Spezifität der quantitativen Resistenz von Blättern und Früchten der Tomate (Lycopersicon ssp. L.) gegenüber Phytophthora infestans (Mont.) de Bary.

Ziel der vorliegenden Arbeit war es, einen Beitrag zur Resistenzforschung bei Tomaten gegenüber P. infestans zu leisten, um erste Grundlagen für eine mögliche Züchtungsstrategie auf Basis unterschiedlicher quantitativer Resistenzen zu erarbeiten. Hierzu wurde untersucht, inwieweit unterschiedliche qualitative und quantitative Resistenzen bei Tomatenblättern und -früchten vorliegen, und ob hierfür verantwortliche Mechanismen identifiziert werden können. Zudem wurde untersucht, ob isolatspezifische quantitative Resistenzen identifiziert werden können. Zu diesem Zweck wurde mit einer erweiterten Clusteranalyse, basierend auf einer modifizierten Sanghvi-T2 Distanz, ein statistisches Verfahren entwickelt, welches die Identifikation von quantitativen, isolatspezifischen Resistenzen unter der Berücksichtigung der Variabilität ermöglicht. Des weiteren wurde geprüft, inwieweit zwischen den Resistenzausprägungen auf dem Blatt und den Resistenzausprägungen auf der Frucht ein Zusammenhang besteht und inwieweit die im Labor beobachteten Resistenzen unter Freilandbedingungen eine Rolle spielen. Im Labortest wurde die qualitative und quantitative Blattresistenz von 109 Akzessionen aus elf Lycopersicon und Solanum Arten gegenüber zwölf unterschiedlich aggressiven und teilweise auch unterschiedlich virulenten P. infestans Isolaten untersucht (Kap. 3). Die Früchte von 38 Tomatensorten wurden auf ihre Resistenz gegenüber drei P. infestans Isolaten geprüft. Zusätzlich wurde der Einfluss der Fruchtnachreife auf die Resistenzeigenschaften der Tomatenfrüchte gegenüber P. infestans analysiert (Kap. 4). Insgesamt 40 Sorten wurden auch unter Feldbedingungen auf Blatt- und Fruchtbefall untersucht (Kap. 5). Die frühen Stadien der Infektion von Tomatenblättern mit P. infestans Sporangien wurden mikroskopisch bei acht Tomatensorten mit unterschiedlichen quantitativen Reaktionsprofilen und drei Isolaten untersucht (Kap. 6). Hierzu wurden die Entwicklungsstadien von P. infestans Sporangien nach 24h, 48h und 60h nach der Inokulation auf und im Blatt mit der Calcofluor und der KOH - Anilin Blau Färbung sichtbar gemacht. Das Auftreten und die Lokalisation von H2O2 im Blatt nach 48h und 60h nach der Inokulation in Reaktion auf die Infektion wurde mithilfe einer DAB (3,3′ - Diaminobenzidine) Färbung untersucht. Es wurden einige, z.T. auch wahrscheinlich neue, qualitative Blattresistenzen gegenüber P. infestans gefunden, jedoch war keine der 109 Akzessionen vollständig resistent gegenüber allen Isolaten. Für die quantitative Resistenz von Blättern lagen in vielen Fällen isolatspezifische Unterschiede vor. Die Sorte x Isolat Interaktionen konnten mit Hilfe der erweiterten Clusteranalyse erfolgreich analysiert werden und die Akzessionen in Gruppen mit unterschiedlichen quantitativen Resistenzprofilen bzgl. der Interaktion mit den Isolaten und des Resistenzniveaus eingeteilt werden. Für die Fruchtresistenz konnten keine qualitativen Resistenzen gegenüber den drei getesteten Isolaten gefunden werden. Im Gegensatz dazu unterschieden sich die Tomatensorten in ihrer quantitativen Resistenz und Sorten und Isolate interagierten signifikant. Auch für die Fruchtresistenz konnten Gruppen mit unterschiedlichen quantitativen Reaktionsprofilen gebildet werden. Insgesamt nimmt die Anfälligkeit von Tomatenfrüchten mit zunehmender Reife kontinuierlich und signifikant ab. Unter Laborbedingungen korrelierten nur die Sporulationskapazität der Früchte und der prozentuale Blattbefall. Im Feldversuch über zwei Jahre und mit bis zu 40 Tomatensorten war der Zusammenhang hoch signifikant, jedoch asymptotisch, d.h. bereits bei sehr geringem Blattbefall war der Fruchtbefall sehr hoch. Bei den Tomatenherkünften, die sowohl im Labor als auch im Feld auf ihre Anfälligkeit getestet wurden, erschienen die Blattanfälligkeiten ähnlich, während kein klarer Zusammenhang zwischen der Fruchtanfälligkeit im Feld und im Labor bestand. Die Entwicklung von P. infestans auf der Blattoberfläche war unabhängig von der Sorte. Sowohl beim Eindringen und der Etablierung von P. infestans ins Blatt als auch bei der damit verbunden H2O2 Aktivität im Wirt wurden deutliche isolat- und sortenspezifische Effekte gefunden, die aber nur zum Teil mit den quantitativen Unterschieden der Blattresistenz korrespondierten. Sorten, die bei hoher Resistenz unterschiedliche Reaktionsprofile aufweisen, sind grundsätzlich interessante Kreuzungspartner, um die quantitative Resistenz gegenüber P. infestans zu verbessern. Hier sind vor allem Sorten, die sich auch in ihrer H2O2 Aktivität unterscheiden von Interesse.

