Use of maternal haploids for improving maize inbred lines
--Chalyk, ST (Cealic, ST)

Spontaneous recurrent mutations result in each maize material, exhibiting a certain amount of harmful mutant genes. Most new mutations are recessive and are slow to be eliminated. They represent so called genetic load (King and Stanfield, 1997). Maize lines, populations and hybrids become less vigorous due to this load.

Cleaning of a breeding material from harmful spontaneous mutations is very desirable. However, elimination of the genetic load is difficult because most of the harmful mutations are of recessive nature. Their appearance is masked by normal homologous alleles in diploid plants. Use of maize haploid plants can be an efficient means for clearing of a breeding material. A possibility of obtaining maize maternal haploids in mass quantity was shown in numerous works. Very important for a breeding program is the fact that the maize maternal haploids are quite viable. They grow normally during the vegetative period. Maternal haploids are usually male sterile. However, some of their female flowers are fertile and function normally. If pollinating an ear from a maize haploid plant with pollen taken from a diploid plant the ear usually forms kernels between several and several dozen. This property of the maize maternal haploid plants allows involving them in a breeding process without doubling a chromosome number.

In our experiment, maternal haploid plants were used to improve two maize lines, 092 and Rf7. Haploids were produced from these lines using a haploid-inducing line. The resulting haploid plants were grown in a field plot. During flowering, well developed healthy haploid plants were pollinated with pollen from diploid plants of the initial line. The seeds produced and the plants grown from them have been designated as the C1 progeny. Well-developed haploid plants are assumed to be free from mutant genes which reduce viability. The use of such plants allows the frequency of harmful genes to be drastically reduced. Therefore, the procedure in our work, in which a line passes through the haploid sporophyte ridding itself of harmful genes, is termed a haploid filter. Thus the C1 progeny is a progeny which has passed through the haploid filter once.

The next year haploids were produced from the C1 progeny. These were grown in an experimental plot and pollinated with pollen harvested from diploid C1 plants. In this way, seeds representing the C2 progeny were produced. That is, a progeny was obtained which had passed through the haploid filter twice. The initial lines, 092 and Rf7, were reproduced per se.

With the aim of comparison initial lines were grown in the field, designated as 092(C0) and Rf7(C0), and their progenies which had passed through the haploid filter once and twice, respectively: 092(C1), 092(C2) and Rf7(C1), Rf7(C2). Plant height is the most informative indicator of plant vigor during early growth of the plant. Therefore, plant growth dynamics was followed during the experiment. Presented in this paper are the results of two measurements. The first one was done at the 6-7 leaves stage (d1) and the last measurement was done after flowering when the plants completed their growth (d6).

To produce seeds for the next cycle of selection, most ears of the plants under study were hand pollinated. Therefore ear measurements and yield determinations were not performed.

The lines studied and their progenies - C0, C1 and C2 - were grown in adjacent experimental plots. The area of the plot was 15 square meters and the density of planting was 60 plants per plot.

The diploid initial lines studied (C0) and their progenies which passed through the haploid filter (C1 and C2) have been found to differ considerably in their growth rate during early developmental stages. Taking line 092 as an example, it can be shown that the tallest plants during early growth were those of C2 (Table 1). The height of these was 49.8±0.9 cm where that of the initial line 092 was only 37.8±0.9 cm. During early growth, the plant height difference was 12 cm, suggesting that the C2 plants exhibited a higher growth rate than those of the initial line (C0).

