Atualmente a procura por produtos sustentáveis têm-se mostrado cada vez mais frequente e promissora. Em espécies de importância comercial, procura-se obter a maior produtividade possível dentro de um curto espaço de tempo aliado à preservação do meio ambiente. Dentro disso, a transformação genética de plantas se mostra uma alternativa atrativa para a geração de variedades de cana-de-açúcar que gerem produtos de maneira mais eficaz. O sucesso da transformação genética está diretamente associada a cultura de tecidos de plantas que precisa ser adequada a cada genótipo e situação de cultivo, sendo a luminosidade um dos principais fatores para a produção de plantas vigorosas. Outro fator importante é a seleção das plantas transgênicas, que precisam ser submetidas a uma quantidade de agente seletivo suficiente para identificar as plantas modificadas geneticamente. Em cana-de-açúcar, a identificação de plantas transgênicas por PCR e a definição do número de cópias é um procedimento de difícil execução e muito oneroso. Isto se dá pois no processo transformação via biolística, a inserção de genes é aleatória, produzindo plantas com variados números de cópias. Em consideração a estes fatores envolvidos na eficiência de obtenção de plantas transgênicas de cana-de-açúcar, os objetivos deste trabalho foram o aperfeiçoamento do protocolo de cultura de tecidos, transformação genética da variedade SP803280 com os genes xth, AtDdm1, como também, definir genes de referência para a quantificação do número de cópias dos genes xth e AtDdm1 inseridos na variedade SP803280 e do gene neo na RB835089, análise de ploidia e tamanho de genoma dos eventos transgênicos comparado com as plantas controle. No estudo a respeito da melhor qualidade de luz durante o cultivo in vitro na fase de regeneração de plantas, tem-se que a luz branca e a junção das luzes LED e branca se mostraram melhores para regeneração e desenvolvimento das plantas enquanto que para plântulas, as luzes LED e branca separadamente foram mais efetivas no crescimento. Para a seleção das plantas uma concentração de geneticina entre 40 e 50 mgL-1 é recomendada. As taxas de sucesso nas transformações genéticas para o gene xth variaram entre 2,5 a 18,3% dependendo do experimento e para AtDdm1 foi de 2,2% em um bombardeamento.Não houveram alterações de ploidia e tamanho do genoma nos transgênicos das duas variedades em relação à planta selvagem. Os genes p4h e prr foram identificados como os melhores para a quantificação relativa de genes inseridos por PCR em tempo real na variedade SP803280 enquanto que para a RB835089 aprt e prr se mostraram mais eficazes. A análise do número de cópias inseridas em eventos transgênicos por PCR em tempo real foi possível através das duas metodologias de cálculo testados por este trabalho, com resultados que concordam com uma tendência nesta determinação de maneira simples e rápida. / Currently the demand for sustainable products has been shown to be frequent and promising. In species of commercial importance, there is an effort to obtain the highest possible productivity in a short time, along with the environment preservation. In this context, genetic transformation of plants appears as an attractive alternative for the development of sugarcane varieties able to generate products in a more effective way. The genetic transformation success is directly associated to plant tissue culture that requires specific condition for each genotype and cultivation process, in which, luminosity is one of the main factors that determines the production of vigorous plants. Another important factor is the selection of transgenic plants, that occur by exposing plants to a sufficient amount of selective agent in order to identify only genetic modified plants. In sugarcane, identification of transgenic plants by PCR and the definition of copy numbers is a difficult procedure to implement and usually is very costly. It is because in the process of genetic transformation by biolistic, the insertion of genes occurs randomly and also produce plants with varied copy numbers. In consideration of these factors directly involved in the efficiency to obtain sugarcane transgenic plants the objectives of this study were the improvement of a tissue culture protocol, the genetic transformation of the variety SP803280 with xth and AtDdm1 genes. Also, the studies include the definition of reference genes for determining the number of copies inserted of xth and AtDdm1 genes into the variety SP803280 and neo gene in RB835089, ploidy analysis and genome size of the transgenic events compared to control plants. In the study related to the best light quality for in vitro plant regeneration, white light and the combination of LED and white lights proved to be better for plants regeneration and development while for seedlings, LED and white light separately were more effective for growth. In order to obtain selection of transgenic plants, geneticin concentration between 40 and 50 mg L-1 is recommended. Success rates in xth genetic transformation ranged from 2.5 to 18.3% depending on the experiment, and for AtDdm1 was only 2.2% in just one biolistic bombardment. There were no changes in ploidy and genome size in transgenic events related to their wild type plant. The genes p4h and prr were defined to be the best for determining the copy number of transgenic events by real time PCR in SP803280 variety, while for RB835089, the genes aprt and prr were the most effective. The analysis of the number of inserted copies was possible using the two calculation methodologies tested by this work, with results that agree with a tendency in a simple and fast quantification methodology.

