Rozprawy doktorskie na temat „Brassica juncea”

Turnip mosaic virus (TuMV, family Potyviridae, genus Potyvirus) has the widest host range amongst potyviruses. Globally it was said to be the second most important virus infecting field vegetables. Brassica juncea (Oriental mustard, family Brassicaceae), is an amphidiploid plant species with the genome AABB, comprising the genomes of the two diploid species, Brassica rapa (AA) and Brassica nigra (BB). It is widely grown and has various uses including as a leaf, stem, or root vegetable, oilseed crop, forage crop, condiment and biofumigant. Most B. juncea cultivars are very susceptible to TuMV, resulting in severe losses. Research on TuMV resistance and the mapping and identification of natural resistance genes would be very useful in order to speed up breeding resistant crops through marker-assisted selection. Sources of resistance to TuMV have been identified in B. juncea. The specificity of the resistances has been determined. A B. juncea DH line for which there is genomic information has been challenged with TuMV and found to be susceptible. This line has been used as a susceptible parent in crosses with resistant plants derived from different sources to develop segregating populations for mapping the resistance gene(s). Two BC1 populations (222 plants and 205 plants) and one F2 population (159 plants) have been phenotyped and segregation ratios were not significantly different from a Mendelian model based on the action of two recessive genes. Parental lines and selected plants in the two BC1 populations have been analysed by SNPs genotyping using the Illumina Infinium Chip. Genetic linkage maps have been constructed and QTLs have been mapped. Additionally, attempts are being made to identify a dominant TuMV resistance gene present in both Brassica napus and B. rapa. Inter-specific crosses have been made in order to introgress this gene into B. juncea. Resynthesised B. juncea plants possessing this dominant resistance have been produced through embryo rescue and polyploidy induction of F1 plants from crosses between resistant B. rapa and susceptible B. nigra plants. BC2 plants have also been developed by crossing B. rapa and B. napus plants possessing the dominant TuMV resistance with a susceptible B. juncea plant line.

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.

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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.

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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 μM, 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 μM 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 μM) 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 μM, 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 μM 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 μM 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 μM 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 μM 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 μM) 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.

Indian mustard (Brassica juncea L.) has recently been identified as a potential and profitable alternative oilseed crop in the grain growing regions of Australia. To date, no research has been reported on adaptation of mustard in water limited Mediterranean-type environments in south Western Australia. Experiments presented in this thesis were undertaken to study adaptation of mustard in the Mediterranean-type environments in south Western Australia, with the hypothesis that mustard would be better adapted to these environments due to its reputation for drought tolerance. Experiments were conducted with three main aims. Firstly, to identify the effects of genotype, environment (times of sowing/seasons/sites) and genotype x environment interaction on the phenology, growth, dry matter production, seed yield, oil and protein contents of mustard and canola. Secondly, to identify phenological, morphological and physiological characters responsible for adaptation and yield improvement of mustard in these environments. Thirdly, to study the response of mustard to soil moisture deficits, especially in the post-flowering period, in comparison to canola. Adaptation of six mustard breeding lines/cultivars varying in maturity, height and oil quality and three canola cultivars varying in maturity were tested at a medium rainfall site (Northam) in the 1999 growing season. These genotypes were sown at four times after the break of the season and their phenology, growth, morphology, dry matter production and partitioning, radiation absorption, seed yield and its components, and seed oil and protein concentrations were measured. Adaptation of mustard to short season, low rainfall areas was tested, in the 2000 and 2001 growing seasons, at three sites (Merredin. Mullewa and Newdegate), by sowing seven genotypes of mustard and canola at three times after the break of the season.Seed yield, oil and protein concentrations were measured at all three sites and detailed measurements of phenology, morphology, dry matter production and partitioning, radiation absorption, seed yield and its components, and seed oil and protein concentrations were taken only at Merredin. The effects of post-flowering soil moisture stress on mustard and canola was studied in detail using rainout shelters at Merredin in the 2001 growing season. Measurements of water use, leaf water potential, osmotic potential, osmotic adjustment, relative water content, and leaf diffusive conductance were taken together with morphology, dry matter production and partitioning, radiation absorption, seed yield and its components, and seed oil and protein concentration. Mustard produced seed yields similar to canola at a medium rainfall site at Northam in south Western Australia. Early sowing (May) was more suitable for mid and late maturing genotypes and mid sowing (early June) was optimum for early maturing genotypes at this site. Dry matter production and seed yield was highest in early sowing due to balanced pre-anthesis and post-anthesis development of the crop and its ability to avoid terminal drought. Very late sowing (after July) significantly reduced the dry matter production, seed yield and oil concentration of mustard and canola due to poor establishment, reduced post-anthesis duration, soil moisture and high temperature stresses which occurred at the end of the season. Mustard did not produce significantly higher dry matter and seed yield compared to canola at the medium rainfall site, Northam. Seed yield and oil concentration of mustard and canola in low rainfall environments (Merredin, Mullcwa and Newdegate) were higher when sown early in the season (May). Longer growing duration and post-anthesis duration were favourable for higher yields.Higher rainfall during the post-anthesis phase, warmer pre-anthesis phase and cooler post-anthesis phase were associated with higher seed yield in these environments. As shown by the Principal Component Analysis and the Finlay Wilkinson Analysis, adaptation of mustard genotypes to low rainfall environments was better compared to canola genotypes. Mustard genotypes, 887.1.6.1, 82 No 2298 demonstrated their general adaptability by producing the highest mean seed yield across all environments and showing average phenotypic stability across all environments. The low yielding canola genotype, Oscar was best adapted to high yielding environments and showed below average phenotypic stability. Low yielding mustard genotypes, JM 25 and JM 33 were best adapted to low yielding environments and showed above average phenotypic stability. Early flowering and developmental plasticity had a significant contribution to yield potential and its stability. All mustard genotypes were more tolerant to soil moisture and high temperature stresses and exhibited early vigour compared to canola varieties. Mustards produced significantly higher dry matter compared to canola under soil moisture and high temperature stresses. Yield reduction due to late sowing VI was greater in canola compared to mustards. Greater dry matter production of mustards under severe soil moisture stress was related to their higher water use and radiation use, which in turn was related to their superior osmotic adjustment.Osmotic adjustment improved dry matter production in mustards as it allowed stomata to remain partially open at progressively lower leaf water potentials and maintained higher stomatal conductance, maintained leaf area and reduced the rate of leaf senescence by increasing both avoidance and tolerance of dehydration and thereby increased radiation use, increased water use by stomatal adjustment, and increased soil moisture uptake by producing deeper roots. Mustard exhibited many agronomic advantages over canola, such as vigorous seedling growth, quick ground covering ability, early vigour, and the feasibility of direct harvesting due to non-shattering pods. Despite all these advantages currently available mustard genotypes do not have the ability to out yield canola due to their lower efficiency of conversion of dry matter to seeds, as indicated by lower harvest indices, and inferior yield component structure. Further breeding in mustard is required to modify its morphology and yield component structure. Mustard plants with more pods and pods with more seeds would produce higher yields. Shorter, compact plant stature and reduced branching would improve harvest index in mustard. Furthermore, development of mustard genotypes with high oil quality and concentration similar to canola would improve its market value as an oil seed crop.

Indian mustard (Brassica juncea L.) has recently been identified as a potential and profitable alternative oilseed crop in the grain growing regions of Australia. To date, no research has been reported on adaptation of mustard in water limited Mediterranean-type environments in south Western Australia. Experiments presented in this thesis were undertaken to study adaptation of mustard in the Mediterranean-type environments in south Western Australia, with the hypothesis that mustard would be better adapted to these environments due to its reputation for drought tolerance. Experiments were conducted with three main aims. Firstly, to identify the effects of genotype, environment (times of sowing/seasons/sites) and genotype x environment interaction on the phenology, growth, dry matter production, seed yield, oil and protein contents of mustard and canola. Secondly, to identify phenological, morphological and physiological characters responsible for adaptation and yield improvement of mustard in these environments. Thirdly, to study the response of mustard to soil moisture deficits, especially in the post-flowering period, in comparison to canola. Adaptation of six mustard breeding lines/cultivars varying in maturity, height and oil quality and three canola cultivars varying in maturity were tested at a medium rainfall site (Northam) in the 1999 growing season. These genotypes were sown at four times after the break of the season and their phenology, growth, morphology, dry matter production and partitioning, radiation absorption, seed yield and its components, and seed oil and protein concentrations were measured. Adaptation of mustard to short season, low rainfall areas was tested, in the 2000 and 2001 growing seasons, at three sites (Merredin. Mullewa and Newdegate), by sowing seven genotypes of mustard and canola at three times after the break of the season.
Seed yield, oil and protein concentrations were measured at all three sites and detailed measurements of phenology, morphology, dry matter production and partitioning, radiation absorption, seed yield and its components, and seed oil and protein concentrations were taken only at Merredin. The effects of post-flowering soil moisture stress on mustard and canola was studied in detail using rainout shelters at Merredin in the 2001 growing season. Measurements of water use, leaf water potential, osmotic potential, osmotic adjustment, relative water content, and leaf diffusive conductance were taken together with morphology, dry matter production and partitioning, radiation absorption, seed yield and its components, and seed oil and protein concentration. Mustard produced seed yields similar to canola at a medium rainfall site at Northam in south Western Australia. Early sowing (May) was more suitable for mid and late maturing genotypes and mid sowing (early June) was optimum for early maturing genotypes at this site. Dry matter production and seed yield was highest in early sowing due to balanced pre-anthesis and post-anthesis development of the crop and its ability to avoid terminal drought. Very late sowing (after July) significantly reduced the dry matter production, seed yield and oil concentration of mustard and canola due to poor establishment, reduced post-anthesis duration, soil moisture and high temperature stresses which occurred at the end of the season. Mustard did not produce significantly higher dry matter and seed yield compared to canola at the medium rainfall site, Northam. Seed yield and oil concentration of mustard and canola in low rainfall environments (Merredin, Mullcwa and Newdegate) were higher when sown early in the season (May). Longer growing duration and post-anthesis duration were favourable for higher yields.
Higher rainfall during the post-anthesis phase, warmer pre-anthesis phase and cooler post-anthesis phase were associated with higher seed yield in these environments. As shown by the Principal Component Analysis and the Finlay Wilkinson Analysis, adaptation of mustard genotypes to low rainfall environments was better compared to canola genotypes. Mustard genotypes, 887.1.6.1, 82 No 2298 demonstrated their general adaptability by producing the highest mean seed yield across all environments and showing average phenotypic stability across all environments. The low yielding canola genotype, Oscar was best adapted to high yielding environments and showed below average phenotypic stability. Low yielding mustard genotypes, JM 25 and JM 33 were best adapted to low yielding environments and showed above average phenotypic stability. Early flowering and developmental plasticity had a significant contribution to yield potential and its stability. All mustard genotypes were more tolerant to soil moisture and high temperature stresses and exhibited early vigour compared to canola varieties. Mustards produced significantly higher dry matter compared to canola under soil moisture and high temperature stresses. Yield reduction due to late sowing VI was greater in canola compared to mustards. Greater dry matter production of mustards under severe soil moisture stress was related to their higher water use and radiation use, which in turn was related to their superior osmotic adjustment.
Osmotic adjustment improved dry matter production in mustards as it allowed stomata to remain partially open at progressively lower leaf water potentials and maintained higher stomatal conductance, maintained leaf area and reduced the rate of leaf senescence by increasing both avoidance and tolerance of dehydration and thereby increased radiation use, increased water use by stomatal adjustment, and increased soil moisture uptake by producing deeper roots. Mustard exhibited many agronomic advantages over canola, such as vigorous seedling growth, quick ground covering ability, early vigour, and the feasibility of direct harvesting due to non-shattering pods. Despite all these advantages currently available mustard genotypes do not have the ability to out yield canola due to their lower efficiency of conversion of dry matter to seeds, as indicated by lower harvest indices, and inferior yield component structure. Further breeding in mustard is required to modify its morphology and yield component structure. Mustard plants with more pods and pods with more seeds would produce higher yields. Shorter, compact plant stature and reduced branching would improve harvest index in mustard. Furthermore, development of mustard genotypes with high oil quality and concentration similar to canola would improve its market value as an oil seed crop.

