XA0101052
INIS-XA--390 Mutation Breeding Review JOINT FAO/IAEA DIVISION OF ISOTOPE AND RADIATION APPLICATIONS OF ATOMIC ENERGY FOR FOOD AND AGRICULTURAL DEVELOPMENT INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA
No. 4 March 1986
MUTATION BREEDING IN PEPPER S. DASKALOV* Plant Breeding Unit, Joint FAO/IAEA Division of Isotope and Radiation Applications of Atomic Energy for Food and Agricultural Development, Seibersdorf Laboratory, IAEA, Vienna
Abstract Pepper (Capsicum sp, ) is an important vegetable and spice crop widely grown in tropical as well as in temperate regions. Until recently the improvement programmes were based mainly on using natural sources of germ plasm, crossbreeding and exploiting the heterosis of F hybrids. However, interest in using induced mutations is growing. A great number of agronomically useful mutants as well as mutants valuable for genetic, cytological and physiological studies have been induced and described.
Acknowledgements: The author expresses his gratitude to Dr. A. Micke, Head, Plant Breeding and Genetics Section, FAO/IAEA Joint Division and to Dr. T. Hermelin, Head, Agriculture Laboratory, Joint FAO/IAEA Programme, Seibersdorf Laboratory, for their critical review of the manuscript and valuable contributions. * Permanent Address: Institute of Genetics, Sofia 1113, Bulgaria
32/ 22 In this review information is presented about suitable mutagen treatment procedures with radiation as well as chemicals, M effects, handling the treated material in M , M and subsequent generations, and mutant screening procedures. This is supplemented by a description of reported useful mutants and released cultivars. Finally, general advice is given on when and how to incorporate mutation induction in Capsicum improvement programmes.
INTRODUCTION
Peppers are important vegetable and spice crops widely grown in tropical as well as in temperate regions.
Peppers (common pepper, sweet pepper, red pepper, garden pepper, chile) are members of the family Solanaceae, The genus Capsicum includes five cultivated species (C. annuum L., C. frutescens L, , C. pendulum klilld., C. pubescens R and P., and C. chinense Jacq.) and approximately 20 wild species. The present review will almost exclusively refer to C. annuum,
The center of diversity of the pepper (C, annuunt) according to Lippert et al. (1966) is Central America (Mexico and Guatemala). Pepper (C. annuuni) is a diploid (2n = 24) species (Huskins and La Cour, 1930) usually cultivated as annual crop through sowing in seed beds and transplanting the seedlings into open fields or glasshouses. Direct seeding in the field is also practiced although to a limited extent. Concerning the flower biology pepper is a facultative self-pollinating species. Depending on the cultivar and location of cultivation, cross pollination varies from 5 to 60%. {
The pepper fruits are used fresh, cooked, pickled, canned, smoked or ground for sweet or hot spice. They are rich in vitamin C (up to 400 mg%, average 150-250 mg%), and contain also some quantities of vitamins A, B. , B and P. The red fruits contain 300-450 mg% rutin. The dry matter content of the sweet fruits is 5-12%, of hot ones 9-20%. The fruits contain 3—7% sugar, 0.7-0.8% protein, and carotenoids like capsantin 0.35%, carotin 0.03%, capsorubin 0.07%, zeaxantin 0.025%. The hot fruits contain up to 0.1% capsaicine (Christov et al. 1966). A great diversity of fruit forms exists, e.g. length varies from 2 to 30 cm, width from 0.5 to 15 cm, weight from 5-300 g., shape may be long, conic, cherry form, tomato form, blocky, "kapia" type. The colour of immature fruits can be dark green, green, light green. yellow or sulfury white, and mature fruits can be red, salmon red, pink, orange, lemon yellow or white.
The plant height varies from 30 to more than 100 cm. Most pepper cultivars have an indeterminate dichotomous growth pattern but determinate bunchy type cultivars are also known and used expecially for red pepper production.
The various kinds of use, the great variability of forms and the different cultivation practices are the reasons that breeding objectives are rather diverse. Rather common are the following aims:
— resistance to economically important diseases, e.g., Phytophtora capsici, TMV, CMV, Verticilium dahliae, Leveillula solanacearum, etc. - suitability for single mechanized harvest - early and high yielding hybrid cultivars using male sterility — cultivars suitable for glasshouse growing under low temperature and light intensity
The economic importance of pepper is increasing and the production expanding. Until recently, improvement programmes were based mainly on using natural sources of germplasm, cross breeding and heterosis effect of F hybrids, but based upon recent successes there is growing interest in taking advantage of induced mutations.
WUTflTIOftl RESEARCH
Research has not yet answered all the questions concerning the most effective mutagenic treatment procedures (e.g. type of mutagenic agent, dose or concentration, treatment conditions, plant part treated), handling of M , M and M generations, screening procedures, etc. Most of the published data are only of preliminary nature and systematic investigations are lacking.
Probably the first mutation experiment in pepper was undertaken by Raghavan and Venkatasubban (19A0). The authors exposed dry seeds to X-rays and succeeded to identify several types of mutations affecting size of plants, branching, chlorophyll content, size of leaves and fruits. Oaska'lov (1968, 1971, 1972, 1973, 1974, 1976, 1977 and 1981) has investigated the mutagenic effect of gamma rays, X—rays and EMS, and obtained several useful mutations, e.g., male sterile mutants, anthocyaninless mutants, gene markers, mutants with changed fruit form and colour, dwarfs, etc., which were used directly, in cross breeding programmes and in heterosis breeding. Terzyan and Sahakyan (1974), investigating the mutagenic effect of X-rays, have found a dose dependent increase of the frequency of morphological mutations and a different mutability of the cultiuars.
The mutagenic effect of chemical mutagens (e.g. El, EMS, DMS, NEU, NMU) has been investigated by a number of other authors like Kalovkyan (1968), Videnin et al (1968), Batikian and Galukian (1971), Solomatin (1973), Videnin and Skripnikova (1971, 1972), Skripnikova (1976, 1978), Patil and Meshram (1981). Batikian and Galukian (1971) have observed a significant difference of mutability of the cultivars used and multiple mutations induced by NEU and NMU treatments. According to Skripnikova (1976) the highest mutation frequency was induced by NEU, however, mutants having economically valuable characters were found more frequently after low doses of NEU and DMS. Patil and Meshram (1981), reported a considerable increase in the variance for quantitative characters in M generation after treatment with EMS and DMS. The' mean values were shifted significantly in a negative direction for plant height, primary branches, girth of fruits and in a positive direction for secondary branches, number of fruits, length of fruits and fruit yield per plant.
