for example. Backcross to the original line may not mean crossbreeding, as almost all the background genotype is maintained as the original line.
In case of vegetatively propagated materials, cleaning by backcross can not be applied in most of the cases. If so, try to
induce mutants as many as possible, then screen a clone with the best performance. One basic suggestion for the bothtypes of the crop, apply minimum of the dosage, physical, or chemical mutagens. It may require large M1 and M2
population. But please understand that the frequency of unfavorable mutant gene is also depend on the mutagen dose
applied, the higher the more.
Suppose about 10,000 genes are active in a plant, e.g. in rice, where only one Wx gene is in a genome andshowing about 1/1000 mutation frequency, if we select a wx mutant, the other genes may have the same
frequency in mutational change, that is 1/1000 x 10,000 = about 10 simultaneous mutant genes. Practicallyof the ten such mutant genes, perhaps only a few of them may be included in the selected mutant line. Thenthe performance test of the mutant may also include this extra mutant genes. If these genes were separately
located on different chromosomes, single back cross may be effective to exclude them. Sometimes, such
simultaneously induced second mutation give a bonus character, e.g. BYMV resistance in an early maturingmutant line found in Japan, or the case of salt tolerance of a malting barley ‘Golden Promise’ in England.
But such characters should be screened as the major purpose of the mutation breeding.

Where to search
In this manual, crop plants are grouped at first by the reproductive pattern, seed propagated or vegetatively propagated.
Further characterization will be described in each crop group or species.
Each item may have following components.
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Practice Suggestions for Mutation BreedingGroups of plants
For mutant induction and screening, reproductive pattern and chimerism in the treated material is very important. The
chimerism may be visualized by use of appropriate genotype material, but before such information is available, safe
strategies will be suggested in the practical description section. For example, a big head of Solghum may becomposed of a few chimeric sectors when a seed is treated. Before the chimeric development is visualized, it may besafe to say that the mutant sectors may be formed rather in radial pattern around the plant axis. In rice, the central axis
panicle may have chimeric mosaic structure, but most of the panicles developed later usually segregate 1 mutant and 3
normals. Such information will be obtained during the experimental procedure.
Although modification in accordance with the chimeric structure of the plant is needed, basic seed numbers in one unit
or panicle/ear/cob may be important in mutant screening. Following lists may suggest some of such groupings.
Each crop species should be separated further by polyploidy or other factors.
The number of seeds per panicle/cob/ear/fruit or putative chimeric unit is important in expecting the Mendelian
segregation.
Mutant selection may depend on the purposes of the breeding, sometimes surviving screening applied to pooled M2materials may be very effective, but especially for academic purposes where homozygotes is difficult to survive,
harvesting of heterozygotes are also important, then M1 panicle/spike/cob family system should be considered.
1.
Self pollinating crops
1-1.
Diploid
1-1-1 Very many seeds per unit more than 300
Millet, Solghum
1-1-2 Many seeds per unit
more than 100
Rice, tomato, sesame
1-1-3 Good seeds per unit
more than 50
Mung-bean,
1-1-4 Small number per unit
around10barley, soybean, peas
1-2.
Polyploid
macaroni wheat (4x), bread wheat (6x), triticale(rye-wheat : 6x or 8x)2.
Outcrossing crops
2-1.
Self-compatible
2-1-1 Very many seeds per unit more than 1000
Orchids
2-1-2 Very many seeds per unit more than 100 Cucumbers, maize, melons
2-1-2 many seeds per chimeric unit
more than 50 maize (in terms of chimerism)
2-2.
Self-imcompatible
2-2-1 Very many seeds per unit more than 1000
certain orchids
2-2-2 Small number of seeds per unit
Rye, buckwheat, radish,3.
Vegetatively propagated
3-1. Herbaceous : Chrysanthemum, Canna, some orchids
3-2. Arboreals : Rose, Fruit trees (incl. Oranges (partheno-carpy?))
To develop a new variety, cross breeding is effective, but with established varieties
Cutting, grafting etc are used not to disturb the variety genotype.
4. Seedless plants
Banana, (Lycoris radiata Herb. ), Seedless grape, Some oranges
Mutation or transformation may be the only method to modify genotype.
5. Polyploidy and other plants
Sugar cane,High chromosome number and vegetative reproduction, also allows aneuploidy.
Wheat
Very important cereal, but hexaploid species giving difficulty in mutation breeding.
Orange group
Seeds formed in fruit is very often developed from percenogenic maternal tissue so that cross118

Practice Suggestions for Mutation Breeding

breeding is not so effective method to improve.
6. Other organisms
Diploid/haploid generation and mushroom/hypha stages may need some extra strategies but
basic genetics will be applicable.
6-1. Mushrooms
6-2. Yeast, Molds and other fermentation organisms

Extra Suggestions
Visualization of mutant tissue
If you are going to work on your materials for more than a few years, keep
an appropriate color mutant and produce a heterozygote to study the
chimerism pattern of your material. In case of rice, viable yellow green(chlorina) mutant may be used. If heterozygotes are available in asegregating family, it may be used also. In case of self incompatible
buckwheat, seeds may be considered to have chlorophyll genes accumulated
as heterozygotes in most of them, as they very often give white or yellow
sectors after mutagen treatment like a picture shown here.
Somatic mutation sectors appear after mutagen treatment, according to its growth pattern.
In maize, mutant sectors become countable from leaf 3 after seed treatment. Generally,
mutated cell might not be disturbed its growth, giving very clear sector against normal
tissues and showing typical growth pattern of the tissues (stripes in cereal plants, andfan-shaped sector in buckwheat).
Visualization of chimera will be important in melon/cucumber group. After mutagen
treatment, usually tertiary vine developed after pruning twice, may be free of chimera, sothat pollination between male and female flowers on the same tertiary vine may givemutant segregation in the seeds thus obtained.

Selection method (Visualization of mutant segregation)
Take rice or barley as an example. The genotype is composed of two setof chromosomes, one from pollen parent and the other from maternal egg
cell. Each component has corresponding genes. Induced mutation will
occur only one of those corresponding pair. Almost all the induced
mutant genes are broken or inactivated (recessive) state. So, in M1
generation, somehow the normal gene may show non-mutant character.
In order to find and screen the mutant, homozygous plant must be
produced. This is the fundamental Mendelian segregation.
M2 planting pattern
In most of seed propagated crops may segregate in M2 generation at the rate of one mutant to three normally looking
plants as illustrated above. This suggests that if ten M2 plants out of one mutational unit (panicle or ear in cereals) two
mutants may appear among the sister plants. About ten to 15 plants may be suggested as the segregating family size.
In the Institute of Radiation Breeding in Ohmiya Town in Japan, they used 15 plants for a M1 panicle family for rice.
In Fukui, after integrating special machine transplanter, 14 plants for a panicle family were set (see attached pictures).
Duplicate or triplicate appearance of mutant phenotype assures us that they are the induced mutants.
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Practice Suggestions for Mutation Breeding

After experiencing the segregation of mutants, each researcher may develop and proceed his/her own system dependingon the purpose of the experiment. If lethal mutation like albino or disease susceptible mutant are the target of theacademic studies, this panicle/ear family planting is a necessity to keep the viable heterozygotes in order to maintain the
mutant gene.
Because this panicle/ear family planting needs both field area and sowing/transplanting labor, if the mutagenesis isintegrated in the ordinary plant breeding program, one seed (or a few seeds) from one panicle/ear may be adopted,
because, perhaps one mutant plant may be good enough to start up the improvement.
Survival Screening
In an extreme case, mass sowing or planting and selection of a few survivors may be effective e.g. cold tolerance,
disease resistance, insect and other pest resistances. Planning of the screening system of only a few plants out of
100,000 M2 plants should be considered.
In microbial (bacterial) genetics, this is an ordinary method of screening, but in higher plants, even a breeder may feelsome guilty to discard almost all the plants.
Stress application
Not only in the survival screening, but in some screening a light stress may be used in many phases of screening, e.g.
day length or lodging. Selection by yield and other selection pressures may be another human factor of stress placing.
In case of survival screening, it may be natural to place all the M2 seeds under the stress pressure, but in case of lighter
pressure, it may be advised that repeated screening with the light pressure may concentrate the mutant gene(s).
Basic studies
The purpose of plant breeding is to obtain and disseminate favorable varieties to the farmer, hobbyist or other users.
For this purpose, any resources may be used, and even contamination may give good results. In some cases, thebreeding may be described as a kind of fine art, deeply depending on the breeder’s personal sense. However, tobecome a reproducible technology, the method should be scientifically sound. The difference between science andfine art may be in that, in science there may be no room for human being, but in the fine art, the results is the creative
work of a human being. Gregol Mendel made clear the rule of the segregation in the nature. But he did not set theratio, but the NATURE set the ratio 3 to 1. In other words, even without Mendel, dominant and recessive will
segregate in 3 to 1 ratio. (Actually the segregation rule was re-discovered 35 years later by three scholars in 1900.)
It may be better not to mix the science with practical application of science, but scientific back ground may be a goodguaranty of reproducibility of the next good variety.

Additional Information (See the following websites)
Radiation sensitivity (Amano's collection)
Excel file

http://www.fnca.jp/english/mb/mbm/pdf/1_radslst.pdf

http://www.fnca.jp/english/mb/mbm/pdf/2_radiation_sensitvty.xls

Mutant varieties in the World (based on IAEA publications)
http://www.fnca.jp/english/mb/mbm/pdf/3_mutant_varieties.pdf
Excel file http://www.fnca.jp/english/mb/mbm/pdf/4_mutant_varieties.xls
This list is the output from the Mutant Cultivar Database by E.A., based on the IAEA publications. Key-word search
was applied to analyze the data.
Please note that this is a personally written table and there may be problems in the plant materials (use of hybrid seedsetc.), or reaction of the crop plants (radiation against Zea mays (maize) etc.). Further more, in the USA, the use ofmutant in cross breeding are often “Not Reported to IAEA” so that some mutant cultivars are missing.
However, still some trends may be seen from this table.
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Structure of higher plants has been reported in 1980’s, to have exons (meaningful part) and introns in the gene, the locus
on DNA chain.

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Practice Suggestions for Mutation Breeding

The gene locus, or its genetic information, is written in the four letters A, T, G and C. In this picture, small letters(lower case letters) mean intron and capital letters indicate the exons.

Information written on DNA is transcribed to messenger RNA, then introns are cut off and exons are spliced together tobe translated to proteins or enzymes by the function of transfer RNA which carry amino-acids to the protein synthesis
sites. Proteins synthesized may look like the right colored figure. The left simplified figure may suggest the pockets
for enzymatic function.
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Practice Suggestions for Mutation Breeding

Amino-acids are selected and carried by t-RNA by the list of
triplet codes. The second and/or third letters may have lessmeaning, called redundancy.

The gene disturbed or inactivated by mutation is recessively
inherited or expressed, as the normal gene which still has
function will be appeared as the dominant gene. In the table,
normal dominant gene is shown as “A” and the recessive
mutant gene as “a”.

This is shown in the garden of Mendel Museum in Brunn, Czech
Republic.
This might be only for decorative explanation. The purple Pansy
at the top left is dominant parent and the right white as recessive.
Then F1 hybrid is purple, expressing the dominant gene. Afterself pollination, segregation of three purple and one white occur.
Heterozygotes segregate again in 3:1 ratio, but homozygotes at the
far left (dominant) and far right (recessive) stay as pure line. The
Display also shows up to F3 Generation. The lady at the right is
an IAEA trainee from Portugal.

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Practice Suggestions for Mutation Breeding

1. Rice1) How to prepare starting materials
The breeding method using induced mutant(s) will improve only a few characters, e.g. semi-dwarf (for lodging
resistance) or early flowering or waxy starch, leaving other characters unchanged at all. So, this method may be
applied best to a local varieties. Many kinds of mutant characters will be found during screening procedures.
Therefore, it may be necessary to have clear objectives of the expected improvement. Otherwise, many newly
induced characters may draw interests of the observer, finally giving too much work on unnecessary characters.
To prepare the starting population (materials), if possible, isolation by paper bagging etc may be strongly suggested.
Use of hybrid seeds in mutagenesis is not a good idea, unless specific purpose of chromosomal translocation isintended.
“Hybridization enlarges the possibility of mutation by enlarged number of target gene” is erroneous, asalmost all the recessive mutant gene is already inactivated so that further mutation will not give mutant geneat all (only giving double mutant).
Furthermore, hybrid of mutant gene wx-a and mutant gene wx-b may produce completely normal Wx geneby normal (ordinary) intra-cistronic recombination even without mutagen treatment, at the rate of one in
1,000 or so, depending on the distance between wx-a and wx-b. Such result may be mistaken as adominant mutation.
Keep some original seeds for future backcross.
Use some seeds for treatment control to examine the treatment dose and other procedures. This may be importantwhen you send out seeds to other institution for treatment/irradiation. A small control seeds to examine thetransportation condition is important sometime.
2) Mutagens suggested
This depends on the purpose of the work.
If any kind of mutation is accepted for crop improvement, irradiation by X-rays or gamma-rays, or if you have a small
chemical laboratory, chemical mutagen e.g. EMS may give good results. In case of Sodium Azide1, please consult toappropriate literature for necessary pH and/or exact growth stage to apply.
If Deletion mutation is wanted, intended to extract a specific mutant gene, ion beam, reactor neutrons or otherappropriate high-LET radiation is suggested2. In some radiation, e.g. fast and/or thermal neutrons, small but
appreciable amount of radioactivity will be induced by irradiation. Special care should be taken in practical handlingand also to meet regal restrictions.
If intermediate or leaky type mutation is wanted, chemical mutagen, e.g. EMS, which may cause base-pair
substitution mutation may be suggested3, 4.
The idea will be extended to other special type mutation, like temperature dependent mutations5, which may be
explained by temperature dependent 3-dimentional folding of protein, the gene product, although it is not proved yet.
A comparable rice mutant was also detected in Vietnam, but the critical temperature was a little different from that ofJapanese. If the explanation proved to be true, chemical mutagens to induce base-pair substitution may yield suchmutation as temperature dependent mutations, perhaps.
3) How to decide treatment doseThis may be the very important procedure in the mutagenesis experiment. The sensitivity tests are different forradiations and chemical mutagens, the latter is usually applied as water solution, needing one more step of preliminary
test.
Irradiation
In case of irradiation treatment, sensitivity differentiation is reported in some materials perhaps by a few sensitivity
genes. Generally speaking, long grained indica rice is more resistant to radiations than short grained japonica varieties.
124

Practice Suggestions for Mutation Breeding

To decide appropriate dose, seedling height measurements from 100 Gy to 700 Gy, to cover almost no effect to heavily
damaged dose should be tested at first, in 5 or more dose classes with up to 20 seeds each. About 20% reduced dose
may be suggested as recommendable dose, where M1 seed set is not much reduced. For radio-sensitivity comparisons,LD-50 (50 %lethal dose) or RD-50 (50 % Reduction dose) are often used as critical values. However, for mutant
induction work, such doses are too high for better seed fertility and less accompanied mutation.

About 20 % reducing dose for seedling height may be recommended.
For chemical mutagens, another curve to show the water imbibition is important to decide the treatment.
Dry seeds show very fast water uptake in the initial several minutes, then it reaches to a plateau, where chemicaltreatment (soaking) is suggested. The strength (concentration) test of the water solution may be comparable to theradiation dose test shown above.

