New morphotype of vegetable corn obtained by the method of experimental mutagenesis

Kulish O., Parii M.

30 Vasylkivska Str., Kyiv, 03022, Ukraine

Goal. To obtain genetic mutations using strong chemical mutagens, which have a broad spectrum of action (sodium azide (NaN3) and 5-bromuracil (5BrUra)), with the aim to involve in the selection process; to improve existing and create new high-yielding hybrids of vegetable corn with improved qualities. Methods. Analysis of literature sources, use of strong chemical mutagens, instrumental and statistical. Results. It was established that 5BrUra can be used as a chemical mutagen on corn plants, and point mutations, which are formed under the influence of that mutagen, are a valuable genetic material in selection practice. A new promising genotype of vegetable corn was obtained. According to the results of comparative evaluation of the phenotypes of the obtained mutant form and the studied collection samples, their significant differences were established. The branching of the obtained form of plants occurs from the base of the rod to the apex, while for known mutant forms -- partially from the base to the middle of the rod, or the rod does not branch at all, and several lateral branches are formed. Conclusions. The possibility of using 5-bromuracil as a chemical mutagen on plants has been established. Point mutations, which are manifested under the influence of this mutagen, are valuable genetic material in selection practice. According to the results of comparative evaluation of the obtained mutant forms with collectible samples, it was found that the phenotype of the studied forms have significant differences. A new form of vegetable corn was obtained on the basis of cob branching, which is promising for further research and introduction into production as a new generation of baby corn.

Key words: chemical mutagen, sodium azide (NaN3), 5-bromuracil (5BrUra), mutations ra1, ra2, ra3.

Introduction

Vegetable (sweet) corn is in demand in Ukraine and is characterized by high yields; its cobs have excellent taste and nutritional properties. Vegetable corn first entered the world market in the late XVIII - early XIX centuries in the United States of America. Since then, for 100 years, there have been only 12 varieties of this culture in the world. Sweet corn is a valuable food product that has a unique composition of easily digestible carbohydrates: fructose, sucrose, glucose, maltose, raffinose; contains 12-15% of starch, about 3% of protein, including essential amino acids lysine and tryptophan, 1% of fats, C, B1, B2, PP vitamins, as well as mineral salts containing Ca, K, Mg, Fe, Na, P, Cl and S. In 2015, 67 varieties of vegetable corn were included in the Register of varieties allowed for distribution in Ukraine [1].

Analysis of recent research and publications. Polymorphism of sweet corn is not genetically diverse enough, so artificial mutagenesis is used in breeding programs to expand it. The source of genetic diversity of corn are mutations that, in turn, are the original (starting) material in breeding of this crop. Stadler used first radiation to obtain artificial mutations in corn in the 30s of last century, and Rapoport and Auerbach discovered the existence of powerful chemical mutagens in the late 1940s [2, 3]. Among the modern approaches using artificial mutagenesis are Tilling, CRISPR/cas and insertional mutagenesis. Tilling is a method of molecular biology that allows to identify targeted mutations in a particular gene. Tilling was first used on the model object Arabidopsis thaliana in 2000. Since then, it has been used as a method of reverse genetics for other organisms (such as corn, wheat, rice, soybeans, tomatoes and lettuce). This method combines a standard effective mutagenesis method with the chemical mutagen ethyl methanesulfonate (EMS) with a sensitive DNA screening method that identifies one baseline mutation (so-called point mutations) in the target gene [4-6].

It is known that many different chemical mutagens are used for mutagenesis of crops. The chemical mutagen sodium azide (Na№) causes persistent mutations in the DNA sequence of the plant genome [7].

The classic mutagen 5-bromuracil (modified thymine, 5BrUra) has been used for more than half a century to obtain mutants in bacteria [3, 5, 6, 8-10], but there is no information in the available scientific data on the use of 5-bromuracil in plants. Numerous studies have shown experimentally that 5BrUra intensely causes point mutations, in particular, transitions of inclusion and replication in bacteriophages, bacteria and some higher organisms [3, 5, 6, 8-11]. In most cases, these are the transitions guanine-cytosine (Gua-Cyt) ^ adenine-thymine (Ade-Thy), but this pattern has many exceptions. Usually 5BrUra is incorporated into DNA randomly in small amounts, but in some cases it can almost completely replace Thy. However, the physicochemical mechanisms of 5BrUra mutagenic action remain insufficiently studied [11-16]. In particular, there is no universal hypothesis that would quantitatively explain both the origin of inclusion errors and replication errors from a physicochemical standpoint.

The purpose of research. The aim of the study was to obtain genetic mutations using strong chemical mutagens that have a wide spectrum of action - sodium azide (№N3) and 5-bromuracil (5BrUra), to be involved in the breeding process; to improve the existing and to create the new high-yielding hybrids of sweet corn with improved qualities.

Materials and methods of research. In our studies we used the corn line PYA-3 (parental component of hybrids from the All-Ukrainian Scientific Institute of Breeding) and samples 727 G, 708 A, 308 E, 725 A, 725 B from the collection of Maize Genetics Cooperation Stock Center [1].

As a mutagenic factor was used 5-bromuracil (5BrUra) in concentrations: 0.0191, 0.0955, 0.191, 0.573 and 1.91 g/l, respectively; and sodium azide (Na№) - 1.95, 2.6 and 3.25 g/l [17].

Corn grains were germinated at a temperature of 24⁰C on moistened filter paper until the first roots up to 0.4 cm long. Then eight samples of 100 seeds were taken and soaked in solutions of chemical mutagens 5BrUra and NaN in the above concentrations. Exposure time was two hours. The PYA-3 line, the grains of that were not treated with mutagens, was used as a control [17].

Obtained in generations M0, M1 and M2 plants self-pollinated. The 272 families of the M1 generation were sown. The number of branches on the panicle and cobs, the length of the branches on the cobs, the productivity of the cob and individual branches were determined for the mutant form. Statistical analyses of obtained experimental data were carried out using Microsoft Excel (p> 0.05).

Results and discussion

A decrease in seed germination was observed in the M0 generation. Seed germination decreased with increasing concentration of mutagenic factor. Phenotypic changes of plants - mutations of maize plants (curved stem, dwarfism, lack of generative organs) were detected in all variants during subsequent observations of their development (Table 1).

Table 1 Phenotypic effect in Mo generation