Zubrzycki and von der Pahlen (1972) have compared the efficiency of X-rays and EMS. The chemical mutagen proved to be more efficient in the induction of chlorophyll mutations while no significant difference in the induction of morphological mutations was observed. In another study, however, the same authors (1973) have obtained data showing that EMS induces higher frequencies than X-rays of both cholorophyll and morphological mutations. A study of the efficiency of recurrent X-ray treatments with alternating treatments by X—rays and OES has been undertaken by Sethupathi Ramalingam (1977). The data did not indicate any of these treatment procedures to increase the frequency of chlorophyll mutations appreciably but an alteration of the mutation spectrum was observed.
fiuni et al. (1978) applied gamma irradiation to various development stages - dry seeds, germinated seeds, male gametophyte (mature pollen grains), both gametophytes (flowers a.t the stage of mature pollen grains), zygotes, 15 day-embryos, and 30 day-embryos. The highest frequency for both chlorophyll and morphological mutations has been obtained following treatments of either dry seeds or both gametophytes. Saccardo (1983) and Saccardo and Monti (1984) have reported data concerning the gametophyte irradiation techniques indicating the following advantages: 1) the M plants are non—chimeric, therefore there is no intrasomatic competition; mutations which &re associated with haplophase or embryonic lethality are eliminated. 2) seeds for the M generation can be harvested from the whole M plant and thus a sufficient large offspring for detecting mutants snd observing segregation can be obtained even in the case of severe M sterility. 3) the possibility to obtain a large number of M seeds allows mutant selection in combination with different background mutations, as segregation of most other mutations induced in the same cell will be independent.
The breeder however, must be aware also of the following disadvantages not mentioned by the author: 1) one additional generation is required. 2) the stage of development of the gametophytes and the condition of treatment can not be as easily monitored as in the case of seed treatments.
Numerous investigations are confined to the effect of tnutagenic agents in the M. generation. Kalovkyan (1968) has studied the frequency of chromosome aberrations after treatment with chemical mutagens. Gukasyan and fikopyan (1974) have established a positive correlation between the frequency of chromosome aberrations and the NMU concentration applied. Terzyan et al. (1974) found that the higher the X-ray dose the higher the frequency of chromosome aberrations up to 300 Gy.
Morgan (1963) has made cytological investigations of the tneiosis in two sterile M plants derived from X-ray irradiation of pollen, in order to study the causes of sterility. He has not observed any large chromosomal structural changes but failure of association of homologous chromosomes at diakinesis and metaphase (asynapsis). Despite repeated crosses with normal pollen, both asynaptic plants have remained entirely sterile. Katiyar (1977) has investigated desynaptic behaviour of chromosomes in a deviant from an fl population following 200 Gy gamma irradiation.
Ilieva and Molhova (1975, 1979) have studied the effect of gamma irradiation on microsporogenesis, microgametogenesis, pollen germination and early embryogenesis. 3-20 Gy gamma irradiation at various stages of flower formation caused degenerative changes of the pollen such as multinuclearity, variation in number of pores, outgrowth of the exine, incomplete maturity of the generative nucleus, varying extent of cytoplasmal vacuolization. The greatest degenerative changes were observed following treatments at early stages of anther development - meiosis and pre-meiosis. Pollen germination was inhibited only at doses higher than 2000 Gy. Pollination of non-irradiated flowers with pollen irradiated with doses of 20-30 Gy caused degenerative changes in the embryo and endosperm development, e.g. absence of fertili- zation, delayed embryo and endosperm development, presence of pycnotized nuclei in the endosperm, etc.
Sethupathi Ramilingam (1977), investigating the meiotic behaviour of two desynaptic mutants, has come to the conclusion that induction of mutations in the genetic system governing the meiosis is the cause for the observed desynapsis. The same author (1978) has investigated the relationship between M chlorophyll deficiency and M chlorophyll mutations. Fruits harvested from branches with chlorophyll deficiencies yielded more frequently M chlorophyll mutations than those from normal branches of chimeric M plants.
Subhash and Nisam (1979) studying meiotic abnormalities in M generation after gamma irradiation of dry seeds with doses of 100—250 Gy have observed univalents, U-shaped fragments, bridges, micro-nuclei, polyads and multi- spindles, their frequency increasing with dose. Earlier the same authors (1975) reported X-ray induced aneuploid forms which are characterized by increase in size of flower, height and yield. Rao and Lakshmi (1980) also carried out studies on meiotic abnormalities after gamma irradiation of dry seeds using the strains Cft 960 (red) and Cfi 1968 (yellow) and doses between 100 and 400 Gy. They observed stickiness, clumping, multivalents, univalents, breakage, non-orientation of chromosomes, laggards and abnormal microstores. Pollen sterility seems to be a cumulative result of various meiotic aberrations as well as of physiological and genetic damages caused by the breakage of chromosomes.
Hertnelin et al. (1983) have investigated the chimera pattern of M plants in three species, including pepper, aiming at improving the seed sampling system and thus making mutation breeding more efficient. The position of every M fruit (H seeds) was precisely recorded and each fruit progeny tested for chlorophyll mutation in M . The topography of the chimerism in 54 M plants is given in Fig. 1. The authors concluded that out of the M fruits those at the main bifurcation should preferably be harvested since their seeds segregate more often for induced mutations. In case no fruits are formed at that position, the next ones from each main branch should be harvested.
MUTAGENIC TREATMENT PROCEDURES
Seed treatment
For induction of mutations mainly seed treatments have been used. It is recommended to use seeds of uniform size, possessing 96-100% germinability and standardized moisture content (about 13%) to obtain good reproducibility of results.
It is difficult to determine the mutagen and dose that will yield the highest frequency of useful mutations. Therefore 3 doses should be applied which are supposed to assure survival of 40-60% (LDAO_6O^ after ionizing radiation treatments and 70-80% (LO ) following treatments with chemical 20—30 mutagens. As there is evidence for deviations in mutation spectra for different mutagens, the use of several, different mutagens is recommended, e.g., one physical and one chemical mutagen. Doses and treatment conditions used in mutation experiments are listed in Table 1 for general guidance. The dose range for gamma and X—rays is 60-400 Gy. The sweet cultivars usually are more radiosensitive than the hot ones.
Due to the large range of mutagen sensitivity it seems appropriate to make a pilot test to determine reduction, survival and sterility of H plants before starting mutation breeding.
After gamma or fast neutron irradiation the tt plants are characterized by narrow cotyledons having lots of small dots that can not be estimated quantitatively, but still are indications of effective treatment.
Gametophyte treatment
Male gametophyte (mature pollen) and both male and female gametophyte (flowers at the stage of mature pollen) treatments have been used so far in rare cases and only with radiation as mutagen (Saccardo and Sree Ramulu, 1977; Auni et al. 1978; Saccardo, 1983; Saccardo and Monti, 1984,).
Mature pollen grains at binucleate stage (easy check can be made using aceto-carmine microscopic slides) are to be collected from already dehiscent anthers for mutagenic treatment. The dose range for gamma or X-rays is 5-15 Gy (see Table 1). Immediately after irradiation the pollen must be used for pollination of emasculated non-irradiated flowers. The pollination must be carried out in insect—proof glasshouses or the pollinated flowers have to be isolated with paper bags or cotton to avoid outcrossing. An alternative is the irradiation of the flowers with anthers before dehiscence (binucleate stage) and the use of pollen for pollination of non-irradiated flowers.