Ethyl methanesulfonate (EMS) treatment was very effective in rice. One example of a recipe may be as
follows :Presoaking in distilled water for two hours.
Soaking treatment in 1 % EMS at 25℃ for 5 hours while shaking gently.
Post wash a few times then keep in distilled water over night.
After final washing, seeds may be sown to a seedling bed.
All paper and glass wares and rinsing water used in the EMS treatment should be put into a bucket of
appropriate NaOH solution (1 % or so) to hydrolyze the carcinogenic mutagen.

125Practice Suggestions for Mutation Breeding4) How to grow and harvest the M1 generation
After the mutagen treatment, rice seeds of M1 generation may be planted in field according to the local procedure.
Genetically speaking, each treated seeds should be a separate unit in terms of mutagenesis, so one seedling per hill/hole
may be suggested. If rice transplanter machine is employed, plan well for the number of plant per hill. If one plant
per hill can not be attained, use much more seeds for mutagen treatment and harvest one panicle per hill, neglecting
other seeds in the same hill. (This worked well in the author’s experiment.) The purpose of planting M1 generation
is to advance the generation to M2. So, plants should be maintained to obtain good seeds by application of various
pest controlling procedures. Do not attempt to put any kind of screening in the M1 generation, because the induced
mutant gene (recessive) is not fully expressed at this stage. Occasionally chlorophyll mutant stripe may be observed infew plants among thousands of M1 plants. This may be a double mutation at the same chlorophyll gene locus. Mostof it will be expressed only on some leaves, but it may be a good indication of successful mutagenesis treatment.
Suppose a mutation frequency of a gene as 0.1 % or one in 1000, a possibility of two mutations in thepaternal and maternal gene at the same time will be 0.1 % x 0.1 % = one in 1,000,000. Leaf primodial cells
in a seed embryo may amount to 1000 or more. Thus this double mutant sector may be visualized in
1,000,000 treated cell population or a few stripes in thousands of plant.
The central panicle may have chimerism, but most of later panicles seem to be practically free from chimerism.
Supposing the panicle-wise screening in M2 generation, suggested harvesting system is as follows, each group should
carry name tags accordingly :1) The First Round Harvest : Harvest only one panicle per hill, all through the M1 population. In all the harvested
materials, don’t forget to place name tags in the way available in the local condition, cultivars used, treatment dose, thefirst round of harvest etc. The first round harvest may be used to examine endosperm characters, waxy, sugary, giant
embryo etc. It may be difficult to keep the seeds separately after seed character examinations as you may need
thousands of envelopes, normally looking seeds may be discarded.
2) The Second Round Harvest : In the same way, second round harvest should be made again from the beginning of
the M1 population. Some of the panicles may genetically duplicate from the First Round Harvest, but if you use theSecond Round Harvest in a different category like plant type characters expressed after germination, duplication is not a
problem.
3) The Third Round Harvest : Because M1 generation planting is laborious work, this 3rd round harvest may besuggested to preserve for future work. Store the panicles in dry and cool condition for possible future use. In theauthor’s experiences, out of such stored materials, another research institution found a TGMS mutant, and a few
recessive tall mutant with sd1 (Reimei) background, both may be useful in HYBRID RICE system.
5) How to screen the wanted traits
Induction of mutant gene will occur at random, but mutant character can be used as the screening element. For
example, early flowering mutant can be found in M2 population rather easily. If appropriate screening method isestablished, resistant mutant plant(s) may also easily be found as a survivor in disease or pest inoculated screening field.
This screening field should be developed before actual screening work. The survival screening may be very effective,
but panicle to row planting may still be suggested.
As for a planting pattern, 1) a methodology developed by plant breeder, one grain, or a few grains, from one M1 panicle
each may be practical, however, 2) if the researcher is on a starting stage of the mutant induction experiment, panicle torow planting is strongly suggested to have good experiences in the segregation in M2. After the visual experience on
the Mendelian Segregation, researcher’s may establish his/her own methodology. Theoretically, after mutagen
treatment of multi-cellular seed embryo, the central stem may show chimerism. However, after full growth of a rice
plant, chimerism might be much reduced. Among almost 20 or more panicles from a single treated seed, most of
panicles show segregation ratio of one mutant to three normal plants.
126

Practice Suggestions for Mutation Breeding

For practical screening, the knowledge on where the expected mutant character will be expressed is very important.
1) If endosperm character, e.g. waxy starch, (also, sugary, floury, and giant embryo etc) is the target character, it will
be expressed on the seeds produced on the M1 panicle. These seeds are in M2 generation (see files of PictureWord inthis CD-ROM). The details may depend on the variety and the planting custom of the region, but in Japan, if a seedpopulation is treated by a mutagen in spring, after harvesting and drying, screening or observation of the characters is
possible in November or December of the same year. (Please note that this screening is made on the M2 seeds, so thatthe rule of M2 screening is still applied here.)2) On the other hand, plant structure (semi-dwarf etc), physiological characters (early flowering etc) which are
expressed in M2 plant or maternal tissue mutant like hair-less (glabrous) the target characters need to be examined in
M2 field, where panicle family planting (at least 10 or more plants per panicle) is suggested. Segregation of more than
two mutant plants in a panicle family gives good confidence to the observer. If the purpose is for academic studies
where lethal mutant(s) are also important, then, to maintain the mutant gene as heterozygotes, this panicle family
planting will be a necessity.
3) Some seed characters depend on the plant structure. A mutant character with twin pistils in each flower should besearched in the M2 plant population, or M3 seed population (twin grains may be seen as strange twin grains within a setof hulls). Some of seed shape of short round (in japonica) or a length mutant in indica lines, may be resulted frommutant characters of the housing of hulls. For these maternal tissue characters should be searched in the M2 plantgrown on the 2nd year.
6) How to brush up the mutant
The number of genes in a species is said to be more than 10,000. Suppose the number of genes important inagriculture, as only 10,000, and mutagen treatment induces a gene at 0.1 % frequency, the one treatment may induce
10,000 x 0.1% (=1/1000) = 10
that is about ten mutant gene will be occurred simultaneously after one mutagen treatment. So, if you were successful
in inducing a sd1 mutant or a good wx mutant, the promising mutant line may carry also nine other mutant genes
induced simultaneously. Sometimes simultaneous mutant gene gives another very significant character like virus
resistance in a barley early mutant in Japan or salt resistance in Milns Golden Promise (barley in UK). However, inmost of the cases, they are albino or reduces seed fertility, that is unfavorable elements.
Rice plant has 12 chromosomes. So, it may be highly possible that the simultaneously induced genes may bedispersed on the different chromosomes. Actually one backcrossing to the original line easily excludes theunfavorable gene and seed fertility may be returned to normal level. Such backcrossing is not a cross breeding, but anecessary procedure before yield tests. It is only to retrieve the original genotype except the favorable new mutant
gene.

Some Mutants for Hybrid Rice System
Induced mutations are recessive. So, it will not expressed in F1 hybrid generation. As far as the mutant is used in
one side of the parent, any mutant may be used. Contrary, if one
want to express a phenotype, both parent must carry the same gene,even if the mutant site (within the locus) may be different. For thispurpose, mutagenesis will be a powerful means to have independent
origin for the same gene/cistron.
Nuclear gene male sterility is relatively easy to get, but usually very
difficult to apply to the Hybrid Rice system. Temperaturedependent TGMS mutant in rice may be one good possibility (cf.
Tripletcode.doc in Introduction).
127

Practice Suggestions for Mutation Breeding

Fertile twin grain mutant has two sets of pistils,
suggesting better external pollen reception. This
recessive mutation will not be expressed in the F1
hybrid generation, when ordinary seed shape isnecessary.

Flower of the twin grain mutant has two pistils, bothare fertile, if condition allows.

In rice the flowering time of the day is not long, a few
hours in the morning. This open hull mutant keeps
open its flowers, or can not close. However, thismutant has good seed fertility as shown in this picture.
As for the pollen parent, tall plant for better pollen
dissipation is desired in sd1 background. For thisspecial purpose, leaf color mutant as a marker gene was
successfully used to induce “tall mutant but with sd1
background (cf. markergene.doc).

Finally, hybrid rice systems, three lines by cms and two lines by tgms or pgms(photoperiod dependent ms) may beillustrated as follows. For seed harvesting crops like rice, dominant Rf (fertility restorer) is a necessity in the three lines
using cms, but such dominant gene can not be induced by mutagenesis. Tgms or pgms does not require Rf, but
practical problems may need solutions.

128

Practice Suggestions for Mutation Breeding

Lodging has been a big problem in many crop plants. After heavy
wind, near harvest time, highly valued “Koshihikari” often lodges.
. The first Lodging resistant mutant variety was developed from‘Fujiminori’(left) and registered as ‘Reimei’. The new variety wasnamed in the hope of successful application of mutation in crop breeding,meaning ‘the dawn’ or ‘day-breake’Ten years later, a comparable American variety “Calrose-76” followed as the same
lodging resistant variety developed by Dr. Rutger (Picture.) and his group. The
semi-dwarf gene sd1 was identified in “Reimei”, “Calrose-76” and the famous“IR-8”.
(Copied from IAEA publication)

The use of Mutation Breeding in Japanese Rice.
After successful release of lodging resistant ‘Reimei’, so called direct use of mutant amounted to about 20 andindirect use (use of mutant line in cross breeding) amounted to almost 50 varieties. For examples, low amylose (sticky
but not fully waxy) line ‘Milky queen’ is bred as direct use of mutant, and strong culm semi-dwarf ‘Kinu-hikari’, giant
embryo cultivar ‘Hai-minori’, and low gluterin (good for allergic patients) ‘LGC-1’ are examples of use in crossbreeding.
Recent low amylose rice cultivars in Japan
MutantProgeny
Mutagen
NM391
Gamma-ray induced‘Asa-tuyu’‘Aya’‘Hanabusa’‘Ayahime’74wx2N1
EMS induced‘Snow Pearl’‘Takitate’
74wx8N1
EMS induced‘Akane-Fuji’‘Soft 158’
EMS induced‘Milky Queen’
NMU induced‘Yawara-Komachi’
NMU induced‘Silky Pearl’NMU induced
129

Practice Suggestions for Mutation Breeding

Mutants screened in M1 panicle.
When multi-cellular seed embryo is treated withmutagen (top left), the central axis panicle may havechimeric structures, but later coming panicles may
segregate within the entire panicle. Please note thatthe seeds on the panicle are the following generationand the seeds are in M2 generation, so endosperm
characters or seed embryo characters are segregated on
the M1 panicle as shown in the top right green panicle.
Lower figure suggest also the presence of someheterozygotes. In case of academic studies, where
lethal mutant may have some importance, panicle
family planting is important to keep the heterozygotes
for maintenance of the lethal mutant gene. A few
examples are shown here.

Typical example of endosperm mutation may be waxy starch mutant (white endosperm(‘Miyuki-Mochi’) at right induced from Wx (‘Toyo-Nishiki’). Glutinousness or
amylose deficiency can be confirmed by iodine staining (see Starchmutant in this ricefile).
Lighting behind the seeds shows normal (left two grains) and giant embryo (right upper two
grains) and turbid wx grains (lower two grains). Comparable mutant line of giant embryo
has been registered as ‘Hai-Minori’ in Japan. Half way germinating seeds contains many
excellent nourishing components, good for health. The character is relatively frequent
after chemical mutagen treatments.
The giant embryo mutant has various nourishing components in the enlarged
embryo. The mutant has registered as “Haiminori” in Japan (Left picture.
Right picture is ordinary Koshihikari for comparison).

This picture may be comparable to sugary mutant in maize, the sweet corn, although use inrice has not developed yet.

Mutant character selectable in M2 generation.
Albino, abnormal dwarf etc. may be easily come to ones mind. Here othercases of important characters are illustrated as examples.
Early flowering mutants may also come to the mind. These are plantcharacters detectable in M2 generation. This picture shows two early
flowering mutant lines segregated after callus culture mutagenesis, one inleft-back and the other right front.
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Practice Suggestions for Mutation Breeding

Some tissues are maternal
tissues, so that the mutant
phenotype of them can bescreened only in M2 generation.
This mutant keeps the flower
open, or does not close at all, but
still seeds are fertile as seen in
the next picture.

Many “open hull” mutants areseed-sterile, but in this case,seeds are seen in the picture. It
may be a promising mutant for
hybrid-rice system for better
pollen reception. It is recessive
and will not expressed in F1.

Another case of promising mutant for hybrid rice system may be this “twin pistil” mutant.
At first, strange shape of seeds were noticed. The shape is not good for ordinary grain
polishing. But the detailed observation showed that the mutant plants have two sets ofpistils, both seemed to be fertile if condition allows.
The male organs (anthers) are normal and the female organ (pistil)is basically fertile to give twin seeds. It suggests that the pollen
reception may be twice as much as normal single pistil line. Seed fertility of more than 130 %had been nominally recorded on the fertile seed embryo per flower. This character is recessive
and will not be expressed in the F1 generation, suggesting this may be a good character as thematernal line in the hybrid rice system. These characters expressed in the maternal tissues may
be screened in the M2 generation field (in case of the “twin grains” flowers in M2 plants, andseeds shown above are in M3 generation).
Sometimes, purely academic research demands special mutant line which may not be
useful in agriculture. For such academic purposes, mutants are very often requested
from researchers. This picture is a rice panicle mutant,the panicle branches seemed to continue branching
resulting in no flowers at all on the panicle. It has been
induced by gamma-rays and also by EMS. The sterilemutant can be maintained as heterozygotes.

This picture shows two types of the abnormal panicle mutants. The right one is thesame as shown above, but the left one was induced after callus culture.

Use of viable and fertile mutant as marker gene.
Rice is basically a self pollinating plant, however, the mutational
event may be much lower than the assured self pollinating rate.
Therefore in the M1 planting field like this picture (upper left lightgreen(variety ‘Reimei’) and lower right light green(variety‘Norin-8’)) previously induced and increased light green and fertile“chlorina” mutants were used to assure the line purity.
131

Practice Suggestions for Mutation Breeding

Even surrounded by other normal varieties, the gene-marked lines may give the clear
characteristics of the line, in this case, light green color. Contaminated plant with normal
green color may be taken out from the seedling bed. In the right side, purple seedlings areseen, but these are dominant purple line in which contaminated normal green plants may notappear or hidden by the dominant purple gene.
When you can get a promising mutant, this recessive chlorina marker gene may be excluded
easily from the promising mutant lines, by a single back-cross to the original line.

After transplanting to the main field, still floating seedling may come fromneighbor group. Such normal green plant may be excluded from the selection
field. By such very strict field maintenance, a tall mutant (recessive to sd1)
with sd1 (‘Reimei’) background was selected.

Some examples of Rice Mutation Experiments.
Segregation of waxy starch grains (the lowest line) in a japonica variety.
Generally, later panicles segregate 3 normal and 1 mutant grains. In japonica
variety, some heterozygotes look half-waxy, e.g. 2nd and 3rd grains in the top
row. The next fourth grain has a defect embryo.

The left two grains are the original normal grains. Upper right two grains are
giant embryo (perhaps same type as “Hai-Minori” variety). The lower two
grains are waxy starch mutant. This picture was taken by back-illumination.