Estudios de las diferencias existentes entre las medidas de los parametros craneales, en una población de Rallus Rallus SSP. Frugivurus, debido a la presencia de B-cromosomas

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Measuring root traits in barley (Hordeum vulgare ssp vulgare and ssp spontaneum) seedlings using gel chambers, soil sacs and X-ray microtomography

Root characteristics of seedlings of five different barley genotypes were analysed in 2D using gel chambers, and in 3D using soil sacs that were destructively harvested and pots of soil that were assessed non-invasively using X-ray microtomography. After 5 days, Chime produced the greatest number of root axes (similar to 6) and Mehola significantly less (similar to 4) in all growing methods. Total root length was longest in GSH01915 and shortest in Mehola for all methods, but both total length and average root diameter were significantly larger for plants grown in gel chambers than those grown in soil. The ranking of particular growth traits (root number, root angular spread) of plants grown in gel plates, soil sacs and X-ray pots was similar, but plants grown in the gel chambers had a different order of ranking for root length to the soil-grown plants. Analysis of angles in soil-grown plants showed that Tadmore had the most even spread of individual roots and Chime had a propensity for non-uniform distribution and root clumping. The roots of Mehola were less well spread than the barley cultivars supporting the suggestion that wild and landrace barleys tend to have a narrower angular spread than modern cultivars. The three dimensional analysis of root systems carried out in this study provides insights into the limitations of screening methods for root traits and useful data for modelling root architecture.

The influence of winter oilseed rape (Brassica napus ssp oleifera var. biennis) canopy size on grass weed growth and grass weed seed return

Four experiments conducted over three seasons (2002-05) at the Crops Research Unit, University of Reading, investigated effects of canopy management of autumn sown oilseed rape (Brassica napus L. ssp. oleifera var. biennis (DC.) Metzg.) on competition with grass weeds. Emphasis was placed on the effect of the crop on the weeds. Rape canopy size was manipulated using sowing date, seed rate and the application of autumn fertilizer. Lolium multiflorum Lam., L. x boucheanum Kunth and Alopecurus myosuroides Huds. were sown as indicative grass weeds. The effects of sowing date, seed rate and autumn nitrogen on crop competitive ability were correlated with rape biomass and fractional interception of photosynthetically active radiation (PAR) by the rape floral layer, to the extent that by spring there was good evidence of crop: weed replacement. An increase in seed rate up to the highest plant densities tested increased both rape biomass and competitiveness, e.g. in 2002/3, L. multiflorum head density was reduced from 539 to 245 heads/m(2) and spikelet density from 13 170 to 5960 spikelets/m(2) when rape plant density was increased from 16 to 81 plants/m(2). Spikelets/head of Lolium spp. was little affected by rape seed rate, but the length of heads of A. myosuroides was reduced by 9 % when plant density was increased from 29-51 plants/m(2). Autumn nitrogen increased rape biomass and reduced L. multiflorum head density (415 and 336 heads/m(2) without and with autumn nitrogen, respectively) and spikelet density (9990 and 8220 spikelets/m(2) without and with autumn nitrogen, respectively). The number of spikelets/head was not significantly affected by autumn nitrogen. Early sowing could increase biomass and competitiveness, but poor crop establishment sometimes overrode the effect. Where crop and weed establishment was similar for both sowing dates, a 2-week delay (i.e. early September to mid-September) increased L. multiflorum head density from 226 to 633 heads/m(2) and spikelet density from 5780 to 15 060 spikelets/m(2).