For treatment of both gametophytes flowers just before anther dehiscence must be labelled, all other buds and flowers removed and then the whole plants may be irradiated with gamma or X-rays. The dose range for gamma and X-rays in this case is 5-15 Gy (see Table 1). After the treatment, the plants must be kept in a glasshouse or if grown in open fields the flowers must be isolated to avoid outcrossing.
Handling the H generation
M plants must be raised on isolated plots (at least 700 m apart from other pepper plantings) to prevent cross pollination. The use of recessive gene markers (e.g. lack of anthocyanin, marbled first leaves, yellow cotyledons) would help to detect contamination from cross pollination. The safest procedure would be bagging of the M flowers to avoid outcrossing but the procedure is laborious.
Subramanya and Ozaki, 1983, described a mutant whose flowers remain closed during anthesis so that cross pollination is prevented. Utilization of such mutants should be very useful in plant breeding and genetics research.
The recommendations given in the Manual on Mutation Breeding, (2nd Edition IAEA 1977, Technical Report Series No. 119) should be taken into consideration for the calculation of the required M population size. The applied mutagenic treatment, the expected frequency of a desired mutant character, the kind of screening to be undertaken, and land and labour resources available have to be considered. In any case, at least 3000—5000
M1 plants must be raised per experiment. It is recommended to harvest the fruits from the main bifurcation or those from each main branch, (Saccardo and Sree Ramulu, 1977, Hermelin et al., 1983).
The mutagenized population require a longer vegetation period (2-3 weeks) than the untreated material depending upon mutagen and dose. M plants are supposed to be grown under optimal agronomic conditions to increase survival rate, secure optimal development and obtain sufficient seed setting and fruit maturation.
Handling the M generation
Pepper plants require comparatively large space for growing (depending on cultivar and location, approx. 1 m for 10-20 plants). The most commonly used agronomic practice is to raise seedlings in seed beds and then to transplant them into the field. This requires a lot of manual labour or special mechanization which may limit the size of mutagenized populations.
The M plant or fruit progeny method (M plant or fruit-to-row method) has the advantage that several individuals of the same type will segregate in one M family and they can be more readily detected than if they were scattered over the whole M population. In addition mutant plants with only small phenotypic deviations can be recognized more safely as mutants (Kawai, 1975).
Using this method, 20-25 M, plants per M plant or 10-15 I"! plants per ft fruit (with 2-3 fruits per M plant ) have to be grown. The "M plant or fruit-to-row method" is used by Daskalov (1968, 1972, 1973), Saccardo and Sree Ramulu (1977, Bansal (1973), Skripnikova (1976), and others.
If the desired mutant character is very easily and distinctly recognizable on a single plant basis, the M bulk method may be used. It will save labour in M harvest, seed preparation, and labelling during planting. In this case, from each M plant 2-3 fruits from the main bifurcation and the main branches should be harvested and up to 15 seeds taken from each fruit to form the M bulk.
The size of the M field population that can be taken care of by one person is approx. 70,000-100,000 plants but it depends, of course, on the kind of selection to be performed and the number of observations to be made.
Pepper is a diploid species so that selection of mutants is carried out mainly in the M- generation. When monoploid material is being used, selection of mutants may start in M generation (Pochard 1970). To allow progeny testing all discovered mutants must be selfed, usually by bagging the flowers. Handling the M generation
A progeny test must be undertaken from each suspected M mutant to confirm the mutation and examine the potential usefulness of the mutant. Micro—trials may be started, if enough seeds are available.
SCREEWIMG TECHNIQUES FOR THE SELECTION OF PARTICULAR MUTANTS
1. For CMV resistance (Saccardo and Sree Ramulu 1977):
Inoculation of M- plants at four—leaf stage using the spraying technique, according to flarrou and Mogliori, (1965). All plants which do not show any symptoms roust be inoculated again by the abrasion technique. Resistant plants are screened 10-20 days after inoculation.
2. For resistance to Phytophtora capsici Leonian (fruit rot) (Saccardo and Sree Ramulu, 1977): Inoculation of M seedlings at the cotyledonary stage by dipping the roots in the inoculum prior to transplanting them into pots. Screening for resistant plants may start after 10-12 days.
3. For resistance to Verticillium dahliae, Kleb (Saccardo and Sree Ramulu, 1977): Inoculation of M seedlings at cotyledonary stage grown under glasshouse conditions at 21 +1 by dipping the roots into the inoculum prior to transplanting them in pots. Screening for resistant plants may start after 20-30 days.
4. For resistance to Leveillula solanacearum, Gol. f. capsici Berg (powdery mildew) (Todorova and Daskalov, 1979): Inoculation of M, plants at 6-8 leaf stage grown under glasshouse conditions at 23-25°C by spraying conidiospores on the upper surface of the leaves. Screening for resistant plants may start after 10-12 days. All plants which show no symptoms must be re-inoculated.
5. For male sterility (Daskalov, 1968, 1973):
20000-25000 H, plants (approx. 1000 M- families) obtained after gamma or X-irradiation of dry seeds must be screened during the flowering period. The male sterile mutants have flowers with very reduced and
10 shrunken anthers containing no viable, pollen grains, fit the end of the vegetation period the male sterile plants are usually still flowering and possess lots of small parthenocarpic fruits. This makes them easily recognizable.
For anthocyaninless mutants (Daskalov, unpublished):
30,000-40,000 M seedlings (approx. 1500-2000 M families) are required. Screening can be done already at very early cotyledonary stage (2-3 days after emergence) under glasshouse conditions at 23-25 C for lack of anthocyanin (blue colour) on the hypocotyl. Such seedlings develop later into anthocyaninless plants.
USEFUL MUTANT CHARACTERS OBTAIMED AFTER HUTAGENIC TREATMENTS
Numerous mutants have been found and described as a result of mutation induction experiments. Some were valuable for direct use or cross breeding, others are useful in genetic and physiological studies. The gene symbol, mode of inheritance, as well as a short phenotypic description of the known mutants (spontaneous and induced) are given in the gene list (Lippert et al., 1965, 1966) supplemented by Daskalov in 1973. Csillery (1980, 1983) has described 113 additional spontaneous mutants.
The origin and the breeding value of some of the most interesting induced mutants is shortly discussed below.
Male and female sterile mutants
Daskalov (1968, 1973) obtained two male sterile mutants after irradiation of dry seeds from the cultivar, Pazardzhishka Kapia with 120 Gy and selection in M generation (620 families with approx. 15500 plants). In another experiment, the same author (1973) discovered three male sterile mutants in the cultivar Zlaten medal after screening 58500 M, plants (1170 families) following dry seed treatment with 135 Gy gamma rays. Genetic studies revealed that the mutants possess recessive non—allelic monogenic mutations which were denoted as ms-3, ms-4, ms-6, ms-7 and ms-8 respectively. The mutant genes ms—3 and ms—8 are being used directly and in cross breeding for developing male sterile lines with good combining ability (Daskalov 1976, 1978; Milkova and Daskalov, 1977). The ms-3 gene was used for testing a method of hybrid seed production that was later successfully introduced into practice (Daskalov 1973, 1976).
