Influence of Gamma Irradiation on Pollen Viability, Pollen Tube Growth, and Fruit Development in Tomato (Solanum lycopersicum L.)

Abstract

The goal of this study was to assess whether irradiated pollen technology could be used in tomato breeding research. The effects of irradiation on pollen viability, fruit set rate, and embryo formation were investigated. For this purpose, pollens were exposed to gamma rays of 0, 50, 100, 200, 300, and 400 Grays (Gy). The effect of irradiation on pollen viability and tube growth was found to be significant based on counting and measurements performed under in vitro conditions at 24, 48, 72, and 144 h after irradiation. Fruit set and embryo formation in seeds were evaluated 30 days after pollination with pollen irradiated at different doses. It was determined that increasing the irradiation dose resulted in reduced pollen viability and tube length. Endosperm formation was detected in all seeds after 50 Gy of irradiation. However, 50 Gy had no effect on gynogenesis stimulation. Therefore, 100, 200, and 300 Gy doses stimulated embryo formation without endosperm, while 400 Gy of irradiated pollen did not support fruit to set. These results indicate the importance of harvesting time to obtain viable embryos. It should be retracted to an earlier time since late harvest resulted in necrosis of globular embryos on the 30^th day after irradiation. Pollination with one-day-old irradiated pollen was more suitable for gynogenesis induction. The results showed that the irradiated pollen technique can be applied in tomato breeding studies, especially in terms of purifying the obtained breeding lines in a shorter time. In particular, determining the appropriate induction dose for gynogenesis depending on the genotype is important for stimulation efficiency.

Introduction

The tomato (Solanum lycopersicum L.) is a member of the Solanaceae family and is an important crop in Türkiye and around the world. Türkiye is the fourth-largest producer in the world, with an annual production capacity of about 13 million tons (https://data.tuik.gov.tr/Bulten/Index?p=Bitkisel-Uretim-Istatistikleri-2021-37249). It is in first place among vegetable species grown in greenhouses and fields. Owing to the negative effects of global warming, an increased number of diseases, and pests, various breeding programs are being undertaken for tomatoes. They are aimed at developing new varieties that can withstand all of these challenging conditions. In this context, a wide range of methods have been proposed. The breeding process is being speeded up in studies using traditional crossbreeding by using tissue culture techniques such as double haploidization (Sauton, 1989), embryo culture (Bosemark, 1993; Sharma, 1999; Taner, 2002), and anther culture (Sawhney et al., 1994). Additionally, due to the effectiveness of recombinant DNA technology, research on transgenic (GMO) varieties is proceeding rapidly (Frary et al., 2000; Sharma and Prasad, 2020; Zorzoli et al., 2007). Studies have progressed in recent years, especially with the use of gene editing methods like Crispr-Cas9 for mutant breeding (Açar and Aka Kaçar, 2021; Chaudhuri et al., 2022). Many tomato varieties have been developed to date as a result of integrating specific biotechnological methods into breeding studies. These cultivars can combine the tolerance mechanisms for biotic and abiotic stress factors. Consequently, new varieties with qualified tolerances are used in agricultural production. However, as the cost of advanced biotechnological methods in practice is high, classical breeding methods have maintained their importance in tomato breeding studies in many countries. However, crossbreeding studies continue to play a significant role in current research. Because tomato is an open-pollinated species, it is critical to purify the lines developed via breeding as quickly as possible. Currently, F₁ hybrid varieties are extensively used in tomato production, both in greenhouse and field cultivation. F₁ hybrid varieties can overcome the disadvantages of adverse agricultural conditions and achieve superior results compared to standard varieties because they have unique characteristics. However, rapid