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.

L'objectif de cette étude a consisté à obtenir et étudier des autotétraploïdes chez trois espèces de Brassica apparentés: B. Nigra (l. ) Koch, B. Rapa L, B. Juncea (l. ) Czern. Deux voies d'obtention ont été abordées: polyploïdisation somatique par traitement à la colchicine et polyploïdisation gamétophytique par l'intermédiaire de gamètes non réduits. La culture de bourgeons axillaires sur un milieu in vitro additionne de colchicine a permis d'obtenir des autotétraploïdes chez les trois espèces étudiées. Les autotétraploïdes se caractérisent par une augmentation de la masse des graines et de la teneur en sinigrine mais la fertilité est réduite. L'autotétraploïdie entraine une augmentation de la taille des grains de pollen et du nombre d'apertures. Cependant, le critère nombre d'apertures n'est pas suffisant pour détecter des diplogamètes mâles chez les plantes diploïdes. La réalisation, chez B. Nigra de croisements 4x avec 2x et 2x avec 4x permet d'obtenir des autotétraploïdes par l'intermédiaire de gamètes non réduits mâles ou femelles

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.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 17, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Thesis advisor: Dr. Reid Smeda. Includes bibliographical references.

[Truncated abstract] In order to expand the gene pool of canola-quality rapeseed (Brassica napus) reciprocal interspecific crosses were made between B. napus cv. Mystic and near canola-quality B. juncea breeding line JN29. F1 progeny from these crosses were used to make backcrosses to both parents in all possible combinations and directions, and were selfed to form F2-derived lines. The highest frequencies of viable F2 and BC1 progeny were obtained when B. napus was the maternal parent of the interspecific hybrid. BC1 and F2 progeny (and subsequent generations) were grown under field conditions to identify agronomic improvements over the parents. Transgressive segregation was observed in F2 and BC1 and in subsequent generations for agronomic traits (seed yield under high or low rainfall conditions, plant biomass, harvest index, height, branching and days to anthesis) and seed quality traits (oil, protein, glucosinolates, oleic acid). The majority of progeny conformed to B. napus morphology, and a minority segregated to B. juncea morphology in subsequent generations. Some of the B. juncea morphotypes had lower glucosinolates and higher oleic acid than the parent JN29, with no detectable erucic acid, and thereby conformed to canola quality. Methods were developed for tracing B-genome in interspecific progeny. A repetitive DNA sequence pBNBH35 from B. nigra (genome BB, 2n = 16) was used to identify B-genome chromosomes and introgressions in interspecific progeny. Specific primers were designed for pBNBH35 in order to amplify the repetitive sequence by PCR. A cloned sub-fragment of 329 bp was confirmed by sequencing as part of pBNBH35. PCR and hybridisation techniques were used on an array of Brassica species to confirm that the pBNBH35 subfragment was Brassica B-genome specific. Fluorescence in situ hybridisation (FISH) in B nigra, B. juncea (AABB, 2n=36) and B. napus (AACC, 2n=38) showed that the pBNBH35 sub-fragment was present on all eight Brassica Bgenome chromosomes and absent from A- and C-genome chromosomes. The pBNBH35 repeat was localised to the centromeric region of each B-genome chromosome. FISH clearly distinguished the B-genome chromosomes from the A-genome chromosomes in the amphidiploid species B. juncea. This is the first known report of a B-genome repetitive marker that is present on all Brassica Bgenome chromosomes. ... The results suggest that novel B. napus genotypes have been generated containing introgressions of B-genome chromatin from B. juncea chromosomes. B. juncea morphology occurred in interspecific progeny with a chromosome complement similar to B. napus (2n = 38) and without the entire Bgenome present. It also is highly likely that recombination has occurred between the A-genome of the two Brassica species. This research has demonstrated that the secondary gene pool of B. napus may be accessed by selfing interspecific hybrids, and without sacrificing canola quality, if the B. juncea parent is near canola-quality. Interspecific progeny may be screened to enhance the proportion with B-genome positive signals. Some progeny with B. junceatype morphology had improved seed quality over the JN29 parent.

O objetivo do presente estudo foi avaliar a capacidade das espécies de mostarda Sinapis alba e Brassica juncea quanto à capacidade de bioacumulação e translocação de manganês, visando conhecer seu potencial de utilização para fitorremediação de solos. O experimento foi realizado em casa de vegetação na Universidade de Taubaté (UNITAU), no campus das Ciências Agrárias, utilizando Latossolo Vermelho Amarelo de classe textural franco argiloso, nas dosagens de 0, 50, 100, 200, 300, 450, 600 e 800 mg kg-1 de Mn, ajustados através de solução de MnSO4.4H2O. Após 50 dias da semeadura foi feita a colheita, lavagem, secagem e moagem da biomassa, e determinação de Mn na parte aérea e raiz. A produção de biomassa mostrou uma correlação negativa com os teores de Mn no solo, tendo a partir da dose de 200 mg kg-1 o crescimento severamente afetado tornando visíveis os sintomas de toxicidade como: manchas amarronzadas de necrose, encarquilhamento das bordas das folhas e perda da cor verde (clorose). A massa seca da parte aérea apresentou diferença significativa entre os tratamentos e na interação tratamento-espécie, sendo a Brassica juncea superior nas doses de 200 e 300 mg kg-1. A produção de massa seca de raiz também mostrou correlação negativa com os teores de Mn no solo, diferença significativa entre os tratamentos e entre as espécies sendo a Brassica juncea superior a Sinapis alba. A bioacumulação de Mn na parte aérea mostrou correlação positiva com o teor de Mn no solo e diferença estatística significativa somente entre os tratamentos. As duas espécies tiveram o mesmo nível de bioacumulação de Mn na parte aérea. Nas raízes houve diferença estatística significativa para os tratamentos nas duas espécies, e correlação positiva com o teor de Mn do solo. Apesar da Sinapis alba apresentar índice médio de translocação maior, a Brassica juncea mostrou maior conteúdo de Mn por planta, com rendimento máximo nas doses de 200 mg kg-1 e 300 mg kg-1 de Mn no solo. As duas espécies mostraram potencial para uso em fitorremediação de solos devido à alta bioacumulação de Mn (hiperacumuladoras) e translocação. A Brassica juncea mostrou-se mais promissora que a Sinapis alba, por apresentar maior tolerância a altas dosagens de Mn no solo.
The aim of this work was to evaluate the manganese uptake and translocation capacity of two species of mustard, Sinapis alba and Brassica juncea, in order to know their potential for use in the phytoremediation process. The experiment was conducted in greenhouse conditions, at the Agricultural Sciences Department of Taubaté University (UNITAU), using samples of Red Yellow Latosol, argillaceous franc textural class soil. The tested soil manganese doses were 0, 50, 100, 200, 350, 450, 600 and 800 mg kg-1, using manganese sulfate in water solution. The sample soil was taken from 60 cm depth, it was bolted, made homogeneous, chemically analyzed, and nutrients adjustments were made. The harvest took place 50 days after sowing; the biomass was washed, dried until constant weight was reached, ground in type Wiley mill the manganese concentration was determined. The biomass production showed negative correlation with soil manganese concentration, having its growth rate strongly affected from 300 mg kg-1 soil manganese dosage on. Also from this soil manganese level on, toxicity symptoms were visible, such as brown speckles that started on the tips of the older leaves and spread down the leaf until the entire blade was covered; chlorosis and necrosis that effectively reduced the photosynthetic capacity of the plants. The tissue dried biomass showed statistical difference among dosages and in dosage-specie interaction, with Brassica juncea showing greater difference than Sinapis alba in 200 and 300 mg kg-1 soil Mn doses. The tissue manganese bioaccumulation showed positive correlation with soil manganese level and significant difference among treatments. Both species had the same tissue bioaccumulation level. The manganese root concentration also had positive correlation with soil manganese, and statistical difference for treatments was present in both species. Although Sinapis alba translocation index was better, Brassica juncea had greater manganese content level, with best results in 200 and 300 mg kg-1 manganese soil dose. Both species showed good potential for use in soil phytoremediation processes, due to high manganese bioaccumulation and translocation level.

Blackleg, caused by Leptosphaeria maculans is one of the most devastating diseases of canola (Brassica napus) in North Dakota. A study was conducted to characterize prevalence of pathogenicity groups (PG), identify population structure of L. maculans and identify sources of resistance among B. juncea accessions. Approximately 56% of the isolates belonged to PG-4, 13% to PG-3, 11% to PG-T, 5% to PG-1, and 2% to PG2. The remaining 13% of isolates could not be identified. The 605 single-spore cultures used to study the population genetics of L. maculans in ND were grouped according to their county of origin in five regions (NE, NC, NW, WC, and C) and each region was considered a population. These populations were tested for genetic variation at 7 microsatellite, 4 minisatellite, and for mating type loci. High levels of genetic diversity (H = 0.63 to 0.70) and significant gametic disequilibrium (P < 0.001) was observed in all populations. The ratio of mating type idiomorphs deviated significantly (P < 0.05) from 1:1 ratio for four populations. Highly significant (P < 0.001) G''ST between pairs of populations indicated a strong population differentiation. STRUCTURE analysis identified three distinct genetic populations in which the majority of the isolates from WC and nearly half of the isolates from NC were assigned to subpopulation one. The remaining half of NC isolates were assigned to subpopulation two and rest were assigned to subpopulation three. To identify sources of resistance against PG-2, PG-3, PG-T, and PG-4, a set of 298 B. juncea accessions were screened in greenhouse trials. Six accessions were resistant to PG-2 and PG-3, nine accessions were resistant to PG-T and PG-4, and two accessions were resistant to PG-2 and PG-4. DNA extracted from these accessions was screened using 766 DArT markers to identify QTL associated with resistance. Thirteen DArT markers were significantly (P<0.05) associated with resistance to PG-2 with variability ranging from 0.9% to 6.4%. Of these, three were also found significantly (P<0.05) associated with resistance to PG-3 with variability of 4.4%. No markers were found to be significantly associated with resistance to PG-T or PG-4 at (P<0.05).