11 Pochard (1970) succeeded to obtain three recessive male sterile mutants after treatment of monoploid material with EMS. The mutants were denoted as mr 9, me 705 and me 509. The latter is being used for establishing hybrids (Breuils and Pochard, 1975). In a short communication, Rubzov and Solomatin (1974) have reported male sterile mutants obtained after treatment of dry seeds with 0.05% OMS.
A problem in using male sterile mutants in hybrid seed production is sometimes a high percentage of self—pollinated plants due to instability of male sterility (in case of cytoplasmic male sterility) or non-proper separation of the male sterile and male fertile plants in the female parent rows (in case of genetic male sterility). To overcome this, Oaskalov and Mihailov (1983) have developed a new method of hybrid seed production based on the use of a female parent combining induced male sterility with a recessive lethal gene which can be made ineffective by a simple specific treatment. In F all plants resulting from self-pollination of the female parent die at the cotyledonary stage ensuring 100% purity of the hybrid plantation.
Bapa Rao et al. (1980) found in the M generation of gamma ray irradiated plants of variety CA 960 a floral mutant with female sterility. The mutant is characterized by a remarkable increase in the size of the flower, multiplication in the number of floral parts, decrease in the size of anthers and difference in the shape and size of gynoecium. Daskalov (unpublished) has induced two partially female sterile mutants with no changes of the floral morphology and characterized by excessive flowering. Such mutants may be utilized in hybrid seed production as male parent.
Disease resistant mutants
Attempts to induce disease resistance have been undertaken and some encouraging results have been published.
Karasz (1974) has reported induction of a CMV resistant mutant which was released as a cultivar under the name Horgoska Slatka—X—3 (Tab. 1). Saccardo and Sree Ramulu (1974) have screened for resistance to CMV 2600 M plants from 174 M progenies treated with fast neutrons and 2000 M plants from 160 M plant progenies treated with EMS. The frequency of plants showing no symptoms was 0.54 and 0.05 respectively. In another experiment the same authors (1977) have screened 6188 M, plants derived from 280 M. plant after treatment with fast neutrons and 6108 M plants derived from 427 M 12 plants following gamma irradiation for resistance to Verticillium dahliae Kleb. The frequency of plants showing no symptoms was 0.73 and 0.49 respectively.
Todorova and Daskalov (1979) have investigated the effect of gamma rays, fast neutrons and EMS on inducing resistance to powdery mildew (Leveillula solanacearum Gol. After screening 65153 M plants only 3 (0.0046%) plants in the gamma series were found to be resistant. Totally, 331 (0.51%) plants possessing some degree of resistance were observed. Such plants were not found in the 14217 control individuals. The progenies of the resistant plants consist of plants expressing different degrees of resistance. A subsequent selection for resistant plants up to M generation was performed and eight o resistant lines from the cultivars Albena and Kurtovska kapia were developed (Todorova, 1982). One of these lines is under official testing for variety release.
Sotirova and Daskalov (1983) have irradiated dry seeds of the cultivar Kourtovska kapia with 60, 80, 100 and 120 Gy gamma rays, ft considerable number of M plants at the 3—4 leaf stage were inoculated with a monosporial suspension of Phytophtora capsici or M fruits were inoculated by a prick with mycelium. Resistant plants were selected and retested in M and M generations, and some resistant M lines were established. Fruit colour mutants
Daskalov (1974) has found in M generation, after treatment of dry seeds from the cultivar Pazardzhishka kapia with 120 Gy X—rays, mutants with orange mature fruits. In this case mutation from y to y has occurred and the interaction of the genes y_ and c determines the orange colour.
A mutant with sulfury white immature colour (gene mutation from the allele series sju) was reported by Oaskalov (1974). It is interesting to note that this mutation has occurred in an anthocyaninless cultivar possessing aj[ gene. A linkage exists between the genes al_ and sw (Peterson, 1959) and recombinants al sw have not been reported so far.
Dwarf and compact type mutants
Raghavan and Venkatsubban (1940) obtained a dwarf mutant after X-ray treatment of dry seeds. Sethupathi Ramalingam (1977) has induced a compact type mutant with determinate growth pattern by gamma irradiation of the strain
13 K.I. The mutant has a number of desirable agronomic features and is released as cultivar under the name MDU. 1 (Tab. 1). Daskalov (1973, 1974) described two dwarf mutants, denoted dw and dw-2 respectively, which were obtained after gamma irradiation of dry seeds from the cultivar Zlaten medal. Skriprikova (1976) also reports having obtained a compact type mutant following IMEU treatment.
The compact type mutants are suitable for mechanized cultivation and once—over harvest, finother possible use of such mutants as well as dwarfs is to serve as a tool in genetic and mutation research. Plant breeding, in general, requires a lot of land, labour and other costly inputs. This holds true also for mutation breeding as it is particularly crucial to work with very large populations. One way to reduce the costs is to use compact type and dwarf mutants by which the land or greenhouse space required could be substantially reduced and the number of individuals increased. Any desired mutant character selected could later on be transferred to normal type breeding material by crossing. Estimating the costs involved in a 2 year experiment (M and M ) it was found that utilization of compact type or dwarf mutants as experimental material could save approx. 80% of the cost (Daskalov, 1981). One must be aware, of course, that such material can be used only for selecting qualitative mutant characters with simple inheritance.
Mutants with changed fruit form
ft mutant with short conic fruits was obtained by Daskalov (1972) after irradiation of dry seeds of a "kapia" type cultivar (Pazardzhishka kapia) with 120 Gy X-rays. Rubzov and Solomatin (1974) and Skripnikova (1976) have described mutants with conic fruits after treatments of dry seeds with 0.025% NEU. ft tomato form mutant was obtained by Skripnikova (1976) following treatment of dry seeds with 0.02% ethylene imine.
