Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Oilseed and oilseed meal extracted from members of <i>Brassicaceae</i> release broadspectrum biocidal isothiocyanate when ground and exposed to moisture. The compounds are released when the seed enzyme myrosinase catalyzes the hydrolysis of glucosinolates producing glucose, sulfate, and pesticidal isothiocyanates. Allylisothiocyanate (AITC), the predominant isothiocyanate of <i>Brassica juncea</i>, has broad-spectrum biological activities against plants, animals and fungi. Knowledge of the concentration of AITC arising from a treatment with mustard and AITC toxicity to many target and non-target species is not known. Therefore, factors affecting AITC release and assays of mustard toxicity were conducted. The rate of AITC release from mustard meal was affected by temperature and pH. Current isothiocyanate extraction and quantification methods measure a change in the concentration of glucose (a predominant product of myrosinase-catalysed glucosinolate hydrolysis) to determine myrosinase activity. The objectives of this work were to study: 1) factors affecting myrosinase activity in mustard (<i>Brassica juncea</i>), 2) the effects of AITC on seed germination and 3) the toxicity of AITC and mustard meal.<p> Attempts were made to improve the Herb and Spice Method, the only available industrial method to measure total isothiocyanate production in mustard meal. The effects of a wide range of reaction temperatures (7 to 97°C) and incubation times (0 min to 2 h) on myrosinase-catalyzed conversion of sinigrin (a glucosinolate) to allyl isothiocynate (AITC) were studied. Significant inhibition of enzyme activity was observed at all temperatures over 57°C, and at 97°C no myrosinase activity was found. It was concluded that myrosinase-catalyzed conversion of sinigrin to AITC was a rapid process and detectable amounts of AITC could be found in samples in two min, and that higher temperatures inhibited myrosinase activity. The pH of the reaction mixture significantly affected myrosinase-catalyzed conversion of sinigrin to AITC. A change in pH did not affect the substrate, but severely affected the activity of myrosinase. Furthermore, other compounds viz., boric acid (H3BO3), succinic acid (C2H4(COOH)2),calcium chloride (CaCl2) and ethanol (C2H5OH), were explored for their ability to inhibit myrosinase activity. Calcium chloride and ethanol were particularly effective.<p> It was hypothesized that AITC might act as a plant growth promoter/regulator based on the fact that AITC and ethylene, a plant growth regulator, exhibit structural similarity (R-CH=CH2, where R is -CH2SCN and -H in AITC and ethylene, respectively). Therefore, AITC might act as an ethylene analogue. Ethylene is known to promote seed germination and overcome seed dormancy in a dose- and species-dependent manner.Flax and tomato seeds were used as model systems to test the germination enhancing properties of AITC. It was concluded that AITC promoted flax and tomato seed germination and thus might be used for this application in agricultural practice.<p> An assay was developed for testing AITC toxicity in ground seed by exploring HSP70 expression in <i>Caenorhabditis elegans</i> as a marker of toxicity. <i>C.elegans</i> strain N2 was exposed to different concentrations (0 to 10 ìM) of AITC for 2 h at room temperature. Western blotting with anti-<i>HSP70</i> antibody showed a marked increase in the expression of <i>HSP70</i> protein in a dose-dependent manner. Assays of the expression of <i>HSP70A</i> mRNA by quantitative real time reverse transcriptase (RT) PCR revealed no significant change in the expression of <i>HSP70A</i> mRNA at low concentrations of AITC (< 0.1 ìM). However, treatment with higher concentrations (>1ìM) resulted in four- to five - fold increase in expression of <i>HSP70A</i> mRNA over the control. To understand if mustard toxicity was due to AITC alone, or if other compounds in mustard ground seed affected <i>HSP70</i> transcript production, <i>C. elegans</i> was exposed to AITC or <i>Brassica juncea</i> cv. Arrid ground seed (Arrid is a mustard variety with a lower level of sinigrin (<3 ìM per gram of seed), or both. ELISA revealed increased expression of HSP70 protein in C. elegans treated with AITC + ground seed, but the level of protein was less than that observed with AITC alone. These results indicated that mustard ground seed toxicity was contributed primarily by AITC, and that some ground seed components antagonized AITC toxicity in <i>C. elegans</i>.