Introduction The work carried on in the triennium 2007-09 put itself in the field of research known as phytoremediation. This term identifies several techniques (phytoextraction, phytostabilization, phytovolatilization) based on using different plant species able to survive on substrates contaminated by many pollutants derived from human activities and to accumulate them in own tissues or volatilize by evapotranspiration or avoiding their leakage troughout the groundwater . This ability varies strongly trough species and inside a species between genetic pools. About the ability in heavy metals removing , researchers have identified two classes of plant species : Hyperaccumulators : species tipical of metalliferous soils, able to carry on their life cycle without showing symptoms of stress as heavy metals high concentration. (Baker et al., 2000). To be classified as hyperaccumulator a plant species must accumulate a minimum of : 100 mg/Kg d.w. of Cd, 1000 mg/Kg d.w. of Pb, 10000 mg/Kg d.w. of Zn ; ranges for As are not yet determined. (Baker and Brooks 1989, Baker et al., 2000). Some of most rapresentative hyperaccumulator species are Thlaspi caerulescens, Arabidopsis halleri and Viola calamarina, Astragalus spp and several other species of following families : Cariophyllaceae, Fabaceae, Poaceae, Chenopodiaceae, Brassicaceae e Cyperaceae. About As the best accumulator is Pteris vittata. The main limit of hyperaccumulators is little biomass production. Accumulators: in this group there are herbaceous and woody species able to couple a good accumulation with a good biomass production. Between them we can find Brassica juncea, end the genus Salix. This kind of accumulators are the best solution for pollutants removal programs in short and medium period. An high biomass production allows to plan a recycle of it in energy production and to recover heavy metals from ashes. ((Kabata-Pendias and Pendias, 1992 ; Pullford and Dickinson 2004 ; Paulson et al 2003; French et al 2005; Ebbs et al 1997 ; Ebbs et al 1998). This work, divided in several steps, as described below, wants to increase the knowledge on some of previous species, from a physiological point of view and evaluating performances of accumulation in order to replace, when it’s feasible, invading and expensive remediation techniques with phytoremediation that is, in its different applications, more respectful of landscape and more cost-effective. Trials to evaluate phytoextraction performances of four species of willow (Salix spp) , Phragmites australis and Brassica juncea grown on polluted sludge dredged from Venice Lagoon. In collaboration with Department I.M.A.G.E. of Faculty of Engenering of University of Padova, in the field of PRIN 2005 (National Significant Project) “Remediation of sea, lagoon and river sediments” experimental trials were planned in order to understand remediation and biomass production potential of Salix matsudana, S. babylonica, S. alba, S. fragilis, Brassica juncea and Phragmites australis growing on salty polluted sludge , dredged from Venice Lagoon (closest to Marghera harbor) and to think about a phytoextraction installation. Sludge samples were kept in four sites (North industrial channel, West industrial channel-two samples-, and Evolution Basin), then labeled and characterized. To dry, homogenize and ameliorate their drainage capability of sludge, samples were mixed with river sand in a 1:1 ratio. Trial on 50 L tanks : After preliminary trials that underlined impossibility to grow up in field conditions Salix babylonica, fragilis , alba, , Thypha latifolia and Zea mais, seven 50 L tanks were filled up with a bottom layer of expanded clay and sludge up to 35 L (2 with Phragmites, 2 with Salix Matsudana, 2 with Brassica juncea and one without plants ) each one with a tube coupled with a filter, linked in the bottom to collect water samples and favorite drainage. Willow cuttings and Indian mustard seedlings were planted with a universal soil bulk to attenuate transplanting shock and negative effect on young root system of direct contact with polluted substrate, Phragmites rhizomes instead, after an initial steady state, showed to be able to grow on direct contact with sludge. After two months from start of trial, EDTA was added to one of Phragmites tanks to increase phytoextraction performances. Hydroponic trials : hydroponic livestocks of S. Matsudana, S. Alba, S. Fragilis, s. Babylonica and Brassica juncea were prepared mixing Hoagland nutrient solution with same Ni, Cd, Zn, Cu and Pb concentrations detected in first water sample collected from tanks. For each willow species, were prepared one becker as control with Hoagland solution only, one with Hoagland plus metals and one with Hoagland, metals plus NaCl, supplied at increasing concentrations (from 0.05 M to 0.2 M). About brassica, five beckers with 40 seedlings for each one were prepared. One becker as control with Hoagland solution, two beckers with Hoagland plus metals in the same concentration as described above, and two beckers with metals plus raising up Na concentration as described above. At the end of experimental period, cuttings and seedlings were harvested, weighted and metals and Na contents were detected. Twenty L tanks trials : five 20 L tanks with a tap on the bottom were filled up with a layer of fine gravel, a layer of sludge and a top layer of universal soil. They were placed in growth chamber. Each tank hosted three cuttings (one species for each tank) and eight Paspalum vaginatum plants, the fifth one hosted a second series of S. Babylonica cuttings and eight Paspalum vaginatum plants. Trials were 50 days long. A week after trial start, in fifth tank EDDS chelant was added, in order to increase metals bioavailability. After plant harvesting, biometric and metal content measurement were done. Willow root deepening evaluation: to see possible differences in sediment exploration by root systems of willow species twenty four 2 L plastic bottles were prepared. Sixteen of that filled up with same 20 L tank content, and eight (two for species) with grave’, universal soil on the top and soil kept from Experimental farm “L.Toniolo”. Each bottle hosted a cutting (six bottles for species). At the end of trial, willow shoots were removed, then bottles were frozen and sawed in three sections corresponding to universal soil layer and two part of sludge layer corrispondenti alla sezione contenente il terriccio e a due parti conteneti il sedimento. All root systems were extracted, washed up, weighted, scanned and analyzed to determine total length and diameter. Results : Phragmites : we can see an EDTA effective action on Cd, Ni, Pb Zn and Na accumulation in shoots while in rhizomes, there are significative differences only about Pb and Na. No differences in term of biomass production. EDTA became more available Cu and Zn in soil solution; nevertheless metals concentrations remain pretty low. Willows in growth chamber : no differences in leaves accumulation between species while Babylonica EDDS treated became the best performer in Cu, Ni, Pb and Zn accumulation, while it diminuish Na concentration. In stems babylonica appears the best, with and without EDDS for Cu, Zn and Na. About roots Babylonica is again one of best performer in Na, Zn and Cd accumulation. No differences between species or treatment in Ni accumulation. Comparing growth chamber willows with S. matsudana grown in field conditions, this one shows best performaces in leaves accumulation for Na (followed by “growth chamber matsudana”, fact that confirms the ability of the species in Na translocation), and for Cd and Ni with babylonica EDDS treated. In stems, “field matsudana” is the best accumulator of Na, Pb and Ni and it remains the best performer for root system except for Na. hydroponic trials confirm in leaves the strong competition of Na versus accumulation of other anions, particularly versus Cd, Ni, Pb and Zn ; competition versus Cu appears lighter instead. In stems le migliori performances di S. babylonica is the best accumulator of Cd, Cu, Zn and Ni, nevertheless it appears at the same time the best Na accumulator. In root system too babylonica is the best accumulator of Cd, Ni Zn and Na. The roots deepening evaluation trial shows in comparison with control of each species that babylonica root system should be the less suffering (according to accumulation data), while between four species grown on sludge Salix alba shows the deepest root system coupled with smallest diameters. This two characteristics should be a signal of less stress as found by Vamerali et al. 2008. Brassica juncea hydroponic trial show clearly interference of Na in metals accumulation. Anyway accumulation performances in hydroponic are clearly better than that ones of brassica grown in field conditions, also in presence of Na. It should mean that : Ahydroponic trial in this case isn’t reliable in a comparison with field conditions B) probably other limiting factors, as nutrients could affect metal accumulation and biomass production that is pretty low also because the quick flowering, another symptom of stress. Interaction of selenate and molybdate with the transport and assimilation of sulfate in Brassica juncea Recently MOT1, a transporter with an high affinity for Mo was identified in Arabidopsis thaliana (Tomatsu et al., 2007), but it’s common opinion plants could uptake Mo also using S transporters because the two anions are analogs and could compete in binding of active site of the same transporters (Dudev e Lim 2004; Alhendawi et al 2005). Once inside the cell, Mo can interfere with S methabolism becoming substrate for reactions catalyzed by ATP sulfurilase (APS), as is selenate, but in contrast to selenate no Mo containing products are formed in the reaction (Reuveny 1977). Given the significant impact that S fertilizers application may have on Mo and Se distribution and accumulation in plants (Shinmachi et al., 2010), this work studied interactions of sulfur nutrition with molybdenum and selenium in Brassica juncea. To this aim Brassica juncea seedlings were grown with different combinations of S and Se or S and Mo. Experiments were carried out in a short time (24h) as selenate and molybdate were given to plants at a very high concentration (200 µM), which was the same of sulfate in the S-sufficient condition. Chemical and physiological analyses were performed, including quantification of elements and plant growth. Tolerance index (% of fresh weight relative to the control) and root/shoot ratio show a greater stress for plants grown in S deficiency and in presence of Se and Mo. Se and Mo were more absorbed in minus S condition. Both for total S and sulfate Se and Mo competition becomes clear only between 6 and 24 h of treatment, while within an hour there are not significant changes in S uptake. Study on potential effects of Se accumulation on drought stress in Brassica juncea e Stanleya pinnata . Some Se accumulator species (Brassica spp, Stanleya spp, Astragalus spp.) uses Se accumulated as a weapon against herbivors (Freeman et al., 2007; Galeas et al. 2007 ; ) Brassica juncea , differently from other toxic or potentially toxic trace elements, accumulates Se also in flowers and seeds giving value to the hypothesis of an happening evolutionary in the use of Se against parassites and herbivors adaptation (Quinn C. et al., 2008). This two species were grown up in gravel and in hydroponic in presence (20 µM) and in absence of Sodium selenate. The water deficit was experimented at first on gravel by irrigation stopping for 8 days, then in hydroponic by adding polietilenglicol (PEG 10000), to modify Ψw of nutrient solution. In two hydroponic trials’ Ψw was brought to -1.3 / -1.6 Mpa. Measures of photosintetic efficency, biomass and water and osmotic potential show that there are no evidences of a positve effect of Se on drought tolerance except for the non stress condition or light stress (-0.5/-0.6 Mpa). This action, actually not very strong, increase biomass production in plants treated with selenate according to trials on Triticum aestivum (Yao Xiaoqin et al. ; 2009). Study of potentials of Se-accumulator Astragalus racemosus and non accumulator Astragalus convallarius as Ni, Mo, Cr, V, W, Te, Se and As accumulators. The genus Astragalus is composed by many species distributed in northern america, some of that adapted to live in seleniferous soils, so able to develop an high tolerance versus Selenium (Cowgill, 1990). Concentration of Arsenic, Nichel Chromium, Vanadium, Tellurium, Tungsten and Molibdenum raised up in the environment because human activities ( Bamhart 1997 ; Zarchinas et al 2004 ; Zoller et al. 1973 ; Gott and McCarthy 1966 ; ) . the aim of this work is to understand if adaptation to Se accumulation is effective also versus other elements, some of them analogs of Se. These two species of Astragalus germinated in Petri dishes then grown up in growth chamber for 3 months in Murashige & Skooge addicted agargel plus salts of elements mentioned above , mixed in different concentrations based on their degree of toxicity, in order to avoid a too quick death. Results showed that Se hyperaccumulator is not always more tolerant than the non accumulator (A. Convallarius showed more tolerance in V accumulation). Effects of Cu deficiency on Cu-proteins activity and on expression of laccases in leaves and stem of Populus trichocarpa Cu has a role of cofactor in function of several dismutases (enzymes catalyzing formation of H2O2 from R.O.S.) and in laccases enzyme (Pilon et al. 2006). Many clones of P. Trichocarpa were multiplyed and grown up in MS addicted agargel with two different Cu concentrations (0.1 µM e 5 µM) to verify trough quantification of Cu-enzymes activity (western blot and native gel) if and how much these two Cu concentrations (the first closest to the limit of deficiency and the second pretty high but far from toxicity ), could affect Cu-enzymes activity. Once quantified activity of Cu-SODdepending on Cu concentration, a preliminary investigation on Laccases regulation was done. Because Laccases needs four Cu ions and because in a condition closest to Cu deficiency, cell inhibits all Cu requiring enzymes except that necessary to make photosintesis like plastocianin, we wanted verify if Laccases faith was exactly the same. Primers were drawn (RNA short sequences neded to make a Polimerase Chain Reaction in vitro ) to amplify the target sequence of a miRNA (RNA short sequences that, coupled with RISC complex, cut down mRNA inactivating it) (Yamasaky et al. 2007; Abdel Ghani et al 2008) and to obtain after an electrophoretic run, bands of different intensity, depending on the down regulation action made by cell. Preliminary results show different levels of expression for several kinds of laccase in stem and leaves.
Introduzione generale L’attività di ricerca svolta nel triennio 2007-2009 si colloca nell’ambito della fitodepurazione. Con questo termine si identifica una serie di tecniche (fitoestrazione, fitostabilizzazione, fitovolatilizzazione) basate sull’impiego di specie vegetali diverse (l’elenco delle quali è in continua evoluzione) ma accomunate dalla capacità di sopravvivere su substrati contaminati da diversi inquinanti prodotti dall’attività umana e di interagire con il substrato in modo tale, a seconda dei casi, da depauperarne il contenuto di inquinanti accumulandoli nei propri organi e/o liberandoli nell’atmosfera attraverso l’evapotraspirazione fogliare, oppure impedendone la lisciviazione. L’attitudine ad un’azione di fitorimedio varia fortemente da specie a specie e all’interno della stessa specie in base al corredo genetico . Relativamente alla capacità di rimozione di metalli pesanti, sono state individuate due classi di specie accumulatrici: Iperaccumulatrici : sono specie tipiche di suoli metalliferi, in grado di portare a termine il proprio ciclo vitale senza manifestare sintomi di deperimento dovuti all’alta concentrazione di metalli (Baker et al., 2000). Per essere definita iperaccumulatrice, una specie deve rientrare nei seguenti limiti inferiori di bioaccumulo: 100 mg/Kg s.s. di Cd, 1000 mg/Kg s.s di Pb, 10000 mg/Kg s.s. di Zn ; i ranges per quanto riguarda l’arsenico non sono ancora stati ben definiti (Baker and Brooks 1989, Baker et al., 2000). Tra le specie iperaccumulatrici le più rappresentative sono quelle appartenenti al genere Thlaspi, Arabidopsis halleri e Viola calamarina, Astragalus spp oltre a diverse specie appartenenti alle famiglie delle Cariophyllaceae, Fabaceae, Poaceae, Chenopodiaceae, Brassicaceae e Cyperaceae. Per quanto riguarda l’iperaccumulo di arsenico spicca Pteris vittata. Il limite delle specie iperaccumulatrici è la scarsa produzione di biomassa. Accumulatrici: a questo gruppo appartengono specie, ad habitus sia erbaceo che arboreo, in grado di ottenere buone concentrazioni di metalli e metalloidi nei propri organi, pur non arrivando a livello delle iperaccumulatrici, e una buona produzione di biomassa. Fra esse sono state individuate Brassica juncea, e il genere Salix. Le caratteristiche di queste accumulatrici le rendono idonee a programmi di disinquinamento a breve e medio termine, soprattutto per suoli poco o mediamente inquinati. La possibilità di avere una considerevole produzione di biomassa permette di pensare, come seconda fase, al reimpiego di questa per la produzione di energia ed il recupero dei metalli pesanti di interesse industriale ((Kabata-Pendias and Pendias, 1992 ; Pullford and Dickinson 2004 ; Paulson et al 2003; French et al 2005; Ebbs et al 1997 ; Ebbs et al 1998). Il lavoro triennale, suddiviso in diverse fasi, come descritto in seguito, si propone di approfondire la conoscenza su alcune delle specie suddette , sia dal punto di vista della risposta fisiologica sia , con un taglio maggiormente applicativo, dal punto di vista della valutazione di performaces di bioaccumulo al fine di sostituire, ove possibile, tecniche di bonifica invasive e costose con la fitodepurazione che, nelle sue diverse applicazioni, si dimostra piu’ rispettosa del territorio, anche dal punto di vista estetico e considerevolmente piu’ vantaggiosa economicamente. Prove di valutazione della capacita’ di accumulare metalli pesanti da parte di quattro specie di Salice (Salix spp) , di Phragmites australis e Brassica juncea. Date queste premesse, in collaborazione col dipartimento I.M.A.G.E. della Facoltà di ingegneria dell'Università di Padova, nell’ambito del PRIN 2005 ““Risanamento di sedimenti marini, lagunari e fluviali” sono state messe a punto delle prove sperimentali finalizzate a comprendere le potenzialità di Salix spp, di Brassica juncea e Phragmites australis nel mettere in atto un'azione di fitorimedio su sedimenti provenienti dal dragaggio della laguna di Venezia nella zona di porto Marghera. Il campionamento dei fanghi è stato effettuato in quattro siti (canale industriale nord, due punti del canale industriale ovest, bacino dell'Evoluzione), quindi i campioni sono stati catalogati e caratterizzati. In vista dell’allestimento di possibili impianti finalizzati al fitorisanamento dei fanghi dragati dall’area di Porto Marghera, sono state allestite delle prove sperimentali con l’intento di definire le potenzialità fitoestrattive di Salix matsudana, S. babylonica, S. alba e S. fragilis, Phragmites australis e Brassica juncea e valutarne quindi l’idoneità ad essere messe a dimora su questi sedimenti, anche in base alla capacità di sopravvivenza ed alla produzione di biomassa. I campioni provenienti dai 4 siti, sono stati miscelati tra loro ed in rapporto 1:1 con della sabbia di fiume, sia per ottenere un substrato con valori omogenei relativamente alle concentrazioni di inquinanti, sia per aumentare la capacità drenante di un substrato altrimenti totalmente asfittico. Prova su mesocosmo : Dopo test preliminari che hanno evidenziato l’impossibilita’ di condurre prove, ceteris paribus, anche su Salix Babylonica, Fragilis ed Alba, su Thypha latifolia e su Zea Mais, sono state allestite 7 vasche (2 dedicate a Phragmites australis, 2 a Salix Matsudana e 2 a Brassica juncea ed una priva di vegetazione, della capienza di 50 L (riempite fino a 35 L) ciascuna, munite di un tubo per il drenaggio del percolato alla base. Sul fondo delle vasche è stato depositato uno strato di argilla espansa e l’ingresso del tubo è stato protetto dall’intasamento con un filtro Le talee di salice ed i semenzali di brassica sono state messe a dimora con un piccolo pane di terriccio universale al fine di attutire gli effetti dello stress da trapianto e di permettere all’apparato radicale di accrescersi a sufficienza prima di esplorare il sedimento contaminato, mentre i rizomi di Phragmites hanno attecchito a diretto contatto col substrato oggetto di analisi. Dopo due mesi dall’inizio della prova, ad una delle vasche ospitanti la Phragmites e’ stato addizionato il chelante EDTA. Prove in idroponica : prendendo come riferimento le concentrazioni di 5 tra metalli pesanti riscontrati nell’analisi dei campioni di percolato (Ni,Cd, Zn, Cu, Pb,) e la concentrazione media del Na (intorno a 0,2 M) sono state allestite degli allevamenti in coltura idroponica con talee, oltre che di S. matsudana, anche di S. alba, S. babylonica , S. fragilis e di Brassica juncea. Per quanto riguarda i salici, la soluzione contenente inizialmente una soluzione nutritiva di tipo hoagland, è stata arricchita dai suddetti 5 metalli, in concentrazione pari a quella riscontrata nelle acque di percolazione. Per ogni specie di salice è stato allestito un beacker di controllo con sola soluzione nutritiva, un becker con soluzione nutritiva, più i metalli ed uno con le caratteristiche precedenti più NaCl a concentrazioni crescenti (da 0,05 M fino a 0,2 M). Per le brassiche sono stati allestiti 5 becker con 40 piantine ciascuno. Un beacker di controllo con soluzione nutritiva, due con concetrazione di metalli pari a quella del percolato, due con concentrazione di metalli pari al percolato più NaCl 0,05 M (aumentato fino a 0,2 M nel corso di 3 settimane). Alla fine del periodo di trattamento sono state separate e pesate parte aerea ed apparato radicale dei semenzali e ne sono stati analizzati i contenuti in metalli e Na. Prove su microcosmi : sono state allestite 5 taniche da 20 L ciascuna, dotate di rubinetto alla base e sistemate in cella climatica, tutte dotate di uno strato drenante di ghiaino alla base, un orizzonte costituito solo da sedimento ed uno, quello più superficiale, di terriccio universale. Quattro taniche hanno ospitato ciascuna tre talee (una specie per ogni tanica) e otto piantine di Paspalum vaginatum, la quinta tre talee di S. Babylonica e otto piantine di Paspalum. L’insediamento delle talee e della graminacea è durato 50 giorni. Nella settimana successiva alla messa a dimora dei S. Babylonica nella quinta tanica è stato aggiunto l’ EDDS, un chelante che ha lo scopo di rendere più biodisponibili i metalli pesanti presenti nel substrato. Studio dell’approfondimento radicale dei salici : per verificare la capacità delle quattro specie di salice, di esplorare il sedimento contaminato sono state allestite 24 bottiglie in pet da 2 L, di cui 16 riempite con lo stesso contenuto delle taniche e 8 (due per specie) riempite con del terreno proveniente dall’Azienda Sperimentale “L. Toniolo”. In ogni bottiglia è stata messa a dimora una talea di salice (6 bottiglie per specie). Al termine dei 50 giorni di prova condotti in camera di crescita, è stata asportata la parte aerea della talea, le bottiglie sono state congelate e poi segate in tre parti corrispondenti alla sezione contenente il terriccio e a due parti conteneti il sedimento. Tutte le radici presenti sono state estratte , lavate e pesate e sottoposte a scansione digitale. Risultati : Phragmites : azione efficace del chelante EDTA per quanto riguarda l’accumulo di Cd, Ni, Pb Zn e Na nei fusti di Phragmites, mentre differenze significative nei rizomi sono state riscontrate solo per Pb e Na. Nessuna differenza in termini di biomassa prodotta. Il chelante ha reso maggiormente disponibile nella soluzione circolante Cu e Zn anche se le concentrazioni restano molto basse. Salici nelle taniche in cella climatica : gli accumuli a livello fogliare non mostrano differenze significative tra le quattro specie non trattate. Mentre l’EDDS incrementa le prestazioni del babylonica rispetto a se stesso ed alle altre specie per quanto riguarda Cu, Ni, Pb e Zn mentre riduce la concentrazione di Na. A livello di fusti la suddetta differenza si esplica nelle concentrazioni di Cu, Zn e Na; questi ultimi risultano maggiormente accumulati dal babylonica sia con che senza chelante. A livello radicale il babylonica risulta ancora uno dei migliori accumulatori del lotto per quanto riguarda Na, Zn e Cd. Nessuna differenza ne tra specie ne tra presenza/ assenza di chelante per quanto riguarda il Ni. In un confronto tra questi salici ed il matsudana cresciuto piu’ a lungo e in condizioni di T , umidita’ e fotoperiodo variabili, a livello fogliare il salice cresciuto in mesocosmo presenta valori di Na significativamente piu’ alti degli altri salici, seguito dal matsudana cresciuto in cella, a conferma dell’atitudine di questa specie a traslocare piu’ facilmente il Na nella chioma. Anche riguardo Cd e Ni il matsudana di campo denuncia tra i valori piu’ elevati assieme al babylonica trattato con EDDS. Nei fusti, per Na ,Pb e Ni ci sono differenze significative in favore del S. matsudana di campo. A livello radicale il S. matsudana cresciuto in mesocosmo presenta valori significativamente piu’ alti rispetto alle prove in cella climatica ad eccezione del Na. Le prove in idroponica confermano, a livello fogliare che l’accumulo di Na compete fortemente con l’uptake dei metalli, in particolare nei confronti di Cd, Ni, Pb e Zn ; piu’ sfumata invece l’azione competitiva verso il Cu. Da rilevare, a livello di fusti le migliori performances di S. babylonica per Cd, Cu, Zn e Ni, nonostante si rilevi nel contenpo il primato nell’accumulo di Na. Anche a livello radicale il babylonica accumula piu’ delle altre specie per quanto riguarda Cd, Ni Zn e Na. La prova sullo sviluppo radicale mostra, nella comparazione col rispettivo controllo, che l’apparato radicale di S. babylonica risulta il meno sofferente tra le quattro specie (in accordo quindi con i dati di accumulo), mentre comparando le quattro specie cresciute sul sedimento, il Salix alba risulta avere l’apparato piu’ sviluppato in profondita’ e con i diametri minori , segno questo che potrebbe essere considerato di minor sofferenza, come riscontrato anche da Vamerali et al. 2008. L’allevamento in idroponica di Brassica juncea evidenzia differenze significative tra l’accumulo di metalli in presenza di Na rispetto all’accumulo in assenza di quest’ultimo. Le performances di accumulo rispetto alle brassiche cresciute su fango sono nettamente migliori, anche in presenza di Na , a testimonianza del fatto che A) l’idroponica non offre termini di paragone affidabili rispetto alle condizioni di campo B) probabilmente intervengono altri fattori, come la carenza di nutrienti, a condizionare l’accumulo di metalli e la produzione di biomassa che risulta bassa anche per la precocita’ della fioritura, sintomo della condizione di stress. Interazione del selenato e del molibdato con il trasporto e l’assimilazione di solfato in Brassica juncea Recentemente e’ stato individuato in Arabidopsis un trasportare (MOT1) con elevata affinita’ per il Mo (Tomatsu et al., 2007), ma si ritiene che le piante assorbano Mo anche attraverso i trasportatori del S poiche’ i due anioni hanno caratteristiche simili e potrebbero competere nell’occupare il sito attivo degli stessi trasportatori (Dudev e Lim 2004; Alhendawi et al 2005). Una volta all’interno della cellula, Mo puo’ interferire col metabolismo dello S divenendo substrato per le reazioni catalizzate dall’ ATP sulfurilasi (APS), come accade per il Se ma, a differenza di quest’ultimo non si riscontra la formazione di prodotti contenenti Mo derivanti da questa reazione (Reuveny, 1977). A causa del significativo impatto che l’applicazione di fertilizzanti a base di S puo’ avere sulla distribuzione di Se e Mo e sul loro accumulo nelle piante (Shinmachi et al., 2010), si e’ cercato di capire, in questo lavoro,quale sia l’interazione tra l’uptake di S e il Se e Mo in Brassica juncea. A questo scopo semenzali di Brassica juncea sono stati allevati con differenti combinazioni di S e Se o S e Mo. Gli esperimenti sono stati condotti nel breve periodo (24h) poiche’ selenato e molibdato sono stati somministrati ad alte concentrazioni (200 µM), le stesse del solfato in condizioni di normale apporto di S. Sono state condotte analisi relative all’accumulo di S, Se e Mo e ai paramentri di crescita. L’indice di tolleranza (% del peso fresco sul controllo) e il rapporto radici/fusti denuncia un maggiore stress per le piante cresciute in carenza di zolfo e in presenza di Se e Mo. Se e Mo sono risultati maggiormente assorbiti in carenza di S. Sia per quanto riguarda lo S totale , sia riguardo al ssolfato, la competizione con Se e Mo si nota solo tra le 6 e le 24 h di trattamento, mentre nel brevissimo periodo (entro 1 h) non si apprezzano cambiamenti significativi nell’assorbimento di S. Studio dei potenziali effetti del Selenio sulla tolleranza allo stress idrico da parte di Brassica juncea e Stanleya pinnata . Talune specie accumulatrici di Se (Brassica spp, Stanleya spp, Astragalus spp.) usano questa peculiarita’ per difendersi dai predatori (Freeman et al., 2007; Galeas et al. 2007 ; ) e recentemente si e’ visto che Brassica juncea , a differenza di quello che succede per altri elementi tossici o potenzialmente tossici, accumula selenio in grandi quantita’ anche nei fiori e nei semi, avvalorando l’ipotesi, per certe specie o sottospecie o addirittura per alcune popolazioni in via di speciazione, di un adattamento evoluzionistico nell’uso del selenio contro parassiti e predatori erbivori (Quinn C. et al., dati non ancora pubblicati).Su queste due specie sono state condotte prove su substrato ghiaioso e in idroponica in presenza (20 µM) e in assenza di Selenato di sodio. Il deficit idrico e’ stato applicato prima su substrato ghiaioso attraverso la mancata irrigazione, poi in idroponica attraverso l’aggiunta di polietilenglicol (PEG 10000), un composto organico che altera il Ψ idrico della soluzione nutritiva. Nei due esperimenti condotti in idroponica il potenziale e’ stato portato fino a -1.3 / 1.6 Mpa. Dalla misurazione della capacita’ fotosintetica sotto stress idrico, della biomassa e del Ψ idrico fogliare nei diversi esperimenti, si e’ arrivati alla conclusione che non vi e’ una evidente azione positiva del Se nei riguardi della tolleranza allo stress idrico se non per quanto riguarda la condizione di controllo (non stress) e di stress lieve (-0.5/0.6 Mpa). Questa azione, pur non marcata, si esplica nell’aumento di biomassa superiore in piante trattate con selenato rispetto a quelle non trattate in accordo con quanto riscontrato per semenzali di Triticum aestivum (Yao Xiaoqin et al. ; 2009). Studio delle potenzialita’ di Astragalus racemosus (iperaccumulatore di Selenio) e Astragalus convallarius (non accumulatore) nel ruolo di accumulatori di Nichel, Molibdeno, Cromo, Vanadio, Tungsteno, Arsenico, Tellurio e Selenio. Il genere Astragalus comprende diverse specie distribuite in america centro settentrionale, alcune delle quali adattate a vivere su suoli seleniferi e quindi in grado di sviluppare un’elevata tolleranza al Se, divenendo cosi’ iperaccumulatrici (Cowgill, 1990). Arsenico, Nichel Cromo, Vanadio, Tellurio, Tungsteno e Molibdeno sono tutti elementi la cui concentrazione nell’ambiente e’ aumentata a causa dell’attivita’ umana ( Bamhart 1997 ; Zarchinas et al 2004 ; Zoller et al. 1973 ; Gott and McCarthy 1966 ; ) . e l’obiettivo di questo studio e’ capire se gli adattamenti fisiologici nei confronti dell’accumulo di Se siano efficaci anche nei confronti di altri metalli pesanti e metalloidi, in particolare Cromo e Tellurio che presentano caratteristiche steriche simili al Selenio. Queste due specie di Astragalus sono state fatte germinare in piastra petri e quindi fatte crescere in camera di crescita per 3 mesi su substrato di agargel piu’ Murashige & Skooge e addizionato con sali degli elementi sopra indicati dosati in concetrazioni differenti in base al loro grado di tossicita’ (affinche’ le piante potessero crescere ed accumulare metalli senza patirne la tossicita’ fino a morirne troppo precocemente). I risultati hanno dimostrato che non sempre la specie iperaccumulatrice di Selenio ha le caratteristiche per tollerare l’accumulo anche di altri elementi (A. Convallarius si e’ dimostrato piu’ tollerante nell’accumulo di Vanadio). Studio degli effetti del Rame nella fisiologia di Populus trichocarpa ed in particolare sull’attivita’ della proteina Laccasi, coinvolta nella formazione della parete cellulare. Il Rame e’ coinvolto come cofattore, nel funzionamento di diverse dismutasi (proteine che trasformano i ROS in H2O2) oltre che essere cofattore per il funzionamento delle laccasi (Pilon et al. 2006). Allevando in agargel e moltiplicando diversi cloni di Populus trichocarpa generati a partire dallo stesso callo, con due diverse concentrazioni di Cu (0.1 µM e 5 µM) si e’ voluto verificare attraverso analisi dell’attivita’ proteica (western blot e native gel) se e quanto due concentrazioni di rame (una prossima al limite inferiore che ne decreta la carenza ed una piuttosto elevata ma ben entro i limiti di tossicita’), influenzino l’omeostasi cellulare. Una volta verificato la diversa intensita’ dell’attivita’ delle proteine in questione (MnSOD, Cu/ZnSOD, FeSOD, CCS ecc. In base alla concentrazione di rame fornita, si e’ passato ad uno studio preliminare dell’espressione della Laccasi in base alle due diverse concentrazioni di rame. Poiche’ la laccasi necessita anch’essa del Cu come cofattore (4 ioni Cu ) e poiche’ in uno stato di carenza o prossimo alla carenza di rame, la cellula inibisce l’espressione di tutte le proteine che richiedono rame e che non sono strettamente necessarie alla fotosintesi come invece lo e’ la plastocianina, si e’ voluto verificare se la laccasi subisse esattamente questo destino. Sono stati quindi disegnati dei primers (sequenze innesco per amplificare col metodo PCR il DNA retrotrascritto da RNA) che contemplassero l’amplificazione della sequenza bersaglio dei microRNA (sequenze brevi di RNA che in coppia col complesso proteico RISC tagliano e quindi inattivano l’RNA messaggero) (Yamasaky et al. 2007; Abdel Ghani et al 2008) in modo da avere, dopo la corsa elettroforetica su gel di agarosio, bande di intensita’ diversa in base all’azione di downregulation operata o meno dalla pianta. I risultati preliminari mostrano diversi livelli di espressione per diversi tipi di laccasi analizzati presenti nel fusto e nelle foglie,