Recessive gene markers
Easily recognizable seedling markers are very useful in hybrid seed production, mutation breeding, genetic investigations, etc., since they may help to detect and eliminate contamination from uncontrolled cross pollination or mechanical mixtures and to maintain the authentity of breeding material. Mutation induction offers good possibilities for obtaining such seedling markers and large M_ populations are easy to screen in the seedling stage. 14 TABLE 1: EXAMPLES FOR MUTAGENIC TREATMENT PROCEDURES USED IN PEPPER
Plant Part Cultivar Mutagen Used doje or concentration References Treated and treatment conditions
dry seeds P. kapia X-rays 120 Gy; 1.2 Gy/min (LD5o= l0° Oaskalov (1968, 1971) (9% moisture) Kalinkov dry seeds K-1 X-rays 200, 300 and 400 Gy; 5Gy/sec Sethupathi Ramalingam (11% moisture) (1977) dry seeds California wond X-rays 100, 200 and 300 Gy; 11 Gy/min 7ubrzycki and von der Pahlen (LD50 = 199 Gy, L0100 = 400 Gy) (1972, 1973) dry seeds Zlatan medal gamma rays 135 Gy; 3.7 Gy/min Daskalov (1973) (9% moisture) dry seeds NP46A gamma rays 150-400 Gy Bansal (1973) (5-6% moisture) dry seeds Albena gamma rays 60, 80, 120 and 140 Gy Auni et al. (1978) moisture) (I.D50 = «8 Gy) dry seeds Albona gamma rays 60, 80 and 120 Gy Todorova and (9% moisture) K. kapia Oaskalov (1979) dry seeds NP46A fast neutrons 2-8 Gy Bansal (1973) (5—6% moisture)
dry seeds Pimiento fast neutrons 24 Gy; 0.20 Gy/min Saccardo et al. (1976) Quadrate d'Aati Saccardo and Sroe Corno di Toro Ramulu (1977)
dry seeds Albena fast neutrons 20,30 and 40 Gy Todorova and Oaskalov K. kapia (1979)
dry seeds California wonc EMS 0.2; 0.4; 0.6% water solution, 20 hr Zubrzycki and von der Pahlen treatment, shaking, postwashing (1972, 1973) •— TABLE 1: (continued)
Plant Part Cultivar Mutagon Used dose or concentration References Treated and treatment conditions
dry seeds Corno di Toro EMS 0.2% water solution, 23°C, 24 hr Saccardo et al. (1976), treatment, postwashing Saccardo and Sree Ramulu (1977)
dry seeds Sathur Samba EMS 7 x Id"2 mol Sethupathi Ramalingam (1977)
dry seeds Albima EMS 1% water solution, 3,6 and 9 hr treatment, Todorova and Daskalov K. kapia shaking, postwashing (1979)
dry seeds Bjala kapia OMS 0.05% water solution, 20 hr treatment Rubzou and Solomatin Rotund 449 (1974) Michurinskji Krasnji
dry seeds Michurinskji 41 DMS 0.05; 0.02; 0.005% water solution, Skripnikova (1976) 20 hr treatment
dry seeds Bjala kapia El 0.05% water solution, 20 hr treatment Rubzov and Solomatin Rotund 449 (1974) Michurinskji Krasnji
dry seeds Michurinskji 41 El 0.02; 0.01; 0.005% water solution, Skripnikova (1976) 20 hr treatment
dry seeds Bjala kapia NEH 0.025% water solution, 20 hr treatment Rubzou and Solomatin Rotund 449 (1974) Michurinskji Krasnji
dry seeds 0.05; 0.025 and 0.012% water solution, Skripnikoua (1976) Michurinskji 41 NEH 20 hr treatment
dry seeds 6 mM Sethupathi Ramalingam Sathur samba NEH (1977)
dry seeds K.I X-rays 200, 300 and 400 Gy (5Gy/sec) Sethupathi Ramalingam recurrent (1977) treatment dry seeds K.I X-rays and DES 200, 300 and 100 Gy. Sethupathi Ramalingam alternating 4.8 and 12 tnM water solution, 22+l°C, (1977) treatment 8 hr pre-soaking, 6 hr treatment, DES solution changed every 30 min
prc-soaked seeds Astrahan3kji A60 El 18 hr pre-soaking, 0.02; 0.01 and 0.008% Batikian and Galukian Novocherkaskji 35 18 hr treatment (1971)
pre-sonked seeds Aatrahanskji A60 N£H 18 hr pre-soaking, 0.05; 0.025 and Batikian and Galukian Novocherkaskji 35 0,012%, 18 hr treatment (1971)
pre—3oaked seeds A3trahanskji A60 NMU 18 hr pre-soaking, 0.012; 0.010 and Batikian and Galukian IMovochorkaskj i 35 0.008%, 18 hr treatment (1971)
pre-soaked seeds NP46A EMS 12 hr pre-soaking, 0.3% water solution, Bansal, (1973) 6 hr treatment, shaking, postwashing
pre-soaked seeds NP46A NPIH 12 hr pre-soaking, 0.01-0.03% water Bansal (1973) solution, 6 hour treatment, shaking, postwashing
germinated seeds Albona gamma rays 12, 14, 16, 18, 20 Gy, (L050 = Auni et al. (1978) (1 mm radicle) 12.6 Gy)
male gametophyte Floral Gem X-rays 5 and 20 Gy; 1.7 Gy/min Morgan (1963) (pollen)
male gametophyte Albena gamma rays 5, 7.5, 10 and 15 Gy, (LDc,o = 1.29 Gy) Auni et al. (1978) (pollen)
both gametophytes Pimiento gamma rays 7.5 Gy; 10 Gy/min, Saccardo and Sree (whole flower) 23°+lC Ramulu (1977)
both gametophyte3 Albona gamma ray s 5, 7.5, 10 and 15 Gy, (LD50 = 8.9 Gy) Auni et al. (1978) (whole flower)
zygotes Albena gamma rays 5, 7.5, 10 and 15 Gy, (L050 = 9.5 Gy) Auni et al. (1978)
15 days embryo Albena gamma rays 5, 7.5, 10 and 15 Gy, (LD50 = 11.5 Gy) Auni et al. (1978)
30 days embryo Alb«na gamma ray s 5, 7.5, 10 and 15 Gy, (L0b0 = H.O Gy) Auni et al. (1978) Oaskalov (1973, 1974) has induced two anthocyaninless (alj mutants after gamma irradiation (13S Gy) of dry seeds from the cultivars Zlaten medal and Kalinkov by screening a M population of approx. 58500 plants. After fast neutron treatment (30 Gy) of dry seeds from the cultivar Kurtovska kapia and screening 67500 M plants a third mutant of that type was obtained (Daskalov, unpublished). The mutant phenotype (lack of blue stain on the hypocotyl, nodes, immature fruits and anthers) is easily recognizable already after emergence as well as throughout the whole vegetation period. Mutants with marbled (distinct white and green spots) leaves were obtained both after gamma and fast neutron irradiation by Daskalov (1972, 1974). The mutant phenotype is transient and is normally expressed only on the first leaves. Furthermore, mutants with yellow green spots on the first leaves, with yellow green dots on the leaves, with yellow cotyledons, and with light green leaves were reported by Daskalov (1974). Some of them may have interest as gene markers.
A very interesting spontaneous round leaf (rl.) mutant was described by Greenleaf and Hearn (1976). The gene rl has no deleterious effect and the mutant phenotype is easily distinguishable, which makes this mutant a very promising seedling marker.
Mutants affecting quantitative characters
Mutants affecting quantitative characters have also been reported in the literature but data concerning size of the mutagenized population, screening procedures, genetic investigations etc., are insufficient.
