The development and refinement of maize mutagenesis techniques in Moldova
--Lysikov, VN

Early development of maize mutagenesis techniques in Moldova was based on the use of conventional methods employing ionizing radiation. The most common procedure was to expose dry maize kernels to X-ray or gamma radiation. Preference was given to gamma radiation since many types of gamma-ray sources (gamma-ray guns) appeared at the time. The development of new methods of exposure to radiation occasionally depended on specific features of certain gamma-ray units.

The majority of gamma-ray sources were charged with radioactive cobalt 60 (60Co) and provided hard gamma radiation. The half-life of cobalt 60 is 5.3 years, the gamma radiation flux energy being 1.71 MeV. In recent years, a proportion of commercially available gamma-ray sources have been charged with a cesium 137 (137Cs) isotope producing a relatively softer gamma radiation. Use of cesium 137 yielded better results since, unlike cobalt 60, its half-life was longer (some 30 years), making frequent recharges unnecessary. Admittedly, the energy of beta rays emitted by cesium 137 was lower (0.51 MeV).

Our early work largely relied on a comparatively small and low-power unit, GUT- 60Co-50-1, designed for gammascopy of hardware. Its capacity was some 100 Gy/h. The effective irradiated surface was about 10cm in diameter, i.e. a Petri dish with kernels could be placed in it.

Initially, only dry kernels were irradiated to produce mutations. By this time, the mutagenic irradiation dose was already known for maize kernels (100 to 150 Gy). This was within the so-called critical dose whose lethal values ranged from 50 to 60% (LD - 50-60). This irradiation dose consistently induced heritable changes, i.e. mutations. The absolute number of the resulting mutants was, however, low, about 1 to 5%. Such a low rate of mutations, of which far from all could be classed as favourable mutations of any interest to the breeder, could hardly be a satisfactory one. Therefore, the immediate task was to increase the quantity of irradiated material and, hence, the output of mutant forms. However, the small size of the irradiated spot and the comparatively long period of time required to produce a mutagenic dose (10 to 15 h per sample) were a major obstacle to increasing the amount of irradiated material. These difficulties could be overcome in one of two ways: (1) by purchasing a more sophisticated and powerful gamma-ray source, or (2) by developing new methods for irradiating seeds. In due course, we did purchase more powerful gamma-ray sources: GUBE - 60Co-4000-1 and RHM -_-20.

In order to save time, new methods of irradiation were proposed, carefully studied and widely applied.

Among the first irradiation methods to be proposed was that of irradiating water-soaked, rather than dry, maize kernels. In doing so, the mutagenic dose was halved, thus reducing the irradiation time per sample by one half and leaving the rate and range of mutations practically unchanged. Subsequently it was demonstrated that by irradiating not only soaked but also slightly sprouted seeds, the required mutagenic dose could be further reduced by a factor of the order of 2, with the rate and range of mutations remaining virtually unchanged.

Concurrently, a method was developed of irradiating not only water-soaked seeds but also seeds presoaked in solutions containing growth-promoting substances, chemical mutagens, amino acids, etc.

It is essentially the need for irradiating larger samples (and, hence, quantities of plant material) while using the same comparatively low-capacity gamma-ray source of the GUM - 60Co-50-1 type that led us to propose the maize pollen irradiation technique. This technique offered a number of advantages among which the following are worth noting: (1) the considerably extended productive time of the gamma-ray source: the seeds were irradiated prior to sowing, i.e. in spring, and pollen in summer, during maize anthesis, (2) a significantly reduced (by a factor of 2 to 3) required mutagenic dose for irradiating pollen, down to 20 to 30 Gy, and (3) the dramatically increased possibilities for irradiating greater numbers of pollen grains which, being much smaller in size than maize kernels, could be placed in larger numbers in a Petri dish in the gamma-irradiated spot.

It should also be noted that the pollen irradiation technique had another important implication to the geneticist. The sperm in a pollen grain is actually a single cell whereas maize kernels are multicellular structures. Each embryo cell can have a different function. Therefore, the possibility of a mutation occurring in a unicellular structure, such as the sperm, is much larger than in a multicellular one, such as the embryo. In an embryo, there is no way of knowing in which cell a new growth will occur and whether this particular cell will be directly involved in the formation of a new, i.e. mutant, organism.

