FEATURES
Plant breeding: Induced mutation technology for crop improvement
Scientists at the IAEA's Seibersdorf Laboratories are helping breeders to develop crops having more desirable traits
present forms of life are the product of found suitable for domestication; humans have by F.J. Novak three factors: used about 3000 of these for food, fibre, spices, and • mutation, the fundamental source of heritable etc., with 200 ultimately domesticated as crops. H. Brunner variation, Today, only 15-20 of these are food crops of • environmental factors, which influence the major importance. selection of those mutations that survive and The means of developing new plant varieties reproduce, and for cultivation and use by humans has come to be • time, during which the genotype and environ- called plant breeding. Early on, it primarily in- ment constantly interact and evolutionary volved selection, the choice between good and change is realized. bad plants. People learned not to eat all the "best Genetic variation found in nature does not fruit" but to plant the seed from some of them. represent the original spectrum of spontaneous Genetics became a fundamental science of mutations. Rather, this is the result of genotypes plant breeding after the Moravian monk J.G. recombining in populations and continuously in- Mendel discovered the laws of heredity in the teracting with environmental forces. mid-19th century. Plant breeding further ad- Green plants are the ultimate source of vanced when the methodology of hybridization resources required for human life, food, clothing, was developed. Its aim was to combine various and energy requirements. Prehistoric people, desirable properties of many plants in one plant, who depended on their skills as hunters, drew instead of just choosing between good and bad upon abundant natural vegetation to collect plants. This method, often supplemented by nutritious and nonpoisonous fruits, seeds, tubers, germplasm derived from induced mutation, has and other foods. As human populations in- become the most common one for breeding creased, greater and safer supplies of food had to plants through sexual reproduction. be found, and gradually production systems However, some crops—including bananas, based on plant domestication were developed. apples, cassava, and sugar cane—reproduce The domestication of crops historically has vegetatively, especially those that are fully been influenced by ecological and agricultural sterile without seeds. For this important group, conditions, as well as by food gathering alternative approaches had to be developed, preferences. Genotypes that have adapted to a namely techniques of manipulation with somatic wide range of climatic and edaphic conditions tissue: mutation breeding and biotechnology. typically have been selected for cultivation. The achievement of higher yielding crops facilitated population growth, sedentary settlements, and Mutation breeding further development. Which crops were domes- ticated depended not only on the number of Plant breeding requires genetic variation of seeds or the size of fruits, but also on taste, useful traits for crop improvement. Often, how- palatability, and other factors. ever, desired variation is lacking. Mutagenic Only a small fraction of the world's ap- agents, such as radiation and certain chemicals, proximately 200 000 plant species have been then can be used to induce mutations and generate genetic variations from which desired mutants may be selected. Dr Novak is Head of the Plant Breeding Unit at the IAEA's Seibersdorf Laboratories, and Dr Brunner is a senior scientist Mutation induction has become a proven in the Unit. way of creating variation within a crop variety.
IAEA BULLETIN, 4/1992 25 FEATURES
One natural evolutionary product of genetic variation: a mutant of dwarf coconut palm.
It offers the possibility of inducing desired at- Major efforts were devoted during this initial tributes that either cannot be found in nature or phase of mutation induction to define optimal have been lost during evolution. When no gene, treatment conditions for reproducibility. Re- or genes, for resistance to a particular disease, or search focused on changing "random" mutation for tolerance to stress, can be found in the avail- induction into a more directed mutagenesis to able gene pool, plant breeders have no obvious obtain more desirable and economically useful alternative but to attempt mutation induction. mutations. However, it did not lead to the desired Treatment with mutagens alters genes or alterations in the mutant spectrum. Limitations breaks chromosomes. Gene mutations occur were the concomitant increase of plant injury naturally as errors in deoxyribonucleic acid with increasing radiation dose and the low fre- (DNA) replication. Most of these errors are quency of economically useful mutations. This repaired, but some may pass the next cell led scientists to search for potentially better division to become established in the plant off- mutagens. As a result, new methods of radiation spring as spontaneous mutations. treatment, as well as chemical agents with Although mutations observed in a particular mutagenic properties, were found. gene are rare, there are probably 100 000 genes in a cell of a higher plant. This means that every plant may carry one or more spontaneous