Table 1. Plant height values for diploid plants of lines 092 and Rf7 and their C1 and C2 progenies.
Plant height, cm
Genotype d1 Differences from initial line d2 Differences from initial line
092 (C0) 37.8±0.9   163.7±1.0  
092 (C1) 43.0±0.7 5.2*** 164.8 1.1
092 (C2) 49.8±0.9 12.0*** 171.1±0.9 7.4***
Rf7 (C0) 53.8±1.3   171.6±1.3  
Rf7 (C1) 56.5±1.3 2.7 177.7±1.1 6.1***
Rf7 (C2) 58.9±1.3 5.1** 176.3±1.4 4.7***
*,**,*** Differences from initial line are significant at 5%, 1% and 0.1%

The differences observed between C0 and C2 plants presumably reflect the efficiency of application of the haploid filter for improving the line. It can be assumed that the frequency

of harmful genes reducing viability is indeed lower in C2 than in the parental line. The C1 progeny showed an intermediate plant height value between the initial line and C2. The height of C1 plants was 43.0±0.7 cm taller than 092 plants. By the end of the growth season, the differences between the line studied and its C1 and C2 progenies decreased while remaining significant. The height of C2 plants at the end of the growth season was 171.1±0.9 cm, or 7.4 cm taller than the parental line 092.

The same tendency was observed in line Rf7 (Table 1). At the beginning of the growth season, the tallest plants were those of C2. Their height was 58.9±1.3 cm. This was 5.1 cm taller than the plants of the initial line Rf7. The C1 progeny showed intermediate values, being 2.7 cm taller than the initial line. That is, applying the haploid filter resulted, here too, in the plants of this line showing higher rates of growth during early development. This tendency continued throughout the growth season, although by the end of the season the differences leveled off. Thus, at the end of the growth season, C2 plants measured 176.3±1.4 cm in height, being 4.7 cm taller than the plants of initial line Rf7. At the end of the growth season, C1 plants were taller than C2 plants, the difference being insignificant and attributable to random factors.

The results from lines Rf7 and 092 are evidence in favor of an assumption that using
selection at the haploid sporophyte level appears to be a powerful tool for improving breeding material.

A useful indicator to the value of a genotype may be the haploids obtained from this
genotype. Therefore, besides comparisons of diploid plants of initial lines and their C1 and C2 progenies, haploid plants obtained from initial lines and their C1 and C2 progenies were also used for comparisons.

Plant height measurements of haploid plants are presented in Table 2. It has been found that the general tendency observed in diploid plants persists in haploid plants. At the beginning of the growth season, haploids from C1 and C2 progenies were taller than the plants of initial lines 092 and Rf7.

Table 2. Plant height values for haploid plants of lines 092 and Rf7 and their C1 and C2 progenies
Plant height, cm
Genotype of haploid plants d1 Differences from haploids of initial line d6 Differences from haploids of initial line
n092 (C0) 25.1±1.2   105.4±2.6  
n092 (C1) 30.0±0.8 4.9** 108.5±1.6 3.1
n092 (C2) 28.4±1.0 3.3* 105.6±3.3 0.2
nRf7 (C0) 35.9±1.3   108.3±1.4  
nRf7 (C1) 39.7±1.3 3.8* 110.2±2.0 1.9
nRf7 (C2) 39.3±2.3 3.4 109.1±3.4 0.8
*,** Differences from haploid of initial line are significant at 5% and 1%

It is worth noting that in this case, unlike that of diploid plants, the C1 progeny did not
show intermediate plant height values. Haploids of progenies C1 and C2 of line Rf7 were practically of the same height at the beginning of the growth season. In line 092, haploids from the C1 progeny were even taller than those from C2. This is attributable to the fact that in our experiment haploids obtained from C1 passed through a haploid filter twice. We suppose this was quite sufficient to rid the lines under study of harmful genes reducing plant viability. Therefore, haploid plants from progenies C1 and C2 were almost uniform in height. In diploid plants, the C1 progeny resulted from pollination of haploid plants with pollen harvested from initial lines. It is conceivable that with pollen from parental lines, part of a genetic load found its way into the C1 progeny. Therefore, in diploid plants the C1 progeny exhibited plant height values intermediate between the parental line and the C2 progeny which had passed through the haploid sporophyte twice. By the end of the growth season, the differences between the parental lines and their C1 and C2 progenies became less pronounced. However, in some cases they continued to be significant.

It may be concluded that in ridding inbred lines of harmful recessive genes the use of maternal haploid plants appears to be a tool that may be of interest to the breeder.

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