Les conséquences des flux de gènes et de l'introgression entre les cultures transgéniques et leurs apparentés sauvages sont encore au cœur des débats associés à la commercialisation des plantes génétiquement modifiées. J'ai développé mon étude sur les conséquences écologiques et évolutives du flux de gènes entre le colza (Brassica napus) et ses apparentés, la moutarde brune sauvage (B. juncea) et la ravenelle (Raphanus raphanistrum), en réalisant une série d'expériences en serre, au jardin et au champ à Beijing et à Dijon. En premier, j'ai présenté une revue synthétique de la littérature publiée sur les flux de gènes et ses effets sur la fitness chez les Brassicées. En second, j'ai cherché à mettre en évidence le rôle de la taille des semences hybrides entre du colza transgénique Bt et la moutarde. La petite taille des semences a réduit les capacités de croissance et de reproduction, mais l'effet sur la fitness était variable en fonction des fonds génétiques ou spécifiques. Les rétrocroisements sur le colza étaient plus faciles et productifs que pour les autres types de descendants. La plupart de ces plantes avait une morphologie de colza. Liée à la résistance à l'herbicide, cette caractéristique pourrait permettre aux descendants de survivre dans les champs et de disséminer les transgènes aux repousses et aux autres colzas, ce qui serait peut être plus gênant que de voir l'introgression réelle dans le génome du parent sauvage. Troisièmement, j'ai simulé le phénomène d'herbivorie chez la moutarde pour étudier la compétition entre des plantes résistantes et des plantes sensibles indépendamment des problèmes de fitness des hybrides interspécifiques. Les plantes résistantes ont un avantage compétitif évident sous la pression d'herbivorie, et cet avantage est exacerbé sous des conditions difficiles telles que de faibles ressources du milieu et l'intensité de l'herbivorie. L'utilisation d'insectes pour attaquer des populations mixtes composées de rétrocroisements sensibles et Bt-résistants aux insectes a confirmé ce résultat et a montré que le transgène n'avait pas de coût en l'absence d'insectes. La productivité totale des populations a augmenté avec la proportion de plantes résistantes. Quatrièmement, des populations de ravenelles ont été échantillonnées dans quatre régions éloignées entre elles, dont une ayant une longue histoire de coexistence avec le colza et donc ayant plus de chance d'avoir été soumise à l'hybridation interspécifique avec le colza. J'ai interprété la divergence des traits et leur polymorphisme dans le cadre d'une hypothèse d'introgression stabilisée en opposition au simple hasard, bien que les différences avec les autres populations n'étaient pas assez marquées pour faire sortir ces populations du domaine de variation décrit pour les ravenelles. Ces études soulignent plusieurs facteurs qui peuvent accroître le risque des flux de transgènes et l'introgression entre les cultures génétiquement modifiées et leurs apparentés sauvages, et cela doit être pris en compte dans les procédures d'évaluation des risques de l'usage de ces plantes. A savoir : la morphologie cultivée qui rend confuse l'identification des introgressants dans le cadre de la bio-surveillance, les petites semences hybrides avec une dormance et une dispersion supérieures, et l'intensité de l'herbivorie et de la compétition qui exacerbe l'avantage adaptatif des plantes transgéniques résistantes aux insectes. Cependant, l'hypothèse de la formation de " super mauvaises herbes " ne semble pas justifiée.