Albino seedlings indicate that the mutagenic treatment had beeneffective. Fourteen holes were used for one M1 panicle family,
skipping a row of the next seven holes as separation between families.

An example of M2 field planted with panicle torow system. Indicator poles show some kind of
mutants were segregated in the family line.

Two early flowering mutants are shown here as
they segregated in panicle to row planting in a
M2 field.
132

Practice Suggestions for Mutation Breeding

Two or three dwarf mutants aresegregated as indicated by the pole.

This is a case of disease susceptible mutants shown by the pole. In a practicalbreeding, this may be selected out, but for academic research work, this may be
important material to study the infection system of the disease.

When harvesting in a panicle to row field, normally looking(possible-)heterozygotes are important material if academic research work is
intended.

Starch mutant in rice
In rice, seeds produced on M1plant can be used for starch type mutation
screening. The result shown here may look continuous variation fromnon-waxy (left) to complete waxy (right). Extra grains in top row
indicate Controls.

Starch can be stained byiodine solution, normal non
waxy blue and waxy mutant

Starch components can be easily
separated by a simple paper
chromatography run by 1M KOH.
The normal non-waxy starch has
amylose, the bluish spot in the upper
part, but waxy mutant (Right lane) lacks
it.

Intermediate phenotypes were further studied and genetic
analyses made clear that 74wx8N1 was a wx locus mutant,but 74wx5N1 was low amylose mutant but other than thewx locus.

133Practice Suggestions for Mutation Breeding

2. Barley and wheat
Barley is diploid and ordinary (bread) wheat is hexaploid. In a hexaploid crops, albino may not show up in M2
generation by the function of triplicated homeologous genes in the three genoms (A, B, and D). Except suchdifficulties, handling of the seeds may be quite the same way in the two cereals. So, here, barley may be the example.
In wheat, ionizing irradiation often induces chromosome aberrations. Unless such aberration is the target of theexperiments, watch the seed fertility, especially after backcrossing to the original line. Translocation line may continue
lower seed fertility in the BF1, but if the line was aberration free, seed fertility may return to normal.
Basically the method for rice may be followed, but number of seeds per ear/spike, and the number of tillers per M1plant, will be much smaller than those of rice plant.
1) How to prepare starting materials
The breeding method using induced mutant(s) will improve only a few characters, e.g. semi-dwarf (for lodging
resistance) or early flowering or waxy starch, leaving other characters unchanged at all. So, this method may be
applied best to a local varieties. Many kinds of mutant characters will be found during screening procedures.
Therefore, it may be necessary to have a clear objective of the expected improvement. Otherwise, many newly
induced characters may draw attention of the observer, finally giving too much work on unnecessary characters.
To prepare the starting population (materials), if possible, isolation by paper bagging etc may be strongly recommended.
Use of hybrid seeds in mutagenesis is not a good idea, unless specific purpose of chromosomal translocation isintended.
“Hybridization enlarges the possibility of mutation by enlarged number of target gene” is erroneous, asalmost all the mutant gene is already inactivated so that further mutation will not give mutant gene at all.
Furthermore, hybrid of mutant gene wx-a and mutant gene wx-b may produce completely normal Wx geneby intra-cistronic recombination even without mutagen treatment, at the rate of one in 10,000 or so,
depending on the distance between wx-a and wx-b. Such result may be mistaken as a dominant mutation.
Keep some seeds for future backcross.
Use some seeds for treatment control to examine the treatment dose and other procedures. This may be importantwhen you send out seeds to other institution for treatment/irradiation. A small control seeds to examine thetransportation condition is important sometime.
Many of barley and wheat varieties may need some kind of cold treatments (vernalization) to give flower buds. The
need of such treatment is needed in M1 and M2 generations (unless vernalization free mutant is sought in M2
generation (even if such was the case, M1 must be still in the favorable condition).
2) Mutagens suggested
This depends on the purpose of the work.
If any kind of mutation is accepted for crop improvement program, irradiation by X-rays or gamma-rays, or if you havea small chemical laboratory, chemical mutagen e.g. EMS may give a good result. In case of Sodium Azide1, please
consult to appropriate literature for necessary pH and/or growth stage to apply.
If Deletion mutation is wanted, intended to extract a specific mutant gene, ion beam, reactor neutrons or otherappropriate high-LET radiation is suggested. In some radiation, e.g. fast and/or thermal neutrons, small but
appreciable amount of radioactivity will be induced by irradiation. Special care should be taken in practical handlingand also to meet regal restrictions.
If intermediate or leaky type mutation is wanted, chemical mutagen, e.g. EMS, which may cause base-pair
substitution mutation may be suggested.
The idea will be extended to other special type mutation, like temperature dependent mutations, which may be
explained by temperature dependent 3-dimentional folding of protein molecules, the gene product, although it is notproved yet. If the explanation proved to be true, chemical mutagens to induce base-pair substitution may yield suchmutation, perhaps.
134Practice Suggestions for Mutation Breeding3) How to decide treatment doseThis may be the very important procedure in the mutagenesis experiment. The sensitivity tests are different forradiations and chemical mutagen which is usually applied as water solution.
Irradiation
In case of irradiation treatment, sensitivity differentiation is reported in some materials perhaps by a few sensitivity
genes. In barley, the two types of “hulled” and “naked” type seems also different in the radio-sensitivity, perhaps due
to radio-sensitivity gene(s).
To decide appropriate dose, seedling height measurements from 100 Gy to 700 Gy, to cover almost no effect to heavily
damaging dose should be tested at first, in 7 or more dose classes with up to 20 seeds each (see the same in “1rice”).
About 20 % reduced dose may be suggested as recommendable dose, where M1 seed set is not much reduced. For
radio-sensitivity comparisons, LD-50 (50 %lethal dose) or RD-50 (50 %Reduction dose e.g. by fresh weight) are often
used as critical values to compare. However, for mutation induction, such doses are too high for better seed fertility
and especially for less accompanied mutation.

For chemical mutagens, another curve to show the water imbibitions is important to decide the treatment.

135

Practice Suggestions for Mutation Breeding

Dry seeds show very fast water uptake in the initial several minutes, then reach to a plateau, where chemical treatment(soaking) is suggested. The strength (concentration) test of the water solution may be comparable to the radiation test
shown above. Please be aware of the temperature. In barley and wheat, 15 to 20 ℃ presoaking and soaking
treatment may be suggested.
Ethyl methanesulfonate (EMS) treatment may be very effective. One example of a recipe may be as
follows :Presoaking in distilled water at 15℃ for two hours.
Soaking treatment in 1% EMS at 15℃ for 5 hours while shaking gently.
Post wash a few times then keep in dist. water over night.
After final washing, seeds may be sown to a seedling bed.
All paper and glass wares and rinsing water used in the treatment should be put into a bucket of
appropriate NaOH solution (around 0.1 %) to hydrolyze the carcinogenic mutagen.
4) How to grow and harvest the M1 generation
After the mutagen treatment, barley or wheat seeds of M1 generation may be planted in field according to the local
procedure. Genetically speaking, each treated seeds should be a separate unit in terms of mutagenesis, so one seedlingper hill/hole may be suggested. If one plant per hill can not be attained, use much more seeds for mutagen treatment
and harvest one spike per hill, neglecting other seeds in the same hill. The purpose of planting M1 generation is toadvance the generation to M2 by self pollination. So, plants should be maintained to obtain good seeds using various
pest controlling procedures. Do not attempt to put any kind of screening yet in the M1, because the induced mutant
gene (recessive) is not fully expressed at this stage. Occasionally chlorophyll mutant stripe may be observed in few
plants among thousands of M1 (diploid) plants. This may be a simultaneous two mutations at the same chlorophyll
gene (paternal and maternal) locus. Most of it will be expressed only on some leaves, but it may be a good indicationof successful mutagenesis treatment.
Suppose a mutation frequency of a gene as 0.1 % or one in 1000, a possibility of two mutations in thepaternal and maternal genes at the same time will be 0.1 % x 0.1 % = one in 1,000,000. Leaf primodial
cells in a seed embryo may be amount to 1000 or more. Thus this may be visualized in 1,000,000 treatedcell population or a few stripes in thousands of plants.
The central panicle may have chimerism, but most of later panicles seem to be practically free from chimerism.
Supposing the panicle-wise screening in M2 generation, suggested harvesting system is as follows, (the same assuggested in rice) each group should carry name tags accordingly :1) The First Round Harvest : Harvest only one panicle per hill, all through the M1 population. In all the harvested
materials, don’t forget to place name tags in the way available in the local condition. This harvest may be used to
examine endosperm characters. It may be difficult to keep the seeds separately after seed character examination,
normally looking seeds may be discarded.
In Washington State Univ. (Pullman), Prof. Nilans group was once successful to get several waxy barleyendosperm by iodine staining in the half grain system, where embryonic half is kept for germination.
2) The Second Round Harvest : In the same way, second round harvest should be made again from the beginning of
the M1 population. Some of the panicles may genetically duplicate from the First Round Harvest, but if you use thisin a different category like plant type characters expressed after germination, duplication is not a problem.
3) The Third Round Harvest : Because M1 generation planting is laborious work, this 3rd round harvest may besuggested for storing for future work. Store the spikes in dry and cool condition for future use. If seeds are easily
dropping off from spikes, many paper bags may be needed to store the spike families separately.

136

Practice Suggestions for Mutation Breeding5) How to screen the wanted traits
Induction of mutant gene will occur at random, but mutant character can be used as the screening element. For
example, early heading/flowering mutant can be found in M2 population rather easily. If appropriate screening
method is established, resistant mutant plant(s) may also easily be found as a survivor in disease or pest inoculated
screening field. This screening field should be developed before actual screening work. This survival screening may
be very effective, but still panicle to row planting, description of which is just following, may still be suggested.
As for a planting pattern, 1) a methodology developed by plant breeder, one grain, or a few grains, from one M1 panicle
each have been proposed, 2) however, if the researcher is on a starting stage of the mutant induction experiment, panicle
to row planting is strongly recommended. After the visual experience on the Mendelian Segregation, researcher’s
own methodology may be developed. Theoretically, after mutagen treatment of multi-cellular seed embryo, thecentral stem may show chimerism. However, after full growth of the plant, chimerism might be much reduced,
among almost five or more spikes from a single treated seed, most of panicles may show segregation ratio of one
mutant to three normal plants.
For practical screening, the knowledge on which tissue the expected mutant character will be expressed is very
important.
1) If endosperm character, e.g. waxy starch, (also, sugary, floury etc) is the target character, it will be expressed on theseeds produced on the M1 spike. These seeds are in M2 generation. The details may depend on the variety and theplanting custom of the region, but in Japan, if a seed population is treated by a mutagen in autumn, after harvesting anddrying, screening or observation of the characters is possible in May or June of the following year.
2) On the other hand, plant structure (semi-dwarf etc) or physiological characters (early flowering, no-need ofvernalization etc) which are expressed in M2 plant, the target characters need to be observed in M2 field, where spike
family planting (at least 10 or more plants per spike) is suggested. Segregation of more than two mutant plants in a
spike family gives good confidence to the observer. If the purpose is for academic studies where lethal mutant(s) arealso important, then, to maintain the mutant as heterozygotes, this panicle family planting will be a necessity.
6) How to brush up the mutant
The number of genes in a species is said to be more than 10,000. Suppose the number of genes important inagriculture, as only 10,000, and mutagen treatment induces a gene at 0.1 % frequency, the one treatment may induce
10,000 x 0.1 % (=1/1000) = 10
that is about ten mutant gene will occur after one mutagen treatment. So, if you were successful in inducing asemi-dwarf mutant or a good quality mutant, the promising mutant gene may carry also nine other mutant genes
induced simultaneously. Sometimes simultaneous mutant gene gives another very significant character, e.g. virus
resistance in a barley early mutant in Japan, or salt resistance, in Milns Golden Promise, in UK. However, in most of
the cases, they may be albino or reduces seed fertility, which is unfavorable element.
Barley plant has seven chromosomes, and wheat has 21 in haploid states. So, it may be highly possible that thesimultaneously induced genes may be dispersed on the different chromosomes. Actually one backcrossing to theoriginal line easily excludes the unfavorable gene and seed fertility may be returned to normal level.
Such backcrossing is not a cross breeding. It is only to retrieve the original genotype except the favorable new mutant
gene.
As previously mentioned, chromosome aberration might occur in wheat. If BF1 is still shown low seed fertility, it
may have chromosome aberration.

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Mutant segregation and screening work
Bread wheat is hexaploid (6X), but for academic studies diploid species are used like here at the
National Institute of Genetics, Misima in 1970s. In hexaploid species, albino seedling isdifficult to induce and to show, but diploid species shows albino seedlings.
The spikes (or ears) harvested from M1 plants are put in the soil, so that spike family will show
up together like the following two pictures.

The second picture is the case oflight green seedling mutants segregated.
The albino, also shows red
pigment still it contains.

Barley is a standard study plant for mutagenesis. This picture was taken in 1960s
at Brookhaven National Laboratory in the USA (by E.Amano). The barley spikes
harvested from M1 (seedlings are in M2) were put into the sand bed like the case of
the diploid wheat shown previously. The mutagen used by Dr. R. Heiner(previously Washington State Univ.) was dES.

After many preliminary tests, practical disease resistant mutant may be screened likethe one done at the Institute of Radiation Breeding, Ohmiya-machi, Japan.. About
120 million M2 seedlings were germinated and used in screening of “mildew” resistantmutant and several resistant lines could be selected by this screening.