purification of parents for use in hybrid breeding programs is important for introducing new hybrid varieties to the market. Ovary and anther culture methods for in vitro techniques are crucial in breeding studies. Therefore, although anther culture, a tissue culture method, is used in tomato breeding studies, this method can be applied to tomatoes with lower success rates than in pepper and eggplant. As a result, research is being conducted to improve various techniques to speed up the breeding cycle. Haploid plants are obtained through cellular mechanisms such as gynogenesis, androgenesis, semigamy, polyembryony, and chromosome elimination (Blasco et al., 2016; Kambale et al., 2023; Sarı et al., 1994; Sauton, 1987, 1989; Segui-Simarro, 2021; Yang et al., 2004; Zamir, 1983). The irradiated pollen method is a technique used to induce gynogenesis. In particular, in experiments on the induction of gynogenesis, it was discovered that the irradiation dose and irradiation sources, depending on the genotype, have an impact on pollen viability and tube growth. Although studies have shown that pollen tube formation occurs, irradiation inhibits the development of the generative nuclei. The elongation of the pollen tube can be provided only by the activation of the vegetative nucleus (Pandey, 1980; Sauton, 1989; Taner and Yanmaz, 1996). Through this activation, the embryo formation mechanism can be stimulated in egg, synergid, or antipode cells. As a result, induction of the vegetative nucleus stimulates embryo formation even if the pollen tube does not penetrate the embryo sac (Boom and Nijs, 1983; Cuny et al., 1993; Denissen and Nijs, 1987; Kurtar and Seymen, 2021; Sarı and Solmaz, 2021; Taner and Yanmaz, 1996). This stimulation leads to the creation of a haploid embryo. Double haploidization is a technique that accelerates breeding and is useful for genetic mapping studies, especially for tomatoes (Zamir, 1983). Researchers have indicated that after using the irradiated pollen method, the allele frequency of the tomato species in segregation progeny provides a simple species for analysis in terms of traceability of the targeted genes (Tanksley and Rick, 1980; Zamir, 1983). Furthermore, this process has been successfully used to purify selected lines of species, such as melon, watermelon, pumpkin, cucumber, and snake cucumber, particularly in the Cucurbitaceae family (Cuny et al., 1993; Dal et al., 2016; Güler et al., 2017; Gürsöz et al., 1991; Ivanova, 2020; Kurtar et al., 2002; Lotfi et al., 1997; Sarı et al., 1994; Taşkın et al., 2013; Yanmaz et al., 1998; Yıldız et al., 2020). Other studies have revealed that this method is efficient for barley (Powell et al., 1983), apples (James et al., 1985), pears (Sniézko et al., 1987), kiwifruits (Musial and Przywara, 1998), cotton (Savaşkan, 2002), citrus (Kundu et al., 2014, 2016), and sesame (Audu et al., 2021). According to these findings, the irradiated pollen technique helps speed up the reproductive cycle. Although the induced pollen technique is not appropriate for all species, a few studies on tomatoes have suggested that it has the potential for use in breeding programs (Akbudak and Seniz, 2009; Cresti et al., 1977; Zamir, 1983). In mutation breeding studies, it is important to develop an irradiated pollen method to purify the obtained mutant lines and shorten the breeding period. The aim of this study was to demonstrate the effectiveness of the irradiated pollen technique for tomato breeding studies. In this regard, pollen viability, pollen tube development, and fruit set rate were followed after irradiation at various absorbed doses (0, 50, 100, 200, 300, and 400 Gy). This technique has been successfully applied to species of the Cucurbitaceae family (Kurtar and Seymen, 2021). However, intensive breeding studies are being conducted on tomato species worldwide. The results of previous studies on irradiated pollen techniques were found to be promising for tomato (Akbudak and Seniz, 2009; Zamir, 1983; Zorzoli et al., 2007). Unfortunately, there is currently no practical application in breeding programs. As a result, it is critical to incorporate this method into tomato breeding programs.