For the work on maize pollen irradiation to be successful, a special procedure had to be developed and strictly adhered to. In essence, the procedure was as follows: (1) the plants chosen to be studied in the field were, prior to the study, isolated, i.e. the ear and the tassel were each covered with an isolating parchment bag, (2) the plants and both isolating parchment bags were assigned the same number, so that during pollination the pollen could be applied to its own female generative organs, i.e. stigmata, (3) the pollen was harvested in the field in the morning, immediately delivered to the gamma-ray source and, without delay, irradiated, so that the irradiated pollen could be delivered back to the field and applied to its own stigmata before noon. This is a very important prerequisite since, in Moldova, pollination of maize is generally ineffective after 12 o'clock in the noon when insolation is increased and the air becomes very hot. Selfing is to be strictly ensured.

Before long, the maize pollen irradiation technique was modified. In essence, the modofication consisted in that it became common practice to irradiate immature pollen, right in the tassels, rather than the harvested mature pollen. To this end, 4 to 5 days before heading, the tassels were detached from maize plants by an abrupt movement of the hand and immediately placed in a jar with water. Meiosis was found to be already completed in immature tassels, but tetrads continued to be formed. It was demonstrated, also experimentally, that for irradiating pollen in immature tassels the mutagenic dose could be reduced to 10-11 Gy (1000-1100 r.). This allowed the mutagenic dose to be achieved in immature tassels as soon as within an hour even with our low-power gamma-ray source.

Following pollination, the immature tassels were again placed in jars with water. Each tassel was covered with a parchment "isolator" and assigned the same number as the respective plant in the field. The female generative organs (stigmata) in a plant were also covered with parchment "isolators" in advance, to prevent the foreign pollen from alighting on them. The irradiated tassels were held in jars with water for 4 to 5 days to allow the pollen to reach maturity. Then, for the pollen to shed readily, the covered tassels were exposed to the bright sunlight for a few minutes. Thereupon, the pollen was taken to the field and applied to the respective (same number) plant, ensuring strict selfing. This technique gained wide recognition and was extensively used in various experiments.

Interestingly, it is the technique of immature tassel irradiation that was used as the basis for another, also promising technique, that of incorporating radioactive isotopes in maize pollen grains. For incorporation in pollen grains, solutions of radioactive isotopes of phosphorus 32 (32P) and sulfur 35 (35S) were employed. Precisely these isotopes were used because both phosphorus and sulfur were present in sufficiently large amounts in generative organs of maize. Another reason for choosing these particular isotopes was that there are significant differences between them. Thus, 32P produces rather hard beta radiation of the order of 1.7 MeV, whereas sulfur, on the contrary, yields soft beta radiation of the order of 0.13 MeV.

In essence, the above technique was as follows: immature maize tassels, previously covered with parchment "isolators" (bags) and properly numbered, were placed in solutions of radioactive substances rather than being placed in water. In doing so, preference was given to solutions in which radioactive isotopes were contained in a rather mobile form. Most commonly, these were orthophosphoric acid for 32P, and Na235SO4 and other solutions for 35S.

The immature tassels were held in radioactive solutions for 4 to 5 days. Then the pollen was harvested from them and applied to its own stigmata. The incorporation of radioactive isotopes, e.g. of 32P, in tassels was detected, using a Geiger-Muller counter, as soon as 15 minutes later. The applied pollen was also examined for the presence of radioactive isotopes using the appropriate instruments. While the radioactive isotopes finding their way into pollen grains were relatively few in number, their irradiation of pollen from within the pollen grains was quite appreciable and the efficiency of the method proved to be very high. This was probably due to the fact that the half-life of 32P is 14.3 days and that of 35S, 81 days, i.e. what actually occurred here was a long exposure to either hard or soft beta radiation.

The mutations resulting from incorporation of radioactive isotopes in pollen grains were quite unique: they exhibited an unusual variation range and, in a number of cases, many mutants with economic traits were identified. Thus, cytoplasmic male sterility mutations were obtained which were of high potential economic value in the maize seed industry.

