Experimental mutagenesis of corn
--Lysikov, VN

Studies to explore the possibilities for applying experimental mutagenesis to plant breeding in Moldova go back to the latter half of the 60s. The period coincided with the organization in the Republic of introduction of maize line varieties with a view to developing high-yielding, heterotic hybrids.

As a matter of fact, there was neither scientific nor practical experience in applying the experimental mutagenesis technique to produce new parental material of maize line varieties. This was due to difficulties in working with maize as a cross-pollinated crop and specific features of reproduction of its selfed lines. Besides, one should take into account the level of development of classical genetics at the time, i.e. strict adherence to Johanssen`s theory of pure lines, resulting in a number of leading geneticists, including those working with maize, declaring the impossibility of obtaining new mutant lines of maize from its pure lines, such as American selection lines introduced at the time by the VIR.

In view of the above, work on maize in Moldova had, as it were, to be started anew in order to consistently prove that (1) it was possible, using the experimental mutagenesis technique, to produce new mutant lines from true homozygous selfed lines of maize, (2) new mutant lines would exhibit a range of agronomic traits essential for breeding work, (3) novel mutant lines of maize, reproducing by selfing, would transmit their valuable traits to their progeny, (4) new mutant lines would, upon intercrossing with other selfed lines, e.g. with an American selection line, yeld hybrids showing enhanced heterosis.

With respect to the above four points, the new mutant maize lines were clearly shown, on the basis of extensive experimental evidence, to be of considerable interest to breeders. They offer large numbers of valuable gene carriers which not only possess new traits and properties ensuring the broadening of the variability spectrum, but also speed up the development of high-yielding and highly heterotic maize hybrids resistant to adverse environments, diseases and pests.

As a result of this work, a collection of maize mutants was established comprising over 500 mutant lines. Each of these lines differed significantly from their parental stocks and possessed a number of new valuable traits useful in breeding. These mutant lines include forms with shortened growing season, drought-resistant forms, dwarfs showing high grain and silage productivity and forms exhibiting a number of other features essential for applied breeding. Concurrently and in parallel with examination of morphological and physiological traits, mutants were studied in detail with respect to quantitative traits.

Of special interest turned out to be mutant maize lines showing increased grain protein content, including those forms which exhibited high content of amino acids, such as lysine, tryptophane, methionine, etc. Sometimes, these forms contained no less and even more lysine than the forms carrying the opaque2 (o2) gene. It is known that the transfer of the o2 gene into maize lines encountered great difficulties since together with the o2 a number of genes linked to it were being transferred which were undesirable and even harmful in terms of breeding. For example, the "ramosa2" (ra2) gene causing ear separation, and others. Mutant lines exhibiting high content of essential amino acids do not have these undesirable genes.

A collection of promising mutant lines of maize was established and a catalogue published describing and characterizing over 500 forms. Mutant lines from our collection were freely distributed to breeders in Russia, the Ukraine, Kirghizia and other regions of the former Soviet Union (FSU). This enabled plant breeders at various institutions to use the lines in their work and, more importantly, identify among them those which showed valuable traits and properties in that particular locality and to attempt their further improvement.

Thus for example, 125 mutant lines from our collection were donated to the All-Union Maize Research Institute in Dnepropetrovsk (the Ukraine). A detailed study of these under the environmental conditions of the central Ukraine enabled Prof. G. V. Grizenko to identify 21 forms resistant to root rot. Root rot resistant maize lines did not exist until that time even in the VIR collection. We could not identify these forms ourselves since in Moldova the disease was not expressed at all due to aridity.

Similar encouraging and interesting results were obtained when our mutant maize lines were used to identify forms resistant to various maize diseases. This work was carried out by Dr. E. N. Kobeleva, a plant pathologist, at the Zherebkovskaya experimental station (the Ukraine). She even published a monograph concerned with the problem.

