PROPAGATION AND TISSUE CULTURE HORTSCIENCE 50(11):1671–1676. 2015. significant way to increase yield per unit area is to develop cultivars that are resistant to such problems. Effect of Pepper Types on Obtaining Haploid plant production methods accel- erate plant breeding studies and thus, play an Spontaneous Doubled Haploid Plants important role in developing new cultivars. Obtaining homozygous pure lines using con- via Anther Culture ventional methods takes a long time: 10–12 years for open-pollinated and 6–7 years for Davut Keles¸ self-pollinated plants. This time can be short- Alata Horticultural Research Institute, 33740 Erdemli, Mersin, Turkey ened up to 2 years using tissue culture techniques. Gynogenesis (ovule and ovarium Hasan Pınar culture), parthenogenesis (pollination with Department of Horticulture, Faculty of Agriculture, Erciyes University, irradiated pollen), and androgenesis (anther 38039 Kayseri, Turkey and microspore culture) are commonly used to obtain haploid plants via tissue culture. Atilla Ata The success of these methods varies accord- Alata Horticultural Research Institute, 33740 Erdemli, Mersin, Turkey ing to the species. Positive results in pepper have been achieved with anther culture (Al Hatıra Tas¸kın1 Remi et al., 2014; Buyukalaca et al., 2004; Department of Horticulture, Faculty of Agriculture, Cxukurova University, Comlekcioglu et al., 2001; Ercan et al., 2006; Gemesne Juhasz et al., 2001; Niklas-Nowak 01330 Adana, Turkey et al., 2012; Olszewska et al., 2013; Ozkum€ Serhat Yıldız and Tıpırdamaz, 2002; Tasxkın et al., 2011). The advantage of this method is that there are Alata Horticultural Research Institute, 33740 Erdemli, Mersin, Turkey thousands of microspores in each anther and Saadet Buy€ ukalaca€ numerous haploid plants can be obtained x from a single anther. The main principle of Department of Horticulture, Faculty of Agriculture, Cukurova University, anther culture is the prevention of pollen cell 01330 Adana, Turkey development, which normally results in for- Additional index words. anther culture, Capsicum annuum, pepper, spontaneous chromosome mation of the male gamete. Instead, the imma- ture pollen cells are induced to form embryos doubling similar to somatic cells. Haploid plants have Abstract. The most successful technique used to obtain haploid plant in pepper is anther one set of chromosomes and therefore they are culture. The chromosome content of haploid plants can be doubled spontaneously or using not fertile. Haploid plant chromosomes can be colchicine. In this study, we compared the rate of spontaneous doubled haploidy of different doubled spontaneously or using chemicals pepper types. Seven charleston, six bell, eight capia, and seven green pepper genotypes were such as colchicine. Colchicine treatment is used as plant material. Murashige and Skoog (MS) nutrient medium with 4 mg·LL1 expensive and harmful to both human health naphthaleneacetic acid (NAA), 0.5 mg·LL1 6-benzylaminopurine (BAP), 0.25% activated and the environment. The International Car- L1 L1 charcoal, 30 g·L sucrose, and 15 mg·L silver nitrate (AgNO3) was used. Ploidy levels of nivorous Plant Society suggests that toxic plants obtained through anther culture were detected using both flow cytometry and simple colchicine should only be applied by in- sequence repeats (SSR) markers. The results showed that different spontaneous doubled dividuals wearing gloves and other personal haploidy rates were obtained from different pepper types. The highest rate was observed in protective equipments (Cahill, 2015). Plant bell pepper type with 53.4% (mean of six genotypes) of haploid plants undergoing lossesmayalsobeobservedduringcolchi- spontaneous chromosome doubling. This was followed by charleston and capia types with cine application. 31.9% and 30.4% doubling, respectively. Green pepper type gave the lowest spontaneous The disadvantages of colchicine treat- doubled haploidy rate with 22.2% doubling. The results obtained from this study will be ment and requirement for an intensive qual- useful both for future work on haploidy in pepper and for breeding programs. ified labor force led breeders to use of spontaneous dihaploidization. So far, studies have shown that different pepper types react Peppers are a commonly consumed veg- of a Turkish traditional food, ‘‘Dolma.’’ Green to anther culture differently. However, no etable worldwide and are especially popular and charleston pepper types are consumed study has been found in the literature on the in the Mediterranean basin including Turkey. raw, cooked, and pickled. Capia pepper type difference between various pepper types for Peppers are grown in almost every region of can be used smoked and as pepper paste. spontaneous doubled haploid production. Turkey and are consumed in various forms Pepper, is known to have originated in central These types of studies are available for barley (fresh, pepper paste, sauce, pickle, and as and southern America and belongs to the and wheat (Kahrizi and Mohammadi, 2009; spices). There are different consumption Solanaceae family with Capsicum annuum L. Mirzaei et al., 2011a, 2011b). Thus, it is methods for different pepper types. For exam- as the most commonly grown species. Pep- important for breeders to determine the spon- ple, bell peppers are used for the preparation pers have a high nutritional value and are taneous doubled haploidy rate for pepper, especially rich in vitamin C with 103 mg/100 g which is one of the most important vegetables (IBPGR, 1983). In addition, peppers are in the world in terms of both cultivation and rich in minerals. A 100 g portion of green consumption. In this study, we investigated Received for publication 16 July 2015. Accepted fresh pepper contains 29 calories, 1.1 g pro- the rate of spontaneous doubled haploidy for for publication 8 Sept. 2015. tein, 0.2 g fat, 92.6 g water, 4.2 g carbohy- different pepper types. We would like to thank the Alata Horticultural drate, and 1.4 g cellulose. After China and Research Institute and the Republic of Turkey Mexico, Turkey ranks third in pepper pro- Ministry of Food, Agriculture and Livestock for Materials and Methods supporting this research. We are also grateful to duction in the world with 2.1 million tons in Anne Frary for critical reading of the manuscript 2012 (FAOSTAT, 2013). Problems such as Plant material. In the current study, seven for English. low and high temperatures, pests, and dis- charleston, six bell, eight capia, and seven 1Corresponding author. E-mail: hatirataskin1@gmail. eases have a negative effect on the quality green pepper genotypes were used as plant com. and yield of pepper production. The most material (Fig. 1). Pepper seeds were planted HORTSCIENCE VOL. 50(11) NOVEMBER 2015 1671 photoperiod conditions. Embryos obtained pepper type on obtaining spontaneous dou- from anthers were transferred to 15-cm glass bled haploid plants via anther culture. Seven tubes containing hormone-free MS nutrient different genotypes were used to determine medium (Fig. 2). the rate of spontaneous doubled haploidy for Flow cytometry studies. The ploidy level green peppers. For each genotype, 300 an- of plants was detected using a flow cytometer thers were examined. The highest plant num- (Partec, Munster,€ Germany). Young leaves of ber obtained from anther culture was found to plants obtained from anther culture were put be 6.7 plants per 100 anthers in green type 2. into petri dishes containing 400 mL extraction This genotype was followed by type-4, type-5, buffer (Partec 05–5002 CyStainÒ ultraviolet type-6, and type-3 with 5.7, 5.3, 4, and 3.7 Precise P), chopped using a sharp razor blade plants per 100 anthers, respectively. Rates of in petri dishes for 30 to 60 s and incubated spontaneous doubled haploidy were 20%, between 30 s and 5 min. These samples were 25%, 9.1%, 23.5%, 25%, 8.3%, and 44.4% filtered using a Partec 50 mm CellTrics in types 1 to 7, respectively. The mean sponta- disposable filter. The filtrate was combined neous doubled haploidy rate for green peppers with 1.6 mL staining buffer, incubated for 30 was calculated as 22.2% (Table 1; Fig. 5). to 60 s and analyzed in the flow cytometry in In charleston pepper type, seven different the blue fluorescence channel. The ploidy genotypes were tested. The fewest plants level of each plant was determined according were obtained from charleston type-4 with Fig. 1. Representative (A) bell, (B) charleston, (C) to the peaks (Fig. 3). A diploid commercial 7 plants from 300 anthers; however, three of green, and (D) capia pepper types. cultivar was used as control. these plants were doubled haploids. Plant SSR analyses. Embryo type (diploid/doubled number per 100 anthers varied with values haploid) was determined using Hpms1-117 of 6 (type-1), 4.7 (type-2), 7 (type-3), 2.3 in plugs containing two volumes peat and one SSR marker (Fig. 4). More than 20 SSR (type-4), 6.7 (type-5), 6.7 (type-6), and 10.3 volume perlite. In the study conducted by markers derived from the pepper genome (type-7), depending on genotype. Spontane- Buyukalaca et al. (2004), plants grown in the (Sol Genom network) were tested in this ous doubled haploidy rates varied between greenhouse were found to have a better re- study and Hpms1–117 was selected based 14.3% and 44.4%, and the average was found sponse to anther culture than those grown in on its clear polymorphism. Polymerase chain to be 31.9%. The mean plant number was the open field. Therefore, the plants used in reactions (PCR) were performed in 15 mL 6.23 per 100 anthers (Table 1; Fig. 6). this study were grown in the greenhouse. volume containing 4.8 mLwater,2mLDNA, Eight pepper genotypes were tested to Normal horticultural cultivation practices 1.5 mL10· PCR buffer, 0.2 mL Taq DNA assess the performance of the capia pepper such as fertilization, irrigation, and plant pro- polymerase (5U/mL), 1 mL deoxynucleotide type.