Selective Elimination of Gametes during Pollen Storage at Low Temperature as a Way to Improve the Genetic Structure of Sporophytic Population for Cold Tolerance

Introduction

It is known that many genes are expressed at the level of gametophyte at different stages of its development, including pollen maturation, mature pollen grain, pollen germination and tube growth. Many of these genes are expressed both at the gametophytic and at the sporophytic levels (Mulcahy, 1979; Hormaza and Herrero, 1992). This is the basis for the selective elimination of gametes, which may lead to a change in the structure of segregating sporophytic populations.

One of the first who found the selective elimination of gametes was Brink (1925). He stored F₁ maize pollen over calcium chloride at 40°C and revealed the change in Mendelian segregations in F₂ populations in the direction of increasing the number of plants with waxy seeds.

Subsequently, there was a lot of work concerning the selective influence of various agents on the pollen of different plant species. It was shown that maize pollen storage for a long time not only at low but also at room temperature could change the structure of sporophytic populations due to selective elimination of male gametes. In this case, both the changes in monogenic ratios for some marker genes and in evaluation of recombination frequency between them were observed (Lyakh and Soroka, 1992). Heather J. Clarke et al. revealed the change in F₂ population structure for flower color after growing of F₁ chickpea hybrid plants at low temperature (Clarke et al., 2004). Significantly modified monogenic and digenic ratios for two DNA markers were found after pollen selection for resistance to toxins of wilt pathogen in Cicer arietinum L. (Ravikumar et al., 2006).

It is now known that pollen selection for tolerance to many abiotic factors, including low temperature, is quite effective. Mature pollen storage at low temperature increased the cold tolerance in tomato (Kravchenko et al., 1988), rape, flax (Lyakh et al., 2000) and other crops. Pollen selection for cold tolerance during pollen germination and tube growth has been successfully used in tomato (Zamir et al., 1982; Dominguez et al., 2005) and chickpea (Heather J. Clarke et al., 2004). There were also positive results on the macrogametophytic (ovule) selection for cold tolerance in tomato (Kravchenko et al., 1988).

Acreage expansion of sunflower to the north is limited by unsufficient cold tolerance of plant, especially at early stages. For a successful early sowing of sunflower, it is important to increase the cold tolerance during seed germination, at the seedling and 2–3 pairs of leaves stages. To cultivate sunflower at high altitudes and in cold regions, frost tolerance during the plant ripening should be increased. Some wild species of Helianthus, growing in cold conditions, could serve as the sources of frost tolerance. However, sunflower breeding for cold tolerance was not almost conducted (Skoric, 2009). At the same time, some cold tolerant varieties of sunflower using cold seed germination test have been revealed (Sirotin et al., 2007).

The aim of this paper was to investigate the influence of F₁ pollen storage at low temperature on the genetical structure of F₂ segregating populations including the monogenic ratios for some marker traits and cold tolerance of sporophytic generation.

Materials and methods

F₁ sunflower hybrids of “dichotomous venation” × “xantha”, “xantha” × “dichotomous venation”, “xantha” × “dwarf” cross combinations were used as the material for research. The parental lines of these hybrids were contrasting in cold tolerance.

“Xantha”, “dichotomous venation” and “dwarf” lines were obtained through experimental mutagenesis. “Dichotomous venation” mutant sample has the marker trait of modified leaf venation. In contrast to the original line, which has reticulate venation, the mutant is characterized by a dense network of the fan-shaped veins. “Dwarf” mutant has shorter internodes, compact habit, serrate leaf margin and possesses the xeromorphic traits. Both mutant traits are easily identified at early stages of plant development (Lyakh et al., 2005).

F₁ hybrids were grown in the field conditions during 2013. Pollen mixture of several F₁ plants was placed in parchment packages (1 cm³ per package) and stored in a refrigerator at 3±1°C for a period of 7 days for “dichotomous venation” × “xantha” and “xantha” × “dwarf” plants and 8 days for “xantha” × “dichotomous venation” cross combinations. Viability test showed that pollen treatment significantly decreased pollen germination on the artificial nutrient medium. After that previously emasculated F₁ plants of the same cross combination were pollinated with stored pollen. F₁ plants pollinated with fresh pollen were used as the control.

Cold resistance of F₂ sporophytic populations was evaluated by the seed germination at low temperature. For this purpose seeds were treated with 1% KMnO₄ solution for a period of 10 min. The seeds were then placed in Petri dishes on a filter paper previously moistened with distillated water. It was boiled beforehand for a period of 5 min, and then nystatin (250 thousand units/L) and Previkur (2 mL/L) were added. Closed Petri dishes were placed in a refrigerator at 5±1°C. After 7 days, the percentage of seed germination was calculated (Polevoy et al., 2001).

Germinated and not germinated seeds were separately planted in wooden boxes in the phytotron at an optimum temperature. The genetic structure of F₂ segregating populations for “dichotomous venation” and “dwarfness” marker traits was analyzed at the stage of the second pair of true leaves. The following comparisons were performed: (a) experimental (stored pollen) to control (fresh pollen) F₂ populations, composed of seeds germinated and not germinated in Petri dishes at low temperature; (b) experimental (stored pollen) to control (fresh pollen) F₂ populations, composed of seeds germinated in Petri dishes at low temperature; (c) F₂ population, composed of seeds germinated in Petri dishes at low temperature, to F₂ population, composed of seeds not germinated in Petri dishes at low temperature, both experimental and control.