Oilseed and oilseed meal extracted from members of <i>Brassicaceae</i> release broadspectrum biocidal isothiocyanate when ground and exposed to moisture. The compounds are released when the seed enzyme myrosinase catalyzes the hydrolysis of glucosinolates producing glucose, sulfate, and pesticidal isothiocyanates. Allylisothiocyanate (AITC), the predominant isothiocyanate of <i>Brassica juncea</i>, has broad-spectrum biological activities against plants, animals and fungi. Knowledge of the concentration of AITC arising from a treatment with mustard and AITC toxicity to many target and non-target species is not known. Therefore, factors affecting AITC release and assays of mustard toxicity were conducted. The rate of AITC release from mustard meal was affected by temperature and pH. Current isothiocyanate extraction and quantification methods measure a change in the concentration of glucose (a predominant product of myrosinase-catalysed glucosinolate hydrolysis) to determine myrosinase activity. The objectives of this work were to study: 1) factors affecting myrosinase activity in mustard (<i>Brassica juncea</i>), 2) the effects of AITC on seed germination and 3) the toxicity of AITC and mustard meal.<p> Attempts were made to improve the Herb and Spice Method, the only available industrial method to measure total isothiocyanate production in mustard meal. The effects of a wide range of reaction temperatures (7 to 97°C) and incubation times (0 min to 2 h) on myrosinase-catalyzed conversion of sinigrin (a glucosinolate) to allyl isothiocynate (AITC) were studied. Significant inhibition of enzyme activity was observed at all temperatures over 57°C, and at 97°C no myrosinase activity was found. It was concluded that myrosinase-catalyzed conversion of sinigrin to AITC was a rapid process and detectable amounts of AITC could be found in samples in two min, and that higher temperatures inhibited myrosinase activity. The pH of the reaction mixture significantly affected myrosinase-catalyzed conversion of sinigrin to AITC. A change in pH did not affect the substrate, but severely affected the activity of myrosinase. Furthermore, other compounds viz., boric acid (H3BO3), succinic acid (C2H4(COOH)2),calcium chloride (CaCl2) and ethanol (C2H5OH), were explored for their ability to inhibit myrosinase activity. Calcium chloride and ethanol were particularly effective.<p> It was hypothesized that AITC might act as a plant growth promoter/regulator based on the fact that AITC and ethylene, a plant growth regulator, exhibit structural similarity (R-CH=CH2, where R is -CH2SCN and -H in AITC and ethylene, respectively). Therefore, AITC might act as an ethylene analogue. Ethylene is known to promote seed germination and overcome seed dormancy in a dose- and species-dependent manner.Flax and tomato seeds were used as model systems to test the germination enhancing properties of AITC. It was concluded that AITC promoted flax and tomato seed germination and thus might be used for this application in agricultural practice.<p> An assay was developed for testing AITC toxicity in ground seed by exploring HSP70 expression in <i>Caenorhabditis elegans</i> as a marker of toxicity. <i>C.elegans</i> strain N2 was exposed to different concentrations (0 to 10 ìM) of AITC for 2 h at room temperature. Western blotting with anti-<i>HSP70</i> antibody showed a marked increase in the expression of <i>HSP70</i> protein in a dose-dependent manner. Assays of the expression of <i>HSP70A</i> mRNA by quantitative real time reverse transcriptase (RT) PCR revealed no significant change in the expression of <i>HSP70A</i> mRNA at low concentrations of AITC (< 0.1 ìM). However, treatment with higher concentrations (>1ìM) resulted in four- to five - fold increase in expression of <i>HSP70A</i> mRNA over the control. To understand if mustard toxicity was due to AITC alone, or if other compounds in mustard ground seed affected <i>HSP70</i> transcript production, <i>C. elegans</i> was exposed to AITC or <i>Brassica juncea</i> cv. Arrid ground seed (Arrid is a mustard variety with a lower level of sinigrin (<3 ìM per gram of seed), or both. ELISA revealed increased expression of HSP70 protein in C. elegans treated with AITC + ground seed, but the level of protein was less than that observed with AITC alone. These results indicated that mustard ground seed toxicity was contributed primarily by AITC, and that some ground seed components antagonized AITC toxicity in <i>C. elegans</i>.