This mutant line shows disease resistance against the soil born
BYMV (barley yellow mosaic virus). The surrounding lines wereclearly damaged by the virus disease. This resistant mutant was atfirst selected as early maturing mutant, but later it was found to be adisease resistant line to the virus as seen here by good growth. Thismight be a favorable case of simultaneously induced mutation. The
mutant was registered later as ‘Masakado-mugi’ in Japan.
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3. Maize
Maize is basically out crossing crop having male flower tassel at the top of a stem and female cob(s) are in
the lower stem node(s). The seed embryos are already multi-cellular in its structure, so that the seed treatment gives
chimerism, separating the male tassel and the female cob. Therefore special strategy should be set in mutagenesis andscreening.
Another very strange characteristics of this crop plant may be the fact that ionizing radiations usually does notgive good mutant. It induces deletion mutations of various sizes, generally large and unfavorable size for agricultural
performances (Stadler and Roman 1948, Mottinger 1970 and Amano 1968, 1972). The chimerism in the M1 plantand very strange reaction to the ionizing radiation make this important crop a very difficult material for mutation work.
However, if the chimerism pattern is understood and appropriate mutagen (chemical mutagen, EMS, may besuggested) could be used, mutation work in this crop may give good results, both in academic work and agronomical
applications. In the IAEA mutant cultivar data bases, on Internet system and the attached database based on the IAEA
publications, many mutant cultivars were registered, as the system accepted the reported content without confirmation.
However, perhaps only chemical mutagen induced data might be dependable (refer to Mottinger 1970 Genetics
64:259-271, or Amano 1985 Gamma-Field Symposia 24:81-96).
The basic handling of this plant in pollination system should be followed in the mutation work.
1) Removal of male tassels may be depend on the project purpose, but detasseling and pollination by use of a paintbrush may be helpful in mass pollination work.
a) If marker gene system can be used, remove male tassels (detasseling) for better pollination work.
For example, if new waxy starch mutant is wanted in an inbred line, mix waxy line pollen with the original inbredline in equal amount (e.g. by weight) and place pollen onto the isolated silk with dry painting brush. Mutatedwaxy female cob will show 1 waxy and 3 non-waxy seeds. The non-waxy seeds may be composed of 1)+/wx-line, 2) +/+(original) and 3) wx-induced (with original background)/+(the original). The last one will give
inbred waxy mutant line by self pollination in the next generation.
It may sound complicated, but this kind of strategy is very effective if it should be well planned beforehand.
b) If a new character is sought, leave male tassel and perhaps free open pollination may be used in M1, if isolated plot
can be used for the M1 generation.
Observation of chlorophyll sectoring in the male tassels which suggests that each chimeric unit might be as large asa single brunch of a male tassel. So, about 10 or more mutant sectors in a tassel.
In a seed-EMS treatment experiments, female cobs, most of the mutated seeds, e.g. colored (from C-I to C) seeds
distributed rather evenly on the cob. However, there was one case, where only one clear mutant seed was foundat the bottom of a cob. This may mean that the small mutated sector was only at the base of the cob and one
grain was shown as 1 mutant against 3 normal seeds. Therefore, circular sampling at the base of a cob may
contain more mutant sectors than a row sampling from bottom to top.
1) How to prepare starting materials
In maize, commercial seeds are either hybrid seeds or inbred. Hybrid seeds will give good performance incommercial cultivation, but for plant breeding work, only inbred line may be meaningful. For mutation work, perhaps
improvement of local (inbred) varieties may be a good target.
Well controlled hand pollination at least one generation and uniform appearance of seeds on any cob, should
be confirmed. The improved line then may be used as parental line for “hybrid seed system”.
The idea of “Hybrid seeds enlarge the target gene number” is erroneous. Mutant genes are mostly
inactivated, or in other words they are already broken, so the effective target genes of a hybrid is reduced than pureinbred line. AA genotype has two targets, A and A, but Aa has only one target A.
Isolated experimental plot may be needed for mutation work. Please be noted that maize pollen grains may
fly away as far as hundreds of meters.
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Practice Suggestions for Mutation Breeding2) Mutagens suggested
As described before, maize reacts to physical mutagens in a strange way. Maize can repair thechromosomal damages (e.g. chromosomal breakage) as other plant species, but the timing of the repair seems to bedifferent from other plants. This phenomenon has been reported by L. J. Stadler (1948 Genetics 33:273-303 etc.), J. P.
Mottinger (1970 Genetics 64: 259-271) and E. Amano (2001 Gammer Field Symposia No. 40 35-47). Personally,
during my more than 20 years of experiences, no good viable mutants could be available in maize by gamma-, X-,
fast-neutrons and thermal neutron irradiations. Germicidal UV to pollen grains was successful but details are still left
to be studied.
Compared to these radiations, chemical mutagen tested, e.g. EMS was very successful in Wx, Sh, C-I loci togive various good and viable recessive mutant lines. In these induced mutants, intermediate phenotype (mis-sense or
leaky type) mutations were often observed, but in most of the cases they were clear “complete” type mutants.
3) How to decide treatment dose
The methodologies and evaluation of the results are same as those for rice or barley. So, refer to thosemethods, shown elsewhere. But please be noted that the dose may differ from rice, or often among the maize lines.
So, if you are starting the mutagenesis work, it will be better to examine the dose-response curve by yourself on yourmaterial. Ten to 20 seeds per dose class may be good enough, but the number of dose classes being five to ten,
extending from 10Gy to significant damaging, e.g. 400Gy or so.
4) How to grow and harvest the M1 generation
Isolation plot may be used, or if it is not available, isolation by bagging should be done. However, otherhandling in the M1 field may be planned only to obtain M2 seeds. Depending on the purpose and strategies, open
pollination may be accepted if an isolated plot is available.
4-1) Use of genetic marker : If marker-use-pollination (e.g.
searching of waxy mutant) is used to induce the same mutant
character in an inbred line, the mutant character may be examinedon the cob of M1 plant or on M2 seedling/plant. Perhaps you aretreating inbred dominant line. Prepare both the tester line (e.g.
inbred wx/wx line) and the inbred original line. Collect pollen
from both tester and the inbred parental line. Mix equal amountand put on the silk of the treated female. By this way, you candetect the intended mutant kernels on the female cob. On thecob, normal looking kernels are either non-mutant kernel (+/testerwx) or inbred kernels (wx/+ or +/+). In the following generation,
by self pllonation within the plant these normal looking kernels
may give segregation of wx or +/+. If the plant give good
growth by heterosis, it might be hybrid seeds with the tester. If
the same growth as the inbred parent and segregating the intended
mutant gene, you are successful in obtaining theinbred-line-mutant.
4-2) Unless the marker-use-pollination is utilized, mutant screening may be done in the M3 generation after self
pollination of M2 plants. The chimeric mutant sector might be most possibly locate in the base of the cobs.
So, plant the M2 seeds collected from the base of the cobs, two rows around the cobs. The total population
will amount to about 20 grains times M1 cobs. Each M2 plants must be self pollinated within the plant. If
this was the method you are adopting, then the M1 plants may be open pollinated, provided if you get good
isolation environment.
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Practice Suggestions for Mutation Breeding5) How to screen the wanted traits
If marker-use-pollination is used to induce the same mutant character in an inbred line, the mutant charactermay be examined on the cob of M1 plant (in cases of endosperm chracters) or on M2 seedling/plant.
Unless the marker-use-pollination is utilized, mutant screening may be done in the M3 generation after self
pollination of M2 plants. As described before, circular sampling at the bottom of M1 cob may be recommended to
grow M2 plants, which should be self pollinated to give M3 seeds/plants for screening. Screening techniques may
differ from character to character, e.g. disease resistance needs active inoculation or disease affected environment.
6) How to brush up the mutant
Simultaneous mutation might occur in the treated materials. Therefore, at least one back cross should be
tried to exclude unfavorable additional mutation. As far as the original parental line is used, general genetic
background is maintained as the inbred line. This “back-crossing” is not disturbing the genetic background much, so it
should be separated from cross breeding. The use of the mutant line may be still the direct use of the mutant (notindirect use).

Mutagenesis in maize (Chimerism)
Mutagenesis in maize needs some special considerations. There may be two important problems regarding maize
mutagenesis 1) appropriate mutagen and 2) chimerism. For mutagens suggested, see the other file “Mutagenmaize”.
In cases of rice orbarley, self
pollination of theM1 plant may bemostly real self
pollination within
the flower.
However, inmaize, when theseed was treatedby mutagen, theembryo is alreadyin multi-cellular
structure. The mutant tissues may have developed already, the top male tassel
primodial cells had been separated from the lower node female flower primodial cells, making self pollination within
the plant as not-real-self-pollination in the sense of mutagenesis. This is shown in the color pictures in this file.
Seed embryo had been developed in multi-cellular structure.

This picture shows the presence of mutant tissue development by the leaf color. When apure line was treated, generally no such mutant tissues develops in the M1. But
sometimes at a rate of a few cells in the millions of mutational unit (cells) double mutationof the same gene locus might occur to give mutant sector like this. The frequency of suchsectors are very low, but it suggest successful treatment, as it occurred in rice or tomato,
too.

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Practice Suggestions for Mutation Breeding

In this picture, pure white sectors
developed on a tiller or branch, meanwhile
the plant shows light green sectors on oneof the male tassel branch. Thesemutation might be independent events asseen in the color difference.

Here the mutant sectors on the maletassel is albino or white.
This is the detected C mutation from C-I, color inhibitor gene. All
dominant (C-I, Sh, Bz, Wx) was treated and pollinated with allrecessive tester. This picture shows the cases of C (color) mutations.
Please note the cob at the center has one purple colored kernel at thebase of the cob, suggesting that the mutant sector came in only here,
and the rest left un-mutated. The two cobs at right might be thecases of intermediate (leaky or mis-sense) mutation giving lighter but
clearly light purple color.
Waxy gene is a precious marker gene in genetics and mutagenesis
work, as it is expressed both in the endosperm and in pollen grains.
Further more the waxy starch has been sought in the East Asianpeople, in rice, sorghum, millet and other cereals. The waxy geneused in the maize genetics are said to be found in the collection from
North-East region of China. Here in this picture, the treatment andthe detection was done as the case of the C mutation, andsegregating 1 to1, as explained above.
Thus detected mutant grains are the hybrid between the induced wx
and the tester wx. This “heterozygous status make theisolation/extraction of the newly induced gene very difficult.
Therefore special pollination methodology was developed to isolate
the “imbred line mutant” by use of the marker gene.
The mutagenesis treatment to the all dominant line and pollination
with a tester were basically the same. But the pollen grains used todetect the mutant was an equal mixture of pollen grains of the testerand non-treated imbred line. The tester pollen, 639, and the pollen
from original non-treated 6311R were measured, using a smallbalance, equal weight, mixed and used to pollinate using a dry
painting brush. The detected mutant cob produced 3:1 segregation,in stead of 1:1 as shown previously. Among the normal segregants,
some were heterozygote with the tester, but others were inbred line
(sib pollination) and some segregated the induced wx seeds which
will be further isolated as an “inbred line mutant”.

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Problems here may be the use of specific marker gene. In case of such marker gene is not available like a case of new
disease resistance, this system may not be applied. Please refer to the description in the text part.

Mutagenesis in maize (mutagen)Mutagenesis in maize needs some special considerations. There may be two important problems regarding maize
mutagenesis, 1) appropriate mutagen and 2) chimerism. For Chimerism problems, see the other file “Maize Chimera etc.”
This list shows some personal results of E.
Amano. In maize, ionizing radiations had
produced several detectable mutations, but nomutant line survived in later experiments asshown in the cob pictures shown here. Aftermutagen treatment of top left material, C-I, Sh,
Bz, Wx (all dominant), the M1 plant waspollinated by the top right tester (C, sh, bz,wx). Detected wx mutants were planted andself pollinated. Lower left three cobs are fastneutron induced wx and right six cobs are
EMS induced wx mutants. The segregation
pattern and seed sets are quite normal in theright group, but the radiation induced threecobs show less seed fertility and modifyed
segregation, suggesting retarded survival of theradiation induced wx gene/chromosome (Amanoet al. 1965Mut.Res., Amano 1972 GFSymp).
This strange reaction of maize plant had been
pointed out around 1950 by the pioneer researcher
L. J. Stadler. Compared to fails to get goodmutant by radiations, chemical mutagens,
EMS, was very effective producing evenintermediate phenotypes (leaky or
mis-sense mutation). Nitroso-guanidine
is said to be a highly effective mutagen inother organisms like bacteria, did notshow promising results although it was
only a preliminary trial.
Further detailed report was referred here
the one by J.P. Mottinger in 1970 (inGenetics). The lower part illustration ismade (by E.AMANO) according to thelist shown in the report (upper part). The
report suggested that out of several
analyzable mutant lines showed that they
were still some size of deletion mutants.

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4. Tomato (also Capsicum peppers etc)
Tomato and Capsicum peppers may look very different from rice, but genetically it is diploid, self-compatible
and propagated by seeds, i.e. comparable to rice. Insects may mediate the pollen but basically they are self compatible
plant. The difference from rice may be the objectives of breeding. The target of production of rice is the seeds, andfruits for tomato. In terms of mutagenesis and mutant selection, fundamental part for tomato and hot-pepper（Capsicum pepper）may be common with rice, e.g. reduction of growth by mutagen treatment may be comparable,
except in rice, seedling height (i.e. length) is a good indication for treatment damage, but in tomato, fresh weight or dry
weight (i.e. weight) increase after certain growth period should be evaluated.
In Japan, a variety of tomato was registered and released as “In vitro propagated variety”. This variety`Koshi-no-Rubi` is “a half-way variety from cross to fixation”, so that the seeds still segregate between the two parents,
ordinary tomato and so called mini-tomato. This high quality fruit can be sold in big cities, but the seeds of it willshow further segregation and can not reproduce the same high quality. Thus the major production area can continue to
produce the high quality harvest as a local specialty, if in vitro produced young plants are restricted to the region.
Unfortunately, ordinary mutant variety has no such restriction mechanism. But the idea of limitation tocertain region may be a good argument topic.
1) How to prepare starting materials
Some nurseries/seed-companies sell F1 hybrid seeds for practical cultivation. They show very goodagronomical performance by its heterosis as a hybrid (F1). For mutation experiments, these hybrid seeds should
not be used. Two independent recessive mutants of a specific locus may produce normal (dominant)recombinant seed at a rate comparable to spontaneous mutation frequency. Such recombinant normal seed may
be easily misunderstood as “Dominant Mutation”. Crop breeding by use of mutagenesis has its power inmodification of only a few genes in the original genotype. Use of homozygous pure line is strongly advised.
Ideally, like in rice or in barley, the expected material line should be planted in isolation plot or isolation
harvest by bagging isolation. Handling of harvested isolation seeds may follow the usual laboratory system. A
dropped seeds should not be picked up, because such performance is the source of contamination in experiments
in genetics. Breeding is a practical work and if good result is obtained regardless of the sources, contaminatedseeds or well maintained, it is considered as a success. However, to keep high reproducibility, contaminatedseeds should be carefully separated from the materials for mutagenesis.
If you continue the mutagenesis work for several years, a yellow green viable mutant detected in earlier
experiment may be a very good genetically MARKED material to avoid possible contaminant seedling. If you
are using homozygous yellow green mutant line and don’t have any other yellow green line around, thecontaminated seed/seedling will show normal green color at the seedling stage. The you may exclude from yourexperiments as contaminant. This method had been successfully used in rice (at Inst. Rad. Breed./Japan). Suchmarker gene may be easily excluded from the promising mutant line by back crossing.
Condition of the seeds, i.e., whether they had been kept in a desiccator of specific condition or not, may
be important, if reproducibility of the experiment is important like the case of a pilot experiment of a project.
However, if you simply want to have a favorable mutant line in a specific variety, such condition may not be
important, if compared to the purity of the mutagenized materials.
In case of hot pepper, seeds should be carefully handled, perhaps by use of tweezers to avoid
contamination of fingers by the very hot component of the fruits.
2) Mutagens suggested
The materials represented by tomato, dry and dormant seeds may be sent out to irradiation facility allover the world, if so wanted. In another words gamma-ray may be the most convenient mutagen for the seeds ofthis group. The flat seeds may also fit for the ion-beam irradiation where penetration of ion beams is often arestriction factor. As described in rice, in case of gamma-ray or X-ray irradiation, the secondary electron
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equilibrium should be considered. Usually three mm thick plastic plate (acryl-plate etc.) covering may be goodenough.
If you have good aeration in your chemistry laboratory you may want to use chemical mutagen by
yourself. In case of chemical mutagen, e.g. Ethyl methanesulfonate (EMS), very high mutant induction rate may
be expected. But good care should be taken as most of the chemical mutagens are also known as carcinogenic
agents. Please refer to the introduction, or description for maize.
In case of EMS, alkaline degradation of the agent may be suggested. Prepare a plastic bucket in an
aerated fume chamber and make NaOH solution of about 0.1% or so. Put all the tissue papers, glass wares,
tweezers and waste/rinsing solutions in it for at least one week. Then discard the alkaline solution after appropriate
neutralization with acids (HCl etc).
3) How to decide treatment dose
Basically the same way as described in RICE, but instead of seedling height, fresh weight of two week
seedlings may be used. For official presentation, use of fresh weight or dry weight will depend on the facility
available. For quick assessment, fresh weight will be good enough. Good growth and survival should beguaranteed by small but clear decrease of the growth, by 20 % or less.
In case of chemical mutagen treatment, solution uptake by the seeds may not be much when compared tobeans (they imbibe very large amount of water). But the damage by the soaking treatment to the mutagen
solution should be carefully examined.
In case of irradiation, dry seeds may be left for a few days before sowing, but wetting of seeds bychemical mutagen treatment may need immediate sowing. There are reports describing about drying back after
soaking treatment, but without making preliminary tests under your own conditions, it will be better to sow the
wet seeds immediately. Please remember that after pre-treatment soaking and mutagen treatment seeds might
have started to germinate.
4) How to grow and harvest the M1 generation
In any mutagenesis work, M1 generation is important only to advance the generation to M2 (or M1V2 incase of vegetatively propagated plant). Therefore, as far as good amount of seeds can be harvested from M1
plant, high density planting or any other savings in the field work may be admitted. Usually each M1 plantshould be recognized as different individual M1. Pest control and other good maintenance should be performed
to assure good harvest of seeds from the M1 plants. Market quality of the produce (fruits) is not important.
The population size of M1 may be more than a few thousands, in most of the cases. Suppose a mutation
frequency of 0.1%, treatment of 1000 seeds will produces one mutant or not at all. There was a survey in Japanshowing that in most of the successful mutagenic treatment, the M1 populations were more than 10 thousands. It
may sound very large, but it may be achieved if market quality of the harvest is not considered, and high density
planting can be practiced.
Depending on the reproductive nature of the plant species, i.e. self-compatible or not, pollination system
may differ, but most of tomato and hot peppers are self compatible. If insect shield is available and seed/fruit
can be obtained under such condition, it will be the best. Self pollination in M1 generation is the fundamental
factor in seed crops for mutant induction and selection.
M1 plant may often show somatic mutation sectors in its leaf. Even if the seeds might be complete pureline or in other words, complete homozygotes, it may still show such somatic mutant sectors, albino, yellow orlight green color. Suppose a case of somatic mutation frequency of 0.1%, also occurs in the other counterpart ofthe same locus, as a possible simultaneous mutation. Occurrence might be 0.1 % x 0.1 %, that is one in
1,000,000. This may suggest that in every one million treated cells, the two simultaneous mutation may occur.
In case of M1 plant, it may correspond to one in 100 plants or so. Such mutant sectors in earlier leaves will be
wasted before incorporation to M2 seeds, but they may be a very good indication of successful mutagenic
treatment.
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Practice Suggestions for Mutation Breeding