Another technique of maize seed or pollen irradiation was that of combining low (stimulative) and high (mutagenic) radiation doses. This technique was developed with a view to increasing the relatively low viability of the mutants produced. Moreover, allowance was made for the fact that the irradiated object (seed or pollen grain) was composed of morphologically, physiologically and genetically heterogeneous tissues exhibiting differential responses to various low and high radiation doses. Two versions of the technique were studied: the direct, under which a low (stimulative) dose was applied first to be followed by a high (mutagenic) dose, and the reverse, where a high (mutagenic) dose was applied followed by a low (stimulative) radiation dose.

An important feature of this technique was variation in the timing of low and high dose application. This time interval - a peculiar kind of relaxation - varied from one maize cultivar to another, ranging from 5 min to 1 h in "best" treatments. In most cases, the best results were obtained with the direct method of irradiation combined with the relaxation time around 5 min.

Somewhat unique among the maize irradiation techniques is that of irradiating maize pollen with high (lethal) and superhigh (superlethal) doses. This technique was developed as a result of ingenious studies of Prof. V.S. Syomin (Moldova Institute of Viticulture and Fruit Growing) on interspecific hybrids of fruit crops and came to be known as Pandey method. It is apparent that high (lethal) and superhigh (superlethal) radiation doses all but kill maize pollen making it inviable, i.e. incapable of pollination and fertilization. Very high doses of the order of several hundred and even thousand Gy affect the genetic basis of pollen, i.e. DNA. They actually cause fragmentation of the DNA into stretches of varying length, or oligonucleotides. This peculiar fragmented DNA is incapable of growth within pollen tubes on maize stigmata and, consequently, can not reach the site of fertilization and participate in the fertilization process.

That is the reason why a special procedure was developed for induced delivery of these stretches of DNA (oligonucleotides) to the site of fertilization by means of a peculiar "vector". Acting as such a vector can be non-irradiated own pollen which readily germinates on maize stigmata. While germinating, this pollen entraps and carries along individual oligonucleotide stretches, occasionally so long that they contain genetic information corresponding to intact genes and, of course, a large number of small stretches carrying little genetic information. Our studies employed marker lines with a set of specific genes. In mixing irradiated donor pollen (containing fragmented DNA) with non-irradiated recipient pollen, incorporated at times are individual small DNA fragments and at other times larger oligonucleotide stretches carrying intact genes. A peculiar process of parallel involvement of these fragments in fertilization occurs. Furthermore, it has been demonstrated experimentally that in this case two processes occur simultaneously and in parallel: transfer of individual genes and experimental mutagenesis. Transfer of intact genes is controlled by using marker lines. The occurrence of new mutations, i.e. mutagenesis is attributable to small DNA fragments acting as mutagenic agents.

Two more maize irradiation techniques are currently being studied with a view to experimental production of new mutations. One involves irradiation of embryos isolated from maize endosperm, the other of female generative organs on plants growing in the field.

As is known, the maize embryo is diploid, whereas the endosperm is triploid. Since very early in its development the diploid embryo depends on the triploid endosperm for its nourishment, the latter may have a pronounced effect on the developing organism. In order to reduce or even eliminate the influence of the triploid endosperm, the embryo was isolated prior to or immediately after irradiation.

The isolated irradiated embryo is placed on a specially selected nutrient medium and grown according to the in vitro tissue culture procedure. From the resulting callus, regenerants are removed which are initially also grown on an artificial medium and then transplanted to pots with soil.

Already early results suggest increased somatic variability and even previously unobserved traits, such as expansion of the embryo scutellum, in some genotypes.

The other novel irradiation technique, that of irradiating female generative organs on a plant growing in the field, became feasible with the purchase of a portable X-ray apparatus, REIS-1. This instrument can be carried in a briefcase and is a battery- or mains-operated unit. It is mounted on a tripod in the field right in front of the chosen plant at the height corresponding to the position of the developing female generative organs (ears) of the maize plant. The apparatus is capable of autonomous operation. The X-ray beam produced by the apparatus is narrow and can be focused on a particular spot. The dose is determined based on the irradiation time.
 


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