The influence of winter oilseed rape (Brassica napus ssp oleifera var. biennis) cultivar and grass genotype on the competitive balance between crop and grass weeds

Three experiments conducted over two years (2002-04) at the Crops Research Unit, University of Reading, investigated competition between autumn sown oilseed rape cultivars (Brassica napus L. ssp. oleifera var. biennis (DC.) Metzg.) and Lolium multiflorum Lam., L. x boucheanum Kunth and Alopecurus myosuroides Huds., sown as indicative grass weeds. Rape cultivar (cv.) had a substantial effect on grass weed seed return. Over the six cultivars tested, L. multiflorum spikelet production ranged from just under 400 spikelets/m(2) in the presence of cv. Winner to nearly 5800 in competition with cv. Lutin. Cultivar competitiveness was associated with high biomass, large dense floral layers and early stem extension. There was some evidence of differential competitive tolerance between rape cultivars. The results suggested that rape cultivars could be screened for competitiveness by measuring floral layer interception of photosynthetic active radiation. L. x boucheanum cultivars varied in ability to compete with rape. In the absence of inter-specific competition, spikelet density was similar for Aberecho and Polly (circa 31000 spikelets/m(2)) but when grown with rape Polly outyielded Aberecho (i.e. 12 090 and 7990 spikelets/m(2) respectively).

The influence of winter oilseed rape (Brassica napus ssp. oleifera var. biennis) cultivar and grass genotype on the competitive balance between crop and grass weeds

Four experiments conducted over three seasons (2002–05) at the Crops Research Unit, University of Reading, investigated effects of canopy management of autumn sown oilseed rape (Brassica napus L. ssp. oleifera var. biennis (DC.) Metzg.) on competition with grass weeds. Emphasis was placed on the effect of the crop on the weeds. Rape canopy size was manipulated using sowing date, seed rate and the application of autumn fertilizer. Lolium multiflorum Lam., L.rboucheanum Kunth and Alopecurus myosuroides Huds. were sown as indicative grass weeds. The effects of sowing date, seed rate and autumn nitrogen on crop competitive ability were correlated with rape biomass and fractional interception of photosynthetically active radiation (PAR) by the rape floral layer, to the extent that by spring there was good evidence of crop: weed replacement. An increase in seed rate up to the highest plant densities tested increased both rape biomass and competitiveness, e.g. in 2002/3, L. multiflorum head density was reduced from 539 to 245 heads/m2 and spikelet density from 13 170 to 5960 spikelets/m2 when rape plant density was increased from 16 to 81 plants/m2. Spikelets/head of Lolium spp. was little affected by rape seed rate, but the length of heads of A. myosuroides was reduced by 9%when plant density was increased from 29–51 plants/m2. Autumn nitrogen increased rape biomass and reduced L. multiflorum head density (415 and 336 heads/m2 without and with autumn nitrogen, respectively) and spikelet density (9990 and 8220 spikelets/m2 without and with autumn nitrogen, respectively). The number of spikelets/head was not significantly affected by autumn nitrogen. Early sowing could increase biomass and competitiveness, but poor crop establishment sometimes overrode the effect. Where crop and weed establishment was similar for both sowing dates, a 2-week delay (i.e. early September to mid-September) increased L. multiflorum head density from 226 to 633 heads/m2 and spikelet density from 5780 to 15 060 spikelets/m2.