muta- Plant biotechnology tions into the next generation. Gene mutations without phenotypic (visible) expressions are Breeding for improved plant cultivars is usually not recognized. Consequently, genetic based on two principles: genetic variation and variation appears rather limited, and scientists selection. The process is extremely labourious have to resort to mutation induction. There are and time consuming with high inputs of intellec- no other economic ways of altering genes, ex- tual and manual work. (See box.) However, the cept to wait a long time for spontaneous muta- development of plant cell and tissue culture over tions to occur. the last 20 years has made it possible to transfer Artificial induction of mutations by ionizing part of the breeding work from field to laboratory- radiation dates back to the beginning of the 20th conditions. century. But it took about 30 years to prove that Extensive research has resulted in new areas such changes could be used in plant breeding. of plant breeding, namely "plant biotechnology" Initial attempts to induce mutations in plants and "genetic engineering". They are based on mostly used X-rays: later, at the dawn of the cellular totipotency. or the ability to regenerate "Atomic Age", gamma and neutron radiation whole, flowering plants from isolated organs were employed as these types of ionizing radia- (meristems). pieces of tissue, individual cells. tions became readily available from newly estab- and protoplasts. The isolated plant parts are lished nuclear research centres. aseptically grown in test tubes on artificial media
26 IAEA BULLETIN, 4/1992 FEATURES
Some tools and products of plant breeding (clockwise from top left): a mutant of paddy rice induced by ionizing radiation; yams and other root and tuber crops can be genetically improved by mutation breeding; tissue culture and in vitro mutagenesis are basic methods of biotechnology for improving crops; "Golden Maidegg", an apple mutant with improved market value, was induced at the Seibersdorf Laboratories by irradiation of cuttings from "Golden Delicious" apples; mutation breeding has improved the tolerance to environmental stress of Azolla, a water fern used as biofertilizer in rice paddies.
IAEA BULLETIN. 4/1992 27 FEATURES
General scheme of mutation breeding Breeding a new variety of crop takes anywhere from 12 to 15 years of intensive effort The steps in- clude:
Generation Characterization
Seeds, pollen, vegetative parts, or tissue cultures treated by physical (radiation) or chemical mutagens.
Mi(MiVi) Plants grown from treated seeds (Mi) or vegetative propagula (MiVi).
M2(MiV2) Population of plants grown from seeds (M2) or vegetative parts (MiV2> harvested from Mi or MiVi respectively. Selection of desired mutants may start in this generation or later.
MS - MS Continuing selection, genetic confirmation, mulitphcation
(MiV3 - MiV8) and stabilization of field performance of mutant lines.
Next 2 - 3 generations Comparative analyses of mutant lines during different years and in different locations.
Next 2 - 3 generations Official testing before release as a new variety.
Applications of nuclear techniques in plant breeding Cross Breeding Mutation Induction (using mutants)
Genetic Engineering
| | Mutation breeding
Tracer techniques
Both
Biochemical-and DNA Markers
Crop improvement is based on two basic principles: genetic variation and selection. Disease and Pest Serving as invaluable Resistance tools are mutagenic irradiation and isotope tracer techniques, which are incorporated into the Yield (Photo- various breeding synthesis Studies) methods.
28 IAEA BULLETIN, 4/1992 'Nll3TinS
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The mixed planting Middle East & Europe I Latin America of a main crop with specific cover crops (e.g. forage legumes or grasses) minimizes the use of herbicides. 28.3° 26.4% 22.6 24.5 Role of the Seibersorf Laboratories The Plant Breeding Unit of the IAEA Laboratories at Seibersdorf was set up in the mid-1960s to support the Joint FAO/IAEA 15.1% 23.6% Division's programme of genetic crop improve- 34.0% 25.5% ment. Nuclear techniques in plant breeding are developed and transferred to countries by re- Fellows Trainees search and development in mutation breeding Total: 53 from 26 countries Total: 212 from 73 countries and related biotechnological techniques, training scientists from developing countries, and provid- ing irradiation services and technical advice. Training activities scientists and plant breeders already has iden- Initial research in the Plant Breeding Unit in the Plant tified desirable genotypes in grain legumes focused on the development of mutation induc- Breeding Unit of (soybean, garden bean) and other plant species, tion methods with ionizing radiation and chemi- the Seibersdorf including trees. cal mutagens. The aim was to achieve high Laboratories, For many developing countries, breeding mutagenic efficiency, i.e., a high frequency of 1982-92 crops for tolerance to salinity and acidity in soils desirable mutations at minimal plant injury and is of high priority. Current breeding strategies the highest possible reproducibility. This re- (including mutation induction and in vitro selec- quired a definition of radiation source charac- tion) have clearly been successful in incorporat- teristics in terms of dose homogeneity