A study by Z. A. Orinshtein specifically aimed at inducing, by experimental mutagenesis techniques, resistance in maize lines to boil smut (Ustilago zeae), a major maize disease in Moldova, also merits special attention. Since nobody had previously succeeded in obtaining such mutations, the experiment involved maize forms highly sensitive to boil smut under normal conditions, including the GelberLand-Mais cultivar. All the work was performed with artificial infection, such that each plant was recurrently inoculated at the growing point with germinated spores of boil smut. It turned out that treatment with a combination of ionizing radiation and Rapoport`s supermutagens allows maize forms to be identified which are almost completely resistant to boil smut. A test for heritability of this resistance carried out by assistant professor K. I. Kuporitskaya of the Plant Pathology Department at the Kishinev Institute of Agriculture showed that the most resistant forms (genotypes) consistently transmit boil smut resistance from generation to generation for 7 to 10 years.

Current breeding and seed-growing work aimed at producing interstrain hybrids of maize is performed on material showing cytoplasmic male sterility (CMS) thus eliminating the need to cut the tassels away from female parent plants and improving the quality of hybrid seed material. This led academician A. E. Kovarsky to explore the possibility of developing by experimental mutagenesis new mutant lines of maize exhibiting CMS. The experiments (by A. I. Konotop, Yu. S. Orlov and S. G. Byrka) very soon allowed identification of 9 mutant lines of maize of which 8 showed CMS and 1 nuclear sterility. All lines exhibiting CMS resulted from treatment with a combination of ionizing radiation and chemical super mutagens, the line showing nuclear sterility having been produced by sonication.

A subsequent test for type of sterility, which was performed using indicator lines, showed that we did generate maize lines carrying CMS, of the Moldavian type at that, the so-called S type. Among these, the mutant line derived from line VIR-49 appeared to be a very promising one, since no such line with CMS existed in previous collections. At the time, the line proved to be very useful to breeders in producing new maize hybrids. A special study of this line by A. F. Palii, one of the post-graduate students (now a professor) of the corresponding member of the Academy of Sciences of Moldova T. S. Chalyk, provided a complete support to CMS of the Moldavian (S) type having been generated by experimental mutagenesis.

Considering the fact that maize mutant lines themselves are not used directly for harvesting (except in seed production) and only interstrain hybrids are used for major plantings, an objective was set to study new mutant lines for combining ability. In other words, it was necessary to demonstrate the possibility of using mutant lines in breeding work to produce high-yielding hybrids, especially as there were no such data in the world at the time.

The formulated problem was addressed along two lines simultaneously. On the one hand, a large number of mutant lines of Moldavian selection were examined for general combining ability (GCA) by the classical topcrossing technique and on the other hand, best mutant lines in diallel crosses were studied for specific combining ability (SCA) with subsequent mathematical treatment of the results according to the method of Griffing and Khotyleva. The experimental evidence of O. V. Blyandur and V. G. Mordvinova clearly showed that mutant lines of maize can exhibit both high GCA and high SCA.

Concurrently, the same problem was solved in somewhat different but very ingenious way by A. I. Konotop. He chose mutant lines from among classical American selfed lines of maize comprising the double interstrain hybrid VIR-42 the most common among the introduced maize lines in Moldova at the time. Derived from these lines were first simple mutant hybrids of the "Slava" type (VIR44 x mutant VIR38) female parent of the double hybrid - and the simple hybrid of the "Svetoch" type (VIR40 x mutant VIR43) - the male parent of the double hybrid. Then these simple mutant hybrids were used to produce a double hybrid of the VIR42 type (mutant Slava x mutant Svetoch).

A comparison by A. I. Konotop of the double hybrid of the VIR42 type, derived from a cross of mutant lines, and simple hybrid VIR42 showed the former to be undoubtedly superior to the latter. Thus, it was demonstrated that even a "simple assemblage" of the double interstrain hybrid VIR42 performedf according to the traditional scheme but involving mutant maize lines results in the already high-yielding, highly heterotic maize hybrid showing a considerable, mathematically provable yield increase.

Thus, experimental mutagenesis was definitively shown to be a promising tool for producing increasingly high-yielding heterotic hybrids from maize lines. This was subsequently very well confirmed by work on developing new high-yielding interstrain maize hybrids which were introduced into cultivation and came to occupy vast acreages. The academician V. V. Morgun and Dr. I. G. Chuchmii (Ph.D. in agriculture) have been successfully working along these lines in the Ukraine. In Moldova, this work has been rather successfully carried out by Dr. O. V. Blyandur.