La phytoremédiation constitue une nouvelle technologie permettant de dépolluer les sols contaminés par l'utilisation de plantes. Parmi les différents aspects possibles de cette méthode, figure la phytoextraction basée sur l'absorption et l'accumulation du polluant dans les parties aériennes. Pour être efficace, il est nécessaire de disposer de plantes présentant une biomasse élevée. L'objectif de ce travail visait à sélectionner des variétés de moutarde indienne (Brassica juncea L.) tolérantes et accumulatrices de cadmium.
La première partie du travail a consisté à mettre au point et à optimiser une méthode de régénération in vitro de plantes de moutarde indienne, à partir de couches cellulaires minces transversales (CCMTs) (influence de l'organe utilisé, de AgNO3 et de la benzylaminopurine). La régénération a été réalisée en appliquant une pression de sélection par le cadmium pour modifier la tolérance au métal.
La deuxième partie aborde l'effet des traitements par le cadmium : in vitro et en serre, sur le développement des plants. Une analyse des perturbations physiologiques et biochimiques observées a permis d'évaluer la tolérance des plants vis à vis du cadmium et indiquent que les plantes néoformées en présence de cadmium mettent en place un système d'exclusion du métal.
Dans la troisième partie, pour compléter l'étude précédente sur des plantes néoformées, l'effet du cadmium a été testé sur des plantes de B. juncea directement issues de semis. Plusieurs paramètres physiologiques et biochimiques, caractéristiques des stress, ont été étudiés (activité gaïacol peroxydase, peroxydation des lipides, pigments, acides aminés libres, proline glucides, polyamines libres et conjuguées). En raison de leurs propriétés anti-oxydantes, une attention particulière a été portée aux polyamines dont l'application exogène permet d'envisager son utilisation pour améliorer la capacité d'accumulation du cadmium par les plantes.

