<p>Plant microbiomes are increasingly recognized for their potential to help plants with critical functions such as nutrient acquisition. Nitrogen is the most limiting nutrient in agriculture and growers apply substantial amounts to meet crop needs. Only 50% of N fertilizers are generally taken up by plants and the rest is subject to loss which negatively affects environmental quality. Organic fertilizers such as cover crops and animal manure can help reduce this loss, though these materials must mineralize via microbial mediated processes before they are available for plant uptake, which makes managing fertility using these sources difficult. Some plants can scavenge nutrients from organic materials by stimulating positive priming processes in soil. Carrot (<i>Daucus carota.</i> L) is known as an N scavenging crop, making it an ideal model crop to study these interactions. In a greenhouse trial, soils were amended with an isotopically labeled corn residue to track N movement, and planted with one of five carrot genotypes expected to differ in nitrogen use efficiency (NUE). Changes in soil b-glucosidase activity, ammonium (NH₄⁺-N) and nitrate (NO₃^--N) concentrations, soil bacterial community composition, weight and carbon and N concentrations, and total δ¹⁵N of above and below ground carrot biomass were determined. Results indicate that there are genetic differences in the ability of carrots to promote priming under N limited conditions, which could be exploited to enhance NUE in carrots. Soil microbial communities differed between genotypes, indicating that some of these microbes could play a role in the differential N scavenging responses observed, and/or contribute to other important functions such as resistance to pests. Endophytic microbes residing inside carrot taproots also have potential to contribute to NUE and other benefits, but are notoriously difficult to isolate and culture. New next generation sequencing technologies have revolutionized the study of microbiomes, though using these tools to study bacterial endophytes in plants is still difficult due to co-amplification of plant organelles. Consequently, a second study was conducted to determine if subjecting carrot tissues to hollow fiber microfiltration followed by enzymatic digestion could enhance recovery and amplification of bacterial endophytes. Carrot taproot digests were subject to amplification using a standard V3-V4 16S primer set, as well as two alternative (blocking and mismatch) primer sets that have prevented amplification of plastids/mitochondria in other plant species. Results indicate that the microfiltration/digestion procedure can increase the number of bacterial endophyte OTUs assigned and could be further optimized for use in carrots. The blocking and mismatch primer sets were not as effective in blocking co-amplification of plant products as they are in other studies, possibly due to the presence of a high number of chromoplasts in carrot tissues. Taxonomic assignment of bacterial endophytes differed significantly between the primer sets, indicating that multiple primer sets may be needed to fully characterize these communities in carrots. The enzymatic digestion procedure could artificially inflate certain taxa, which could be helpful if targeting specific taxa. These studies demonstrate that carrots are intimately connected with microbes residing in the soil and within their taproots, and further exploration of these plant-soil-microbial relationships could enhance the yield and sustainability of carrot production systems.</p>

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Orientador: Francisco Luiz Araújo Câmara / Banca: Antônio Ismael Inácio Cardoso / Banca: Roberto Boczko / Resumo: O presente trabalho teve como objetivo avaliar a influência dos diversos ritmos da Lua (sinódico, sideral, anomalístico, tropical e draconiano) sobre o rendimento, a qualidade e a conservação pós-colheita de cenoura, quando semeada em diferentes datas, sob as mesmas condições de manejo, em uma propriedade familiar com manejo biodinâmico no município de Botucatu-SP. O delineamento experimental utilizado foi o de blocos ao acaso com 31 tratamentos em 2005, e 14 tratamentos em 2006. A diferença entre tratamentos foi a data de semeadura, que variou de 5 