The influence of winter oilseed rape (Brassica napus ssp oleifera var. biennis) canopy size on grass weed growth and grass weed seed return

Four experiments conducted over three seasons (2002-05) at the Crops Research Unit, University of Reading, investigated effects of canopy management of autumn sown oilseed rape (Brassica napus L. ssp. oleifera var. biennis (DC.) Metzg.) on competition with grass weeds. Emphasis was placed on the effect of the crop on the weeds. Rape canopy size was manipulated using sowing date, seed rate and the application of autumn fertilizer. Lolium multiflorum Lam., L. x boucheanum Kunth and Alopecurus myosuroides Huds. were sown as indicative grass weeds. The effects of sowing date, seed rate and autumn nitrogen on crop competitive ability were correlated with rape biomass and fractional interception of photosynthetically active radiation (PAR) by the rape floral layer, to the extent that by spring there was good evidence of crop: weed replacement. An increase in seed rate up to the highest plant densities tested increased both rape biomass and competitiveness, e.g. in 2002/3, L. multiflorum head density was reduced from 539 to 245 heads/m(2) and spikelet density from 13 170 to 5960 spikelets/m(2) when rape plant density was increased from 16 to 81 plants/m(2). Spikelets/head of Lolium spp. was little affected by rape seed rate, but the length of heads of A. myosuroides was reduced by 9 % when plant density was increased from 29-51 plants/m(2). Autumn nitrogen increased rape biomass and reduced L. multiflorum head density (415 and 336 heads/m(2) without and with autumn nitrogen, respectively) and spikelet density (9990 and 8220 spikelets/m(2) without and with autumn nitrogen, respectively). The number of spikelets/head was not significantly affected by autumn nitrogen. Early sowing could increase biomass and competitiveness, but poor crop establishment sometimes overrode the effect. Where crop and weed establishment was similar for both sowing dates, a 2-week delay (i.e. early September to mid-September) increased L. multiflorum head density from 226 to 633 heads/m(2) and spikelet density from 5780 to 15 060 spikelets/m(2).

The influence of winter oilseed rape (Brassica napus ssp oleifera var. biennis) cultivar and grass genotype on the competitive balance between crop and grass weeds

Three experiments conducted over two years (2002-04) at the Crops Research Unit, University of Reading, investigated competition between autumn sown oilseed rape cultivars (Brassica napus L. ssp. oleifera var. biennis (DC.) Metzg.) and Lolium multiflorum Lam., L. x boucheanum Kunth and Alopecurus myosuroides Huds., sown as indicative grass weeds. Rape cultivar (cv.) had a substantial effect on grass weed seed return. Over the six cultivars tested, L. multiflorum spikelet production ranged from just under 400 spikelets/m(2) in the presence of cv. Winner to nearly 5800 in competition with cv. Lutin. Cultivar competitiveness was associated with high biomass, large dense floral layers and early stem extension. There was some evidence of differential competitive tolerance between rape cultivars. The results suggested that rape cultivars could be screened for competitiveness by measuring floral layer interception of photosynthetic active radiation. L. x boucheanum cultivars varied in ability to compete with rape. In the absence of inter-specific competition, spikelet density was similar for Aberecho and Polly (circa 31000 spikelets/m(2)) but when grown with rape Polly outyielded Aberecho (i.e. 12 090 and 7990 spikelets/m(2) respectively).

Evaluation of diagnostic molecular markers for DUS phenotypic assessment in the cereal crop, barley (Hordeum vulgare ssp. vulgare L.)

The deployment of genetic markers is of interest in crop assessment and breeding programmes, due to the potential savings in cost and time afforded. As part of the internationally recognised framework for the awarding of Plant Breeders’ Rights (PBR), new barley variety submissions are evaluated using a suite of morphological traits to ensure they are distinct, uniform and stable (DUS) in comparison to all previous submissions. Increasing knowledge of the genetic control of many of these traits provides the opportunity to assess the potential of deploying diagnostic/perfect genetic markers in place of phenotypic assessment. Here, we identify a suite of 25 genetic markers assaying for 14 DUS traits, and implement them using a single genotyping platform (KASPar). Using a panel of 169 UK barley varieties, we show that phenotypic state at three of these traits can be perfectly predicted by genotype. Predictive values for an additional nine traits ranged from 81 to 99 %. Finally, by comparison of varietal discrimination based on phenotype and genotype resulted in correlation of 0.72, indicating that deployment of molecular markers for varietal discrimination could be feasible in the near future. Due to the flexibility of the genotyping platform used, the genetic markers described here can be used in any number or combination, in-house or by outsourcing, allowing flexible deployment by users. These markers are likely to find application where tracking of specific alleles is required in breeding programmes, or for potential use within national assessment programmes for the awarding of PBRs.