Materials and Methods

Plant material

Commercial tomato seedlings of Nalan cv. were used in this study. The seedlings were planted in 30 cm-diameter pots containing a 1:1:1 mixture of garden soil, peat, and burnt manure. The macro- and micronutrients needed for plants grown in a fully automatic greenhouse were supplied to the plants under controlled conditions of the irrigation system in accordance with the existing automation system at every stage of plant development.

Pollen irradiation and pollination

During anthesis, flowers were emasculated and isolated in isolation bags. Simultaneously, flower buds were collected from the plants at the anthesis stage for irradiation. The buds were exposed to radiation by using a cobalt (⁶⁰Co) source at the Turkish Energy Nuclear and Mineral Research Agency, Nuclear Energy Research Institute (TENMAK-NÜKEN). The buds were irradiated to doses of 0, 50, 100, 150, 200, 300, and 400 Gy with a dose rate of 223 Gy·h⁻¹ and at ambient temperature. Polination was performed immediately on previously emasculated flowers using irradiated and non-irradiated pollen to determine the difference in the fruit and embryo formation rate.

In vitro pollen viability and pollen tube growth determination

Emasculated flower buds were pollinated with 1- and 2-day-old irradiated and non-irradiated pollen. After first day pollination, irradiated and non-irradiated flower buds were soaked in n-pentan commercial chemical solution (CH₃(CH₂)₃CH₃) to collect the pollen. Then n-pentan was evaporated. The pollen was placed in Petri dishes containing silica gel capsules and stored in a refrigerator for further use. Measurements as described by Taner and Yanmaz (1996) were conducted to determine the effect of irradiation on pollen viability and in vitro pollen tube development under laboratory conditions. Both irradiated and non-irradiated pollen were sown on a nutritional medium containing 15% sucrose, 5 mg·L⁻¹ boric acid (H₃BO₃), and 1% agar (Gürsöz et al., 1991) in a laminar flow cabinet to avoid external contamination. The pollen was sown daily and incubated at 24 ± 1°C. The pollen viability and pollen tube growth experiments were conducted in four replicates. One-, 2-, 3-, and 6-day-old pollens under a binocular microscope (at 1.6 × magnification) were counted to determine the pollen tube formation rate of 100 pollen grains in each replicate (Fig. 2). The pollen tube length was measured after 144 h on one-day-old pollen incubated in vitro under a light microscope (20 ×/0.45) using an ocular with an objective micrometer. Measurements were performed in four replicates on 40 pollen tubes for each replication.

The effect of irradiation on fruit set and embryo formation

Based on the developmental stage of the flowers, 41 emasculated flowers were pollinated with irradiated and non-irradiated pollen. Thirty days after pollination with both irradiated and non-irradiated pollen, fruit counts were performed on plants cultivated in a thoroughly controlled greenhouse. The impact of different radiation exposures on fruit set was determined. The fruits were collected on the 30^th day and sterilized by surface burning with 96% ethanol before embryo resque. After sterilization, the seeds were removed from the fruits and opened in a sterile laminar flow hood cabinet to determine the effects of different radiation doses on endosperm and embryonic development. The seeds were then examined under a binocular microscope at 2.5 × magnification.

Statistical analysis

Statistical analysis of obtained data was performed using MINITAB and MSTATc statistical programs within a 0.01 error limit.

Results and Discussion

The effects of irradiation on pollen viability

The pollen vitability and tube length following the irradiation procedure were followed in this study to determine the effects of different radiation doses. It was found that the highest viability and pollen tube formation rate was 85% in control (one-day-old) pollen. It was determined that the next best average obtained from the control was that following a 50 Gy treatment with 54.33%; the ratio declined linearly with increasing irradiation doses (40%, 31%, and 25%). Accordingly, it was found that the lowest germination rate was 5.67% at 400 Gy (Fig. 1). Statistically, it was determined that there was no difference between the viability rates obtained in the control and 50 Gy applications and that the difference between 200, 300, and 400 Gy applications was not significant. However, there was a difference between the 100 Gy application and other applications. It was statistically determined that pollen viability was influenced by the interaction between pollen age and irradiation dose.

Fig. 1

The effects of irradiation on the pollen germination rate (%) (The differences between means were determined with letters according to the DUNCAN test (a, b, c, and d) at P < 0.01 and LSDv: 0.4354, Sx: 0,0183).

The dose-dependent effect of irradiation on pollen viability was found significant at 0.01 probability. Pollen viability drastically declined with age, as seen in a comparison of the vitality of irradiated and non-irradiated two-day-old pollens. Except for the control and 50 Gy treatments, the viability rate was determined to be 0.00% in three-day-old pollen. The six-day-old pollen’s vitality was observed to be 0.00% in both the control and at all doses. Akbudak and Seniz (2009), found similar results for tomato and they reported that the viability rate decreased as the irradiation dose increased. In studies conducted on several species, including melon and pumpkin, pollen age and radiation dose were found to have a significant influence on pollen viability. It was found that one and two-day-old irradiated pollen was suitable for pollination research to encourage gynogenesis (Gürsöz et al., 1991; Taner and Yanmaz, 1996). In parallel, Kurtar (2009) discovered that depending on the radiation dose, pollen age, and growing season, the vitality of pollen exposed to radiation at doses between 50 and 300 Gy was reduced.

The effects of irradiation on pollen tube elongation

To ascertain the effect of different irradiation doses on pollen tube growth and tube length, they were examined in vitro (Fig. 2). As can be seen in Figure 3, the average pollen tube length of the control pollen was 3.67 mm according to measurements taken 144 h following the in vitro sowing of the one-day-old pollen, and this value decreased to 3.22 mm with 50 Gy treatment. At 100 Gy onwards, it was seen that the pollen tube length decreased as the irradiation dose increased. The average pollen tube lengths were 2.28, 1.50, 1.39, and 1.12 mm at increasing doses, respectively (Fig. 4). Statistical analysis showed that the difference in pollen tube development between the treatments was significant within a 0.01 error value. Other trials for watermelon (Sarı, 1994) and melon (Taner and Yanmaz, 1996) had similar results. Therefore, it can be inferred that pollen tube growth was negatively affected by higher radiation exposure.

Fig. 2

In vitro pollen tube formation with the 50 Gy treatment (magnification 20×/0.45).