Made available in DSpace on 2015-03-26T13:36:42Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1139190 bytes, checksum: 7d7600e51fc96aab96c671593415f23b (MD5) Previous issue date: 2011-07-29 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Arsenic is the more dangerous element to human health. Phytoremediation is an important technology to reduce the antropogenic effect in the environmental contamination, but it is necessary a detailed knowledge of tolerance mechanism, which is the goal of this work. We choose Brassica juncea, tolerant plant to several heavy metals, such as cadmium, and a plant of rapid growth. The arsenate was added to nutritive solution at final concentrations of 10, 50, 200 and 500 &#956;M, and data was collected after 6, 10, 13, 14, 15 and 16 days of exposition. Higher the arsenic in the treatment, higher the level found in leaves, stalk and in roots reach the highest level (5457 mg Kg-1). This amount was 26 and 34 times higher than that found in stalk and leaves, respectively. No alteration in phosphorus level was found at any treatment . This absence of phosphate uptake inhibition seems to have a contribution to the tolerance found in this genotype. In contrast, arsenic concentrations equal or higher than 50 &#956;M were associated with increase in sulphur content in roots in parallel with an increase in total phenol content in these organs, while it reduced sulfur levels in leaves. The two highest doses of arsenate led to the appearance of toxicity symptoms in leaves, characterized by marginal chlorosis and purple coloring in the abaxial faces of old leaves. Despite the highest levels of arsenic in roots, no change in root length and dry weight was detected. In shoot however, we observed reduction in leaf area, leng th and fresh weight in plants treated with 200 and 500 mM arsenate. Additionally, only the highest dose has produced a reduction in net photosynthesis (A) and stomatal conductance (gS) by the 13th day. On the 14th day it was possible to observe the reduction of these parameters in plants treated with 200 mM arsenate. Longer periods in the presence of hight doses allowed us to observe that, besides the reduction in stomatal opening, arsenic could also produce biochemical limitation to photosynthesis, which was associated to reduction in electron transport rate (ETR) and in the quantic efficiency of photosystem II [Y(II)], concomitantly with increase in the level of energy loss as heat [Y(NPQ)]. The second higher dose (200 &#956;M) has not altered the A and other fluorescence parameters, by the 13th day and only negligible effects in leaf area and dry mass in parallel to no effect in roots was observed. Altogether these data support that the genotype of Brassica juncea under study is tolerant to arsenic. Despite of accumulation of high levels of arsenic in roots, no oxidative damage was observed, and no effects in root growth was noted. The fact that high levels of arsenic has occurred in roots, with minor effects in growth indicate the great potential of this plant to be used in phytoremediation as a phytostabilizer. / O arsênio é considerado o elemento mais perigoso para a saúde humana. Fitorremediação é uma importante tecnologia para amenizar o efeito antropogênico na contaminação ambiental, mas para o seu sucesso é necessário a compreensão detalhada dos mecanismos de tolerância de metais pesados, objetivo deste trabalho. A espécie escolhida foi a Brassica juncea, tolerante a vários metais, como cádmio e zinco, e que possui um crescimento rápido. O arsênio foi fornecido a solução nutritiva na forma de arsenato de sódio nas concentrações de 10, 50, 200 e 500 &#956;M, sendo coletados dados aos 6, 10, 13, 14, 15 e 16 dias após a exposição. Quanto maiores os níveis de arsênio presentes no tratamento, maiores os níveis encontrados na planta, havendo um acúmulo de até 5457 mg Kg-1 de As nas raízes expostas a 500 &#956;M de arsenato. Esta quantidade foi 26 e 34 vezes superior aquela observada em caules e folhas, respectivamente. Em nenhum dos tratamentos foram observadas alterações na concentração de fósforo. Esta ausência de inibição de acúmulo de fósforo pode significar a presença de um mecanismo de tolerância à toxidez do arsênio neste genótipo. Em contraste, doses de arsênio iguais ou superiores a 50 &#956;M aumentaram os níveis enxofre nas raízes, em paralelo com um aumento no teor de fenóis, ao passo que reduziu os níveis de enxofre na folha. As duas maiores doses de arsenato levaram ao aparecimento de sintomas de toxidez nas folhas, caracterizados por clorose marginal e coloração arroxeada nas faces abaxiais de folhas velhas. Apesar dos altos níveis de arsênio em raízes, não houve redução no comprimento e na massa seca deste órgão. Na parte aérea por outro lado, foi observada a redução na área foliar, comprimento e massa fresca em plantas tratadas com 200 e 500 &#956;M de arsenato. Adicionalmente observou-se que somente a maior dose reduziu a fotossíntese líquida (A) e condutância estomática (gS) até o 13º dia. A partir do 14º dia já foi possível observar a queda destes parâmetros em plantas tratadas com 200 &#956;M de arsenato. Maiores períodos na presença altas doses de arsenato permitiram observar que alem da redução da abertura estomática, há uma limitação bioquímica contribuindo para a redução da fotossíntese. A diminuição em A esteve associada a limitações na etapa fotoquímica onde ocorreu uma redução na taxa linear de transporte de elétrons (ETR) e na eficiência quântica do fotossistema II [Y(II)], concomitante com o aumento da perda da energia luminosa na forma de calor [Y(NPQ)]. A segunda maior dose (200 &#956;M) não provocou redução em A e nos parâmetros fotoquímicos até o 13º dia, com reduções negligíveis na área e massa foliar, com nenhum efeito no crescimento das raízes. Em conjunto, estes resultados permitem evidenciar que o genótipo de Brassica juncea em estudo possui tolerância aos níveis de arsênio. Apesar de acumular níveis altíssimos de arsênio nas raízes, estas plantas não apresentaram dano oxidativo, nem redução na acumulação de massa seca nas raízes. O fato de que altos níveis de As seja acumulado nas raízes, com efeitos reduzidos no seu crescimento, indica o grande potencial desta espécie para o seu uso na fitorremediação como uma espécie fitoestabilizadora.