Molecular and phenotypic characterization of the alternative seasonal growth habit and flowering time in barley (Hordeum vulgare ssp. vulgare L.)

Barley can be classified into three major agronomic types, based on its seasonal growth habit (SGH): spring, winter and alternative. Winter varieties require exposure to vernalization to promote subsequent flowering and are autumn-sown. Spring varieties proceed to flowering in the absence of vernalization and are sown in the spring. The ‘alternative’ (also known as ‘facultative’) SGH is only loosely defined and can be sown in autumn or spring. Here, we investigate the molecular genetic basis of alternative barley. Analysis of the major barley vernalization (VRN-H1, VRN-H2) and photoperiod (PPD-H1, PPD-H2) response genes in a collection of 386 varieties found alternative SGH to be characterized by specific allelic combinations. Spring varieties possessed spring loci at one or both of the vernalization response loci, combined with long-day non-responsive ppd-H1 alleles and wild-type alleles at the short-day photoperiod response locus, PPD-H2. Winter varieties possessed winter alleles at both vernalization loci, in combination with the mutant ppd-H2 allele conferring delayed flowering under short-day photoperiods. In contrast, all alternative varieties investigated possessed a single spring allele (either at VRN-H1 or at VRN-H2) combined with mutant ppd-H2 alleles. This allelic combination is found only in alternative types and is diagnostic for alternative SGH in the collection studied. Analysis of flowering time under controlled environment found alternative varieties flowered later than spring control lines, with the difference most pronounced under short-day photoperiods. This work provides genetic characterization of the alternative SGH phenotype, allowing precise manipulation of SGH and flowering time within breeding programmes, and provides the molecular tools for classification of all three SGH categories within national variety registration processes.

Sub-tropical urban environment affecting content and composition of non-structural carbohydrates of Lolium multiflorum ssp italicum cv. Lema

This study analyzed the relationship between environmental factors, especially air pollution and climatic conditions, and non-structural carbohydrates (NSC) in plants of Lolium multiflorum exposed during 10 consecutive periods of 28 days at a polluted site (Congonhas) and at a reference site in Sao Paulo city (Brazil). After exposure, NSC composition and leaf concentrations of Al, Fe. Cu, Zn, Pb and Cd were measured. The seasonal pattern of NSC accumulation was quite similar in both sites, but plants at Congonhas showed higher concentrations of these compounds, especially fructans of low and medium degree of polymerization. Regression analysis showed that NSC in plants growing at the polluted site were explained by variations on temperature and leaf concentration of Fe (positive effect), as well as relative humidity and particulate material (negative effect). NSC in the standardized grass culture, in addition to heavy metal accumulation, may indicate stressing conditions in a sub-tropical polluted environment. (C) 2008 Elsevier Ltd. All rights reserved.

Variabilidade de isolados de estirpes de Bradyrhizobium ssp recomendadas para a cultura da soja