Although the somatic mutant sectors may be a good indication, but please do not start selection of mutant
phenotype in M1 generation. Most of the induced mutant genes are still hidden in the tissues as they arerecessively inherited.
Harvest of the fruit may follow the case described in rice. But perhaps, single round harvesting may beappropriate in most of the cases, as the seed characters are not so important like rice and other cereals. Mutations
expressed in the seeds may not be important in these seeds, unless it may be used as a genetic marker. After the
harvest of well ripen fruit, seeds should be separated/washed/cleaned and separately stored in small coin envelops
for every M1 plant, until sowing.
5) How to screen the wanted traits
In this crop group, mutant character expressed in the M2 generation will be important. So, the seeds
harvested from M1 plants should be sown and carefully assessed. Supposing 3:1 segregation ratio, ten to 15
seeds from each M1 plant may be suggested to find more than two mutants in the segregating M2 family. In
mutation breeding procedures, there is a theory suggesting to sow one grain from one M1 unit (ear/panicle) for
better economy in the field work. But to find the same mutant phenotypes in the same family will give good
confidence to the researcher, no doubt.
The sowing and/or transplanting system will depend on the institutional system. But the first mutant
recognizable will be albino (white: Chlorophyll-less) seedling. In practical agriculture, it may be of no use at all,but albino seedling will be a very good indication of the successful mutagenesis treatment. At the seedling stage,
dwarfness may be also recognizable if so intended. But perhaps in most of the cases, disease resistance and/or
fruit quality or fruiting-type/plant-type may be the objectives.
M2 plants should be planted out to the screening fields as much families as possible, but here again,
market quality may not be so important. Suppose a mutation frequency of 0.1%, 1,000 M1 families might notgive promising fruit quality but 1,001th plant might bear interesting fruit. The breeder’s eye to examine the plantcharacter will be, of cause, important. Detailed quality examination should be done after market quality
cultivation in M3 or later generations. If you find a promising mutant, then make backcross to the original line to
exclude possible accompanied “unfavorable and simultaneous mutant gene(s)”.
If you have good screening field, e.g. disease infested field or other abiotic stressed fields, gross survival
screening may be performed. Mix your seeds and sow in the screening field, then critically observe and selectthe survived plant. Often rescue transplanting the survived plants to normal propagation field to harvest the
mutant seeds should be considered.
6) How to brush up the mutant
As often mentioned, mutagenesis treatment might induce also other unfavorable mutations
simultaneously induced in the same materials. In seed propagated crops, they may be cleaned out by backcrossto the original parental line. It requires one more generation to purify the mutant line, but still the effect is clear,no doubt. This kind of cross to the original line is to retrieve the original genotype except for the mutant gene,such may be considered as the case of “direct use of mutant” in breeding project.

Mutagenesis in Tomato and Capsicum
Tomato, capsicum (hot pepper), egg plant are very alike in the mutational
work. Only in the capsicum work, you must be very careful about the very
hot component contained in the fruit material. Otherwise, genetically thefundamentals are the same as in rice.
This picture of tomato M1 seedlings shows light colored leaf sector in M1
plant. As shown in this picture, all the other seedlings are normal. The
occurrence of such mutant sector on M1 plant might be the samemechanism as suggested in other part of the descriptions, e.g. in maize.
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Practice Suggestions for Mutation Breeding

This capsicum seedling also shows white or light green mutant sector on the
M1 seedling. If only part of the leaf (upper side or lower side) was affected,
the color might be looked light green.

The chlorophyll mutant sectors on M1 plant need special genetic
explanation but the one shown here might be understood as some kind ofphysiological mutagen damage, or kind of damage by the agent/drugs (inthis case, EMS). Abnormally developed leaves suggest the treatment was
effective to give damages, but genetic effect is not suggested.
Mutant searching is again a mass screening job. In this experiment, albino mutant in the same genetic line was sought,
and several mutants could be obtained.

Several albino seedling could be detected

Generally, albino seedling may have noeconomic values, but for academic studies orvisual cell markers in case of cell fusion
experiments, it may be a good visual marker.

In tomato or Capsicum albino mutant grafted on normal green plant can keep growing and give seeds if self pollinated.

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5. Cucumber and melons
This group is generally monoecious but male and female flowers are separately developed. In most of
the plants, they are self compatible but when the seeds which has developed to have multicellular seed embryo,mutagen treatment of seed will give severe chimerism, i.e. this side is a mutant but the other side is non-mutatednormal tissue. To overcome this chimerism problems, two systems may be suggested.
1) Seed treatment.
Seed treatment may be done like other seed crops, rice or barley etc., but cut back the vine twice to give
tertiary vine. The chimerism might be reduced in the tertiary vine, so that the self pollination within thesame tertiary vine may be comparable to true self pollination, perhaps segregating mutant in the next
generation (M2).
2) Pollen treatment to suppress chimerism in the seed embryo.
In order to suppress chimerism in the seed embryo, irradiation of pollen/male-flower was successful (Iidaand Amano 1990 Gamma Field Symposia No29 95-111). However, for this method, irradiation facility
should be at hand at the time of flowering.
In this method, higher radiation dose may reduce the fertile seed of the fruit. It may be difficult to assay
appropriate radiation dose, but theoretically most recommendable method, although radiation source must beat hand.
In the following practical suggestions, method 1) above will be described, as the method may becomparable to other seed crops, except for the chimera ceasing method.
1) How to prepare starting materials
In some crops of this group, hybrid seeds may be soled in market. But as explained in the Tomato Section,
inbred line should be selected. If time allows, confirmation of artificial self pollination in an isolation plot is strongly
suggested. In market, young fruit of cucumber is sold, but for harvesting of seeds, the fruit will grow much larger.
The number of seeds needed in preliminary experiment and for the main experiment(s) should be carefully planned.
In watermelon, seedless fruit may be produced from triploid plant, but it may not be a reasonable material formutation induction. If a good mutant line could be selected it may be used in such very special purposes.
2) Mutagens suggested
The seeds of this group are usually uniform and flat, except for some pumpkins and squashes. They
may fit for ion beam irradiation, but perhaps gamma-rays and X-rays may be suggested as convenient mutagens.
In maize, a very strange and special plant, chemical mutagens should be used, but in other materials, like thiscucumber group, gamma-rays or X-rays will give good penetration and results.
If chemical mutagen can be used, the water uptake in the early phase of soaking treatment should be
carefully assessed, although the water uptake will be not so large as in beans. For the method of water uptake
assessment see the RICE Section3) How to decide treatment dose
The dose assessment idea and method are comparable to the RICE Section. But in stead of seedlingheight, fresh-weight or dry-weight may be used as described in the TOMATO Section. Generally, 20 %decreased level after appropriate growth of the seedling, may be used as the dose will show clear decrease as thetreatment damage but still showing good growth to give necessary M2 seeds.
The description here are for the method 1) in the introduction. So a good growth to give the tertiaryvine with good male and female flowers must be expected. Therefore a little less than 20 % decreased dose may
be advised.
In case of chemical mutagens, the same will be applied.
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Practice Suggestions for Mutation Breeding4) How to grow and harvest the M1 generation
As mentioned before, self pollination within the same tertiary vine should be considered. Other
cultivation system may follow the description in the TOMATO Section. One practical difficulty here may be
the plant shape and the management of how to cut back the hundreds or possibly thousands of M1 plants. Thelong vine and the heavy fruit for seed harvesting should be considered in the field management.
As described in other Sections, M1 plants and fruits may not be a market quality, but should be a good M2
seeds producer. Perhaps one self pollinated fruit from each treated seed will be more than good enough to obtainthe M2 seeds. Seeds should be cleaned separately for each fruit i.e. M1 plant and separately stored in (small coin)envelope.
5) How to screen the wanted traits
In this group of crops, the target traits may be plant type, fruit quality, disease resistance, abiotic stressresistance etc. However, M2 planting for examination of the traits are basically the same as TOMATO or RICE
etc. But here the field management/planning is important as it takes much area compared to RICE or BARLEY.
But please be noted that the mutant phenotype is expressed in the self pollinated M2 generation. A possible
economic planting will be dense planting only to detect the mutant phenotype, neglecting the market quality.
In case of disease or abiotic stress screening, dense planting and survival selection may be applied by useof pooled M2 seeds.
In either systems, family planting or pooled M2 seeds, again appearance of albino seedling will be a very
good indication of successful mutagenesis treatment.
If you can obtain viable yellow green plants or spine/hair-less mutant which may be used as a genetic
marker, it may be useful in demonstrating the chimera tissues after the mutagen treatment. It may be also usefulas a marker for a variety newly developed. If not desired in the future breeding procedures, a single backcross
may exclude the marker-mutant gene.
6) How to brush up the mutant
If any promising mutant could be obtained, make at least one backcross to the original line to exclude
unfavorable simultaneous mutations, as often described in other Sections.

Mutagenesis in cucumber
Cucumber and other melon group has male and female flowers separately like maize. The difference may be theflower bearing position on the plant. When a multi-cellular seed embryo was treated by a mutagen, chimericmutational sector might be developed on the M1 plant, making self pollination within the plant not a true self pollinationin terms of mutagenesis work. To
overcome this difficulty, there may be twoplans applicable; 1) mutagenesis treatment
before seed embryo development, and 2)
reduce chimeric tissues by pruning(cutting-back) and pollination within thetertiary vine. Pictures shown here were
examples from the former plan. Male
flowers were collected in the morning,
irradiated by gamma-rays and thenpollinated to virgin female flowers. Thus,
each seeds developed might be different, in
terms of mutational event, but within theseed embryo, all cells might have the same
genotype, so that the self pollination within149

Practice Suggestions for Mutation Breeding

the plant, will be a real self pollination segregating the seedling mutant shown here. Again, albino seedling may be avery good indication of the mutagenesis.
However, for this kind of treatment, the researcher must have irradiator at hand. The second suggestion may be agood possibility for ordinary field breeders, as he/she can ask the treatment to other institution or he/she can treat theseeds by chemical mutagen in their laboratory.

This is a normal leaves of cucumber.

By the Plan 1 method (treatment of half generation upstream)
induced leaf shape mutant.
The mutant is the same line as the lower right of the our pictures,
Fig.4. The quality of the fruit was the same as the original, butby this mutation, we could get a good marker gene for the leaf
shape. The actual effect is unknown yet, but the twines havebranches (seen at the lower left corner for example), compared tothe original line which has no branched twines.

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6. Chrysanthemum
Chrysanthemum may be a good model representing vegetatively propagated crops. Ordinary varieties arehexaploid but having good heterozygosities in its genotype, making mutation work rather easier. In vegetatively
propagated plants, tissue culture techniques are often very useful methodology. In triploid and seedless banana, inwhich ordinary propagule unit is very large and difficult or often impossible to irradiate in a gamma-hot cell by large
numbers, the late Dr. F. Novak of IAEA (Seibersdorf Lab.) developed adventitious embryony techniques, by which
thousands of tiny embryos can be irradiated in a pair of Petri-dish.
Presently, mutagen treatment may be possible in almost all plant species, but in some plants thescreening/selection methodologies may still to be developed, or in others still very laborious or need much efforts.
Here, Chrysanthemum was taken as a model plant.
1) How to prepare starting materials
There had been many successful reports on inducing flower color/shape changes and plant type changes.
However, the change/mutation depend on the genotype, i.e., how much heterozygous, the used line was. The
popular cultivars may be hexaploid, a difficult case in mutation breeding, but if your material is aa, bb, and Cc,
mutation from C to c will give cc color. But if your material was aa, bb, and CC, one C mutated to c will stillshow C color, until you mutate both CC to cc. Therefore, it may be strongly suggested that before starting fullsized experiments, you should make preliminary mutagenic experiments with high dosage and examine if yourmaterial can give mutant color sectors or not. The color sector or patches may be only a few mm or evenmicroscopic size. If high dosage of the mutagen still did not give favorable color SPOT, it may be better to giveup the line and change to other line.
This preliminary examination to see the possibility to change the color of ornamental plants may becommon to all the plant materials of this type. For this preliminary test, high mutagen dose may be applied ashigh as it still give a few flowers, on which small mutant sectors can be examined. In the full scale experiment,
the dosage should be kept as small as possible, and as many mutant as possible should be selected for the best
performances. Please be noted that in these plant group, exclusion of unfavorable mutant genes induced
simultaneously can not be done by back-crossing like the case of rice or other seed crops.
Chrysanthemum is vegetatively propagated plant, so its propagation may need only good labeling work.
One cutting of about 10 cm long may have ten or more axillary buds if fully grown. So, for acute irradiation, atleast one hundred cuttings may be produced for better results.
2) Mutagens suggested
Many good results have been reported by gamma-rays or by ion-beams. In cases of gamma-rays or
X-rays (hard X-rays, more than 150 keV or so) irradiation sample may be bundled, if thickness of the sample isthin enough in terms of distance from the radiation outlet. In case of ion beam radiations, very often penetrationand linear energy transfer, and Bragg Peak position should be considered. For these physical factors, thebreeder/biologist should consult well to the engineer/physicist.
Compared to these ionizing radiations with high penetrability, chemical mutagens may need good
examination of treatment methodology. It may be applied to cultured tissues in the culture bottles, but for that
purpose, sterilization of the mutagen solution perhaps through filtration by Millipore system may be required.
Most of chemical mutagens may be degraded by autoclaving.
Recent studies suggest tissue culture procedures especially when callus culturing for certain period oftime is included, inherent retro-transposon may be activated and produces insertion-mutation in the material.
Such retro-transposon induced mutation may be stable in the traditional cultures, or perhaps simple in vitro
meristem cultures. It may be reactivated only by the next callus induction and culturing.

