Isolated microspore culture experiments were carried out in sweet pepper (Capsicum annuum L.) F 1 hybrid genotypes. In the first experiment, four culture media (W14, B5, MS and NLN) were compared to test their effectiveness in inducing the formation of microsporederived structures in two genotypes. The experiments revealed the superiority of B5 medium. In the second experiment, the effects of different ratios of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.1, 0.2 and 0.5 mg l -1 ) and kinetin (0, 0.2 and 0.5 mg l -1 ) were also investigated in B5 medium with two genotypes. The effect of growth regulators were investigated on the production of microsporederived calli and embryo-like structures (ELSs), the ratio of the two and plant regeneration (number of regenerated plantlets) in microspore culture. The histological experiments revealed the differences between the microsporederived ELSs and calli. The most promising results were obtained on the investigated parameters in the presence of 0.1 mg l -1 2,4-D and 0.2 mg l -1 kinetin producing the highest number of plantlets in both genotypes tested. In the response of 11 genotypes, the androgenesis induction was successful in each sweet pepper genotypes tested using the best basic medium and growth regulators combination. In case of 11 genotypes, the number of ELSs ranged from 20 to 100/Petri dish (an average of 48.1 ELS/Petri dish), while the number of green plantlets varied from 0 to 8 plantlets/Petri dish (an average of 1.5 plantlets/Petri dish) depending on the genotype. The spontaneous rediploidization rate obtained was 25% in isolated microspore.
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.
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