and ing degrees of tolerance in different species. The precise assessment of absorbed dose in biologi- use of genetic engineering for creating environ- cal targets by appropriate dosimetry. Irradiation mental stress-resistant plants will depend on the of seeds with gamma rays and neutrons was identification of specific genes which contribute commonly done, given the ease of handling, the to the adaptation to specific stress environments. simple standardization of factors which modify In tropical countries, agriculture practices radiation sensitivity, and good reproducibility. have maintained the yield level of different crops The establishment of methods for controlling through "intercropping" instead of by increased oxygen-dependent effects in the radiobiological monocrop cultivation. Breeding crops for multi- response to electromagnetic radiation was a ple functions—such as biomass production, im- major achievement. The Laboratory actively contributed to standardizing neutron irradiation Radiation service of seeds in nuclear reactors by developing spe- statistics, 1967-92 cial facilities for this purpose. These were known Treated samples 20329 as SNIP, for Standard Neutron Irradiation Treated species 217 Facility for swimming-pool-type reactors; and as Treated cultivars 1 134 USIF, for Uranium Shielded Irradiation Facility Recipient Member States 108 for Triga-type reactors. Seed samples 17872 This research was the basis for the IAEA Vegetatively propagated plants 1 046 Laboratories' worldwide seed irradiation service Cobalt-60 gamma treatments 14382 using fast and thermal neutrons at a high-dose Fast neutron treatments 5416 precision and reproducibility of induced effects. Other mutagen treatments 531 Moreover, efficient and accurate treatments of seeds with chemical mutagens, mostly alkylating Note: Examples of major plant species treated include: cereals agents and azides, were developed with the aid (rice, wheat, barley, Iridíale, millet, tef); legumes (soybean, peanut, common bean, cowpea. mungbean); root and tuber of isotope-labelled compounds and compared crops (cassava, yam, cocoyam, potato); fruits (citrus, apple, with mutation induction by ionizing radiation. apricot, peach, grape vine): ornamentals (chrysanthemum, an- The Unit has undertaken supportive research on tirrhinum, achimenes, tulip): and others (rape, sesame, mutation breeding in cereals, pulse crops, in- amaranth, quinoa, niger). dustrial crops, and vegetatively propagated crops. 30 IAEA BULLETIN, 4/1992 FEATURES As each crop species has a variable reproduc- ture and in vitro plant regeneration are being tive capacity (number of progenies per plant) and investigated for use in mutation induction and a specific system of reproduction (self- or cross- mutant selection. Somatic embryogenesis and pollinated sexual reproduction or asexual plant regeneration from cell suspensions of propagation), a universal breeding approach Musa are used to develop somatic cell manipula- cannot be developed and species-specific proce- tion procedures for banana and plantain breed- dures have to be applied. Most vegetatively or ing. Methods of screening such plants for resis- asexually propagated species are difficult to im- tance to Panama disease are studied in tissue prove genetically by conventional cross- and culture, and biochemical markers (peroxidase) mutation breeding methods. These breeding are applied for the identification of tolerant problems can be more easily resolved by using genotypes. DNA markers are used for identify- biotechnology in combination with mutation in- ing mutants and characterizing cultivars and duction, and the Unit initiated in vitro mutation species of Musa. Mutant clones identified at the breeding activities during the mid-1980s. Seibersdorf Laboratories are tested in the field in Several tropical food crops of great importance tropical countries. to the food security of developing countries were • Mutation breeding to improve the chosen as the main focus of R&D and training tolerance to environmental stress of Azolla. activities in biotechnological plant breeding at Azolla is a small aquatic fern that lives in sym- the IAEA Laboratories. biotic relationship with the nitrogen-fixing cyanobacterium Anabaena. Under suitable field conditions Azolla can double its weight every Research and development activities 3-5 days. The Azolla-Anabaena symbiotic sys- tem provides green manure for flooded crops, The Unit provides focused support to the particularly rice. Induced mutagenesis has FAO/IAEA's co-ordinated research and techni- produced Azolla variants tolerant to high cal co-operation programmes. Assistance is salinity, toxic aluminium levels, and/or to her- provided to numerous projects in terms of exper- bicides. Tolerant plants are being investigated tise for building facilities for plant tissue culture under field conditions to confirm that heritable and mutagenic treatment, for quality control of changes cause the increased tolerance to en- dosimetry of mutagenic irradiation, and for the vironmental stress. development and transfer of nuclear tech- • Methods of mutation induction and nologies for plant improvement. breeding of tropical root and tuber crops (cas- Ongoing R&D includes the application of sava and yam). Cassava and yam are among the nuclear methods and associated