Мерешко К. М. Вплив oбрoбки кoлхіцинoм на мoрфoмeтричні пoказники рocлин роду гірчиця : кваліфікаційна робота магістра спеціальності 091 "Біологія" освітньої програми "Генетика" / наук. керівник О. А. Бойка. Запоріжжя : ЗНУ, 2020. 77 с.
UA : Дана рoбoта викладeна на 77 cтoрінках друкoванoгo тeкcту, міcтить 18 таблиць та 3 риcунки. Пeрeлік пocилань міcтить 63 джeрeл. Мeтoю данoї рoбoти булo дocлідити вплив рoзчину кoлхіцину в різних кoнцeнтраціях на рoзвитoк рocлин рoду Гірчиця. Oб’єктoм дocліджeння були рocлини гірчиці. Мeтoди дocліджeння – хімічний мутагeнeз, лабoратoрні мeтoди вирoщування рocлин гірчиці, вимірювання мoрфoлoгічних пoказників, cтатиcтичні мeтoди oбрoбки даних. В рeзультаті прoвeдeння дocліджeння булo вcтанoвлeнo, щo oбрoбка кoлхіцинoм в цілoму виявила cтимулюючий вплив, який призвів дo збільшeння уcіх пoказників (виcoти рocлин, кількocті бутoнів, квітoк та cфoрмoваних cтручків) у пoрівнянні з кoнтрoльними рocлинами. Значущіcть цієї рoбoти пoлягає в тoму, щo на cьoгoдні в Україні за відoмими нам літeратурними джeрeлами нe булo прoвeдeнo дoкладних рoбіт щoдo впливу рoзчину кoлхіцину в різних кoнцeнтраціях на рoзвитoк рocлин рoду Гірчиця. Гірчиця є актуальнoю cільcькoгocпoдарcькoю рocлинoю, яка в наш чаc привeртає дo ceбe багатo уваги. Рocлини цьoгo рoду ширoкo викoриcтoвують в харчoвій, фармацeвтичній прoмиcлoвocті та в якocті тeхнічних культур. Cамe тoму гірчиця є культурoю рoбoта з якoю нікoли нe втратить cвoєї актуальнocті. Тeoрeтичнe значeння рoбoти пoлягає у рoзширeнні знань щoдo впливу кoлхіцину на рocлини, а практичнe у тoму щo булo виявлeнo кращі кoнцeнтрації рoзчину кoлхіцину для пoдальшoї рoбoти з культурoю.
EN : Thіs paper іs presented at 77 pages of prіnted text, contaіnіng 18 tables and 3 fіgures. The lіst of references іncludes 63 sources. The purpose of thіs work was to іnvestіgate the іnfluence of the solutіon of colchіcіne іn varіous concentratіons on the development of plants of the mustard specіes. The object of the study were plants of mustard. Methods of research were chemіcal mutagenesіs, laboratory methods of growіng plants, measurement of morphologіcal traіts, statіstіcal methods of data processіng. As a result of the study, іt was found that treatment wіth colchіcіne as a whole showed stіmulatіng effects, whіch led to an іncrease іn all traіts (plant heіght, number of buds, flowers and formed pods) compared wіth control plants. The sіgnіfіcance of thіs work іs that today іn Ukraіne, accordіng to known lіterary sources, no detaіled work has been done on the іnfluence of the solutіon of colchіcіne іn varіous concentratіons on the development of plants of the Mustard's genus. Mustard іs an actual agrіcultural plant, whіch іn our tіme attracts a lot of attentіon. Plants of thіs specіes are wіdely used as food, pharmaceutіcal and іndustrіal crops. That іs why the mustard іs a culture whose іnvestіgatіon wіll never lose іts relevance. The theoretіcal value of the work іs to іncrease knowledge about the іnfluence of colchіcіne on plants, and practіcal іn that the best concentratіons of a solutіon of colchіcіne were found for further work wіth culture.

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.

Two trials compared the effects of canola meal (CM) and juncea meal (JM) with and without dietary enzyme supplementation, on production performance, egg quality, bone quality and liver health characteristics of white- (WSLH) and brown-shell egg laying hens (BSLH). A total of 360 Lohmann LSL-Lite White (Trial 1, WSLH) and 300 Lohmann Brown-Lite (Trial 2, BSLH) laying hens were fed one of 10 isoenergetic and isonitrogenous diets (Soybean meal, 10 % CM, 20 % CM, 10 % JM or 20 % JM with or without a dietary enzyme cocktail of Superzyme OM and Bio-PhytaseTM) for 48 weeks. Based on the results of production performance, incidence of mortality, egg quality, bone quality, and liver health data, up to 20 % CM or JM can be included in diets of WSLH and BSLH without detrimental effects. Enzyme should be included in diets for both WSLH and BSLH.

Two trials were carried out to evaluate the effect of inclusion level of canola meal derived from Brassica (B.) napus and B. juncea on cattle performance and nutrient utilization. Trial 1 consisted of backgrounding (54 d) and finishing (153 d) phases. The control diet for the backgounding (BK) phase consisted of 39% barley silage, 30.4% barley grain, 22.8% brome grass hay and 7.8% supplement (DM). Treatments consisted of B. napus or B. juncea at 15 or 30% (DM) inclusion, replacing barley grain. The finishing control diet consisted of 88.3% barley grain, 4.4% barley silage and 7.3% supplement (DM). Treatments consisted of B. napus or B. juncea at 10 or 20% (DM) inclusion, replacing barley grain. During BK, dry matter intake (DMI), average daily gain (ADG), gain: feed (G:F) increased linearly (P < 0.01) as the level of inclusion of B. juncea meal increased. Cattle fed B. napus meal showed a quadratic response (P = 0.05) in DMI and linear increase (P = 0.02) in ADG with increasing inclusion. During finishing, DMI increased linearly (P = 0.05) for cattle fed B. juncea meal while a quadratic response (P = 0.02) was seen with B. napus meal. Feed efficiency and NEg content of the diet (P ≤ 0.02) decreased linearly with increasing inclusion of both meals. Trial 2 evaluated dietary rumen fermentation and total tract digestibility characteristics in a 5 x 5 Latin Square Design. Diets were similar to finishing phase of Trial 1. There was no effect of treatment on rumen pH, however a linear increase in acetate (P ≤ 0.01), ammonia (P < 0.01) and decrease (P < 0.01) in propionate was seen with both meal types. Crude protein and acid detergent fiber digestibility increased (P = 0.03) linearly with increasing inclusion of B. juncea meal. The results indicate that canola meal derived from B. napus and B. juncea is not suitable as a supplemental energy source replacing for barley grain in finishing diets but canola meal from B. juncea can be fed at levels up to 30% of the DM in backgrounding diets if priced appropriately.

A genetic linkage map of mustard (Brassica juncea) was developed using two double haploid populations produced from crosses between a low erucic cultivar “ZEM1” and two moderate erucic acid lines “Vniimk351” and “Vniimk405” with the use of SSR and SRAP markers. The linkage map of the ZEM1xVniimk351 population included 13 linkage groups with an overall length of 791 cM with an average marker interval of 5.7 cM. The linkage map of the ZEM1xVniimk405 population also contained 13 linkage groups with a distance of 623 cM and an average marker interval of 4.6 cM. Using the linkage maps for the two populations, QTLs were detected for seed oil, protein and fatty acids. QTL analysis for fatty acids indentified QTLs on LG1, 7 and 12 for the ZEM1xVniimk351 population and LG1, 3 and 4 for the ZEM1xVniimk405 population. Analysis for the seed oil and protein content in the ZEM1xVniimk351 population identified 2 QTLs on LG1 and LG4 and 1 QTL on LG1 respectively. The QTL analysis ZEM1xVniimk405 of oil and protein content identified 1 QTL for oil and protein on LG1. The variation of fatty acids was shown to be the result of monogenic inheritance of the FAE1 gene in both populations.

Two experiments were conducted to determine the apparent (ATTD), standardized (STTD) and true total tract digestibility (TTTD) of phosphorus (P) and ATTD of calcium (Ca) in Brassica napus black (BNB) and Brassica juncea yellow (BJY) canola meal (CM) fed to growing pigs. In Experiment 1, eight semi-purified diets containing graded levels of P i.e., 0.8, 1.6, 2.4 and 3.3 g/kg of DM, from either BNB or BJY, were fed to growing pigs with an initial BW of 19.9 ± 0.22 kg (mean ± SEM) in a randomized complete block design. The total and basal EPL estimated with the regression analysis and P-free diet methods were 665 ± 0.03 and 209 ± 96 mg/kg of DMI, respectively. The TTTD and STTD of P were determined to be 33.3 and 31.0% for BNB and 32.0 and 28.3% for BJY, respectively. In Experiment 2, the effect of high level of phytase supplementation on the ATTD of P and Ca and STTD of P in growing pigs was studied. Forty-two growing pigs with an initial BW of 19.8 ± 1.22 kg (mean ± SEM) were randomly allocated to 7 dietary treatments with 6 pigs per treatment according to a completely randomised design in a factorial arrangement with the factors being: 1) 2 types of CM (BNB and BJY) and 2) 3 levels of phytase (i. e., 0, 500 and 2,500 U/kg). The ATTD of P increased from 39.1 to 69.3, and 78.0% in BNB and from 46.0 to 71.4, and 78.0% in BJY as phytase levels were added at 0, 500 and 2,500 U/kg, respectively. The STTD of P increased from 40.0 to 70.0, and 78.3% in BNB, and from 46.3 to 72.1, and 78.5% in BJY as phytase levels were added at 0, 500 and 2,500 U/kg. The basal EPL estimate was 117 ± 23.4 mg/kg DMI. Fecal P excretion in BNB and BJY were reduced by average value of 50.3 and 61.0% with the addition of both 500 and 2,500 FTU phytase II respectively. Results from these two experiments show that the values obtained for STTD and TTTD of P in BNB and BJY were similar.