The worldwide occurrence of sites contaminated with toxic metals and the associated high costs of remediating them using chemical and mechanical methods have led to calls to develop inexpensive and sustainable approaches based on the use of plants that naturally accumulate large amounts of metals in their tissues. The ability of plants to remediate metals has been rigorously studied and some species have been identified as excellent phytoremediators. However, the growth of phytoremediators is often retarded under high soil metal concentrations, rendering them ineffective. Meanwhile, some plants do not have remediating abilities but are capable of growing in contaminated environments with little or no sign of stress. Despite the volume of research dedicated to the screening and evaluation of phytoremediators, major questions remain about why some plants survive but do not remediate while the growth of phytoremediators is mostly hindered. The growth and metal-remediating efficiency of plants exposed to toxic concentrations of metals can be enhanced by inoculating phytoremediating plants with certain bacteria but the mechanisms behind this process remain unclear. Furthermore, the use of leguminous plants to improve the growth of a target plant under a mixed planting system has long been recognised as an effective yield-enhancing cropping system. However, the possibility of a non-remediating but tolerant leguminous plant conferring metal tolerance to a phytoremediator has not been explored. This thesis reports results from repeated glasshouse and lab-based growth experiments on the phytoremediating plant Brassica juncea exposed to 400 – 600 mg Zn kg-1. The aim was to investigate the abilities of two plant growth promoting bacteria (PGPB) species Pseudomonas brassicacearum and Rhizobium leguminosarum, and a leguminous plant Vicia sativa to promote B. juncea growth and enhance remediation of Zn-contaminated soil. B. juncea plant roots were analysed using synchrotron based micro-focus X-ray Fluorescence (μXRF) imaging and X-ray Absorption Near Edge Structure (μXANES) analysis to probe Zn speciation. P. brassicacearum exhibited the poorest plant growth promoting ability, while R. leguminosarum alone and in combination with P. brassicacearum significantly enhanced B. juncea growth and Zn bioaccumulation. X-ray Absorption Spectroscopy (XAS) analysis showed that reduced plant growth was due to root accumulation of Zn as Zn sulphate, Zn oxalate and Zn polygalacturonic acids. The better growth and increased metal accumulation observed in plants inoculated with R. leguminosarum and its combination with P. brassicacearum was attributed to root storage of Zn in the chelated forms of Zn phytate and Zn cysteine. A subcellular analysis of plant root also showed that the PGPB enhanced tolerance to Zn contamination by enhancing epidermal Zn compartmentalisation depending on the nature of root colonization, and induced changes in Zn speciation to less toxic Zn species in the epidermis and endodermis of plant root. The thesis therefore identifies enhanced Zn compartmentalization at the root epidermis and bacterial mediated changes in Zn toxicity through changes in Zn speciation as key complimentary mechanisms of plant growth promotion and enhanced Zn accumulation in plants by PGPB. Further experiments investigating alternative phytoremediation strategies showed that the use of the leguminous plant V. sativa in a mixed planting system with B. juncea plants completely out performed the effects of bacteria in promoting the growth and remediation potential of B. juncea under Zn contamination. By combining PGPB with mixed planting, B. juncea recovered full growth while also achieving maximum phytoremediation efficiency. The novel legume assistedmicrobial phytoremediation method that is reported in this thesis is the first to demonstrate complete plant growth recovery in plants exposed to 400 – 450 mg kg-1 soil Zn contamination for 5 weeks. Survival of V. sativa was attributed to its root storage of Zn in the chelated forms of Zn histidine and cysteine whereas in the roots of stunted B. juncea plants the majority of Zn was present as Zn oxalate and toxic Zn sulphate. Although the use of natural and synthetic chelates has been reported to enhance phytoremediation, this thesis recommends a legume-assisted-microbialphytoremediation system as a more sustainable method for Zn bioremediation.