A produção da soja em escala comercial tem sido viabilizada técnica e economicamente, entre outros fatores, devido a fixação biológica do N2 por estirpes de Bradyrhizobium que podem suprir a demanda de nitrogênio desta leguminosa. No entanto, a variabilidade existente nas estirpes de Bradyrhizobium spp recomendadas para inoculação da soja tem comprometido o processo simbiótico. Variantes espontâneos isolados a partir das estirpes de B. japonicum (SEMIA 5079 e SEMIA 5080) e B. elkanii (SEMIA 587 e SEMIA 5019) foram avaliados quanto ao desempenho simbiótico (eficiência, nodulação em diferentes hospedeiros e competitividade), indução de clorose foliar em diferentes hospedeiros, morfologia colonial, habilidade de metabolização de carboidratos e tolerância à salinidade em diferentes temperaturas de incubação. Nas etapas de eficiência simbiótica e competitividade foi usada a cultivar de soja BR-16 e na avaliação da nodulação e indução de clorose foliar foram usados os seguintes hospedeiros: soja (Glycine max) (cultivares Clark e Peking), caupi (Vigna unguiculata) e guandu (Cajanus cajan). A caracterização genotípica foi realizada através da reação em cadeia da polimerase (PCR), com os oligonucleotídeos iniciadores BOX A 1-R, ERIC e RP01 Os resultados obtidos demonstraram que variantes e estirpes originais diferem quanto a características fenotípicas, e que diferenças nos perfis eletroforéticos de DNA analisados através da amplificação com os oligonucleotídeos iniciadores testados, evidenciaram a variabilidade genética presente entre as estirpes originais e os variantes selecionados. A cultivar de soja Clark, assim como caupi e guandu foram susceptíveis à rizobiotoxina produzida por estirpes e variantes de B. elkanii. Embora não se tenha verificado diferenças na nodulação em diferentes hospedeiros quando variantes ou estirpes de B. japonicum e B. elkanii foram inoculados em soja, caupi e guandu, foi observado uma simbiose eficiente para a soja (cultivares BR 16, Clark e Peking). A partir da variabilidade existente nas estirpes SEMIA 587, SEMIA 5019, SEMIA 5079 e SEMIA 5080 foram selecionados variantes eficientes e competitivos quanto à fixação de N2 em soja.

Características das células espumosas no fígado, linfonodos mesentéricos e intestino de bovinos associados ao consumo de Brachiaria ssp.

Foram estudados fragmentos dos fígados, linfonodos hepáticos e mesentéricos, bem como dos intestinos de 100 bovinos machos, castrados, entre 3 e 4 anos de idade, da raça Nelore e provenientes do estado de Mato Grosso, onde eram mantidos em pastagens de Brachiaria decumbens e/ou Brachiaria brizantha. Macroscopicamente, os fígados apresentavam coloração amarela que se mantinha mesmo após a fixação em formalina, o que não foi constatado nos animais alimentados com outros pastos. O parênquima dos linfonodos apresentava áreas esbranquiçadas em forma de estrias paralelas, especialmente intensas nos linfonodos hepáticos. As alterações histológicas incluíram tumefação de hepatócitos, colangite mononuclear, áreas com proliferação de ductos e a presença de macrófagos espumosos de distribuição multifocal no fígado, nos linfonodos mesentéricos e hepáticos e na submucosa do intestino. Os tecidos com células espumosas foram submetidos a estudos histoquímicos, lectinoistoquímicos e imunoistoquímicos com o objetivo de caracterizar o conteúdo destas células. Através da Coloração de Perls, constatou-se as células espumosas com citoplasma corados de azul-claro, especialmente nos linfonodos e evidenciando a presença de depósitos de sais férricos, possivelmente em conseqüência da fagocitose de eritrócitos extravasados. Na imunoistoquímica, o anticorpo Mac 387, marcador de macrófagos, não marcou as células espumosas. Na lectinoistoquímica, observou-se que a lectina PNA atuou como marcador devido às altas afinidade e especificidade de união com as células espumosas. Com esta lectina, observaram-se células espumosas isoladas, as quais não eram vistas através de outras colorações. Além disto, em estudos feitos em humanos e em nossos animais, a PNA demonstrou ser específica para macrófagos, o que reforça a hipótese de que as células espumosas sejam macrófagos. A comparação do padrão de afinidade das lectinas entre os animais alimentados e os não alimentados com Brachiaria sp., demonstrou que há alterações na composição de glicídios nos animais alimentados por Brachiaria spp.

Cultura de anteras e embriogênese de genótipos selecionados de cevada (Hordeum vulgare L.ssp.vulgare)