Canola (B. napus L.), one of the most valuable oilseed crops in the world, has been reported with significant crop losses up to 100% due to blackleg disease caused by Leptosphaeria maculans. Genetic resistance is a primary method to control blackleg, and the highest levels of resistance can be introduced from Brassica species containing the B-genome through interspecific hybridization. With successful introgression of resistance, the BC3 recombinant lines, derived between resistant B. juncea UM lines and susceptible B. napus L. cv. Westar, showed high levels of resistance to two L. maculans isolates 03-15-03 and PG4-1M at the seedling stage. In the analysis of blackleg resistance in synthetic hexaploid Brassica lines using susceptible B. juncea UM3086, the resistance against L. maculans isolate 03-15-03 in F2, BC1, BC2 and BC2F2 populations indicated that two genes are inherited in backcross populations and each gene can confer the same level of blackleg resistance. The high number of resistant phenotypes and compatibility of interspecific crosses in hexaploids crossed with tetraploid Brassica imply that synthetic lines are a feasible tool for developing blackleg resistance.
October 2016

Canola meal (Brassica napus L.) and oriental mustard bran (Brassica juncea) were subjected to alkali and acid pretreatment to expose pentosan, for enhancing further enzymatic hydrolysis by endo-1,4-β-xylanase from Trichoderma longibrachiatum for the production of oligosaccharides. Pretreatment especially with alkali, effectively increased the relative content of pentosan to about ~ 41% and ~ 72%. Alkali pretreated canola meal and mustard bran resulted in a pentose content of 2.28 ± 0.15 g and 3.20 ± 0.11 g per 100 g substrates at 18 h and 24 h of reaction respectively, which corresponded to ~ 26% and ~ 28% conversion of original pentosan in substrates. UPLC-MS data showed xyloglucuronic acid (XGlcA) as the major oligosaccharide in the hydrolyzates. Reversed-phase HPLC-DAD indicated the principal phenolic compound in the hydrolyzates was sinapine. DPPH radical scavenging assay showed that endoxylananse hydrolyzates of acid pretreated substrates had strong antioxidant activities in comparison to alkali pretreated samples.

碩士
中山醫學大學
生化微生物免疫研究所
106
Nowadays, due to changes in people’s lifestyle and dietary habits, the proportion of metabolic diseases such as obesity, diabetes and fatty liver have increased with each passing day. Non-alcoholic fatty liver disease (NAFLD) is mainly characterized by excessive fat accumulation in the liver. It has been suggested that NAFLD spans a spectrum of disease from hepatic steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Therefore, how to effectively ameliorates NAFLD and reduces the body fat formation becoming a global issue that we should not be underestimated. According to previous studies, Brassica juncea is rich in glucosinolates, which have been proven to possess many potential pharmacological properties, including hypoglycemic, anti-oxidation, anti-inflammatory and anti-carcinogenic activities. However, the potential effects of glucosinolate and the mechanism of its action in against HFD-induced NAFLD and obesity remain unclear. Therefore, this study aim to investigate whether Brassica juncea and its glucosinolate extracts (BGE) have anti-obesity and hepatoprotective effects against HFD-induced NAFLD and further explore the mechanism underlying this process in vivo and in vitro. Brassica juncea treatment significantly reduced body fat, dyslipidemia, hepatic steatosis, liver injury, inflammation and by reversing the decreased expression of antioxidant enzyme activity to attenuate oxidative stress in HFD-fed rat liver. Moreover, Brassica juncea and BGE enhanced the activation of AMPK to reduce numerous lipogenic-related proteins, such as SREBPs, FAS and HMGCoR, but increase the expression of lipolysis-related proteins including CPT-1 and PPARα to attenuate hepatic steatosis. Additionally, Brassica juncea and BGE also could ameliorate NAFLD by inhibiting TNF-α and NF-κB to anti-inflammatory. Furthermore, BGE also efficiently suppressed the expression of SREBP-1c and PPAR-γ proteins to reduce the differentiation of 3T3-L1 preadipocytes. Based on the above results, this study demonstrates Brassica juncea and BGE reverse HFD-induced obesity, hepatic steatosis and liver injury could therefore represent an unprecedented hope toward improved strategies for NAFLD and obesity. We expected BGE can be applied to the development of hepatoprotective or anti-obesity nutrient food and keep close tabs on people''s health in the future.

Zinc is the heavy metal occurring in the greatest concentrations in the majority of wastes in modern, industrialized communities and is a common metal found at metal-polluted sites. Phytoextraction is a plant-based remediation technology aimed at the removal of metals from contaminated sites through the use of metal-accumulating plants. Although Indian mustard (Brassica juncea Czern.) has been identified as a moderate accumulator of Zn, little is known about the Zn-phytoextraction potential of this plant and whether or not mineral nutrition could be used to improve plant growth and Zn accumulation under Zn-contaminated conditions. The objectives of this research were to determine how the Zn phytoextraction potential of B. juncea is affected by: (a) the concentration of Zn in the nutrient medium; (b) the alleviation of Zn-induced Fe deficiency; (c) the molar % ratio of NH 4+ to NO3− and (d) the concentration of N and P in the nutrient medium. The pH of nutrient solutions generally decreased with: increasing Zn supply; decreasing Fe supply; and increasing proportion of NH4+-N in nutrient solutions. The ability of B. juncea to phytoextract Zn increased with increasing Zn supply initially, but then leveled off because the increase in shoot Zn concentration was accompanied by a suppression of shoot growth. Zinc-induced Fe deficiency was found to limit the effectiveness of B. juncea to phytoextract Zn, and increased P fertility appeared to exacerbate this problem. Alleviating Fe deficiency with root applied Fe-EDDHA had a limited effect on increasing Zn phytoextraction because Zn accumulation was suppressed with increasing Fe supply. Plants concentrated more Zn in shoots and roots if plants were supplied a high proportion of NO3 −, and increasing NH4+ nutrition enhanced growth. Zinc phytoextraction was maximized if plants were supplied 90% NO3−-N and 10% NH4 +-N. Zinc accumulation in shoots increased with increasing N and P supply, but the effect of N was greater. To the extent that N was a limiting factor for growth, N additions increased plant growth, but growth was not effected by P nutrition. Increased N fertility can increase Zn phytoextraction, but the effect of P nutrition is limited.

碩士
國立臺灣大學
農業化學研究所
93
Conventional cleanup technology is generally too costly, and often harmful to desirable soil properties (i.e., texture, organic matter) for the restoration of contaminated sites. More recently, increasing attention has been given to the development of a plant-based technology (phytoremediation) to remediate heavy metal contaminated soils without extensive excavation, disposal costs, and loss of topsoil associated with traditional remediation practices.The success of a phytoremediation process is dependent on adequate plant yield and high metal concentrations in plant shoots. The largest numbers of hyperaccumulating species in the world belong to Brassicaceae. The optimum plant for phytoextraction would be able to both tolerate and accumulate high levels of heavy metals and also grow with a high biomass yield. But there are many different species of Indian mustard in the world, and the growth and uptake of those Indian mustard are not clear. Therefore, the object of this study is to compare the growth and heavy metal accumulation of three Indian mustard (Brassica juncea) grown in soils contaminated by Copper, Zinc, Cadmium and Lead. The study site was located at the green house of National Taiwan University. The investigation was conducted from July 2004 to April 2005. Four salt solution were added to the air-dried soil to control the total concentration of four metals at 200, 400 mg Cu kg-1, 100, 200 mg Zn kg-1, 25, 50 mg Cd -1kg, 500, 1000 mg Pb kg-1. Three kinds of Indian mustard (Brassica juncea) grown in soils contaminated by zinc, cadmium, lead or coppe. The soil moisture content was maintained at 60% of the water holding capacity, by weighing and adding deionized water. Soil solution were collected directly by RSMS after seeding 0 , 7, 14, 21, 28, 35 day. Test plants were harvested after seeding 35 days, then harvested plant were digested by the H2SO4/H2O2 digestion method. The concentration of Cu, Zn , Cd, and Pb in soil solution and plant digested solution were determinated by inductively coupled plasma optical emission spectrometry (ICP-OES)(Perkin-Elmer 2000 DV). The total and available concentration of Cu, Zn , Cd, and Pb in soil were digested by aqua regia and EDTA, DTPA extractable methods , then determinated by atomic absorption spectrometry (Hitachi 180-30 type). The results indicated that the soil limiting concentrations of Cu and Zn of the three different species of Indian mustard were lower than 200 and 100 mg kg-1. The soil limiting concentration of Pb of the three different species of Indian mustard was 1000 mg kg-1, and the maximum Pb accumulation of the three Indian mustards was 200 mg kg-1, which was much lower than the accumulation of the Pb hyperaccumulator . The soil limiting concentration of Cd of the three different species of Indian mustard was 25 mg kg-1, and the maximum Cd accumulation of the three Indian mustards was 200 mg kg-1. It reaches the standard level of the Cd hyperaccumulator .The biomass of India and Pakistan Indian mustard were significantly higher than Afghanistan specie (p<0.05). There were no significant different of Cd accumulation between the three Indian mustard species. The Pb accumulation of the three Indian mustard species, the species of Afghanistan of Indian mustard was significantly higher than the species of Indian and Pakistan (p<0.05). The total Cd removal , the species of India and Pakistan were significantly higher than that of Afghanistan species (p<0.05). There were no significant different between the Pb removal of the three Indian mustard species. Harvestd at 7th day after applying EDTA, the Cd concentration of Indian mustard were increased form 200 to 330 mg kg-1, and the total removal of Cd were increased form 97 to 157 μg pot-1. Harvestd at 7th day after applying EDTA, the Pb concentration of Indian mustard were increased form 80 to 700 mg kg-1, and the total removal of Cd were increased form 38 to 250 μg pot-1. In conclusion, the ability of phytoremediation of the three species of Indian mustard are not different, and adding EDTA solution can significantly increase the uptake of heavy metal in contaminated soil.

The research tested the Brassica juncea ability, to phytoextract and phytostabilize lithium from mine tailings in lieu with vanadium and chromium, sown in a heterogeneous acidic rhizosphere. Five different heterogeneous growth media formulations were prepared from lithium mine tailings, homogenized peat and dewatered municipal biosolids. The Brassica juncea was grown for eighty six days, under homogeneous growing conditions, irrigated bi-daily with organic fertilizer, amended with LiCl, harvested and chemically analyzed. The phytoextraction and phytostabilization data revealed that the Brassica juncea was capable of absorbing more vanadium in its physiological parts rather than lithium and chromium. Likewise the monocotyledonous plant was grown homogeneously on the most favorable growth media, amended with lithium chloride and was able to phytoharness and phytostabilize more lithium rather than chromium per dry weight basis. In botanical efficiency parameters the monocotyledonous plant was ten times more efficient than the Brassica juncea in the bioaccumulation and efficiency removal rates for lithium and twice as much as for chromium. The relative growth rate of the monocotyledonous plant was twice as much as the Brassica juncea. Moreover, it surpassed the monocotyledonous plant in translocation indexes for chromium more than six times and twenty times for lithium. The findings revealed the possibility of a three way symbiosis formed between the hyperaccumulant plant grown in a heterogeneous rhizosphere and coupled with EK system at certain growth periods that will result in an increased electromigration and electrophoresis of heavy metals in the growth media solution.