advanced tech- most important staple food crops of the lowland niques, such as in vitro culture and molecular tropics. Mutation breeding technology is being genetics, to improve the production of a wide developed to increase variation in plant stature, range of crops through mutation breeding. The cyanide content, disease, and pest resistance. In development of biotechnological methods for vitro techniques are used for the propagation of breeding vegetatively propagated crop plants of healthy plants and improved clones. Somatic major importance in developing countries has a embryogenesis is being developed for cassava high priority. and yam improvement through in vitro Currently, the following R&D areas are mutagenesis and later on by somatic cell being pursued: manipulation. Mutant and polyploid clones are • Somaclonal and mutagen induced varia- prepared for field testing in Member States. tion. Systematic studies are being conducted to • Tissue culture in cocoa as a system for compare the genetic variation caused by tissue more efficient mutation breeding. Attempts to culture (somaclonal) variation with that induced breed cocoa for disease resistance have yielded by irradiation and chemical agents. Genetic very limited success. A major constraint is that variation is being studied among maize plants little variation exists in currently available cul- derived from in vitro cultured material via tivars. Somatic embryogenesis is being somatic embryogenesis. This is being done to developed for propagation of desirable assess the nature of somaclonal and induced genotypes and, through in vitro mutagenesis and variation and its potential for use in practical pollen mutagenesis, is being applied for induc- breeding. tion of virus-resistant cocoa trees in Ghana. • Mutation induction and breeding tech- Plant breeding research at the Seibersdorf nology for banana and plantain. Low genetic Laboratories is directly problem- and client- variation and sterility handicap genetic improve- oriented. Many positive results of scientific work ment of banana and plantains (Musa spp.) by have been achieved by junior scientists from conventional breeding techniques. Shoot-tip cul- developing countries during their assignments IAEA BULLETIN, 4/1992 31 FEATURES under the IAEA's fellowship training discuss and evaluate the potential role of induced programme. Local cultivars and genetic material mutations and advanced biotechnologies in their from tropical countries are brought to the national breeding programmes for specific crop Seibersdorf Laboratories, transferred to tissue improvement of cereals, legumes, oil crops, culture conditions and used for experimental forages, vegetables, fruits, root and tuber crops, work. Protocols and techniques that are specifi- palms, rubber, and other plants. cally developed for a crop and a particular genotype are then directly used in national programmes. Additionally, breeding material originating from mutant lines and clones which Support for national programmes are ready for field testing are dispatched from Seibersdorf to developing Member States in sup- A radiation treatment service is provided at port of their breeding programmes. no cost to FAO and IAEA Member States to foster the application of nuclear techniques in crop improvement programmes and to render Training of plant breeders direct support to plant breeders in developing countries. Mutagenic treatment is applied to Training in plant breeding represents the seeds, corms, tubers, scions, cuttings, and tissue most active component of technology transfer at cultures ("in vitro materials") with precise doses the Seibersdorf Laboratories. For 20 years the of gamma and fast neutron radiation. The doses Plant Breeding Unit has supported the Agency's are carefully calibrated to assure reproducible fellowship programme and organized inter- effects. Users of the service are requested to regional training courses. Training activities are report on the objectives of the applied mutation closely connected with R&D efforts on crop breeding project and to provide an adequate plant improvement and the application of material (population size) to ensure a high prob- nuclear techniques in breeding. (See graphs.) ability for mutation induction of desired charac- During a period of three to twelve months, fel- ters. Moreover, a prior radiosensitivity test in a lows usually work with radiation or chemical greenhouse is frequently performed to assess induced mutagenesis in plant species cultivated useful radiation doses for the great variety of in their home countries. Whenever possible, biological samples in mutation breeding. The training of small groups of two-to-five fellows is treated materials are dispatched with a detailed organized for solving common problems. The irradiation protocol and with the request to report experiments are individually designed to assure on the induced radiation effects in the first and that laboratory techniques and results will be second mutation generation. This feedback is directly applicable upon return to the home in- required to improve radiosensitivity estimates of stitute. species and cultivars from different environ- As a result of their work, fellows have ments. produced numerous scientific publications in in- Over the last 25 years, the Unit has provided ternationally