Videnin et al. (1968, 1971, 1972), Dolgich (1970), Rubzov and Solomatin (1974), Skripnikova (1978), Batikian et al. (1980) applying chemical mutagens succeeded in inducing higher yielding mutants of interest for plant breeding. Mutants with enlarged fruits were reported by Rubzov and Solomatin (1974), Skripnikova (1976), Batikian et al. (1980). A mutant with increased dry matter content of the fruits has been obtained by Skripnikova (1976) after treatment with 0.012% NEU. Rubzov and Solomatin (1974) have discovered a mutant with thick pericarp and increased weight of the fruits after treatment with 0.025% NEU.
Earlier ripening mutants have been obtained by Videnin et al. (1968), Dolgich (1970), Rubzov and Solamatin (1974), Skripnikova (1976) after treatments with chemical mutagens. Indira and Abraham (1979) have discovered 18 in M (after treatment of dry seeds with 100 Gy gamma rays) an early flowering tricarpellate mutant.
Linkage studies using mutants and gene transfer
Zubrzycki and von der Pahlen (1974) have used some gamma and EMS induced mutations (any, dug 1. brl and aur) and a spontaneous one (m) to investigate genetic linkages on the basis of recombination values.
Pochard et al. (1974) using a trisome tester set have determined the exact location of some mutant genes, e.g., the genes C, xa-3 and xa-8 are localized on chromosome XI (Ja-trisome).
Pollen irradiation for gene transfer was investigated by Daskalov (1984). For facilitating a backcross programme (classical gene transfer) the author has proposed the following procedures:
The pollen of the donor plant 8 must be irradiated with a sublethal dose (15—30 Gy) for induction of high frequencies of gene and chromosome mutations. Then plants of type A are pollinated.
F M plants expressing a high percentage of pollen sterility and, if possible, lacking undesirable dominant characters, must be used as the male parent for the backcross to cultivar A, which is to be improved. In the BC. generation, fertile plants that resemble cultivar ft and also contain useful characters of cultivar B have to be selected for the continuation of the backcross. In some cases pollen may be irradiated again to delete undesirable linked genes. The procedure ends when the breeding objective is fulfilled, i.e. the desired gene or genes of the donor plant B are transferred to the genetic background of cultivar A. Due to the changed segregation pattern and the shift to the maternal parent one may expect less generations than needed usually for the backcross procedure.
19 TABLE 2; LIST OF MUTANT VARIETIES
Name of Variety Place and date of Kind and date of mutagenic Main improved attributes release (or approval) treatment [parent variety] of variety and name of principal or mutant crosses worker and institute (mutant underlined)
Horgoska slatka-X-3 1974 Yugoslavia Gamma ray3 Resistant to CMV Karasz, I.
Albena 197 Krichimski ran 1976 Bulgaria X-ray, dry seeds, 1965 Hybrid variety, high yield, S. Oaskalov and L. Milkova [Pazardzhishka kapia] early, improved fruit quality Institute of Genetics mutant 794 ms-3, source of male Sofia 1113 sterility for line 215 ms-3, the female parent for the hybrid variety MDUl 1976 Tamil Nadu, India Gamma rays Compact plant type, higher R. Sethupathi Ramalingam 1969-1970 yield and cspsaicine content Oept. of Agric. 8otany [K.I] Agric. College and Research Institute, Madurai Tamil Nadu Agric. Univ. Lyulin 1981 Bulgaria 135 Gy gamma rays, Hybrid variety based on induced L. Milkova and S. Daskalov dry seeds, 1970 male sterility, very early, Institute of Genetics, [Zlaten medal] high yield Sofia 1113 mutant Zlaten medal ms-8, the female parent for the hybrid variety RELEASED MUTANT CULTIVARS: (see Tab. 2) So far release of five mutant cultivars was reported in the literature, ft. Obtained by direct use of mutants 1. Horqoska slatka-X-3 (Karasz, 1974). 2. Albena (Daskalov, 1975). 3. HDU 1 (Sethupathi Ramalingam, 1977). B. Obtained by cross breeding 1. Krichimski ran (Oaskalov and Milkova, 1976). 2. Lyulin (Milkova, Oaskalou, 1981, 1983). Oetails for these cultivars are given in Table 2. For up-to-date information on these cultivars and future ones, the Mutation Breeding Newsletter, published twice a year by IAEA, may be consulted. REFERENCES AUNI, S., DASKALOV, S. and FILEV, K. (1978) Radiogenetic effect of gamma irradiation under different ontogenetic states of sweet pepper. C.R. Acad. Bulg. Sci. 31, 1357-1360 BANSAL, H. (1973) Induced mutation affecting flower development in Capsicum annuum L. Curr. Sci. 42, 139-140. BAPA RAO, N., LAKSHMI, IM., and PRAKASA RAO, P.S. (1980) Gamma-ray induced floral mutant affecting female fertility in Capsicum L. Curr. Sci. 49, 944-945 BATIKIAN, G. and GALUKIAN, M. (1971) [A study of recessive visible mutations in Capsicum annuum induced by the effect of chemical mutagens] (in Russian) Tsitologiya i genetika 5, 39—44 BATIKIAN, G., GUKASIAN, L, and TUMANIANE, E. (1980) [Investigations of pepper mutants induced by N-nitroso-N-methyl urea] (in Russian). In: Khimichenskij Mutagenez i Immunitet (Chemical Mutagenesis and Immunity). Moskwa, Nauka. BREUILS, G. and POCHARD, E. (1975) Essai de fabrication de l'hybride de piment "Lamyo—INRA" avec utilisation d'une sterilite male genetique (me 509). Ann. Amelior. Plantes 25, 399-409 CHRISTOV, S., POPOVA, D. and VESELINOV, E. (1965) Piper (in Bulgarian) [Pepper] Zemizdat, Sofia 21 CSILLERY, G. Gene mapping of the pepper needs more initiatives, (contribution to the gene list) In: EUCARPIA Capsicum working group, IVth Meeting, 14-16 October 1980, Wageningen, 5-9 CSILLERY, G. New Capsicum mutants found in seedling, growth type, leaf, flower and fruit. In: Proc. of the 5th EUCARPIA Meeting of the Capsicum and Eggplant working group, 4-7 July, 1983, Plovdiv, 127-130 DASKALOV, S. (1968) A male sterile pepper (C. annuum L.) mutant. Theoret. and Appl. Genetics 3_8_, 370-372 DASKALOV, S. (1971) Two new male sterile pepper (Capsicum annuum L.) mutants. C.R. Acad. Sci. Agr. Bulg. 4, 291-294 DASKALOV, S. (1972) [Investigations on induced mutations in pepper (Capsicum annuum L.) I. Morphological characteristics of mutants in the variety Pazardzhishka kapia 794] Genetika i selektsiya (in Bulgarian) 5, 221-230 DASKALOV, S."