Excessive metal deposition in soil is of major concern to the environment due to the toxicity of metals to animals and plants. Since metals do not degrade, reducing risk of exposure relies in either removing the metals from soil, or changing their speciation which leads to changes in bioavailability, mobility and toxicity. Plants have been shown to provide a cheap alternative to chemical methods for both removing and changing metal speciation, particularly when augmented with plant growth promoting bacteria. The focus of this thesis was to investigate whether the form (speciation) in which a metal contaminant is introduced to soil affects both plant health and the efficiency of metal remediation by the plant, using the well-known hyperaccumulator Brassica juncea (L.) Czern and zinc (Zn) as the metal contaminant. This study also examined the role of plant growth promoting bacteria in changing metal speciation, impact on metal toxicity and phytoremediation efficiency. Brassica juncea was grown in pots containing soil spiked with equal amounts (600 mg Zn kg-1) of soluble Zn (ZnSO4) and nanoparticulate ZnS and ZnO. Plant height, number of leaves, root length, plant biomass and chlorophyll content of Brassica juncea were used to assess Zn toxicity. Zn localisation and speciation in soil and plant tissues was studied using transmission electron microscopy (TEM), synchrotron micro-X-ray fluorescence elemental mapping (μXRF) and synchrotron X-ray absorption spectroscopy (XAS). Growth parameters showed that ZnSO4 was the most toxic form of Zn whilst ZnS and ZnO effects were not statistically different. These differences were linked to differences in Zn content in root and shoot biomass, which was higher in ZnSO4 treatments. Inoculation with Rhizobium leguminosarum and Pseudomonas brassicacearum enhanced plant growth, Zn concentration in plant biomass and translocation of Zn in all Zn treatments. XAS analysis showed that Zn speciation was altered in roots of plants inoculated with bacteria, with Zn cysteine as the most dominant form of Zn in all inoculated Zn treatments, suggesting a role for cysteine in ameliorating Zn toxicity. By also assessing Zn speciation changes across the soilrhizosphere- plant interface, this study established that Rhizobium leguminosarum modified Zn speciation at the rhizosphere. Through this thesis work, metal speciation is a major factor in determining the efficiency of metal phytoremediation and plant tolerance. Hence, this research provides useful information on Zn speciation which will contribute to effective implementation of Zn phytoremediation.

Canola and juncea meals can be used in poultry diets instead of soybean meal. The meals were fed to broilers and turkeys in digestibility trials to measure the apparent metabolizable energy (AMEn). Throughout growth trials, canola and juncea meals were fed at four levels (0, 10, 20 and 30 %) with and without a dietary enzyme cocktail (DEC). This study investigated higher inclusion levels than previously recommended for use in broilers diets. The carcass compositions, fatty acid analysis and liver parameters were measured to investigate the effects of diets on birds. The AMEn of the meals were used in formulating broiler and turkey diets. Growth trials found positive effects of DEC on performance, allowed higher inclusion levels of the meals. No negative effects of diets were observed for liver parameters and carcass compositions. The fatty acid profiles of carcass tissues were improved by increasing levels of canola oil in diets.