A eficiência da técnica de cultura de anteras, em escala comercial, ainda pode ser considerada baixa quando medida em número de plantas duplo-haplóides férteis obtidas para cada antera estabelecida in vitro. Dessa forma, o presente trabalho é pioneiro no estudo detalhado da embriogênese in vitro do micrósporo e do grão de pólen de cevada (Hordeum vulgare L. ssp. vulgare). Com o objetivo de contribuir para o aperfeiçoamento da técnica de cultura de anteras foi analisada a embriogênese, com especial ênfase na etapa da indução, através de análises citológicas e histológicas de anteras cultivadas in vitro. Foram analisadas uma cultivar brasileira de cevada, em comparação com linhagens de duas outras cultivares brasileiras, que foram selecionadas, por seleção divergente para maior ou para menor resposta na indução da rota embriogênica e, respectivamente, para menor ou para maior capacidade de regenerar plântulas verdes. Somente foram estabelecidas em cultivo in vitro as anteras que apresentaram micrósporos e pólens jovens, das linhagens selecionadas da cultivar A-05 (S3A22 e S3A23), e da cultivar BR-2(S3B63 e, apenas na cultura de anteras, S3B61), bem como da cultivar MN-599 (nãoselecionada). Para as análises histológicas, foram fixadas, a cada dois dias, duas anteras, correspondentes a cada fileira da mesma espiga, após o início do cultivo in vitro. As anteras em cultivo e respectivas estruturas multicelulares foram fixadas em FAA 50%, desidratadas em série etílica e incluídas em hidroxietilmetacrilato. Os blocos de resina polimerizada foram secionados longitudinalmente com 3 mm de espessura. Para as análises citológicas foram fixadas, de cada espiga recém-coletada, três espiguetas sendo uma da base, outra do meio e outra do ápice. Após o pré-tratamento à baixa temperatura (5 °C), porém antes do cultivo in vitro, foram fixadas três anteras (amostras utilizadas como controles). A cada três dias, durante o cultivo, três anteras foram fixadas (até 18 dias). As anteras em cultivo e estruturas multicelulares foram fixadas em Farmer e FAA 50%, transferidas após 24 horas para etanol 70%. Na cultura in vitro das anteras houve diferenças entre uma das linhagens da cultivar A-05 em relação a cultivar MN- 599, na produção inicial de estruturas embriogênicas, diferença que desapareceu na produção total. Entretanto, houve diferenças na formação dos xiii embriões: a cv.MN-599 formou embriões bem diferenciados ao passo que a linhagem S3A22 produziu um número aparentemente menor, sendo que os embriões não eram bem diferenciados. A linhagem S3B63 não apresentou embriões até o final da análise histológica. Considerando que a amostra dessa linhagem, mantida em cultura, formou plantas verdes, pode-se propor que a formação de embriões deve ocorrer posteriormente ao desenvolvimento da cv.MN-599. Cabe destacar que houve diferenças significativas entre as cultivares A-05 e BR-2 quanto à regeneração de plântulas verdes. Esses resultados indicam ter havido maior eficiência da seleção em relação à etapa da regeneração. Com relação às categorias classificatórias dos micrósporos e grãos de pólen, constatou-se que desde o início da análise histológica (2o dia de cultivo in vitro) até o final (34o dia), foram observados micrósporos, o mesmo tendo sido observado na análise citológica. Os grãos de pólen multinucleados ocorreram praticamente em todo o período de cultivo in vitro, em ambas análises; não ocorrendo nos controles da citologia (antes do cultivo); os multinucleados foram observados a partir do 3o dia, enquanto que os multicelulares a partir do 4o dia de cultivo. As estruturas multicelulares foram observadas a partir do 8o dia. A quantidade e o tamanho das estruturas multicelulares foram variáveis ao longo da análise histológica, sendo que do 14o ao 20o dia foram encontradas as de maiores dimensões, resultantes da proliferação celular por mitoses sucessivas. A partir do 22o dia (cultivar MN- 599), a ocorrência de estruturas multicelulares no interior dos lóculos da antera diminuiu, predominando o processo de proliferação externo às anteras. Para as linhagens, a partir do 18o dia foram observadas estruturas multicelulares liberadas das anteras. A análise das estruturas multicelulares permitiu classificá-las em quatro categorias: 1. SFD: Sem forma definida; 2. MAC: meristema apical caulinar; 3. MAR: meristema apical radical embrionário adventício; e 4. Embriões. As estruturas amorfas apareceram em maior número, quando comparadas com as outras categorias. Em síntese: as linhagens selecionadas e a cultivar diferiram não apenas no tempo necessário para a formação dos embriões, mas também no desenvolvimento dos mesmos, que foi mais diferenciado na cultivar MN-599, porém sendo observados mais cedo na linhagem S3A22 e S3A23, do que na cultivar MN-599.