Gametic and somatic embryogenesis through in vitro anther culture of different Citrus genotypes

Abstract: In vitro tissue culture represents a useful technique for advancing Citrus breeding and propagation. Among in vitro regeneration systems, anther culture is commonly used to produce haploids and doubled haploids for a fast-track producing homozygous lines, in comparison with the traditional self-pollination approach, which involves several generations of selfing. In addition, anthers culture can produce somatic embryos that can also be used for clonal propagation. In this study, two thermal shocks were applied to the anthers of six Citrus genotypes (two clementine and four sweet oranges), just after they were put in culture. The response obtained was different depending on the genotype: both clementines, namely Hernandina and Corsica, produced homozygous and triploid regenerants (microspore-derived embryos), whereas all of the analyzed regenerants from sweet oranges, three cultivars of Tarocco and Moro, produced heterozygous and diploid regenerants similar to the parental genotypes (somatic embryos)

In vitro anther culture of sweet orange (Citrus sinensis L. Osbeck) genotypes and of a C. clementina 7 C. sinensis 'Hamlin' hybrid

Citrus, and particularly sweet oranges, are very recalcitrant to anther culture. In this paper it was evaluated for the first time the response of 27 genotypes of Citrus sinensis and of one hybrid C. clementina 7 C. sinensis, to in vitro anther culture. Ten genotypes of sweet oranges showed embryogenic callus induction, mostly blood sweet oranges genotypes, such as Tarocco, Moro and Sanguinelli. In vitro microspore developmental switches from the gamethophytic to the sporophytic pathway were shown by DAPI staining in microspores of these responsive genotypes, after 10 months in culture. However, microsatellite marker analyses showed that these calli were heterozygous. The flow-cytometric analysis of these embryogenic calli showed the presence of two peaks, corresponding to haploid (n) and diploid (2n) genotypes. Differently, anther cultures of the hybrid C. clementina 7 C. sinensis produced tri-haploid (3n) embryogenic calli and the embryos obtained were homozygous when analyzed by molecular markers (sample sequence repeats), confirming the more responsive characteristic of clementine to microspore embryogenesis through anther culture. \ua9 2014 Springer Science+Business Media Dordrecht

Somatic embryogenesis from ovaries of sweet orange cv. Tobias

Callogenesis, somatic embryogenesis, and regeneration were obtained from tissues of unfertilized ovaries of sweet orange (Citrus sinensis Osbeck.) cv. Tobias. The influence of two modified basal media, woody plant medium (WPM) and N6 medium, to induce callus formation from pistils was determined. Overall, high frequencies of callogenesis were observed when either medium was used. However, initial culture of explants in WPM medium followed by transfer of callus to N6 medium resulted in higher frequency of callus induction (of 2.30 callus per explant that were larger than 0.5 cm in size), and of subsequent development of embryogenic callus (10%). A total of 125 somatic embryos were obtained. After 6 months of culture, 72% of somatic embryos germinated into plantlets. These plantlets were subsequently micrografted in vitro, and then acclimatized. Ploidy of these plants were determined using flow cytometry and TRAPS molecular markers were used to confirm their maternal origin.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico, CNPqConselho Nacional de Desenvolvimento Cientifico (CNPq

Posição da gema axilar e a indução de mutação em mudas de crisântemo (Dendranthema grandiflora Tzvelev) Position of the axillary bud and mutation inducion in chrysanthemun (Dendranthema glandiflora Tzevelev) plantets

O tratamento mutagênico de meristemas multicelulares em plantas de propagação vegetativa geralmente resulta na formação de plantas quiméricas. Os setores mutados podem ser ampliados e estabilizados por meio de podas repetidas. Este trabalho teve como objetivo estudar a influência de podas em distintas posições (da 1a até a 6a gema axilar) de um ramo M1V1, na freqüência de obtenção de mutantes de coloração de inflorescência e no tamanho dos setores mutados, em plantas de crisântemo irradiadas com raios-gama. Para tal, mudas enraizadas do cultivar ‘Ingrid’ (de coloração rosa escuro) foram tratadas com 20 Gy de raios-gama. Após 40 dias do plantio foram realizadas as podas. Avaliaram-se a freqüência de mutantes e o espectro de mutação para a coloração da inflorescência nos ramos M1V2. Não foram observados mutantes no controle. No total de plantas avaliadas, 22,1% apresentavam mutações na coloração das flores sendo que, do total de plantas mutantes, 1,8% eram mutantes periclinais (com coloração única) e as restantes (98,2%), apresentavam apenas alguns setores mutados. Não se observou diferença significativa quanto ao tamanho do setor mutado e ao número de mutantes nos diversos tratamentos (podas em diferentes gemas axilares). Com relação ao espectro de mutação, observaram-se mutantes com flores de coloração bronze, rosa claro, chá-rosa, variegado, amarelo, branco, vinho e creme. Um mutante de coloração vinho foi selecionado, multiplicado e avaliado em ensaio de produção. Este mutante denominado ‘Magali’ foi multiplicado para lançamento como novo cultivar.<br>Mutagenic treatment of multicellular meristems from vegetatively propagated plants generally results in the formation of chimeric plants. Mutated sectors can be increased and stabilized through the cutting-back method. The objective of the present research was to study the influence of the application of this method in the M1V2 population, originated from six different axillary buds from the M1V1 chrysanthemum branches. For this purpose, rooted plants of the cultivar ‘Ingrid’ (dark pink) were irradiated with 20 Gy of gamma-rays and the prune was carried out 40 days after planting. Frequency and spectrum of flower color mutants were evaluated. No mutants were observed in the control population. In the M1V2 population, 22.1% of the total plants were mutants (white color, dark bronze, pale pink, yellow, wine, variegated and cream). Among them, 1.8% were periclinal chimeras (with only one different color from the original) and the others showed mutated sectors. No differences were observed in mutation frequency and size of mutated sector among six M1V2 populations. The wine colored mutant was selected, multiplied and evaluated in a yield trial. This mutant named ‘Magali’ was multiplied and was released as a new cultivar