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Practice Suggestions for Mutation Breeding3) How to decide treatment dose
Chrysanthemum and other vegetatively propagated crops are difficult materials to examine or describe
the optimum or favorable mutagen dose. In literatures, there may be various suggestions in measurement of thetreatment effect. After the most popular irradiation work, fresh/dry weight may be measured as indication ofretarded growth. Again, like in the case of rice (seedling height after some growth period), the dose which
retarded the growth to half is too strong for mutant screening. In case of seed crops, back-cross to the originalline can exclude unwanted mutant genes, but in vegetatively propagated crops, the procedure can not be applied.
Therefore, the selected promising mutant line may carry the simultaneously induced mutant genes.
One most probable way to escape from such drawback may be 1) low dose treatment to reduce
accompanying mutant gene, 2) select as many (independent) mutants as possible even with the same phenotype.
For example, if a yellow color is wanted from a pink flower line, select two or three or more independent yellow
colored mutant line, e.g. yel-1 from cutting No3, yel-2 from cutting No24 and so on. Then take the best one as
the best performing mutant line.
As for the gamma-ray dose, experienced nursery breeder once suggested the use of only 250 rad (= 2.5
Gy). The value is much far below the RD-50 (reducing dose for 50%). Such small dose may require large
population in M1V1, M1V2 etc. but no doubt the accompanying unfavorable simultaneously induced mutant
genes may be reduced very much.
Please be noted that in the preliminary experiments described in 1) above, rather very high dose issuggested to test the line. But when you start your full scale project, use as low dose as possible in your field
capacity to incorporate as the screening materials. If low dose is used, normal non-mutant (perhaps 99.9% or
more!) plants may still be put to the flower market to save/earn necessary budgets.
4) How to grow and harvest the M1 generation
In vegetatively propagated crops, selection is directly made in the field. In case of pest resistancesearching, inoculation or exposure to the pests (disease or insect) may be required, but plant/flower color,shape/type of plant or flower may be selected directly in the growing field. The selected plant may still show
chimerism in M1V1 generation field, but perhaps in M1V3 or later population chimerism may not be shown. In
most of seed crops, plant generations are renewed rather automatically, but in vegetatively propagated plants, theoriginal M1V1 plants may be still alive in the field. If cut back in rather hard degree, the newly grown shootmay be used as M1V2. Such use of M1 plant depend on the condition/capacity of the field. Anyway,
screening of the chimera-free mutant line should be done in M1V3 or later generations.
5) How to screen the wanted traits
In Japan, the black-spot-disease resistant sport of Japanese pear `Nijisseiki` was found on a tree, planted
in a gamma-irradiation-field and irradiated by chronic gamma-rays for almost 20 years. However, in this case,the period may be considered as the time necessary to be screened as fully grown mutant sport. Mutation occurs
in a single cell, then it grows into larger and detectable size. In vegetatively propagated crops, this kind of time
lag to reach selectable size of the tissue is necessary.
In this black-spot-disease resistant sport of Japanese pear, registered and released as `Gold Nijisseiki` wasthen used to develop toxin-screening-method which accelerated the other two resistant lines in the Japanese pear.
In case of flower colors of Chrysanthemum, petal tissue of a new mutant color sector can be cultured invitro to let it be a single re-differentiated plant.
6) How to brush up the mutant
In this vegatatively propagated crop group, vegetative generation advancing from M1V1 to M1V3 ormore is inevitable. To shorten the period, tissue culture method as a tool for obtaining chimera-free mutant
tissue may be useful.
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Even if mutant tissue can be selected in M1V3 or later generation, the structure of a meristem (growthcorn) may still suggest some kind of instability which may be observed in the practical agricultural field. To
avoid this rather inherent chimerism in the meristem, some people suggest the use of tissue culture to obtainchimera-free mutant line. To obtain chimera-free tissue, callus formation at least once will be the easiest way,
but elongation of the callus period may result in activation of retro-transposon as mentioned above. The periodof the callus culture should be kept minimum.

Chrysanthemum is preferred flower all over the world.
Colors, shapes, growth habits are all breeding objectives for long time
incorporating spontaneous mutations. These are example of large flowers
in Japan.

Some cuttings may be irradiated then put
to soil to give a clone, then often color
mutant sector appears as this picture.
However, please be noted that the mutant sector is a somatic mutation sectors andthe appearance may strongly depend on the genotype. If completely homozygous
and polyploid, mutant sector may not be expressed. To start your work, you
should run a preliminary experiment whether your material can give somatic mutant
sector or not.

This is a case of white mutant sector developed on a pink flower
variety.

Red mutant sector in yellow flower variety.

Left lower portion of theyellow flower variety, redmutant sector/flower is seen.

Yellow mutant sector on white flower. Perhaps this white color might
be a suppressed form (dominant) of yellow color, due to a dominant color
inhibitor gene, inactivation of which revealed the hidden color of yellow.

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An example of yellow flower on white variety. Mutant colored sector may be cultured on
appropriate medium and mutant line may be developed.

A private nursery in Japan developed six color variant having the same plant and flower types. They named them“Bio-giku-Rainbow”. In the close up picture, lower left is the original variety.
The lower picture shows the view of flowering season. Plant type and flowering times all very alike.

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7. Difficult Crops
Genetically and basically there may be two fundamental applications. 1) mutation induction in
homozygous crops through Mendelian Segregation of mutant homozygotes; 2) mutation induction in alreadyheterozygous genotype, by inactivation of dominant gene. In rice or in some variety of Chrysanthemum, it may
easily be applied, however, very often application seemed very difficult, due to 1) very long life cycle to obtain
THE SEGREGATION, 2) unknown genotype; 3) dominant gene/phenotype desired etc. In the last case,induced mutation methodology may not be appropriate, and other method e.g. gene transfer by cross-breeding(and repeated back-crossings) or gene (DNA) transfer by transformation method.
In case of “very long life-cycle”, tissue culture may be helpful but it may not a decisive method.
Special long term project may be needed. For the case of “unknown genotype” a preliminary experiment withhigher mutagen dosage (LD-50 for example) may be helpful to examine the possibility of occurrence ofdetectable mutant sector. If you get some positive reaction out of the high dose experiment, then reduce themutagen dose to ordinary lower level (half or less of the LD50dose).
For self incompatible crops like buckwheat or radishes/Brassica(2x) see the next section of “8 Self
incompatible”. In these crops, Mendelian segregation may be difficult to have. Experiments in Drosophila fly
or mouse and/or mammals (extreme case of self incompatible organism) may be a good reference.
1) How to prepare starting materials
Preparation of the starting materials may be the same as the other cases. To leave the untreated control
will be necessary. Materials should be prepared as much as possible including preliminary experiments. The
researcher should be good in handling of the material plant, both in nursing and observations.
2) Mutagens suggested
X- or gamma-ray irradiation will be the easiest mutagen with its high penetrability to the plant materials.
These radiation may not leave induced radioactivity like the case of neutron irradiation.
Some chemical mutagens are known to have much higher mutagenic action, but application may needvarious know-hows in its application in vivo or in vitro.
3) How to decide treatment dose
In case of the “preliminary experiment” suggested above, high dose of around LD-50 (the dose by which
50 % of the material will be dead after some period) may be suggested. But in the project application, about
20 % down dose, described in “Rice”, that is far less than LD50 dose, should be used.
4) How to grow and harvest the M1 generation
This may depend on the reproduction pattern and genetic system of the material. If your material isheterozygous, or positive reaction in the preliminary experiment, you may start selecting in the M1 generation.
If your material has very long life cycle but self compatible, basically follow the rice plan. In othercases, refer to other plans suggested.
5) How to screen the wanted traits
If you can detect any albino seedling, it may be the first step of the success. Then proceed to screen any
disease resistance or abiotic stress resistance. This general strategy may be applicable in most of the cases,
however, in rice bacterial blight resistance screening, the powerful chemical mutagen, EMS, produced many
albino seedlings, but no resistant seedling was found (Dr. K. Nakai (personal comm.)). With reactor thermal
neutrons he obtained many resistant mutant. This may be an exceptional case and need further studies, but one
possibility may be that with point mutations (induced by EMS) may still produces some nourishing proteins, butwith the high LET radiations, like neutrons, the deletion type mutations may not produce the nourishing proteins155

Practice Suggestions for Mutation Breedingnecessary for the pathogenic bacteria.
6) How to brush up the mutant
Simultaneously induced mutation may occur like other plant group. The same methodology may be
applied here also. But to overcome the difficulty of the very long lifecycle, try to induce and screen as muchmutant as possible, in search of better mutant which may perform well without laborious back cross and otherwork.

Orchid and other examples
Difficulty in Mutation Work may be due to 1) very long life cycle, 2) self
incompatibility, 3) polyploidy and 4) seedlessness, and/or their
combinations.
Den. pharaenopsis is a very important exporting flower in the tropical Asia.
Variety improvement has been done by cross-breeding incorporating color
variants and some remote species. The difficulty mainly is due to theperiod to reach flowering, but invitro culture system is very well developed for these plants and mutant
induction may depend on further endeavor of researcher/breeder.

Most of the ornamental orchids have typical (normal) shape of the species,
but in some oriental orchids,
abnormal double flower like this “Yogo-chou” (above picture) is highly
evaluated.
Normal shape is like the left one, (This variety lacks dark dots, which isusually seen in the wild type).

Self-incompatible plants like buckwheat (Fagopyrum esculentum) may carry somemutated gene in it, so that after mutagenesis chlorophyll mutant sectors may be seen likethis. Usually the border of the mutant sector is very clear and the pattern indicates thedevelopmental pattern of the tissues.

Oilseed rape (Brassica napus) is tetraploid and self compatible, but radish and most of other diploid Crucifer vegetables
are self incompatible. However, old landraces have been developed perhaps by incorporation of natural/spontaneous
mutations. Famous two land-races in Japan are shown here.

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Practice Suggestions for Mutation BreedingThin but very long Moriguchi-Daikon Late, thick but very large radish two
Sakurajima-daikons are seen here (compare the size with the lady’s foot).

Sweetpotato is hexaploid and self incompatible. As a mutagenesis trial for this plant, leaf petiole dipped into chemicalmutagen solution (EMS) seemed to give small root-bulb which may then planted in the field for searching mutant
sport/sectors.

The seedless plant like banana is still possible to improve by mutation, if embryoids can be induced by tissue culture,
hundreds of plant can be irradiated in one Petri dish. The techniques had been developed by Dr. F. Novak in IAEA
laboratory in Seibersdorf, Austria.

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8. Self incompatible crops
Some crop plants have out-crossing nature, which disturbs the usual mutant selection procedure in M2
generation. Cucumber, melons and maize plants are grouped in this out-crossing/out-pollinating, but they are
self-compatible, that is, by artificial pollination, we can expect mutant segregation in the selfed M2 progenies.
However, some plants are SELF-INCOMPATIBLE and need special handling in detection of mutant plant.
Diploid Brassica, radish (Raphanus) and buckwheat (Fagopirum) are such difficult plants. At the last part ofthis section, an example tried to break through this difficulty in feeding crop is attached for your reference.
Self incompatible true out crossing crops will react in the mutagenesis experiments like animals or
insects. Therefore it may be a good Idea to use mutation experiments in these, e.g. Drosophila. The Idea is to
mimic small population cage system. Treated M1 plants may be allowed to pollinate freely, but in a small
population group. The attached reference suggest such small population groupings. By that way, within asubgroup after a few generations, recessive homozygotes will start segregation. This may be observed byproduction of albino seedlings in the progeny. Albino plants are useless in crop improvement work, but it is avery clear indication of successful mutagenesis treatment done and very appropriate generation to detect the
mutant plants.
But, please be noted that the number of subunits in an experiments may amount to more than hundreds.
Suppose a mutant frequency of 0.1 % and one subunit may contain ten treated seeds for starting, 100 subgroup
might have 1000 treated seeds, then you may find one mutation (of a few plants, perhaps) or not at all.
Another possibility may be to induce self compatible mutation at first, then apply the method used in rice.
Theoretically a very good Idea, but at first, you should search a reliable reference.
Buckwheat is well used in Japan making good noodles from the flour of the harvested seeds. Once I
tried to induce waxy starch in buckwheat, expecting the mutant character will be expressed in pollen grain, whichis haploid, so I examined pollen grains in the buckwheat field. Theoretically it should work well, if otherdeskwork businesses did not disturbed me. If such mutant could be found, the branch or the plant will bemarked and subpopulation will be planted to have segregation in the following or in later generations.
In radish, bud pollination may give selfed progeny, but hundreds, or more, of bud-pollination will be a
tough work.
If your target phenotype can be detected in cultured tissues, in vitro culture and direct detection may givesome results. But again, the mutant phenotype may be suppressed by the normal non-mutant dominant gene in
M1 generation. Very often the planning on a paper/desk is very useful in the experiments in genetics.
1) How to prepare starting materials
In this group of crops, very often the line purity is questionable. But if the genotype impurity is so large
to disturb the mutagenesis experiments, ordinary selection breeding will give good results before mutagenesis
work. Please examine the deviation of phenotypes well before starting the mutation induction experiments.
Even if the deviation of the phenotype is not large, perhaps some genes are in heterozygous condition,
especially in chlorophyll genes. Then, the mutagen treatment will give chlorophyll deficient mutant sectors inleaves, much frequently than rice or tomato etc. Mutant sectors on leaf may eventually lost and may not betransmitted to the next generation. But the color mutant sectors on leaves will be a very good indication ofmutagen treatments.
2) Mutagens suggested
The size and shape of the seeds will differ from crop to crop. Small seeds of Brassica may be irradiatedby ion beams. Raphanus seeds are also small but somewhat critical in penetration of low energy ion beams. In
this group of crops, so far no special reaction to ionizing radiation, like the case of maize, is not reported. As forthe mutagens X- and gamma-rays may be the most appropriate.
Chemical mutagens may be used also. In this case, the water uptake by the seeds may not so significant
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like beans, but before starting the experiment, water uptake experiment should be done at least once.
Pollen culture in Brassica should be kept as short as possible, if somaclonal variation is not the purposeof the culture work.
3) How to decide treatment dose
The procedure may be described in other sections, e.g. RICE or TOMATO. Weight may be a goodcomponent to see the treatment damages after a few weeks growth.
As for the experimental planning, many dose classes with relatively small numbers of seeds will do well.
Please be noted that in seeds high in oil contents seemed to be resistant to X- and/or gamma-rays. In someexample, up to 700 Gy gamma-ray are reported as appropriate. So please extend your highest dose up to thatdose or higher (1000 Gy). As usual, the highest dose should be determined as the high dose where most of theseeds are dead. The sensitivity against ionizing radiation seems to depend on the inherent repair system and not
the size of the seeds.
4) How to grow and harvest the M1 generation
This group needs extra care in the experimental design. That is why this section is described.
Basically small population cage work in Drosophilla may be a good model to refer. Plants may be grown in thefield according to your scheme (attached reference may show one idea). Accuracy in Genetics should be
respected than market quality product.
Please do not attempt any selection, until you can observe albino seedling or other true indications ofmutant segregation.
5) How to screen the wanted traits
The last sentence of the foregoing section is important to indicate that you may start selection of mutant.
You may plant out the sub grouping as families to detect and/or select favorable/promising mutant plant.
Gross screening in biotic/abiotic selection field may be done with pooled seeds for several subgroups.
There will be one very important factors, when you find ONE promising mutant in the field. Please
remember that your material is self-incompatible material!! Therefore make pollination to or from the mutant to
the original line or brother/sister plants. The mutant phenotype may be hidden in the next generation, but the
mutant gene will be maintained in the progeny and segregate a few generations later.
6) How to brush up the mutant
The usual procedure of CLEANING should be done somewhere before the yield test. Preferably, themutant should be crossed to the original non-mutant line, as described above. In case of brother/sister
pollination, one more back crossing will be suggested as a real cleaning work.