de maio a 4 de junho em 2005, e de 25 de abril a 25 de maio em 2006, sempre entre 13 e 15hs. A colheita foi feita 82 dias depois de cada semeadura, equivalente a três ciclos da lua sideral, e no ponto que o consumidor de produtos orgânicos e biodinâmicos valoriza, segundo experiência do produtor. Para retirar o efeito da tendência dos dados na avaliação dos tratamentos, foi utilizada a metodologia de avaliação estatística do cálculo do Índice Estacional (IE). Foram avaliadas as seguintes características: massa fresca de raízes e folhas, massa seca, diâmetro, comprimento, teor de nitrogênio, fósforo e boro das raízes e perecibilidade das raízes com 30, 60 e 90 dias póscolheita. Nos dois períodos avaliados, a massa seca de raízes foi a única que, no contraste entre médias, apresentou diferença significativa nos ritmos sinódico tradicional e sinódico caboclo. No ritmo sinódico tradicional, a fase nova foi superior às fases crescente e cheia. No sinódico caboclo, a fase cheia foi inferior às demais. No contraste entre médias, o ritmo sinódico foi o que mais apresentou resultados significativos, e em menor proporção, os ritmos anomalístico, draconiano e sideral; O ritmo tropical (ascendente X descendente) e teor de nitrogênio não apresentaram resultados significativos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The purpose of this work was to evaluate the influence of moon rhythms (synodic, sidereal, anomalistic, tropical and draconic) on yield, quality and postharvest storage of Daucus carota L. roots under biodynamic management sowed in different dates. The experiment was carried out over a two period on a biodynamic farm, in Botucatu, São Paulo State, Brazil. Rhythms were tested observing the effects of seeding at different planting dates. The experiment was performed with four randomized blocks and 31 treatments (different dates) in 2005 and fourteen treatments in 2006. The harvest occurred 82 days after the sowing, when carrot roots show the most desirable aspect for the organic and biodynamic consumers. The magnitudes of effects associated with planting at a specific lunar position were measured by the deviations from the trend curve. The following characteristics were evaluated: fresh mass of roots and leaves, dry mass, diameter, length, nitrogen, phosphorus and boron content of the roots and perishability of the roots at 30, 60 and 90 days post-harvest. Dry mass was the only one that in the contrast between averages showed significant results in the two periods of the experiment. Result was that the synodic new phase was superior to the first quarter, and full phases and in the "caboclo" synodic rhythm, the full phase was inferior to the other. It was clear that the synodic "caboclo" rhythm had the most significant influence followed by the traditional synodic one. The draconic, sideral and anomalistic had less influence and the tropical rhythm had no influence at all considering studied aspects. Nitrogen and tropical rhythm did not present any lunar influence. The two evaluated periods are still not conclusive related... (Complete abstract click electronic access below) / Mestre

by Chan, Yuk Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 166-175). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.v / 摘要 --- p.vii / LIST OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xiv / LIST OF TABLES --- p.xvii / LIST OF ABBREVIATIONS --- p.xviii / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.5 / Chapter 2.1 --- Vitamin A --- p.5 / Chapter 2.1.1 --- General and properties --- p.5 / Chapter 2.1.2 --- Biological importance of vitamin A --- p.6 / Chapter 2.1.3 --- Deficiency symptoms --- p.9 / Chapter 2.1.4 --- Dietary source of vitamin A --- p.10 / Chapter 2.1.5 --- Metabolism of dietary vitamin A and provitamin A in human --- p.12 / Chapter 2.1.5.1 --- Digestion and absorption --- p.12 / Chapter 2.1.5.2 --- Bioconversion --- p.12 / Chapter 2.1.5.2.1 --- "β, β-carotene-15,15'-monooxygenase (BCMO)" --- p.13 / Chapter 2.1.5.3 --- "Transport, uptake and storage" --- p.15 / Chapter 2.2 --- Vitamin A deficiency (VAD) --- p.19 / Chapter 2.2.1 --- Present situation --- p.19 / Chapter 2.2.2 --- Global efforts in dealing with VAD --- p.21 / Chapter 2.2.2.1 --- Vitamin A supplementation --- p.21 / Chapter 2.2.2.2 --- Food fortification --- p.22 / Chapter 2.2.2.3 --- Biofortification --- p.23 / Chapter 2.2.2.3.1 --- Conventional selective breeding --- p.23 / Chapter 2.2.2.3.2 --- Biosynthesis of provitamin A in plants --- p.25 / Chapter 2.2.2.3.3 --- Carotenoids enhancement in major plants and food crops --- p.31 / Chapter 2.3 --- Inherent problems of the present carotenoid enhancement --- p.34 / Chapter 2.3.1 --- Recommended Dietary Amount of vitamin A --- p.34 / Chapter 2.3.2 --- Factors affecting the bioefficacy of provitamin A in human body --- p.35 / Chapter 2.3.2.1 --- Bioavailability --- p.36 / Chapter 2.3.2.2 --- Bioconvertibility --- p.38 / Chapter 2.3.2.3 --- Health and nutrition status --- p.39 / Chapter 2.4 --- Previous study in our lab --- p.41 / Chapter 2.4.1 --- Overexpression of rice PSY1 --- p.41 / Chapter 2.4.2 --- Introduction of carotenoid genes and BCMOs into rice --- p.44 / Chapter 2.5 --- Overview of the project --- p.50 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.52 / Chapter 3.1 --- Chemicals --- p.52 / Chapter 3.2 --- Bacterial strains in regular cloning --- p.52 / Chapter 3.3 --- BCMO genes and carotenogenic genes --- p.53 / Chapter 3.4 --- Expression of BCMOs in bacterial system --- p.54 / Chapter 3.4.1 --- lac promoter system --- p.54 / Chapter 3.4.2 --- pBAD-TOPO® system --- p.56 / Chapter 3.5 --- Construction of gene cassettes for plant transformation --- p.58 / Chapter 3.5.1 --- Gene cassettes for carrot transformation --- p.58 / Chapter 3.5.1.1 --- Construction of gene cassettes for chicken or zebrafish bcmo driven by CaMV 35S promoter --- p.58 / Chapter 3.5.1.2 --- Construction of gene cassettes for chicken or zebrafish bcmo driven by lycopene-β-cyclase promoter --- p.63 / Chapter 3.5.2 --- Gene cassettes for Arabidopsis transformation --- p.67 / Chapter 3.5.2.1 --- Construction of gene cassettes expressing Dcpsy --- p.67 / Chapter 3.5.2.2 --- Construction of gene cassettes expressing mbcmos --- p.69 / Chapter 3.5.3 --- Gene cassettes for Rice transformation --- p.72 / Chapter 3.5.3.1 --- Construction of gene cassettes expressing mbcmos --- p.72 / Chapter 3.5.3.2 --- Construction of gene cassettes expressing Ospsyl and mbcmos --- p.74 / Chapter 3.5.4 --- Confirmation of sequence fidelity --- p.76 / Chapter 3.6 --- Carrot transformation --- p.76 / Chapter 3.6.1 --- Plant materials --- p.76 / Chapter 3.6.2 --- Preparation of Agrobacterium --- p.76 / Chapter 3.6.3 --- Agrobacterium mediated transformation --- p.77 / Chapter 3.6.3.1 --- Seed germination --- p.78 / Chapter 3.6.3.2 --- Co-cultivation with hypocotyls --- p.78 / Chapter 3.6.3.3 --- Callus induction and selection --- p.78 / Chapter 3.6.3.4 --- Liquid cell culture preparation and embryogenesis induction --- p.79 / Chapter 3.6.3.5 --- Regeneration --- p.80 / Chapter 3.7 --- Arabidopsis Transformation --- p.80 / Chapter 3.7.1 --- Plant materials --- p.80 / Chapter 3.7.2 --- Preparation of Agrobacterium --- p.81 / Chapter 3.7.3 --- Agrobacterium mediated transformation --- p.81 / Chapter 3.7.3.1 --- Co-cultivation --- p.81 / Chapter 3.7.3.2 --- Selection --- p.82 / Chapter 3.8 --- Rice transformation --- p.83 / Chapter 3.8.1 --- Plant materials --- p.83 / Chapter 3.8.2 --- Preparation of Agrobacterium --- p.83 / Chapter 3.8.3 --- Agrobacterium mediated transformation --- p.83 / Chapter 3.8.3.1 --- Callus induction from mature rice seeds --- p.84 / Chapter 3.8.3.2 --- Co-cultivation and selection --- p.84 / Chapter 3.9 --- Detection of transgene expression --- p.86 / Chapter 3.9.1 --- Detection at DNA level --- p.86 / Chapter 3.9.1.1 --- Genomic DNA extraction --- p.86 / Chapter 3.9.1.2 --- PCR screening --- p.86 / Chapter 3.9.1.3 --- Synthesis of DIG-labelled DNA probes --- p.86 / Chapter 3.9.1.4 --- Southern blot analysis --- p.87 / Chapter 3.9.2 --- Detection at RNA level --- p.88 / Chapter 3.9.2.1 --- Total RNA extraction --- p.88 / Chapter 3.9.2.2 --- Northern blot analysis --- p.89 / Chapter 3.9.2.3 --- RT-PCR --- p.89 / Chapter 3.9.3 --- Detection at protein level --- p.89 / Chapter 3.9.3.1 --- Antibody production --- p.89 / Chapter 3.9.3.1.1 --- B.CMO protein induction in pET30a-bacterial system --- p.90 / Chapter 3.9.3.1.2 --- Immunization of rabbit and serum collection --- p.93 / Chapter 3.9.3.2 --- Protein extraction and Tricine