In addition to producing usual types, we have paid attention to generating large mutational changes, the so-called macromutations. In macromutations, variability usually affects a large number of important systemic characters. Identification and isolation of a macromutation has always been a very rare event. Moreover, its study encountered numerous difficulties, since not infrequently it was necessary to determine a somewhat unusual pattern of expression of characters and their inheritance which may often be complicated by incomprehensible manifestations.

Therefore, when in 1962 an original radiation macromutation, "Corngrass", was obtained following irradiation of pollen of the VIR44 maize line with gamma rays in a dose of 15 Gry, it immediately became the focus of attention. This was a very stunted (40 cm), bushy, herbaceous plant totally different from normal maize plants and, besides, completely lacking the male reproductive organs. In order to preserve this mutation, whose female reproductive organs were located in axils (two pistil filaments at each leaf), it was pollinated with a mixture of pollen from different maize lines including that from VIR38 and VIR43.

An unusually intense morphogenetic process occured during the second and subsequent generations. Among the resulting forms, many exhibited marked, complex changes in plant morphology, such as a greatly increased number of stems, partially altered stem (even becoming geniculate in shape); altered internode positions; changed leaf morphology and phyllotaxis; significantly altered male and female reproductive organs; etc.

Many mutant plants showed a clear-cut and pronounced expression of some of the characters of putative ancestral forms of maize - teosinte, gama grass (Tripsacum), and even Job`s tears. Prof. F. M. Couperman of Moscow State University reported the case of simultaneous expression of characters of the above three ancestral forms of maize.

Studies of diversity of the "Corngrass" mutation progeny enabled Dr. A. N. Kravchenko to classify them morphologically. He subdivided all the forms into five categories: cultivated homozygotes, cultivated heterozygotes, teopodal forms (resembling mutations Tp1 and Tp2), corngrass forms (resembling the Cg1 mutation), and branched.

Long-term observations showed that the morphogenetic process was very intensive over 25 years, with only brown and teopodal groups making the largest contribution to diversity and serving, as it were, as a source of new forms. Furthermore, it was shown that the entire progeny of the radiation-induced macromutation "Corngrass" segregates into stable, or readily stabilized upon selfing, and unstable forms which, upon long selfing, continuously segregate not only known forms, but completely new ones as well. It was obvious that a mutation exhibiting genetic instability had been obtained.

It is only recently that the properties and features of genetically unstable mutation have begun to be studied. This was due to a number of reasons, such as: first, the previously known cases of genetic instability were discovered by chance, as spontaneous mutations; second, in the majority of cases they occurred in Protozoa rather than in plants belonging to higher organisms; third, before the molecular bases of modern genetics were developed, the very explanation of possible genetic instability, as well as description of well defined cytogenetic factors such as those by Barbara McClintock had not been accepted as serious ones and sometimes even questioned by most researchers.

It is for this reason that many experimenters, having encountered such cases in their work, exercised caution, being reluctant to waste time on studying the yet unclear phenomenon., and simply ignored them. Other researchers restricted themselves to their brief description, "registering" only what lied on the surface and required no large effort to be verified. Still others, while realizing that some new phenomena may be behind this but fearing being misunderstood by others, thought they were not in a position to udertake serious studies along these lines. And finally, there were those who did not know how to start an in-depth study of this phenomenon. The state of knowledge at the time was not high enough to enable this.

Extensive studies by Dr. N. V. Krivov, of the Institute of Genetics (Kishinev), on the pattern of behavior and expression in the progeny of traits characteristic of the Corngrass mutation using the genetic analysis technique allowed a number of important conclusions to be made. Of these, the following are worth mentioning:

1. This mutatation was found to be determined by a dominant gene, designated Cg2, and to be nonallelic to the known American mutation Cg1 (corngrass). It was shown to be located in the short arm of chromosome 3.

2. The expression of the Cg2 gene is strongly dependent on modifier genes, which allows this mutation to be regarded as highly sensitive to the genetic background.