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Practice Suggestions for Mutation Breeding

Buckwheat (Fagopyrum esculentum) is self incompatible plant. The out-crossing nature has served the plant to holdmutant genes in it. So, mutagenesis treatment very often reveals chlorophyll mutant sectors in M1. Such sectors
suggest that mutagenesis treatments has been effective. Induction of self compatible mutant may be a good idea, butinbred-weakness (reverse of heterosis) may give a new problems.

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9. Beans
This group of crop is genetically the same seed propagated diploid crop like rice. Therefore, basicallyrefer to the description on rice. However, there are two distinct differences in their behavior or handling
methodology.
1) When dry seeds are irradiated by X- or gamma-rays, the dose-response curves and suggested dose applied
may be comparable to rice. But if you want to apply chemical mutagen as water solution, the water uptake
by the bean seeds are very much larger than rice or other cereals, very often reaching twice or three times
more than its original weight. The water uptake measurement and “chemical mutagen dose” experimentshould be done thoroughly before the main experiments.
2) Another difference may be the number of M2 seeds obtained from M1 plant. To have a clear
3(wild):1(mutant) segregation, about 10 to 15 M2 seeds from possible mutant sector should be harvested.
The size of “possible mutant sector” may be observed as mutant-colored tissue on M1 plant, if appropriate
numbers of heterozygote seeds are mutagenized. Without that information, this may be just a guess, but afruit/capsule developed in a node may be considered as one M2 seeds family.
2-1) Some crops have long capsule accommodating many seeds in it, like “yard-long-bean” or “azuki bean”. Insuch cases, one fruit/capsule seeds may be considered as a family which may segregate into expected 3:1.
2-2) The other group may have only a few seeds in a fruit/capsule like “soybean” or “peanuts”. In case ofsoybean, fruit/capsule grown on a leaf node may be considered as a “family”, although this should be tested
as described above. In other cases, like “peanut”, the family may be only 1 to 3 seeds etc. In these cases,
the fruit/capsule on the same branch may be grouped (only) for convenience, although 1 to 3 segregation
may not be expected.
1) How to prepare starting materials
Most of this group of seed propagated crop may be obtained as pure line. But it may be still advised toplant them in isolated plot to have the starting materials, the true pure line seeds. The artificial pollination may
be difficult in some of the plant species, e.g. soy-bean, but artificial pollination will be important when a favorable
mutant can be obtained, to clean up the mutant line. For that purpose, some hundreds seeds should be kept away
for future back-crossing work. The seeds may be stored under low temperature.
The starting materials may be kept dry until mutagen treatment. Whether the seeds are kept in ahumidity controlled desiccator or not may be depend on the purpose of the experiment. If it is to see thereproducibility of the irradiation or other mutagen treatment work, all procedures should be well controlled andwell described. But if it becomes a routine treatment in your laboratory, perhaps just air dried seeds may beused.
2) Mutagens suggested
Dry seeds may be sent to irradiation facilities elsewhere in the world, but please respect the plantquarantine regulations in the recipient country. With X-ray or gamma-rays, again, secondary electron equibrium
may be important as described elsewhere (Tomato section). With X- or gamma-rays, perhaps sample seeds may
b e irradiated from outside of the envelope packages. But in ion beam or other delicate irradiation work, where
penetration of the radiation is critical, spherical seeds like soybean will needs extra-care in handling. Also large
seeds like Phaseolus(beans) or Arachis(peanuts) may have penetration problems. Perhaps X- or gamma rays
will be convenient, if mutant seeking is the purpose.
If you want to treat the material by chemical mutagens, water uptake and the actual mutagen doseabsorbed by the seeds must be carefully examined beforehand. The wasting method of the chemical mutagens
needs further extra care for researchers and environmental safeties. This is described in MAIZE or othersections.
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Practice Suggestions for Mutation Breeding3) How to decide treatment dose
Treatment dose may be determined as described in the RICE section. To determine the damages bytreatment dose, fresh weight or dried weight may be used, instead of seedling height. Measurement may bedone after some growth period, e.g. two or three weeks, after the treatment.
If you want to treat the material by chemical mutagens, water uptake and the actual mutagen doseabsorbed by the seeds must be carefully examined beforehand. In the water uptake experiment, about ten to 30
dry seeds may be measured every 30min or one hour for the first day, then perhaps up to the next days. If you
fined a slowed down in the water uptake, that plateau period may be appropriate for soaking treatment for three toseven hours at 25 degree centigrade or so.
To measure the growth suppression by the mutagen treatment, allow the treated seeds, irradiated or seedsoaked in a mutagen solution, to grow for two or three weeks in your nursery bed. In these preliminary
experiments fresh weight or dried weight of the seedlings may be measured like the case of Tomato.
When successfully obtained the preliminary experiment results, either with radiation or chemicalmutagen, the dosage of the mutagen may be determined as 10 to 20% growth reducing dose range, where growthand production of M2 seeds may be expected.
4) How to grow and harvest the M1 generation
M1 plant may be grown, favorably, in an isolated plot. As often suggested, the growth of M1 isnecessary to obtain the M2 seeds safely. Therefore market quality may not be important, but control the vines sothat every M1 plants can be recognized. In soy bean or some of other crops having simple erect plant types, it
may not be so important, but vine type climbers need extra cares in separating each M1 plant when harvesting theM2 seeds. For bushy plants, the same recognizing cares will be needed. Ordinary pest control proceduresshould be applied to have genetically appropriate harvest.
The harvested M2 seeds (on M1 plant) may be put in a coin envelopes separately for each M1 plant, driedthen stored until sowing. Please refer to the M2 family situations described in the 2-1) and 2-2) described above.
5) How to screen the wanted traits
The seed coat derived from maternal tissue, but if you can see through the seed coat, the cotyledons may
be examined first, like the case of rice, where endosperm characters e.g. waxy starch or not, can be examined. In
cases of seed characters, which one is the M1 and which one is the M2 characters must be learned beforehand.
The same M2 planting patterns suggested in the RICE section may be applied to beans also, although M2family definition might be different or not be determined yet. In some cases for bifotic /abiotic stress resistancemutant searching, pooled seeds may be planted in the screening field to select surviving plants.
Even in such cases of gross survival screening, sub grouping of M1 plants may be useful. Suppose you
have successfully selected two disease resistant mutant from subgroup #12, you can not tell that they might become from the same origin/mutation or independent origin. However, if the mutant was found in the subgroup#12 and #15 separately, they are clearly independent mutants of different origins.
6) How to brush up the mutant
Here again, as very often described, to clean up unfavorable simultaneous mutant gene, back crossing tothe original line is an important and necessary procedure. In some plant species, emasculation and artificial
pollination are difficult, but this is a necessary procedure in brushing up the mutant before formal yield testing.
Please name the mutant with appropriate genetic name, and so in the mutant variety name, when it
become the time.

164Practice Suggestions for Mutation Breeding

Some examples of soybean and sesame mutants
Soy-bean is useful in very wide applications, forprotein, oil and starch. In the field, this plantfixes nitrogen from the air as nitrogen sources,
having Rhyzomes in the root. The function
may be increased by “super nodulation”
mutation like this picture (second from left).
The mutant still produced nodules even innitrogen abundant hydro culture (second fromright). Agricultural meanings/effects should be
investigated further.

Sesame usually has one capsule at each leaf-node. But in Egypt, they induced three capsule for each leaf node. The
total harvest might depend on the amount of photosynthesis, but still significant harvest increase was recorded and themutant was registered as a good variety in Egypt. Three capsules per leaf node may be known to some people, but
new induction in good variety may open a new field.

Three capsules per leaf node.

Normal plant165

Practice Suggestions for Mutation Breeding

10. Callus culture
Use of tissue/callus culture method may have several faces to support plant breeding work. Embryo
Rescue may be the most significant contribution for the crop improvement. In relation to the mutation breeding
work, there may be two ways of contributions.
1) One may be to help easier handling of materials in ordinary mutagenesis work. For example a planting
material of banana is too large to irradiate hundreds or thousands of them. The late F. Novak of IAEA,
Seibersdorf Laboratory developed a method of embryoid irradiation system, by which hundreds of separable
embryoids can be irradiated in a Petri-dish. Another useful help from the tissue culture may be solid mutant
tissue/plant production from chimeric tissue.
2) The other important factor is mutant production method known as “somaclonal variation”. Some part of thisphenomenon can be explained by “activation of Retro-Transposon” as reported by H.Hirochika (1995
Gamma-Field Symposia No.34 77-91 and 1999 in Molecular Biology of Rice pp43-58 Ed. K.Shimamoto,Springer-Verlag, Tokyo, Hirochika 1997 Plant molecular Biology 35: 231-240), and an example had beenreported in a IAEA Seminar held in the Philippines in 1999 (Amano et al. 1999 FAO/IAEA Seminar
IAEA-SR-210/22: pp27-30).
The latter one occurs when callus culture is extended for a certain period of time, e.g. for a few months.
During such heavy stresses, otherwise inactive/sleeping DNA sequence start producing messenger RNA, then the
product seems to be modified to change into DNA by the help of retro-replication system producing DNA typetransposon. If the callus period was elongated too long, then too many transposon DNA are spreading in the
genotype. The reason of albino regenerated plants, often found from prolonged callus, may be explained by thismechanism.
Fortunately, such retro-transposon induced mutant seems to be very stable in ordinary agricultural
conditions, until callus induction done again for any reasons. If you do not want to have suchsomaclonal-variation/mutation, the period of callus state should be kept as short as possible. However, thereported example showed several cases of “all mutant families” in some phenotypes. As mentioned in the
introduction section of this Addendum, details are still to be studied. According to the example cases, suchnon-segregating cases seems to be limited to only a few phenotypes. An explanation by methylation of the geneseems to be very likely, although detailed studies are still needed.
In this Section, Callus culture as mutagenesis method will be described. Embryoid culture and otherhelping methodology should be referred tissue culture text books/manuals/recipes.
1) How to prepare starting materials
The preparation of the starting materials is the same way as other mutagenesis work, as far as callus
induction and plant regeneration system is reproducibly established. If your material, particular variety of a crop,
is not sure of the procedures, try to induce a mutant in easier variety, from which you can introduce by pollination,
cell fusion or other methodology.
2) Mutagens suggested
In this section, the mutagen is comparable to a stress given to the plant materials, e.g. callus induction andsome period of callus culture. Callus induction agent may be of any kind, 2,4-D etc.
3) How to decide treatment doseIn this method, culturing period as callus may be comparable to the dosage/strength. If You maintain
only a short period of time, the transposable DNA may not be many, so that the mutant induced will be very few.
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Practice Suggestions for Mutation Breeding

If the culturing period was too long, too many transposable DNA might be produced. If you can have DNA
analysis Laboratory and have a good DNA marker, then you can try direct analysis of the transposable DNA inyour material.
4) How to grow and harvest the M1 generation
If callus culture period is appropriately short and the numbers of transposable DNA is not many, cells of
the regenerated plant have only a few heterozygous recessive insertion mutant genes in it, comparable to theirradiated M1 plants. The method of detecting and selecting mutant plants is basically the same way as inirradiation method. But please be noted that the generation naming is different in the regenerated plants. The
regenerated plant from a callus is in R0 generation, like the case of transgenic plant in T0. The insertion mutant
genes are “inactivated state”, therefore to have a homozygous recessive mutant, one more planting in the field will
be needed for the plant type mutation.
If you want seed character mutant, e.g. waxy starch in rice grain, the first segregating generation will be
the R1 generation which is comparable to M2 seeds in irradiation and other mutagenesis treatment. The namingof the generation may be confusing, but please consider the expected genotypes.
The field maintenance, harvesting of R0 (=M1), and planting of the segregating generation of R1 (=M2)
are the same as ordinary mutagenesis work. Only the generation call is different. Please be careful in reportingyour results.
5) How to screen the wanted traits
If you are thin king of the callus-mutation/retro-transposon-mutation as a method of mutagenesis, thedetection/selection methodology will be the same as ordinary mutagenesis work, family wise detection or grosspooled planting.
6) How to brush up the mutant
In this methodology, the resulting mutant may be of insertion mutant, which may be searched by theinserted DNA sequence among the all genes in the genome(s). This may be a very precious mutant gene, easily
detected by DNA analysis and identified by its phenotype. The mutant plant will be also used as very stablemutant for plant breeding.
Please be noted that ordinary transposon induced mutant is very often unstable and return to the original
non-mutant state at relatively high rate. Also many cases of color spots on the flower petals might be due toordinary transposons. For the academic research work, if the gene can be identified by the inserted transposon, it
has enormous value. For ornamental flowers, it may be also meaningful. However, for cereals and otherpractical agricultural crops, such instability may not be appropriate in the breeding work.
The cleaning of unfavorable simultaneously induced mutant gene should be done as usual. Otherwise,
you can not tell which mutant gene was really effective in the later procedures, breeding or academic work.

Callus activation of retro-transposonCallus/tissue culture often brings aberrant plants when regenerated.
Hirochika reported possible participation of “Retro-Transposon” for such mutagenesis. By stresses of callus culture,
retro-transposon is copied to m-RNA, then reversely synthesized to a piece of DNA which will be inserted to otherlocus. The inserted locus is inactivated like mutations (gene splitting). This new mutation will be stable as far as
ordinary life cycle is maintained.