Carotenoid biosynthesis and quality characteristics of new hybrids between tangor (citrus reticulata x c. sinensis) cv. 'murcott' and sweet orange (c. sinensis) cv. 'pêra'

Phenotypic characteristics, as well as the relation between carotenoid accumulation and gene expression during ripening were determined in fruits of five new hybrids between tangor cv. ‘Murcott’ and sweet orange cv. ‘Pêra’. The genotypes were classified into the orange-like group, showing mainly epoxycarotenoids, oval fruit shape and yellowish color, or in the mandarin-like group, showing mainly β-cryptoxanthin, flattened shape and deep-orange coloration; although some hybrids presented intermediate characteristics. The diversity in carotenoid composition of hybrids and genitors were mostly explained by patterns of gene expression. High carotenoid (250–426 μg/g dry weight [dw]) and β-cryptoxanthin (81–125 μg/g dw) contents, observed in the mandarin-like group, were generally associated with high expression of upstream genes (GGPPS1, PSY, PDS). On the other hand, low expression/repression of these genes and high expression of downstream genes (BCHX and ZEP) were associated with low carotenoid (~158 μg/g dw) and β-cryptoxanthin (5–22 μg/g dw) contents and epoxycarotenoid accumulation, as occurred in the orange-like group. Breeding experiments resulted in hybrids with outstanding higher carotenoid contents than both genitors (up to 426 μg/g dw versus 158–250 μg/g dw in genitors), which was attributed to transgressive segregation. Differences among genotypes have great impact on commercial fruit quality and potential health benefits, such as the provitamin A content122461470CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP465440/2014-2; 402463/2016-12017/08648-0; 2013/09804-5; 2018/00133-4; 2018/23752-1; 2013/07914-8; 2011/18605-

Ploidia e fertilidade de pólen em progênies de citros Ploidy and pollen fertility in citrus hybrids

Este trabalho foi desenvolvido para estimar a fertilidade do pólen e determinar o nível de ploidia em progênies dos cruzamentos da tangerineira 'Montenegrina' (C. deliciosa Ten.) com a tangerineira 'King' (C. nobilis Lour.) e com a laranjeira 'Caipira' (C. sinensis (L.) Osb.). As plantas, de pés-francos e com idade entre 11 e 12 anos, são mantidas na Estação Experimental Agronômica da Universidade Federal do Rio Grande do Sul, em Eldorado do Sul. Avaliaram-se o nível de ploidia, através da contagem dos cromossomos em células em meiose, e a fertilidade do pólen, por coloração do pólen com carmim propiônico, em 2005 e 2006. Todos os híbridos avaliados são diplóides e a fertilidade de pólen variou de zero a 98%. C27 - híbrido de 'Montenegrina' x 'Caipira' - revelou-se praticamente estéril, e a fertilidade de C21, do mesmo cruzamento, é de 10%. A menor fertilidade observada no cruzamento da 'Montenegrina' x 'King' foi de 42%.<br>This work was performed to estimate pollen fertility and determinate the ploidy level of 11 to 12 years old progenies of crosses of 'Montenegrina' mandarin (Citrus deliciosa Ten.) with 'King' mandarin (C. nobilis Lour.) and with 'Caipira' sweet orange (C. sinensis (L.) Osb.) maintained at Estação Experimental Agronômica of UFRGS, in Eldorado do Sul, Brazil. The ploidy level was determined by chromosome countings in meiosis cells and pollen fertility evaluated by staining pollen grains with propionic carmine. All evaluated hybrids are diploid and pollen fertility varied from zero to 98%. C27, a hybrid 'Montenegrina' x 'Caipira', was found is sterile, and C21, from the same cross, showed 10 % fertility. The lowest observed fertility in the progeny 'Montenegrina' x 'King' was 42 %