**2. Haploidization and doubled haploid (DH) technique**

If the number of chromosomes in the somatic cells of the plant species is as much as the number of chromosomes found in their generative cells, these plants are called haploid (with n chromosome, single chromosome set), and the process of obtaining haploid plants is called haploidization. The implementation of chromosome doubling of haploid plants with spontaneous doubling or certain antimitotic chemicals brings the chromosome numbers to the normal chromosome numbers and 100% homozygous plants are produced. This stage is called doubled haploidy (DH) or dihaploidization [12]. With the use of DH lines, providing the desired properties, as a male or female parent in hybrid (F₁) seed technology, the importance of DH technology has become even better understood, and its use has become increasingly widespread.

Haploidy studies, which began with the discovery of spontaneous haploid plants in 1922 [13], gained momentum in the laboratory conditions after the 1960s, and they are becoming increasingly popular nowadays. As far as it is known, spontaneous parthenogenetic haploid plant formation in eggplant has not been encountered so far. In laboratory conditions, the first study to obtain this type of haploid plant is on anther culture and reported by Raina and Iyer [14]. It was later reported by the Chinese Haploid Research Group that developed first healthy haploid and doubled haploid (DH) eggplant plants [15]. In other anther culture studies [16–21] following them, the production of haploid and DH eggplant plants has been successfully carried out.

In the eggplant haploidy studies to date, anther culture among the androgenesis techniques has been mostly used. Protocols used in eggplant anther cultures are based on different versions of the protocol used by Dumas de Vaulx et al. [22] for pepper anther culture. Dumas de Vaulx and Chambonet [17] developed a protocol similar to pepper anther culture and started using it in eggplant and reported successful results. The success of this protocol is based on the treatment of anthers with high-temperature (+35 ⁰C) when they are first introduced into the medium. The positive effects of high-temperature applications on haploid embryogenesis have also been reported in the studies conducted on Brassicaceae family [23, 24].

In eggplant microspore culture studies, the anthers are either cultured after being pre-treated and isolated [25], or different pre-treatments were applied to the isolated microspores [11, 26, 27]. Although the use of microspore culture was quite limited in eggplant breeding until today, the studies on this subject have been continued because of its different advantages compared to anther culture. In particular, the practical microspore culture protocols to be developed for direct embryogenesis have the potential to give great momentum to eggplant breeding studies.

While DH technique is used commercially in vegetable breeding for certain species (pepper, eggplant, melon, cucumber, squash) in Turkey, it is in the process of being improved to an effective level for a variety of vegetables (onion, leek, gherkin, tomato, watermelon, cabbage, carrot, spinach). The use of DH technique is restricted due to the response of haploid plant formation varying on the species. This is because haploid response is under the effect of various factors. For instance, each of the different genotypes within a species reacts differently to the technique used, or several genotypes respond positively, while some genotypes do not respond at all. The basis of these differences is due to genotype. In addition to the genotype, the growing conditions of plants used as donor parents, climatic effect, season, temperature, light intensity, the age of the plant, irrigation and fertilization regimes, type of stress factors and severity of exposure, and the chemicals used for

*Sustainable Crop Production*

the world after China, India and Egypt [6].

China are the secondary gene centers [1]. It was first cultivated in Asia [2, 3]. While the wild forms of the species were small and prickly, and with bitter fruits, current forms of the plant have been achieved through the selections, during the period of cultivation, in the direction of large, thornless and tasty fruits [2, 4, 5]. Of the 51.3 million tons of eggplants produced in the world, 93% is grown in Asia and only 2.5% (about 875.000 tons) across Europe. As for Turkey, with 854.049 tons of eggplant production, it performs almost as much as the eggplant production of all European countries. In addition, Turkey ranks fourth in the eggplant production of

Plants are affected by the positive or negative conditions of their environments and try to adapt to those regions. In this adaptation process, non-adaptable plants perish, and adapters hide or develop some properties in order to survive, and in this way, different variations occur in their regions. The different climatic features in Turkey have contributed to the formation of our local cultivars, which have been cultivated by the people living in Anatolia for centuries, and have caused Turkey to be the second diversity region of eggplant. Different researchers have classified the eggplants according to their being early-late, adaptation to the environmental conditions, origin, shape, color and other characteristics [7]. Zhukovsky collected eggplant samples while traveling in Turkey between the years 1925 and 1926 and identified five different eggplant varieties on the Anatolian soils over those samples [8]. However, *S. melongena* L., which is cultivated as a culture plant in different countries, is generally considered to have four subspecies. These are (1) spp. *esculentum*, (2) spp. *insanum*, (3) spp. *serpentium*, and (4) spp. *depressum*, and they differ in color, shape, habitus, efficiency, usage area, etc. [9]. There are many different uses of eggplant in Turkish cuisine. Besides being consumed as fresh, dried and frozen, it is used in jams, pickles, sauces and salads. In addition, it has recently attracted attention with its use in diet lists due to its fibrous structure and low-calorie value. Eggplant, in contrast to general belief, is rich in vitamins and minerals. It has important nutritional value and contains very valuable antioxidants and phenolic substances for human health. For this reason, it has been used frequently in drug production and alternative medicine in the world since ancient times. While the fruit and leaves of eggplant have a lowering effect on cholesterol levels in the blood, eggplant extract is used in different treatments such

as diabetes, asthma, bronchitis and digestive disorders [10, 11].

Eggplant cultivation in Turkey was being conducted in the field conditions before. Then, with the development of greenhouse and seed technologies, eggplant production was started in greenhouse and became one of the vegetables produced throughout the year. The yield of the cultivars cultivated in greenhouses has

increased by means of the F₁ hybrid cultivars, because they have higher yield values than the standard cultivars and do not show phenotypic variations. Thus, hybrid cultivars have entered the market with great speed. This has led to a rapid decrease in the standard cultivars with phenotypic variations at high rate and even caused some local species to become near extinct. Since the 1970s, genetic erosion has started all over the world, and it is spreading rapidly in Turkey, too. It is compulsory to take serious steps toward the preservation of resources against this genetic erosion and to protect the local populations. It is possible to produce new eggplant hybrids from the local genotypes in the characteristics desired by the market by taking them into breeding programs, selfing and crossing them with the other parent lines. For this purpose, the combined use of classical and modern breeding methods will lead to speed up the progress. The use of haploidy techniques among modern breeding techniques provides tremendous advantages for breeders, especially since it allows the production of 100% homozygous pure lines only in one generation.

**68**

plant protection have also a great effect on success. The healthier the donor plant is grown, the greater the chance of success in obtaining haploid plants. Another important issue is the nutrient medium consisting of macro- and micro-elements, vitamins, plant growth regulators (PGRs), carbohydrate sources and other unidentified substances. On the other hand, the correct determination of the period of taking the buds and the culture of the microspores at the appropriate stage also directly affects the success. Although different staining techniques are used to determine the bud stage, the most practical method used today is the 4′-6-diamidino-2-phenylindole (DAPI) staining method. As the pretreatments prior to culture, different pre-temperature applications (such as 4, 10, 28, 35°C) are applied to the anthers or buds. However, incubation in the dark at +35°C for 8 days is generally used successfully in eggplant anther cultures. To date, a number of factors affecting microspore embryogenesis have been investigated, and their effects have been demonstrated in several eggplant haploidy studies [11, 17–19, 28–34] conducted in the world and Turkey; however, this article will mainly focus on studies in Turkey.