SDS-PAGE --- p.93 / Chapter 3.9.3.3 --- Western blot analysis --- p.94 / Chapter 3.9.4 --- Detection at final product level --- p.95 / Chapter 3.9.4.1 --- UPLC analysis --- p.95 / Chapter 3.9.4.1.1 --- Extraction of total carotenoids and retinoids --- p.95 / Chapter 3.9.4.1.2 --- UPLC identification --- p.96 / Chapter CHAPTER 4. --- RESULTS --- p.97 / Chapter 4.1 --- Modified bcmo genes --- p.97 / Chapter 4.2 --- Expression of BCMOs in bacterial system --- p.102 / Chapter 4.2.1 --- lac promoter system --- p.104 / Chapter 4.2.2 --- pBAD-TOPO® system --- p.106 / Chapter 4.2.3 --- UPLC detection --- p.108 / Chapter 4.3 --- Carrot transformation --- p.110 / Chapter 4.3.1 --- Construction of gene cassettes for carrot transformation --- p.110 / Chapter 4.3.2 --- Seed germination and co-cultivation --- p.112 / Chapter 4.3.3 --- Callus induction and selection --- p.113 / Chapter 4.3.4 --- Embryogenesis induction and regeneration --- p.113 / Chapter 4.3.5 --- Callus induction in the dark --- p.115 / Chapter 4.3.6 --- Detection of native BCMO --- p.116 / Chapter 4.3.6.1 --- Genomic PCR screening of 35Spro - zebcmo transgenic lines --- p.116 / Chapter 4.3.6.2 --- Southern blot analysis of 35Spro - zebcmo transgenic lines --- p.117 / Chapter 4.3.6.3 --- RT-PCR of 35Spro - zebcmo transgenic lines --- p.118 / Chapter 4.3.6.4 --- Detection at protein level --- p.119 / Chapter 4.3.6.4.1 --- Antibody production --- p.119 / Chapter 4.3.6.5 --- Western blot analysis of 35Spro - zebcmo transgenic lines --- p.123 / Chapter 4.3.6.6 --- Genomic PCR screening of later transgenic lines --- p.123 / Chapter 4.3.6.7 --- Western blot analysis of later transgenic lines --- p.125 / Chapter 4.3.6.8 --- UPLC analysis of later transgenic lines --- p.127 / Chapter 4.3.7 --- Detection of modified BCMO --- p.130 / Chapter 4.3.7.1 --- Genomic PCR screening --- p.130 / Chapter 4.3.7.2 --- Northern blot analysis --- p.132 / Chapter 4.3.7.3 --- Western blot analysis --- p.134 / Chapter 4.3.8 --- UPLC analysis --- p.136 / Chapter 4.4 --- Arabidopsis transformation --- p.138 / Chapter 4.4.1 --- Construction of gene cassettes for Arabidopsis transformation --- p.138 / Chapter 4.4.2 --- Selection --- p.139 / Chapter 4.4.3 --- Genmoic PCR screening of Arabidopsis transformants --- p.140 / Chapter 4.4.4 --- UPLC analysis for Arabidopsis transformants --- p.142 / Chapter 4.5 --- Rice transformation --- p.144 / Chapter 4.5.1 --- Construction of gene cassettes for rice transformation --- p.144 / Chapter 4.5.2 --- "Callus induction from mature rice seeds, co-cultivation and selection" --- p.146 / Chapter 4.5.3 --- Genomic PCR screening of Rice transformants --- p.147 / Chapter 4.5.4 --- UPLC analysis of rice transformants --- p.149 / Chapter CHAPTER 5. --- DISCUSSION --- p.151 / Chapter 5.1 --- Bacterial expression of BCMO --- p.151 / Chapter 5.2 --- Analysis of BCMO in plants --- p.153 / Chapter 5.2.1 --- Carrot --- p.154 / Chapter 5.2.1.1 --- Expression of BCMO in carrot transformants --- p.154 / Chapter 5.2.1.2 --- UPLC analysis of carrot transformants --- p.155 / Chapter 5.2.2 --- Arabidopsis --- p.156 / Chapter 5.2.3 --- Rice --- p.158 / Chapter 5.3 --- Proposed explanation for the failure of retinal production --- p.159 / Chapter 5.3.1 --- Retinal sequestration --- p.160 / Chapter 5.3.2 --- Localization of BCMO --- p.161 / Chapter 5.4 --- Future prospects --- p.163 / Chapter CHAPTER 6. --- CONCLUSIONS --- p.165 / REFERENCES --- p.166 / APPENDICES --- p.176

Effect of soil moisture potential (?), temperature (T), genotype, seeding depth (SD) and rate (SR) on seedling emergence (SE), emergence velocity (EV), root yield and grades of cut and peel carrots were studied. SE was reduced at –120 kPa and totally inhibited at -156 kPa. EV was the lowest at – 5 kPa and – 90 kPa. SE was delayed by 33 d at 5°C, reduced at 30°C and totally inhibited at 35 and 40 °C. Heat units 99.75 and 159.60°Cd were the lowest to initiate and complete SE respectively while the optimum was 300 – 350 °Cd. There was no interaction effect between ? and T on SE. Honey snax at 85 seeds/ 30 cm showed the best SE whereas, Triton recorded the highest total yield at 2.54 cm SD and Fancy yield at 85 seeds/ 30 cm implying certain crop ecological and management factors can influence SE, root yield and quality.