3. A specific character of the Cg2 mutation is a peculiar discontinuity in its phenotypic expression. Thus, a proportion of plants strongly resembles the expression of macromutation of the Tp1 and Tp2 (teopod1 and 2) type, another proportion closely resembling the Cg1 macromutation. In addition, many chimeric (mosaic) plants are identified which carry, on a single plant, shoots of both mutant corngrass, or teopodal, and cultivated type.

4. A remarkable feature of this mutation is its genetic instability. Moreover, it has been found that the instability mutation can occur not only in Cg2 ---> normal (+) but in the normal (+) ---> Cg2 direction, i.e. regular transitions of the mutant-normal type occur. The Cg2 --> normal (+) mutations have been shown to occur at a rate of more than 55% in homozygous Cg2/Cg2 plants and at a rate of 15-17% in heterozygous plants. Furthermore, the frequency of theCg2 allele in somatic cells, measured as a proportion of mosaics among the selfed homozygotes, is of the order of 28%. It has also been found that the Cg2 allele in phenotypically normal plants mutates with a higher frequency in somatic cells (5.5-14%) than in generative cells (2.0-2.5%).

Individual comparative analysis of the behavior of hetero- and homozygotes up to generation 6 has shown that (1) in the progeny of heterozygotes, the segregation ratio corresponds to that of the monohybrid type, i.e. 3:1, but in this case nearly always there is an excess of the normal (+) phenotype and a deficit of mutants (Cg2), (2) in the progeny of Cg2/Cg2 homozygotes, solitary normal plants frequently occur. This, in turn, confirms the Cg2 gene instability, since frequent Cg2 ---> normal (+) reversions result in the generative tissue variegation, leading to the appearance, along with homozygous Cg2/Cg2 cells, of the Cg2/normal (+) heterozygotes. And this, in turn, results, on selfing, in a proportion of plants being of the normal, (+)/(+), type.

That the above mutation is unstable is also evidenced by genotypic differences between progenies of two different ears harvested from a single plant. The same is actually indicated by high frequency of mosaic plants. And finally, this is particularly obvious from the analysis of data on the genotypes of mutant and normal shoots of the same mosaic plant.

5. Another significant feature of the Cg2 mutation is its pronounced mutator activity. An intensive morphogenetic process is observed over 25 generations when various maize forms are crossed with this macromutation. In a number of cases, it was even possible to speak of the Cg2 gene ocasionally inducing a kind of outburst of mutator activity. In cases like these, it could even be called a "biological mutagenic factor".

In a number of elegant and carefully performed experiments, Dr. N. V. Krivov obtained six monogenic recessive chlorophyll mutations, one recessive mutation resulting in leaf necrosis, one mutation phenotypically resembling the ramosa (ra) mutation, and one mutation with abnormal development of vegetative plant organs. In addition, two mutations arose upon crossing the Cg2 with marker lines. One of these was allelic to Japonica (j), the other being linked to markers on two chromosomes and designated as Zebra7 (Zb7).

All the above facts can be very well explained in terms of insertional mutagenesis. It is by insertions of mobile genetic elements (TEs) capable of carrying a peculiar kind of "DNA genetic punctuation marks" such as promoters, enhancers, terminators, etc. that changes in gene activity and their reflection in the phenotype can be explained. This can provide explanations for multiple allelism occurring in the progeny of Cg2, frequent allelospecific transitions and just new mutations arising at the loci sometimes far removed from the Cg2 gene locus itself.

Of course, one cannot help mentioning that the Cg2 mutation exhibits a property rarely found with insertional mutagenesis, the one under which a TE insertion results in a dominant rather than recessive mutation. However, here, as with insertional mutagenesis, the original type of phenotypic expression is partially or completely restored upon excision of the TE from the Cg2 locus.

While classical works of B. McClintock and other authors show the genetic instability in maize to be characteristic of only those genes which determine the variegated coloration and the structure of the kernel, studies of the radiation-induced macromutation Corngrass2 discovered in Moldova have shown for the first time that genetic instability of the Cg2 gene is expressed at the earliest stages of zygote division and is capable of strongly affecting the structure of both vegetative (somatic) and reproductive organs resulting in an intensive morphogenetic process.