(1973) [Gene list for the pepper] (in Bulgarian) Genetika i Selektsiya 6, 401-409 DASKALOV, S. (1973) Three new male sterile mutants in pepper (Capsicum annuum L.). C.R. Acad. Agr. Bulg. 6, 39-41 DASKALOV, S. (1973) [Investigations on induced mutations in pepper (Capsicum annuum L.). II. A study on use of male sterile mutants in hybrid seed production] (in Bulgarian) In: Nauchna Sesiya na Instituta po Genetika u Selektsiya na Rasteniyata, Sofia, 15-16 March, 1971) 217-228. DASKALOV, S. (1973) [Investigation of induced mutants in Capsicum annuum L. III. Mutants in the variety Zlaten medal] (in Bulgarian) Genetika i selektsiya 6, 419-429 DASKALOV, S. (1974) Investigations on induced mutations in sweet pepper (Capsicum annuum L.) In: EUCARPIA Genetics and Breeding of Capsicum, Budapest, 1-4 July, 81-90 DASKALOV, S. (1975) Albena — a new mutant cultivar in sweet pepper for early and mid-early field production, (in Bulgarian) Gradinarstvo 12, 22 DASKALOV, S. (1976) Seed setting of male sterile mutants in connection with heterosis breeding in pepper (Capsicum annuum L.) Genetica Agraria 30, 407-417 DASKALOV, S. (1977) Induced mutations in sweet pepper (Capsicum annuum L.). In: C.R. 3me Congress EUCARPIA Piment, Avignon-Montfavet, 5-8 Juillet, 155-160 DASKALOV, S. (1981) Value of dwarf or compact-type mutants as a tool in breeding programmes, exemplified with Capsicum annuum L. In: Induced Mutations a Tool in Plant Research. IAEA, Vienna, 502-503 DASKALOV, S. (1984) Pollen irradiation and gene transfer in Capsicum. Theoret. Appl. Genet 68, 135-138 OASKALOV, S. and MILKOVA, L. (1976) [Krichiminski ran - a new heterosis cultivar in sweet pepper] (in Bulgarian) Gradinarstvo 10, 22—23 22 DASKALOV, S. and MILKOVA, L. (1978) [Pepper hybrid breeding on sterile basis] Genetika i Selektsiya (in Bulgarian) 11, 155-161 DftSKALOV, S. and MIHAILOV, L. (1983) A new method of pepper hybrid seed production based on mutant genes. In: Proc. of the 5th EUCARPIA Meeting of the Capsicum and Eggplant Working Group, 4—7 July, Ploudiv, 131-133 DOLGICH, S. (1970) [Experimental mutagenesis in vegetable plants] (in Russian) Sel'skokhazyajstvennaya Biologiya 5, 803-811 GUKASYAN, L. and AKOPYAN, D. (1974) [The effectiveness of treating pepper seeds (Capsicum annuum L.) with N-nitroso-N-methylurea] (in Armenian) Biologicheskij Zhurnal Armenii 27, 46-51 GREENLEAF, W. and HEARN, W. (1976) A round leaf mutant in 'Bigharf pimiento pepper (Capsicum annuum L.). Hort. Sci. 11, 463-464 HERMELIN, T., BRUNNER, H., DASKALOV, S. and NAKAI, H. (1983) Chimerism in Mi plants of Vicia faba. Capsicum annuum and Linum usitatisiroum. In: Chimerism in Irradiated Dicotyledonous Plants. IAEA TECDOC 289, Vienna, 1983, 35-42 HUSKINS, C. and LA COUR, L, (1930) Chromosome numbers in Capsicum. Am. Nat. 64, 382-383 INDIRA, C. and ABRAHAM, S., (1979) Tricarpellate mutant in Capsicum annuum L.- induced by gamma rays. Curr. Sci. 48, 406-408 ILIEVA, I. and MOLHOVA, E. (1975) [Study on the microsporogenesis of Capsicum annuum L. after gamma irradiation] (in Bulgarian) Genetika i Selektsiya 8, 353-364 ILIEVA, I. and MOLHOVA, E. (1979) [Effect of gamma irradiation on microgametogenesis, pollen germination and early embryogenesis of pepper] (in Bulgarian) Genetika i Selektsiya 12, 242-252 KALOVKYAN, M. (1968) [A study of cytological changes in Capsicum annuum under the influence of chemical mutagens] (in Armenian) Biologicheskij Zhurnal Armenii 21, 59-65 KALOVKYAN, ft. (1968) [Experimental mutagenesis of sweet pepper. I. Effect of the chemical mutagens ethylene imine, nitrosomethylurea and nitrosoethylurea on sweet pepper in the M-^] (in Armenian) Biologicheskij Zhurnal Armenii 21, 53-62 KARASZ, I., (1974) Breeding of spice pepper variety H.S.-X-3 from irradiated material. In: EUCARPIA Genetics and Breeding of Capsicum, Budapest, 1-4 July KATIYAR, R. (1977) Radiocytogenetical studies in Capsicum: Induced desynapsis. Caryologia 30, 347-350 KAWAI, T. (1975) Factors affecting efficiency of selection of mutants in mutation breeding. In: Gamma-Field Symposia 14, 1-8 LIPPERT, L., BERGH, B. and SMITH, P. (1965) Gene list for the pepper. J. Hered. 56, 30-34 23 LIPPERT, L., SMITH, P. and BERGH, B. (1966) Cytogenetics of the vegetable crops. Garden pepper. Capsicum sp. Botanical Rev. 32, 24-55 MARROU, J. and MIGLIORI, A. (1965) Methode dlinoculation rapide pour l'etude de la resistance des plantes aux virus. Ann. Epiphyties 1,119-128 MILKOVA, L. and DASKALOV, S. (1977) [Study of pepper hybrid combinations on male sterile basis] (in Bulgarian) Genetika i Selektsiya 10, 238-241 MILKOVA, L. and DASKALOV, S. (1981) [Hybrid breeding in pepper (Capsicum annuum L.I (in Bulgarian) Gradinarska i Lozarska Nauka 18, 38-44 MILKOVA, L. and DASKALOV, S. (1983) Lyulin - a hybrid pepper cultivar for early field production. In: Proc. of the 5th EUCARPIA meeting of the Capsicum and Eggplant Working Group, 4-7 July, 1983, Plovdiv, 202 MORGAN, D. (1963) Asynapsis in pepper following X-irradiation of the pollen. Cytologia 28, 102-107 PATIL, A. and MESHRAM, L. (1981) Induced quantitative variation in economic characters by chemical mutagens in red pepper (Capsicum annuum L.). In: Fourth Int. SABRAO Cong. 4-8 May 1981, Malaysia (abstract), 29 PETERSON, P. (1959) Linkage of fruit shape and colour genes in Capsicum. Genetics 44, 407-419 POCHARD, E. (1970) [Induction of three male sterility mutations in red pepper (Capsicum