Submitted by DOGLAS BASSEGIO null (doglas14@hotmail.com) on 2017-04-16T22:03:40Z No. of bitstreams: 1 Tese.pdf: 1143011 bytes, checksum: f84f0671d7fde4c59ee46871a3d37883 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-04-18T17:47:05Z (GMT) No. of bitstreams: 1 bassegio_d_dr_bot.pdf: 1143011 bytes, checksum: f84f0671d7fde4c59ee46871a3d37883 (MD5) / Made available in DSpace on 2017-04-18T17:47:05Z (GMT). No. of bitstreams: 1 bassegio_d_dr_bot.pdf: 1143011 bytes, checksum: f84f0671d7fde4c59ee46871a3d37883 (MD5) Previous issue date: 2017-03-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O gênero Brassica é a terceira maior fonte de óleo vegetal do mundo, com oleaginosas cultivadas no Hemisfério Norte. No Brasil, apesar do aumento nos últimos anos, a espécie Brassica napus L., não é um cultivo tradicional, devido as limitações de fotoperíodo e altas temperaturas. Outras espécies do gênero, como é o caso da Brassica juncea L. e Brassica rapa L., amplamente cultivadas em zonas de baixa precipitação e fertilidade, podem ser opções de cultivo em áreas marginais de sequeiro em condições tropicais, visto que possuem alto teor de óleo com características propicias para o biodiesel, além de menor sensibilidade ao ambiente. Os objetivos deste trabalho foram caracterizar germoplasma de Brassica juncea L. e Brassica rapa L., avaliar o potencial produtivo e a divergência genética de acessos em Botucatu – SP, Brasil. Os experimentos tiveram início em outono-inverno de 2012, com a avaliação de 424 acessos de Brassica juncea L. e 209 de Brassica rapa L. quanto ao florescimento e produção de sementes. Em outono-inverno de 2015 e 2016 acessos de brássicas e um híbrido comercial da espécie Brassica napus L. foram avaliados, em experimentos em blocos ao acaso com três repetições, em relação aos componentes da produção, à produção grãos e óleo e a divergência genética. Do total de acessos introduzidos e avaliados, apenas 51 (12%) de Brassica juncea L. e 59 (28%) de Brassica rapa L. produziram sementes, devido ao efeito de fotoperiodo. Os componentes da produção variaram entre acessos e espécies, com destaque para precocidade e teor de óleo de acessos da espécie Brassica rapa L.; e o número de síliquas e a produtividade de grãos e óleo de acessos da espécie Brassica juncea L.. A produtividade de grãos e óleo foi em média 15% superior nos acessos de Brassica juncea L. em relação a espécie Brassica rapa L., com destaque para o acesso PI 180266 com 2056 kg ha–1 de grãos e 805 kg ha–1 de óleo. A variabilidade genética entre os acessos de brássicas possibilitou a formação de grupos de acessos para futuros ganhos em melhoramento genético. Conclui-se que os acessos selecionados confirmam a hipótese do potencial produtivo sem efeito de fotoperíodo em condições tropicais, o que demonstra que podem ser opções para cultivo em áreas onde a Brassica napus L. não está bem adaptada. / Brassica is the third largest source of vegetable oil in the world, with oilseeds grown in the Northern Hemisphere. In Brazil, despite the increase in recent years, Brassica napus L., is not a traditional crop due to limitations of photoperiod and high temperatures. Other species of the genus, such as Brassica juncea L. and Brassica rapa L., widely cultivated in low rainfall zones and fertility, may be cultivation options in marginal rainfed areas under tropical conditions, since they have high oil content with characteristics conducive to biodiesel, in addition to lower sensitivity to environment. The objectives of this work were to characterize the germplasm of Brassica juncea L. and Brassica rapa L. and to evaluate the productive potential, as well as the genetic divergence of accesses in Botucatu – SP, Brazil. The experiments started in autumn-winter of 2012, with the evaluation of 424 accessions of Brassica juncea L. and 209 of Brassica rapa L. regarding flowering and seed production. In the autumn-winter of 2015 and 2016, Brassica napus L. and commercial Brassica napus L. were evaluated in randomized blocks with three replicates, in relation to the production components, grain and oil production, and divergence genetic. Of the total number of accesses introduced and evaluated, only 51 (12%) of Brassica juncea L. and 59 (28%) of Brassica rapa L. produced seeds, due to the effect of photoperiod. The components of the production varied between accesses and species, with emphasis on precocity and oil content of Brassica rapa L.; and the growth and number of siliques of the specie Brassica juncea L. The yield of grains and oil was on average 15% higher in the accessions of Brassica juncea L. in relation to the species Brassica rapa L., with emphasis on access PI 180266 with 2056 kg ha–1 of grains and 805 kg ha–1 of oil yield. The genetic variability among the accessions of brassicas allowed the formation of groups of accessions for future gains in genetic improvement. It is concluded that the selected accessions confirm the hypothesis of the productive potential without photoperiod effect in tropical conditions, which demonstrates that they can be options for cultivation in areas where Brassica napus L. is not well adapted.