Since McClintock`s experiments were substantiated by molecular techniques, it can be assumed in our case that the Cg2 gene also possesses the property of the so-called "jumping gene", or transposon and, due to this, exhibit the features of a "genetic mutagen". It is only in this way that the "explosion" ("blast") of the morphogenetic process observed over the years can be explained. Therefore, studying specific features of the radiation-induced macromutation Cg2 by molecular biological techniques is becoming a very promising approach. Studies like these are already under way at the Institute of Genetics, Academy of Sciences of Moldova (Kishinev).

Even if the Cg2 gene is not assigned to the category of mobile genetic elements as a result of the studies now in progress, its role as a new, unusual mutator system in maize calls for closer attention and further studies. This is needed in order to understand the theory of evolution of the maize plant and for practical breeding involving this mutator system as a new, powerful driving force of the morphogenetic process aimed at producing new parent stocks of maize.

Concluding the description of the radiation-induced macromutation of maize, "Corngrass2", it should be mentioned that in the 1986 Maize Genetics Cooperation Newsletter included the Cg2 gene (i.e. our "corngrass" mutation) in the list of new unlocalized genes of maize.

Another crop which proved to be a very promising one in terms of application of experimental mutagenesis techniques was the garden gladiolus, Gladiolus hybridus hort. Gladiolus is, in itself, a valuable and interesting flower crop to which new genetic methods were previously inapplicable. It is in this Institute that Dr. A. V. Murin launched and has been carrying out serious research on the gladiolus genetics. This work has yielded an extensive and valuable new parent material for applied breeding. Unfortunately, gladiolus has a number of features posing difficulties to obtaining new parent material by experimental mutagenesis techniques. May be it is for this reason that there has been relatively little literature on the subject. Some of the factors complicating work with gladiolus are as follows:

1) the complex and, probably, interspecific hybrid origin of cultivated gladiolus; although this origin of gladiolus may be regarded as its remote past, it may have occasional and unusual effects in the present as well;

2) the tetraploid condition which, presumably, was absolutely necessary in the past for genome stabilization, but whose peculiar features have to be reckoned with at present;

3) relatively large number (2n=60) of small chromosomes with karyotype features scarcely studied in detail;

4) a complex triennial life cycle.

However, gladiolus has some properties that make it a convenient organism to work with. Thus, two modes of reproduction - sexual and vegetative - occur in this plant. This enables the experimenter to preserve and maintain in the progeny any variability present in the parents by switching from sexual to vegetative reproduction. Another equally useful property is the possibility of producing plants with chimeric tissues, since subsequent radiation-induced production of non-chimeric tissues greatly accelerates the breeding process.

A task was set to produce new valuable parent material for breeding, using advanced genetic techniques, primarily those of experimental mutagenesis and experimental recombinogenesis. Use of a set of chemical mutagens based on substances discovered and synthesized by Prof. I. A. Rapoport as well as irradiation of seeds, pollen, bulbs, and bulbils with ionizing radiation resulted in the production of extensive original material.

In doing so, highly decorative, disease and pest resiatant forms showing good transportability were produced which, in addition, exhibited the degree of variability not found in the cultivated gladiolus. The entire range of forms obtained can be classified as four new types of gladioli:

1) fragrant gladioli having the following odors: flower-scented, citrus, carnation, rose, coffee, etc.;

2) remontant, i.e. capable of regrowth and of producing a second and, occasionally, even a third flower after the first one has been cut;

3) double-flowering, carrying flowers with a large, sometimes very large, number of spikelets;

4) moire flowers with petals of motley coloration which occasionally looks like an ornament or fringe.

As a result, an extensive collection of new parental stocks numbering over 6000 specimens was created. Of these, 500 forms were displayed at various exhibitions, including international ones, where they won various prizes. Two varieties were introduced into cultivation in Moldova.

Thus, application of experimental mutagenesis in Moldova showed this technique to be a promising tool both for elaboration of theoretical problems of genetics and for practical breeding.

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