African leafy vegetables have been shown and suggested to have potential to contribute to human diets and alleviate malnutrition; however, their levels of utilisation are currently low especially in South Africa. This is because there is limited access to these crops due to low availability in the market. Limited access is attributed, in part, to the lack of commercialisation as a result of limited agronomic information describing optimum management options for these leafy vegetables. Availability of such information would contribute to successful commercialisation of these crops. The primary objective of this study was to establish optimum agronomic management factors for Amaranthus cruentus, Corchorus olitorius, Vigna unguiculata and Brassica juncea for irrigated commercial production in South Africa. Seeds of Amaranthus cruentus, Corchorus olitorius were obtained from the Agricultural Research Council seed bank; Vigna unguiculata were obtained from Hydrotech and Brassica juncea seeds were obtained from Stark Ayres. The project consisted of three field studies whose overall objective was to evaluate growth and yield responses of the selected African leafy vegetables to agronomic factors under irrigated commercial production. These field studies comprised of two single factors; summer trials (planting density and nitrogen on three selected crops) and a combined winter trial (nitrogen, irrigation, plant density and planting date on a winter crop). Chapter three (3) investigated the effect of plant density on growth, physiology and yield responses of Amaranthus cruentus, Corchorus olitorius and Vigna unguiculata to three plant densities under drip irrigated commercial production. The plant density levels of 100 000, 66 666 and 50 000 plants/ha were used in the 2011/12 and 2012/13 summer seasons. Parameters measured included chlorophyll content index (CCI), chlorophyll fluorescence (CF), stomatal conductance (SC), leaf number, leaf area index (LAI) and biomass. Amaranthus cruentus and Corchorus olitorius showed better leaf quality at lower plant density of 50 000 plants ha-1 than at 66 666 plants ha-1 and 100 000 plants ha-1. These results are based on bigger leaves expressed as leaf area index (LAI), better colour expressed as chlorophyll (CCI) and higher biomass per plant observed in these crops at 50 000 plants ha-1 in comparison to 66 666 plants ha-1 and 100 000 plants ha-1. In Vigna unguiculata there were no responses observed in LAI and CCI. In Amaranthus cruentus, Corchorus olitorius and Vigna unguiculata fresh and dry mass yield of leaves were higher at 100 000 plants ha-1 compared to other treatments. In A. cruentus and C. olitorius, higher leaf quality parameters (CCI, plant height, leaf number, biomass per plant and LAI) indicated that these crops can perform better at lower densities of 50 000 than at 66 666 plants ha-1 and 100 000 plants ha-1 Therefore, using 50 000 plants ha- 1 is suitable for commercial production of A. cruentus and C. olitorius. In Vigna unguiculata, a plant density of 100 000 plants ha-1 produced the highest fresh and dry mass per unit area without compromising quality in terms of the leaf size (LAI) and colour (CCI). Therefore 100 000 plants ha-1 is a density recommended for commercial production in V. unguiculata.Chapter four (4) was conducted to investigate growth, physiology and yield responses of A. cruentus, C. olitorius and V unguiculata to nitrogen application under drip irrigated commercial production. Three nitrogen treatments levels were used viz. 0, 44 and 88 kg N ha- 1 in 2011/12 season and four nitrogen treatments levels viz. 0, 50, 100 and 125 kg N ha-1 were used in 2012/13 summer season. The nitrogen levels selected for each season were based on recommendations for Amaranthaceae species, Swiss chard (Beta vulgaris L.var cicla) derived from soil analysis of the trial (field) site. Parameters measured included chlorophyll content index (CCI), chlorophyll fluorescence (CF), stomatal conductance (SC), leaf number, leaf area index (LAI) and biomass. Results showed that application of nitrogen at 44 kg N ha- 1 in 2011/12 summer season and 100 kg N ha-1 in 2012/13 summer season improved LAI, CCI, biomass per plants and yield in A. cruentus. A similar trend was observed in C. olitorius except that 44 kg N ha-1 improved stem fresh yield. Further increase in nitrogen fertiliser above 44 kg N ha-1 during the 2011/12 season and above 100 kg N ha-1 in 2012/13 summer season reduced leaf quality and yield in both crops. In V. unguiculata, nitrogen application showed a slight increase in yield values from 0 to 44 kg N ha-1 followed by decrease at 88 kg N ha-1 in 2011/12 summer season; however, this increase in yield was not significant. During the 2012/13 summer season, yield in terms of fresh weight was significantly (P<.001) reduced by applying nitrogen at various levels. However, leaf dry matter content increased significantly (P<.001) with increase in nitrogen from 0 kg up to 100 kg N ha-1, then remained unchanged at 125 kg N ha-1. Therefore, the current study recommends that C. olitorius and A. cruentus could be commercialised at 44 kg N ha-1 and 100 kg N ha-1 which were lower nitrogen application rates than those recommended for Amaranthaceae species. In V. unguiculata, 50 kg N ha-1 improved leaf number; however, this did not translate to any fresh yield advantage, implying that the optimum rate for nitrogen application might be lower than 50 kg N ha-1. Therefore, nitrogen rates less than the ones used in the current study are recommended for V. unguiculata. Chapter five (5) was conducted in winter and it was necessitated by observations made primarily in the previous studies which focused on the effects of single factors such as plant density, planting date and nitrogen deficits. Therefore, there was a need to address interactions between irrigation, nitrogen, spacing and planting date. The objective of this study was to evaluate growth, physiology and yield responses of Brassica juncea to different agronomic and management factors in the 2012 and 2013 seasons. The treatments were as follows: two planting dates in main plot (1 June and 18 July, 2012); two irrigation frequency in sub main plot (once and three times a week); three nitrogen levels (0, 50, 100 kg N ha-1) and three plant densities (133 333, 80 000, 50 000 plants ha-1) as subplots. Parameters measured included chlorophyll content index (CCI), chlorophyll fluorescence (CF), stomatal conductance (SC), leaf number, leaf area index (LAI) and biomass. Results from this study showed a significant interaction effect on plant height, LAI, CCI and CF. Crops irrigated thrice or once a week with 50 kg N ha-1 combined with 50 000 plants ha-1 produced tall plants and bigger leaves (LAI) in the early planting date (1 June) compared to other combinations. Irrigating three times a week combined with nitrogen application at 100 or 50 kg N ha-1 improved CF for late planting date (18 July) in comparison to other combinations. Irrigating once a week combined with nitrogen application at 100 kg N ha-1 increased CCI. There was no significant interaction effect on yield. Application of nitrogen at 50 and 100 kg N ha-1 significantly (P>0.05) increased yield in early and late planting dates compared to the control (0 kg N ha-1), in 2012 and 2013 winter season. Irrigating three times a week led to a significant (P<0.05) increase in yield in the late planting date (18th July) and early planting date (1st June) in 2013 season. Higher plant density of 133 333 plants ha-1 resulted in significantly (P<0.05) higher yield in terms of fresh mass and leaf number in the late planting date 18 July in 2012 and 2013 seasons. However, leaf quality parameters such as leaf size and colour was compromised at 133 333 plants ha-1 relative to 50 000 plants ha-1. Therefore, farmers are recommended to plant early, apply 50 kg N ha-1, irrigate thrice a week and utilise a spacing of 50 000 plants ha-1. The current study indicates that growth and yield of traditional leaf vegetables can be optimised through improved agronomic practise.
Agriculture and Life Sciences
D. Litt. et. Phil. (Agriculture)

MENVSC
Department of Ecology and Resource Management
South Africa is highly dependent on coal for production of electricity. The combustion of coal for electricity generation produces waste by-products which include fine ash (fly ash) and coarse ash. Fly ash (FA) is produced in large amounts while its utilization is low due to its classification as a hazardous material. Sasol produces about 7 million tons of FA, while Eskom produces about 28 million tons of coal FA annually. FA is a fine by-product from pulverized coal with detrimental effects on plants, soil or land, animals and human beings, and pollutes the air. The present investigation focuses on the examination of the chemical elements bioaccumulation and phytoremediation potential of Indian mustard (Brassica juncea) and Spinach (Spinacia Oleracea L). X-ray fluorescence (XRF) was used to analyze the elemental composition of FA and soil, Scanning Electron Microscope (SEM) to examine the morphology, while X-ray diffraction (XRD) analysis was used to evaluate the mineralogy of FA. The concentrations of metal and non-metal species that are released from FA on contact with water at different conditions were quantified using ICP-MS. Pot culture experiment was conducted to grow Brassica juncea and Spinacia Oleracea L. using FA and soil as growth medium. Leachates from the pots were collected and analysed using ICP-MS. Plant parts from harvested Brassica juncea and Spinacia Oleracea L. were cut separately and analysed using ICP-MS for the concentrations of different metal and non-metal species in plant parts. Plant parts were also used to estimate biomass and chlorophyll content (leaves). To prepare these plant samples for analysis, the powdered plant sample (0.5 g) was digested through aqua regia (HCL:HNO3 = 3:1 (v/v)) to near dryness using hotplate and filled to 100 mL of MilliQ water. The samples were filtered and directly used to determine the chemical elements concentrations. Blanks and internal standards were used for quality assurance during analysis. Chemical elements that are present in FA and sometimes in higher concentrations are associated with detrimental effects in plants, animals and human beings, hence phytoremediation is vital. Bioconcentration factor (BCF) was used to estimate the metal species accumulation ability of the plants from the FA, FA/soil mixtures, while translocation factor (TF) was used to assess the plant species potential for phytoremediation of coal fly ash dumps. Analysis of Varience (ANOVA) was used to statistically test data using Graphpad software package. Relationship between chemical elements in soil, FA and FA+soil growth media and also different plant parts (root, stem and leaf) of B. juncea and S. Oleracea L were calculated using the t - test, ANOVA-Bartlett test, Mann-Whitney Test and Kruskal-Wallis Statistic (KW) depending on each data set. The physicochemical characterization of coal fly ash showed that FA from Grootvlei power station can be classified as class F with an alkaline pH level of 10.62. It showed that particle morphology of this FA had a lower degree of sphericity with irregular agglomerations of many particles while there were dominant spherical particles and smaller sharp needle like particles. It is also an alumino-silicate material as confirmed by the high SiO2 and Al2O3 content, while soil leachates had an average/neutral pH of 7.22 with very high amounts of Si. In both soil and FA, elements that were expected to be readily available to plants included Ca, Si, K, Ba, Mo, Na, Al, Mg, Sr and non-essential elements (Si, Ba, Na, Al, Sr), which, if uptaken by plants’ roots can have negative impacts in plants. Physicochemical analysis of soil, FA and FA+soil leachates showed that the alkalinity of the FA changed over time and there was also a decrease in the EC due to dissolution iv of soluble major oxides, which was promoted by continuous water irrigation. The soil and FA+soil growth media showed similar results. Chemical elements like B, Mn, Fe and Ba were occurring at higher concentrations in leachates for most weeks in the pot culture experiments. However, it was observed that in the eleventh week of leachate collection all these chemical elements decreased to very low concentrations. This suggest that these chemical elements can be reduced over time as plants are being irrigated which is either due to uptake by plants or washed off with water. Statistically, there was a significant difference for different chemical elements of leachates from different growth media for each plant species (B. juncea and S. Olearcea L.). The overall growth rate shows that S. Oleracea L was better than that of B. juncea especially in the FA media over time, while the biomass of the two plant species showed similar results. After all, even though S. Oleracea L had carotenoid content below detection limits, it had higher chlorophyll b than B. juncea for all growth media in general. But, statistically there was no significant difference between the two plant species in terms of growth rate and biomass; even between the plant parts denoting similar growth performance for the two plant species under study. The bioaccumulation potential of the two plant species showed that chemical elements such as Fe, Mn, Ba, Zn and B were highly accumulated by the different parts of the plants. However, the chemical elements such as of Mo, Ni, Cu, and Cr showed the least concentrations. This trend was similar for all growth media and both plant species but this changes over time for different growth media and plant species as increasing and decreasing trends can be noticed. This led to no significant difference between plant species and also growth media, statistically. High BCF values of Fe, Mn, Ba, Zn and B were observed in the different parts of the plants for both plant species. However, Mo, Ni, Cu, and Cr had BCF values less than 1 for most growth media over time. BCF values in plant parts differed with time, growth media and plant species. Translocation of chemical elements shows that the B. juncea plant proved to be an effective phytoremediation plant species since it is effective in translocation of many chemical elements for different growth media to shoots while S. Oleracea L failed to translocate most chemical elements from stem to leaves although it translocated some from root to shoots. Hence, it can be concluded from the study that both species can be used in phytoremediation of coal fly ash dumps but with B. juncea being the most effective accumulator and translocator of many chemical elements. However, it can be recommended that chelating agents like ethylene diamine tetra acetic acid (EDTA) be introduced to solubilize chemical elements from growth media matrix into growth media solution to facilitate the quick transport of chemical elements into xylem, and increase