annuum L.) by mutagenic treatment of monoploid material] (in French) In: La sterilite male chez les plantes hort. France, EUCARPIA, 93-95 POCHARD, E., BREUILS, G. and FLORENT, A. (1974) Localization of genes in Capsicum annuum by trisomic analysis. In: EUCARPIA, Genetics and Breeding of Capsicum, Budapest, 1-4 July 1974 RAGHAVAN, T. and VENKATASUBBAN, K. (1940) Studies in the South Indian chillies. I. A description of the varieties, chromosome numbers and the cytology of some X-ray derivatives in Capsicum annuum L. Proc. Indian Acad. Sci. B, 12, 29-46 RAO, N.B. and LAKSHMI, N. (1980) Gamma ray induced meiotic abnormalities in Capsicum annuum L. Caryologia 33, 509-518 RUBZOV, M. and SOLOMATIN, M. (1974) [Valuable forms of pepper] (in Russian) Kartofel" i ovoshchi 1, 30-31 SACCARDO, F. (1983) Gametophyte irradiation in some dicotyledonous crop species. In: Chimerism in Irradiated Dicotyledouous Plants, IAEA TECDOC 289, Vienna, 31-32 SACCARDO, F. and MONTI, L.M. (1984) Mutation breeding in higher plants by gamete irradiation technique. In: Induced Mutations for Crop Improvement in Latin America, IAEA TECDOC 305, Vienna, 225-234 SACCARDO, F., SREE RAMULU, K. and TOMARCHIO, L. (1976) Isolation, transfer and induction of disease resistance in Capsicum. Genetika (Yugosl.) 8, 247-254 24 SACCARDO, F. and SREE RAMULU, K. (1977) Mutagenesis and breeding for disease resistance in Capsicum, In: Induced Mutations against Plant Diseases, IAEA, 275-280 SACCARDO, F. and SREE RAMULU, K. (1977) Mutagenesis and cross breeding in Capsicum for disease resistance against Verticillium dahliae. In: 3me Congress EUCARPIA Piment, Avignon-Montfai/et, 5-8 Juillet, 161-169 SETHUPATHI RAMALINGAM, R. (1977) MDU I, a new mutant chilli variety. Mutation Breeding Newsletter IAEA No. 10, 8-9 SETHUPATHI RAMALINGAM, R. (1977) Mutagenic efficiency of recurrent irradiation and alternate treatment in Capsicum annuum L. Ann. INIA, Ser. General 5, 191-196. SETHUPATHI RAMALINGAM, R. (1977) (Vote on the induced asynaptic mutant in Capsicum annuum L. Science and Culture 43, 237-238 SETHUPATHI RAMALINGAM, R. (1978) Induced chlorophyl chimeras and breeding behaviour in chillies. Curr. Sci. 17, 381-383 SKRIPNIKOVA, Z. (1976) [Mutants of sweet pepper induced by chemical mutagens] (in Russian) Genetika 12, 37-44 SKRIPNIKOVA, Z. (1978) [The use of experimental mutagenesis in sweet pepper breeding] (in Russian) In: Khimicheskij Mutagenez i gibridizatsiya (Chemical Mutagenesis and Hybridization) Moskwa, Nauka 137-139 SOLOMATIN, M. (1973) [Study of the effect of chemical mutagens on pepper varieties in the Mj] (in Russian) Nauch. trudy Voronezh Inst. 56, 36-41 SOTIROVA, V. and DASKALOV, S. (1983) Use of induced mutations in developing pepper forms resistant to Phytophtora capsici Leonian. In: Proc. of the 5th EUCARPIA Meeting of the Capsicum and Eggplant Working Group, 4-7 July, 1983, Plovdiv, 123-126 SUBHASH, K. a.nd NIZAM, J. (1975) X-ray induced aneuploidy in Capsicum annuum. Curr. Sci. 4, 62-63 SUBHASH, K. and NIZAM, J. (1979) Chromosomal anomalies induced by gamma rays in Capsicum. J. Cytol. Genet. 14, 80—82 SUBRAMANYA, R. and OZAKI, H. (1983) Cleistogamy in pepper (Capsicum annuum L.) and its inheritance. In: Proc. of the 5th EUCARPIA Meeting of the Capsicum and Eggplant working group, 4-7 July, 1983, Plovdiv, 53-56 TERZYAN, P. BATIKYAN, H. and SAHAKYAN, T. (1974) [The effect of X-rays on raitotic activity and the frequency of structural chromosome rearrangements in root cells of the species C. annuum L.I (in Armenian) Biologicheskij Zhurnal Armenii 27, 35-39 TERZYAN, P. and SAHAKYAN, T. (1974) [The effect of X-rays on mutation in pepper plants (Mj)] (in Armenian) Biologichenskij Zhurnal Armenii 27, 103-106 25 TODOROVA, J. (1982) Pepper resistance to Leveillula solanacearum Go1. obtained by experimental mutagenesis. In: Ookladi na Vtoriya Natsionalen Simpozium po Imunitet na Rasteniyata, Plovdiv, 1982 (Rep. of Second Nat. Symp. of Plant Immunity, Plovdiv, 1982) 3, 91-98 TODOROVA, Y. and DASKALOV, S. (1979) [Possibilities for the utilization of some mutagenic factors in changing sweet pepper susceptibility to powdery mildew (Leveillula solanacearum Gol. f. Capsici Berg.)] (in Bulgarian) Genetika i Selektsiya 12, 174-179 VIDENIN, K. and SKRIPNIKOVA, Z. (1971) [Changes of the morphological and agronomical traits in pepper induced by chemical mutagens] (in Russian) Praktika Khimicheskogo Mutageneza (Application of Chemical Mutagenesis). Moskwa, Nauka, 201-204 VIDENIN, K. and SKRIPNIKOVA, Z. (1972) [Changes of the morphological and agronomical traits in pepper induced by chemical mutagens] (in Russian) Khimicheskij Mutagenez i Sozdanie. Selektsionnogo Materials (Chemical Mutagenesis and Creation of Breeding Material) Moskwa, Nauka, 280-282. VIDENIN, K., RODIONOV, V., KOROVIN, V. and VANIFATOV, 0. (1968) [The use of chemical mutagens in breeding of ornamental, vegetable and fruit plants] (in Russian) In: Mutatsionnaya Selektsiya (Mutation Breeding) Moskwa, Nauka, 149-154. ZUBRZYCKI, H. and von der PAHLEN, A. (1972) [Comparison between effects of ethyl methane sulphonate and X—rays on induction of mutations in Capsicum annuum LI (in Spanish) In: Induced Mutations and Plant Improvement, IAEA, 387-396 ZUBRZYCKI, H. and von der PAHLEN, A. (1973) Frequencies of viable mutants in Capsicum annuum L. induced with ethyl-tnethane-sulphonate and X-ray treatments. Boletin Genetico (Inst. Fitotec. Castelar) 8, 23-28 ZUBRZYCKI, H. and von der PAHLEN, A. (1974) Genetic linkage in pepper. In: Rev. Agron. N.O. Argent XI, 87-91 26 Mutation Breeding Review Joint FAO/IAEA Division of Isotope and Radiation Applications of Atomic Energy for Food and Agricultural Development International Atomic Energy Agency Wagramerstrasse 5, P.O. Box 100 A-1400 Vienna, Austria Printed by the IAEA in Austria March 1986