GENETIC VARIABILITY OF ANTHER DONOR VERSUS SPONTANEOUS DOUBLED HAPLOID DESCENDENTS AND COLCHICINE INDUCED DOUBLED HAPLOID SWEET PEPPER (Capsicum annuum L.) LINES

Abstract

Haploid (n) and doubled haploid (DH) plants were developed in anther culture of sweet pepper (Capsicum annuum L.). Regenerants were analyzed by flow cytometry for haploid (n = 12) and spontaneous doubled haploid (2n = 24) genomes. Haploid plants were forwarded to colchicine-treatment for induced doubled haploid (2n•) plant production. Molecular polymorphism of anther donor plants (2n), the haploid regenerants (n), the spontaneous (2n) and induced (2n•)-DH plants were analysed by RAPD-, SSR-and ISSR-PCR. The analysis of anther-donor plants compared to DH-descendents showed an unexpectedly wide range of molecular polymorphism. Our results suggest that genetic changes occurring during meiotic recombination is higher than those of occurring during colchicine-induced genomic duplication. 1.IntroductionIn general, cultures of anther, pollen, ovary, and ovule produce haploid (n) regenerants as fixed gametoclonal variants resulted from meiosis. The two processes of meiotic recombination, the crossing over between homologous pairs of maternal and paternal chromosomes, and the random assortment of chromosomes generate genetically different haploid gametes. In the case of pepper (2n = 24) the number of possible gametes as a result of random assortment of chromosomes is equal to 2 n = 2 12 = 4.096. This number is higher considering the numbers of crossing over occurring during the first meiotic cell division. By the application of anther culture, as in the present study, male gametoclonal pepper variants could be selected with new genomic constitutions (Dumas de Vaulx et al., 1981, Mitykó et al., 1995, Fári, 1986, Gémesné et al., 1998, Gyulai et al., 2000. Anther-culture-derived DH-lines are the most advanced inbreds in pepper breeding for hybrid development, new cultivar release with new fruit shape, size, colour etc., and for resistance especially to viruses such as tobacco mosaic virus (TMV) and to bacteria Xantomonas vesicatoria. The significance of the DH technique in breeding is, first, the ability to develop monoploid plants with one set of chromosomes, second, to produce homozygous, doubled haploid (DH) pure lines, and third, to reveal meiotic recombinants with unique genome constitutions (Caranta et al., 1996). The aim of the study presented was to select new meiotic recombinants of anther culture origin with a final aim of new cultivar release. Materials and methods Anther cultureAnthers were excised from flower buds of different pepper lines at the uninucleate microspore stage. Fifteen anthers per petridish, 6 cm in diameter, were excised and laid onto a nutritive medium of Dumas de Vaulx et al. (1981). Regenerated (DH-R 1 ) plants were potted and grown in greenhouses for R 2 -seed production prior to flow cytometric analysis.

Effect of Pepper Types on Obtaining Spontaneous Doubled Haploid Plants via Anther Culture

“…Anther culture is a widely used haploidization technique in pepper. Several studies have been carried out on the development of successful protocols related to pepper anther culture (Abak, 1983;Al Remi et al, 2014;Ata, 2011;Buyukalaca et al, 2004;Comlekcioglu et al, 2001;G emesn e Juh asz et al, 2001;George and Narayanaswamy, 1973;Harn et al, 1975;Niklas-Nowak et al, 2012;Novak, 1974;Olszewska et al, 2013;Saccardo and Devreux, 1974;Tas xkın et al, 2011;Wang et al, 1973). In these studies, the rate of spontaneous doubled haploidy was determined via ploidy analyses.…”

“…The success of these methods varies according to the species. Positive results in pepper have been achieved with anther culture (Al Remi et al, 2014;Buyukalaca et al, 2004;Comlekcioglu et al, 2001;Ercan et al, 2006;G emesn e Juh asz et al, 2001;Niklas-Nowak et al, 2012;Olszewska et al, 2013;€ Ozkum and Tıpırdamaz, 2002;Tas xkın et al, 2011). The advantage of this method is that there are thousands of microspores in each anther and numerous haploid plants can be obtained from a single anther.…”

Androgenic studies in the production of haploids and doubled haploids in Capsicum spp.

“…Techniques for the chromosomal duplication of haploids in pepper cultivars are generally based on treatment with colchicine. Gémesné Juhász et al (2001a, 2001b showed that they could increase the efficiency in 95% of doubled haploid production in pepper cultivars (Capsicum annum L.) by applying colchicine (0.04%) to the regeneration medium. In subsequent investigations, Gémesné Juhász et al (2001a) indicated that the medium supplemented with colchicine resulted in an effective diploidization of 50-95% explants.…”

Creation of pepper doubled haploids and morphological characterization of androgenic plants

“…Confirmation of the microspore origin of diploid androgenic lines can be achieved through analysis of plant phenotypic homogeneity and fruit morphological characterization in the R 2 generation and is particularly noticeable in the hybrid donor genotype (Supena et al 2006a;Kisiala et al 2011). Isozyme and molecular analysis has also been used to test the homozygosity (Dolcet-Sanjuan et al 1997;Gemesne Juhasz et al, 2011;Parra-Vega et al 2013a;Keleş et al 2015).…”