The differences in cold tolerance between the control and the experimental populations were defined by the t-test at the levels of probability of 0.001. Differences in the segregation ratio were evaluated using the χ² method.

Results and discussion

As is shown in Table 1, pollen storage at low temperature in F₁ sunflower hybrids changed the genetic structure of F₂ populations for “dichotomous venation” and “dwarfness” marker traits.

As compared to the control low temperature storage of heterogeneous F₁ pollen population significantly increased in F₂ sporophytic populations the number of plants with “dichotomous venation” and “dwarfness” marker traits in “dichotomous venation” × “xantha” and “xantha” × “dwarf” cross combinations, respectively. Thus, we can say that such procedure favors gametes with the named marker traits. As a result, in F₂ populations the number of plants possessing these marker traits was increased. However, the change of the genetic structure of F₂ population was not observed in “xantha” × “dichotomous venation” cross combination.

Segregation ratios for marker traits in F₂ populations, composed only of seeds which germinated in Petri dishes at low temperature, were analyzed in Table 2. This part of F₂ population is the most cold tolerant part.

The data presented in Table 2 pointed out that low temperature pollen storage in F₁ hybrids of “dichotomous venation” × “xantha” and “xantha” × “dwarf” cross combinations increased the number of plants with marker traits “dichotomous venation” and “dwarfness” in the most cold tolerant parts of F₂ populations, respectively. This effect was not observed in “xantha” × “dichotomous venation” cross combination.

Table 3 shows the comparison of the genetic structure of F₂ populations, composed of seeds that were germinated and not germinated at low temperature in Petri dishes, both experimental and control. This will allow to evaluate the influence of pollen treatment on the difference in segregation ratios of analyzed phenotypes between control and experimental F₂ populations.

In the control, the difference in segregation ratios between F₂ population, composed of germinated seeds, and F₂ population, composed of not germinated seeds, was not observed in “xantha” × “dichotomous venation” crossing combination. In the experimental F₂ population however, such difference was evident. The thing was that the storage at low temperature of heterogeneous pollen population of this F₁ hybrid increased the number of plants with the “dichotomous venation” marker trait in F₂ population, composed of germinated at low temperature seeds, compared with F₂ population, composed of not germinated at low temperature seeds. This indicates that treatment of pollen with low temperature increases in F₂ population the proportion of cold tolerant genotypes possessing the “dichotomous venation” marker trait. Despite the fact that the effect of pollen treatment on the genetic structure of F₂ population according to the data in Tables 1 and 2 was not observed, such effect was found in this comparison.

A similar situation of the selective elimination of gametes after pollen treatment was observed in “xantha” × “dwarf” cross combination. The difference in the segregation ratios between F₂ population, composed of germinated at low temperature seeds, and F₂ population, composed of not germinated at low temperature seeds, was revealed in the control in the direction of increasing the number of plants of the “dwarf”-type among the not germinated seeds. However, no difference in segregation ratios was found in the experimental population. Thus pollen treatment, as compared to the control, increased the number of plants with the “dwarfness” marker trait in F₂ population, composed of germinated at low temperature seeds. The data of segregation ratios for both cross combinations show the change in F₂ population structure after low temperature storage of pollen in F₁ hybrids.

Summarizing the data presented in Tables 1–3, it is possible to draw a general conclusion that pollen storage at low temperature influences selectively the male gametophytes of F₁ hybrids that changes the genetic structure of F₂ populations. This selective influence resulted in the increase in F₂ the number of genotypes with “dichotomous venation” and “dwarfness” marker traits.

The change in the cold tolerance of F₂ populations also indicates the changes in the genetic structure of these populations after pollen treatment in F₁ hybrids (Table 4). This cold tolerance was determined by the percentage of seed germination at low temperature.

Pollen treatment in “dichotomous venation” × “xantha” and “xantha” × “dichotomous venation” cross combinations increased the percentage of F₂ seeds germinated at low temperature from 73.7% to 87.7% and from 21.0% to 58.0%, respectively. In “xantha” × “dwarf” cross combination, the percentage of F₂ seed germination at low temperature in the experiment did not differ from the control. In this case, pollen storage was not effective to enhance the cold tolerance of F₂ population.

Taking into account that pollen treatment in F₁ hybrids of “dichotomous venation” × “xantha” and “xantha” × “dichotomous venation” cross combinations increases the cold tolerance of F₂ populations and at the same time the number of plants with “dichotomous venation” marker trait we can assume that the gene, which determines this marker trait, is at least partially linked to the loci(locus) that determine(s) the cold tolerance in sunflower.

The effect of F₁ hybrids pollen storage at low temperature on the structure of F₂ sporophyte populations was earlier studied in sunflower. It was found that such pollen treatment increased the number of plants with the traits of more cold resistant parent (Gasenko and Lyakh, 1997). However, those data did not allow to conclude about the change in the cold tolerance of the experimental population after pollen selection application.

In sunflower, the selective elimination of the gametes was also observed after heating heterogeneous pollen population in F₁ hybrids. Such treatment favored the male gametes possessing the genes which determine heat and drought tolerance (Lyakh and Totsky, 2014).

The obtained results, indicating that during pollen storage at low temperature the selective elimination of gametes is observed, should be taken into account in sunflower breeding programs as many valuable genotypes can be lost due to such procedure.