The major objective of this study was to produce synthetic genotypes of Brassica juncea from its parental species <i> B. rapa </i> and <i> B. nigra </i> via somatic and sexual hybridization. As prerequisites for somatic hybridization experiments, methods were developed to improve the culture of mesophyll and hypocotyl protoplasts of <i> B. nigra </i> and <i> B. rapa </i>, to obtain reliable plant regeneration from mesophyll protoplast cultures of <i> B. nigra </i>, and to fuse protoplasts of <i> B. nigra </i> and <i> B. rapa </i>. A modified Kao's medium (1977), was found suitable for the culture of mesophyll protoplasts of <i> B. nigra </i> and <i> B. rapa </i>. At a density of approximately $110\sp5$ protoplasts/ml within a culture plate insert surrounded by culture medium, mesophyll protoplast cultures of <i> B. nigra </i> accessions R890, R1819, R3392 and U1218 and <i> B. rapa </i> cvs. R500 and Wong Bok formed colonies. Genotypic differences in cell division and colony formation were observed. Hypocotyl protoplasts of <i> B. nigra </i> and <i> B. rapa </i> were successfully isolated from 6 day-old seedlings cultured in a modified Kao's medium (1977). With <i> B. nigra </i> accession R890 and <i> B. rapa </i> cv. R500, cell division and colony formation were optimal when hypocotyl protoplasts were cultured at a density of 0.5 to $1.010\sp5$ protoplasts/ml within a culture plate insert surrounded by a nurse culture of 4 to 6 day-old mesophyll protoplasts of <i> B. nigra </i>. Plant regeneration was obtained from mesophyll protoplast-derived calli of <i> B. nigra </i> accession R890 originally cultured in inserts; a shoot regeneration frequency of 8.1% was obtained on a medium containing the salts and vitamins of medium K3 (Nagy and Maliga 1976) with 3 g/l sucrose, 18.2 g/l mannitol, 2 mg/l ZR, 0.1 mg/l NAA, 10 g/l agarose, pH 5.6. For somatic hybridizatian studies, methods were developed to select out parental protoplasts using iodoacetic acid and to efficiently fuse protoplasts on the bottom of a petri dish using PEG. Twenty-nine plants were recovered from fusion experiments between mesophyll protoplasts of <i> B. nigra </i> accession R890 and hypocotyl protoplasts of <i> B. rapa </i> cv. Tobin. The somatic hybrid plants resembled natural <i> B. juncea </i>, had $2n=36$ chromosomes and had pollen viabilities ranging from 30 to 45%. Twenty-one plants, derived from one callus colony, possessed the mitochondrial and chloroplast genomes of <i> B. rapa </i>, as found in natural <i> B. juncea </i>. Eight plants, derived from another callus, had a novel cytoplasmic combination consisting of the mitochondrial genome of <i> B. rapa </i> and the chloroplast genome of <i> B. nigra </i>. Synthetic genotypes of <i> B. juncea </i> were also produced from reciprocal sexual crosses between <i> B. rapa </i> and <i> B. nigra </i>. Seventy-eight interspecific hybrid plants from the cross <i> B. rapa </i> x <i> B. nigra </i> and six hybrid plants from the reciprocal cross were identified by their morphology, pollen viability and chromosome number. The colchicine-induced allotetraploids resembled natural <i> B. juncea </i> in morphology, had 18 bivalents at metaphase I, and had between 35 and 70% pollen viability.

The potential for an adaptive response to global climatic change was evaluated for an annual C$ sb3$ weed, Brassica juncea, by performing a selection on fecundity for eight generations. During the selection, atmospheric carbon dioxide and temperature were gradually increased from current levels (370 $ rm mu L cdot L sp{-1}$ CO$ sb2$, 20$ sp circ$C) to conditions predicted during the next century by climate models (650 $ rm mu L cdot L sp{-1}$ CO$ sb2$, 23.6$ sp circ$C) including heat stress events at 32$ sp circ$C/26$ sp circ$C day/night), At the end of the selection, a reciprocal transplant experiment was conducted to identify genetic differences between control selection lines of plants and those selected under increasing CO$ sb2$ and temperature. I observed a genetic adaptation of early vegetative growth elevated CO$ sb2$ and temperature, which resulted in to 63% more biomass and 11% higher photosynthetic rates. Reproductive biomass, however, was decreased during the selection, mainly due to temperature stress, which disrupted flower development and induced strong maternal effects, counteracting the selection on fecundity.