recognized journals and symposia radiation services on more than 20 000 samples proceedings. Very often, as continuation of a from the majority of Member States from the fellowship in Seibersdorf, fellows participate in FAO and IAEA. (See table.) Most of these were co-ordinated research and technical co-operation seed samples which were irradiated with cobalt- projects of the IAEA. 60 gamma rays. The FAO/IAEA Interregional Training Recently, however, requests for mutagen Course on "Induction and Use of Mutations in treatment of in vitro materials and for fast Plant Breeding" has been held at the Seibersdorf neutrons have become more frequent. This Laboratories since 1982. Twenty participants reflects the increasing importance of biotechnol- from different Member States of FAO and IAEA ogy and molecular genetics in plant improve- are admitted annually to this intensive training ment programmes. course that usually lasts 6 to 8 weeks. Through Less than 80 mutant varieties were officially lectures, laboratory exercises, field experiment released before the start of irradiation services. evaluations, seminars, and excursions, par- Over the past quarter century, more than 1500 ticipants are made aware of the latest advanced cultivars of crop plants and ornamentals with mutation techniques and biotechnological and significantly improved attributes — increased molecular biology methods for crop improve- yield, improved quality, higher market value, ment. Special training is given in the safe han- disease resistance, and/or stress tolerance — dling of radiation sources, radioisotopes, and have been released. Some of these mutant particularly hazardous mutagenic chemicals. At varieties were derived from radiation services the end of each course, participants are able to provided by the Seibersdorf Laboratory. O 32 IAEA BULLETIN, 4/1992 FEATURES A banana plant developed by mutation breeding using ionizing radiation. At left The schematic represents a banana mutation breeding system. Propagation of shoot Mutagenic tips in test tubes irradiation An initial plant DNA 'fingerprinting' for Selection and plant genetic characterizatioin regeneration in test tubes Acclimatization of plants in soil Plant selection in the field Rapid propagation of a selected plant in test tubes A mutant clone in the farmer's field Breeding Bananas, plaintains, and cooking bananas are varieties are entirely sterile and therefore impossible hardier bananas different cultivars and species belonging to the to improve by conventional breeding techniques. botanical genus Musa. Banana "trees" are actually Research on the induction of mutations in big herbs which produce fruit that are one of the most bananas by exposing them to radiation and support- important foods for hundreds of millions of people in ing tissue culture techniques was initiated at the developing countries. The world's production is Seibersdorf Laboratories in 1985. Shoot tips were more than 70 million tonnes per year and about 90% isolated from several economically important of the total harvest is used as food for domestic banana and plantain cultivars and micropropagated consumption. The banana industry generates an on artificial media in test tubes. Several types of income of about US $1.7 billion annually for export- mutagenic irradiation (gamma rays and fast ing developing countries. neutrons) were applied on actively growing cells of The cultivation of bananas and plantains is apical shoot tips which were regenerated into plants. seriously threatened by several diseases caused by This research resulted in the development of mutant pathogenic fungi, bacteria, viruses, and nematodes. clones of the most important cultivar of the dessert Some of them may be controlled by pesticides; banana. "Grand Nain". These varieties are now however, the most epidemic pathogen. Fusarium, is being tested in several countries for agronomic per- a soil borne fungus which causes Panama disease. formance such as yield, quality of fruit, and earlier There is no effective chemical control against the harvest. spread of this fungus in infested soil. Panama dis- The Seibersdorf Laboratory supports a co-or- ease has devastated several hundred thousand dinated research programme on breeding for im- hectares of banana plantations in Central America provement of Musa crops and assists several tech- and created serious problems in Africa where many nical co-operation projects in establishing national people are dependent on plantains and cooking breeding programmes in Colombia, Panama. Costa bananas as part of their staple diet. The only way to Rica, Cuba. Ghana, Malaysia, and Thailand. resolve this problem is to breed varieties having Recent developments in molecular biology have disease resistance. made it possible to characterize plant genomes and The world's production of bananas is based on to identify markers for practical use in plant breeding. a very limited number of genetically unimproved Genetic "fingerprinting" of banana cultivars and clones that were selected and domesticated from mutants opens new perspectives for breeding these nature. Although cross breeding has contributed a genetically "recalcitrant" crops which are of such vital little to banana breeding, the most important importance to people in developing countries. IAEA BULLETIN, 4/1992 33