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Practice Suggestions for Mutation Breeding

Compared to other transposons, which moves around but the number remained unchanged, the retro-transposon
increases when the callus stage is prolonged, but the original pieces in the chromosome remains at the original position.
In the following electrophoreogramme, the original position shown at the leftmost lane (Control) is maintained
regardless of the culture period.

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Practice Suggestions for Mutation Breeding

Albino seedlings were observed as good index of the retro-transposon
mutagenesis.

Two stable early flowering mutants are segregated in this M2 field, whereM1 panicle families were planted as M2 lines.

Other example of retro-transposon induced waxy starch mutants
segregated in 3:1(34:11). In this material, japonica rice, someintermediate phenotypes are seen, e.g. top-row 2nd and 3rd grains, but thisis ordinary segregation pattern for them.

There are some strange results like this left marking pole, which indicates all
family members shown rolled leaf phenotype in M2. If it were by adominant mutation, normal plants should be segregated about 25% (or 1:3).
Methylation of the locus is suggested, but this should be studied further.

This mutagenesis system is very useful to put a marking tag to the mutant gene. The
diseased mutant in this picture (shown by the marking pole) may not be good forbreeding purposes, but it may be an excellent material for academic research work on
disease infection, as the gene can be isolated by the retro-transposon tag.

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Practice Suggestions for Mutation Breeding

11. Fruit Trees
Definition of Fruit Trees may be a little difficult. Here, a wide range of arboreal crops (including
Palms) and Banana (botanically herbaceous crop) should be included. But then we will hit many problems.
For each target species, the objectives and problems will be different. The only common factors may be thateach genotype is very characteristic to each cultivar, making cross breeding very difficult and laborious work.
However, in such materials the magic of Mutation Breeding will be very effective in one point improvement（Sanada and Amano 1998）. Another common factor will be the fact, that most of the recessive characters canbe modified by mutation PROVIDED if the dominant unfavorable character is in heterozygous condition.
(1) Heterozygocity of unfavorable characterIf the genotype is not known, try with higher doses of mutagens, if it
gives any small mutant sectors or not. If there are some possibility of the improvement, then apply mutagen
for appropriately lower dose.
(2) Ploidy of the plant/species
Many Bananas are triploid, making mutation breeding very difficult, by having
three copies of genomes. However, some mutations have been reported, together with some successful
mutant cultivars e.g. Novaria and Parfitt (from disease resistant but dwarf Parfitt to normal-sized and disease
resistant Parfitt in Australia)(3) Purpose of mutation work
Disease resistance may be wanted in most of the cases, like the successful
cases of Japanese pear mutant varieties. However, most of the disease resistant genes are known as
dominant genes, where mutation work may not be so effective. The preliminary tests by high dose mutagen
described above and quick inoculation tests may be needed e.g. by use of toxin secreted from the diseasecausing organisms.
(4) Widening of genetic resources
In some Seminar Meetings, widening of genetic lines in Mangosteen
which has only a few different cultivars/land-laces are reported, were suggested. Parthenocarpy or othervegetative propagation system has disturbed the widening. However, this rather closed vegetative
propagation system may maintain spontaneous mutant genes in it. So, mutation work may give somedivergence of the phenotype if heterozygous dominant gene has mutated to recessive form.
(5) Seedlessness in Oranges etc.
Seedless crops might be produced by mutation or by disturbance of
meiotic cell divisions. Triploid clone produced by pollination of diploid and artificially produced tetraploid
may often be successful in reducing the seeds, like the case of seedless watermelon. Disturbance of meiotic
division may be also caused by translocation of chromosomes. This may caused by irradiation of thematerials. Homozygote may produce seeds normally, but heterozygotes may reduce their seeds. Seedlessgrape are produced in Japan by treating the inflorescence by plant hormone solution, but if mutation can bring
the seedlessness to the crop, solution treatment may not be needed any more. In the pomegranate, it is said
that softening mutation of the hard seed made the fruit easier to eat.
(6) Cross breeding procedure
Palm trees are very difficult crop to improve. But if a very long term projectis available, irradiation of pollen and ordinary M2 selection may be done, although not only the time but also
the very wide screening field for M2 may be needed. Tissue culture may not be so useful in reducing thetime and the field area, in the experiments.
Basically most of the elements has been described in the other vegetatively propagated crop, forexample : Chrysanthemum. Perhaps the difference may reside in the major interest, that in the fruit tree
improvement, most of the cases are disease resistance, while in the Chrysanthemums color and flower form may
be very much interested. Otherwise, the genetic handling of the materials is comparable even though one isarboreal and the other is herbal.
In some fruit trees, the major crop improvement method of cross breeding has difficulties likeparthenogenesis, as seen in some oranges, where the seeds are not really the hybrid between the parents. In suchcases, reproductive system itself may be the target of improvement. In other cases, like in Mangosteen,widening of genetic variability for any variety development may be the targets.
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1) How to prepare starting materials
Refer to the Chrysanthemum Section. The genotype of the material is also very important. The
successful case of Black-Spot disease resistance in Japanese pear was the facts that their genotype was known
already, i.e. the susceptible gene is dominant and the ‘Nijisseiki’ variety was heterozygous for the gene. Thus,
destruction of the single susceptible gene made the disease resistant twig on the tree. The mutant twig was foundafter 19 years of irradiation in a gamma-field in Ohmiya-town in Japan. Perhaps most of the period of 19 years
might be needed to develop the mutant cell growing to the detectable size. Two other Japanese pear mutant
varieties could be obtained after a few days long irradiation and toxin screening work.
2) Mutagens suggested
Please refer to Chrysanthemum section. If you have large irradiation facility like gamma-field, you may
examine the planted materials from time to time. However in most of the cases, irradiation is practically acuteirradiation. It may be still useful in inactivation of dominant-susceptible, or dominant disturbing gene to resistantgene actions. In your early phase of experiments, please examine your material to examine that if it isreasonable material for mutagenesis work or not. The idea of genotype appropriate for the mutagenesis work or
not has been described in the Chrysanthemum section. Use of X- or gamma-rays will be much easier thanchemical mutagens.
3) How to decide treatment dose
See the Chrysanthemum section.
4) How to grow and harvest the M1 generation
please be noted that here the promising mutant can be detected in the M1 generation, but to have a good
chimera free mutant clone, M1V3 or later vegetative generation, i.e. M1V4, or M1V5 clones should be tested.
Otherwise, market quality product may not be expected in the M1 field.
5) How to screen the wanted traits
As mentioned above, if disease resistance is your target, inoculation field or toxin screening will beeffective. If disease infested field is not so severe to endanger your materials to survive, close observation of thedisease symptoms will be needed. Toxin for mutant screening may not need to be purified, if it gives good
disease symptoms. Even a crude culturing liquid on the leaf discs may be good enough.
6) How to brush up the mutant
Please be noted that these materials do not fit for the back-crossing to exclude the unfavorable andsimultaneously induced genes. In case of ‘Nijisseiki’ variety of Japanese pear, seed propagation will not give
good results, at all.
Perhaps the only way will be that select as much as possible mutant of the same type and select the best
one out of them.
Another way of brushing up will be the production of a solid mutant clone out of chimeric mutant tissue.
This may be done by tissue culture method. But please be careful the mutant character will not be degradedfurther by prolonged callus culturing (somaclonal variations may degrade the mutant clone).

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Induced mutation in fruit tree
Fruit tree improvement through mutation techniques might be comparable to the case of Chrysanthemum flower. If
the material is heterozygous, mutant of the gene may be relatively easy to obtain, but if the material is homozygous,
recessive mutant phenotype may not easily obtained. In case of Japanese pear, dominant disease susceptible gene inheterozygous condition had been known to the researchers. The researchers developed further the screening
methodologies by use of toxin inoculation.

The final cultivar ‘Gold Nijisseiki’.

In the case of apple fruit-skin color, perhaps the heterozygous
condition of the skin-color was just waited for the mutagenesis
treatment.
Comparable results have been obtained in Austria (Europe), too.
For Banana, see 7) Difficult Crops.

172Practice Suggestions for Mutation Breeding

Picture files
12. World facilities……Brookhaven National Laboratory located in the Long Island, New
York, USA had open field for Gamma-rays irradiation while plants grownunder natural conditions, the Gamma Field. But the design of the Gamma
Field had no upper shielding and horizontal shielding bank. The facility
had been closed in 1968.
X-ray is also used widely for short period irradiation. Deep therapy type facilities having
good power of 250 kV 20mA or above are used often. However, small facilities, as
large as the refrigerator, are in the market (mostly soft X-ray type). As far as it has highpenetration and can deliver good dosages, they may be used for mutagenesis work.

Small accelerator type 14MeV fast neutron
generator had been used in some experiment.
Please make attention for the induced
radio-activities when you use any kinds ofneutrons. In some countries, restriction by
the law may be very strict.
IAEA has fast neutron irradiation service, using SNIF (special neutron
irradiation facility) mounted in the research reactor of the collaborating
Austrian Research Institution. In this picture, beside of the reactor core, anorifice of the SNIF is seen at the lower center can be seen. Irradiation may
be done when the reactor is in operation.

Currently operating largest Gamma Field is in Japan. Complete Circular
Field is surrounded by 10 m high shielding bank. In this picture, 12o’clock position, and 57 m from the radiation source at the center produced
the black spot disease resistant Japanese pear, registered as ‘Gold Nijisseiki’.
Toward the right, 1-o’clock are forest trees, like pine or Criptomeria
japonica, 5 o’clock mulberry, 6 o’clock tea, 8 o’clock, apple tree, 11 o’clock
peachs. The yellow patch near the center is a small rice field, which isrelatively radio resistant.
The Gamma Field in Japan, still actively used as of 2004.
The irradiation Tower at the center is shown here. The radiation source
container is mounted in the air, so that the container itself functions as uppershield. Horizontal beam can be adjusted by a heavy ring filter. The
source is remotely operated from the white room on the bank in the
background. During the morning, the radiation source is stored in thecontainer and workers/researchers can enter the inside the Gamma Field.
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Practice Suggestions for Mutation Breeding

Gamma Field or Gamma Green House is very convenient facility while plant can be grown under the natural condition.
However, in the congested area, so called Sky Shine, secondary or tertiary scattered radiation may raise theenvironmental radiation revel. So, the recently developed chronic irradiation facility in the Kasetsart University in
Bangkok, Thailand, utilizes tissue culture technique, which may not need strong illumination on the material plants.
The radiation sources are stored within the heavy wall and sent to the irradiation position through the stainless steel
guide pipe.
Gamma Room of Kasetsart University, a case of Gamma Greenhouse simulator using tissue culture.

In this picture, some potted plants are seen. As far as the period
is less than the start of etiolating potted plant may be irradiated
here. Some culture vessels are seen in the right side shelf.

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13. Plant for fundamental research
Arabidopsis thaliana has been widely used in genetics experiments. It can be cultured in vitro and harvest necessary
number of seeds within a test tube.
The culture is easy on agar medium and the culture condition is genetically in complete isolation like Drosophila
experiments. The mutagenesis work may be almost the same as in rice, in terms of dose of mutagen and segregationin M2 generation. Here, only a few examples are shown.
Ordinary plant type.

Yellow green leaf mutant

Nicknamed as spinach leaved.
Albino seedlings may be the first one you may notice in M2 generation after mutagenesis.

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14. Use of chemical mutagen, EMS.
Ethyl methanesulfonate (EMS) is a convenient mutagen to use and relatively easy to handle. However, like all theother chemical mutagens, post-treatment handlings of various wastes should be carefully done as they may be
carcinogenic.
1) Storage
Refrigeration is advised by the manufacturer rather than freezing. When takenout from refrigeration, dew drops from the air may damage the chemical. So,
use good amount of Silica Gels in a bottle to accomodate the EMS vial. About
one or two hours before the use, take-out the entire bottle like this picture fromrefrigeration and make balance with the room temperature before opening of the
EMS vial.

In this picture, a pipetter (Pipette-man) is connected to a glass pipette. A
Hypodermic Injection Cylinder may be used in the same purpose. Never try tosuck the pipette by mouth for any chemical mutagens.

A water-bath and a shaker will be suggested for uniform treatment. A
simple rid/covering with aluminum foil to cover the treatment flask andentire apparatus should be placed in a chemical food to assure safety in thelaboratory atmosphere. The left side plastic bucket contains several littersof about 0.1% (NaOH) alkaline water to hydrolyze the EMS waste.
Pipettes, flasks, paper-towels, aluminum foils etc. are all dipped into thisbucket for at least a week, then neutralize and discard.

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General reference Books
1－１

Manual on Mutation Breeding Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture
Technical Reports Series No. 119 (1970)
1－２ Manual on Mutation Breeding Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture
Technical Reports Series No. 119 (1977)
2
A. M. van Harten (1998) in Mutation Breeding, Cambridge Univ. Press,
156-158 and others.

0Introduction
1
2
3
4
5
6
7
8

http://www-infocris.iaea.org/MVD/
See file #IAEAvar of this CD-ROM
E.AMANO’s Lecture text in Hanoi, (April, 1998).
R. B. Kloesgen et al (1986) Mol. Gen. Genet. 203 : 237 - 244
J. Moutschen-Dahmen and M. Moutschen-Dahmen (1963) Radiation Botany
3 : 297 - 310
H. Hirochika (1995) GammaField Symposia 34 : 77 - 91
H. Hirochika et al (1996) Proc.N.A.S. 93:7783-7788
H. Hirochika (1997) Plant Molecular Biology 35:231-240

1Rice
1
2
3
4
5

A. M. van Harten (1998) in Mutation Breeding, Cambridge Univ. Press,
156-158 and others.
O. Yatou and E. Amano (1991) Proc. IAEA Symp. Vol2: 385-389
E. Amano (1972) Gamma Field Symposia 11:43-59
E. Amano (1985) Gamma Field Symposia 24:81-96
K. Maruyama et al. (1990) Gamma Field Symposia 29:11-25

2 Barley&wheat
1

A. M. van Harten (1998) in Mutation Breeding, Cambridge Univ. Press,
156-158 and others.

3 Maize12
3
45

L. J. Stadler and H. Roman (1948) Genetics 33 : 273- 303
J. P. Mottinger (1970) Genetics 64 : 259-271
E. Amano (1968) Mutation Research 5 : 41 - 46
E. Amano (1972) Gamma Field Symposia 11 : 43 - 59
E. Amano (1985) Gamma Field Symposia 24 : 81 - 96

5 Cucumber&melon
1

S. Iida and E. Amano (1990) Gamma Field Symposia 29 : 95 - 111

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Practice Suggestions for Mutation Breeding

6 Self-incompatible
1

Y. Ukai (1983) Technical News 25 Inst. Of Radiation Breeding, Ohmiya-machi,
Japan

10 CallusCulture12
3

H. Hirochika (1995) GammaField Symposia 34 : 77 - 91
H. Hirochika (1999) Molecular Biology of Rice Ed by K. Shimamoto Springer-Verlag, Tokyo pp 43-58
E. Amano et al. (1999) FAO/IAEA Seminar IAEA-SR-210/22 : pp 27 - 30

11 FruitTrees
1

T. Sanada and E. Amano (1998) In “Somaclonal Variation and Induced Mutations in Crop Improvement"
Ed S. Jain et al. pp 401 – 419 Kluwer Academic Publ. Dordrecht.

178