Fig. 3

The effect of irradiation doses on pollen tube length and differences between doses (difference determined by letters (a, b, c, d, e, and f) according to the DUNCAN test, LSDv: 0.7565, Sx: 0.1994).

Fig. 4

Fruit formation after polination with irradiated and non-irradiated pollen.

The effects of pollunation with irradiated pollen on fruit set, seed and embryo formation

Fruit set rates were evaluated after pollination with irradiated and non-irradiated pollen on the 30^th day after pollination. During counting, it was observed that the control had a 100% fruit set rate. Other fruit set rates according to doses were 33.33% for 50 Gy, 40% for 100 Gy, 12.5% for 200 Gy, 83.33% for 300 Gy, and 0% for 400 Gy. Depending on the irradiation dose, the seeds that had been removed from the fruits were inspected under a binocular microscope to determine whether embryonic or endospermic development occurred. Radiation exhibited no detrimental impact on endosperm development at a dose of 50 Gy. Since the 50 Gy gamma rays did not inactivate generative nuclei in the pollen tube, the endosperm developed at a similar level to the control. At the other irradiation doses (100, 200, and 300 Gy), the endosperm did not form in the seeds, but necrotic embryo formation occurred (Fig. 5). This result confirmed that irradiation induces gynogenesis. The fruits formed following pollination at doses other than the control and 50 Gy treatments had fewer seeds. As shown in Figure 4, fruit development decelerated with increasing irradiation doses. However, the quality of the seeds developed in the fruit is the most important factor in this situation (Kundu et al., 2014; Yıldz et al., 2020). In a similar study conducted by Zamir (1983) on tomato, it was reported that the number of seeds in the fruit decreased dramatically after pollination with irradiated pollen with increasing doses. The same finding was obtained in this research at a 300 Gy dose. The fruit set rate was 83.3%, but the mean seed number was 30 in fruits (Fig. 4). In another study on Cucurbit species, it was found that the fruit harvest period is critical to obtain viable globular embryos (Kurtar, 2009; Yanmaz et al., 1998). Appropriate harvest timing allows embryos at the globular stage to survive without necrosis, and they can be regenerated as in vitro plantlets using the embryo rescue technique (Blasco et al., 2016; Ivanova, 2020; Kurtar et al., 2002; Sarı et al., 1994; Yanmaz et al., 1998). Similarly, based on the results of this study, it is considered that harvesting on the 30^th day after pollination was late. Seeds have to be removed from the fruits 15–21 days after pollination. By this time, embryos that have been rescued in this manner can survive without necrosis. The radiation doses to be used in the induced gynogenesis method using irradiated pollen for species such as melon, cucumber, and acur have been established in studies that have been completed to date, but they vary according to the species. To date, no effective dose has been determined for tomatoes. It is thought that irradiation at doses of 200–300 Gy would be effective for early embryo rescue, as reported in this study. Applications below 200 Gy have been ineffective.

Fig. 5

Embryo formation with endosperm with the 50 Gy dose and necrotic embryo (ne) formation at higher irradiation doses (magnification 2.5×).

Conclusion

This research was carried out to determine the efficacy of the irradiated pollen technique in inducing gynogenesis in tomato breeding studies. In this study, the impact of various radiation doses on pollen viability, pollen tube development, fruit set, and embryo formation rate were examined. According to research conducted to determine the effects of different irradiation doses on pollen viability and the ability to form pollen tubes, pollen viability decreases with increasing radiation dose and pollen age. The viability rate of the one-day-old control pollen was 85%, which declined to between 54.33% and 5.67% when the irradiation dose was increased. Based on in vitro experiments to confirm the effects of irradiation on pollen tube growth, it was determined that the length of the pollen tube decreased, and development decelerated, as the irradiation dose increased. The average pollen tube length was 3.67 mm, measured 144 h after the control pollen was incubated in the in vitro nutrient medium, while the pollen tube length linearly decreased in other applications based on the doses. In contrast, the pollen tube was measured at 1.15 mm at a dose of 400 Gy. To better understand gynogenesis induction, the effects of doses on fruit set rate were investigated after pollination with different doses of irradiated pollen. It was observed that the expected effect was not achieved with the application of 50 and 400 Gy doses. Endosperm-free seeds were obtained at doses of 100–300 Gy. Irradiation doses in the 200–300 Gy range were found to be promising doses for embryo induction. However, late harvest applications have a negative effect on embryonic development. From the data obtained in this study and other reported studies, it appears that this technique is promising, can be used in tomato breeding, and will shorten the breeding process.

Acknowledgements

The author is thankful to Prof. Dr. Ş. Şebnem ELLIALTIOGLU for her support with tomato seedlings.

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