A double sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify the proteinaceous inhibitor of tissue plasminogen activator (t-PA) which occur in the tissue of Erythrina caffra Thunb. Using the ELISA the t-PA inhibitor could be detected in nanogramme quantities on the micro titer plate. The concentration of the t-PA inhibitor was determined in different tissues of Erythrina caffra. t-PA inhibitor concentrations in the order of 1 000 microgrammes per gramme protein were found in the seeds. Relatively small quantities of t - PA inhibitor, in the order of 10 to 50 microgrammes per gramme protein, occurred in root, shoot, leaf and living bark material. The t-PA inhibitor was found to accumulate in a similar way to the storage proteins in developing seeds. The accumulation of the inhibitor is at a relatively low level during the early period of seed development but increases exponentially just before the seeds reach their maximum size. The t-PA inhibitor content of the cotyledons decreased drastically during the process of germination and subsequent seedling development. The disappearance of the inhibitor be the result of total degradation of the molecule can or partial proteolysis with the modified molecule still being present in the tissue. An attempt was made to increase the t-PA inhibitor content of excised leaves of Erythrina caffra with protein inducing substances such as polyamines, precursors of ethylene and phytic acid. The protein inducing compounds included cell wall hydrolysates of Erythrina caffra, the marine alga Ecklonia maxima Osbeck (Papenfuss) as well as Lycopersicon esculentum Mill which induced the, synthesis of proteinase inhibitors suggested to be involved in the defense mechanism of plants. None of the substances used, increased the t-PA inhibitor content of excised leaves or in vitro cultures of Erythrina caffra. It is suggested that the t-PA inhibitor is probably not involved in a defense mechanism of Erythrina caffra. A callus and suspension culture derived from shoot tissue was developed to determine the occurrence of the t-PA inhibitor in vitro. The optimal nutrient medium for the growth of callus was the salts and vitamins of MURASHIGE and SKOOG (1962). The medium was supplemented with 3 % sucrose, 0. 1 gramme per litre meso - inositol, 10 micromoles per litre benzyl adenine and 5 micromoles per litre 2,4- dichlorophenoxyacetic acid . Different auxins and cytokinins had a similar growth stimulatory effect on the growth of callus derived from a number of organs of Erythrina caffra. The callus from different organs did however, grow at different rates on the same nutrient medium. Callus derived from leaf, shoot, and cotyledonary tissue grew at similar rates on the nutrient media of MURASHIGE and SKOOG (1962), SCHENK and HILDEBRANDT (1972) and B5 (GAMBORG, MILLER and OJIMA, 1968) despite large differences in the concentration of the nutrients in the three nutri.ent media. The source of nitrogen and ratio of nitrate to ammonium was critical to the growth of callus cultures . The optimal concentration of nitrate and ammonium was 30 millimoles per litre . The growth of callus from different organs was significantly affected by the concentration of sucrose in the nutrient medium. A concentration of 3% was optimal for callus growth. Temperature had a significant effect on the growth of callus. The optimal temperature for callus growth was 25 °C. A shoot cell suspension culture was established and maintained at the same temperature and on the same medium as the callus cultures but with a ten times lower concentration of growth regulators. A low shake speed was essential for the growth of the suspension culture. Maximum growth was obtained at a shake speed of 60 rpm. Relatively low quantities of t-PA inhibitor, in the order of 1 to 5 microgrammes per gramme protein, was detected in the suspension cultures. An attempt was made to increase the t-PA inhibitor content of the suspension cultures with the pro te in i nduc i ng compounds used on excised leaves, but without success. However, the t-PA inhibitor content of the suspension culture was significantly increased with a ten times increase in the sulphate content of the nutrient medium. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1990.

Please open electronic version for Abstract. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

Components of the South African indigenous flora are disappearing at an alarming rate, due to pressures on land use. The flora is protected by proclamation of reserves and conservation legislation, however these measures can never be wholly successful. For these reasons, methods for propagting Clivia miniata, Gloriosa superba and Sandersonia aurantiaca using in vitro techniques were investigated. The highly sought after Clivia miniata var citrina can be successfully cultured using fruit and floral explants. Use of these explants may limit the number of plants produced in culture due to the seasonal nature of flowering. Gloriosa superba and Sandersonia aurantiaca can be propagated using corm explants, with subsequent in vitro stimulation of cormlet formation. To establish a successful tissue culture procedure an integrated approach to all aspects of the culture is necessary. Sterilization techniques should be empirical and specific for each species and explant. The most critical factor in establishing a culture technique is the choice of a suitable explant. Without a suitable explant the success of the culture procedure may be severely limited. Nutritional and environmental variation may modify the explant response in culture, but initial culture response can be directly related to the origin of the explant, particularly, size, time of the year, age and physiological status. Since the discovery of colchicine in Gloriosa by CLEWER, GREEN and TUTIN (1915) a number of researchers have put forward the idea that Gloriosa would serve as a source of colchicine. The present trend in biochemical production is via artificial synthesis, however many desirable compounds still have to be extracted from plant material for biochemical production. The utilization of plant cells that are cultured in vitro provides a viable alternative to the problems involved in the production of chemical compounds. Levels of colchicine in Gloriosa and Sandersonia are very similar, in the range of ± 0,9%. From evidence presented by BELLET and GAIGNAULT (1985), levels of colchicine in the two study species is much higher than the recorded level (0,62%) of Colchicum. This higher level of the alkaloid makes these two plants a viable source for commercial colchicine production. Levels of colchicine recovered from in vitro grown roots and callus was 10 - 20 times lower than that found in -in -viv-o tissue. Levels of colchicine extracted from plantlets grown in vitro was the same as that normally recorded for parent tissue. Higher levels of colchicine in malformed roots adds to the evidence that differentiation increases colchicine production in Gloriosa tissue in vitro. It has been shown that Gloriosa and Sandersonia tissue can synthesize colchicine in vitro. The extent to which the cells synthetic capacity can be enhanced has yet to be determined. However, research into speedier and more wide ranging methods for metabolite production in culture is receiving attention throughout the world. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1988.

Mammalian estrogens and estrogen-like compounds known as xeno-estrogens are being found in and excreted into the environment in ever increasing amounts. The xeno-estrogen DDE has been found at high concentrations of 1-5 mg/kg of soil (Aislabie et. al, 1997). These estrogens and xeno-estrogens are having a devastating effect on animal-life, yet little is known or understood on the effects of estrogens on plant-life. Thus it is important to determine what effects (if any) estrogens may have on plants. Other research has shown that estrogen has an effect on plants grown in vitro (Janeczko and Skoczowski, 2005). This research aims to help increase the amount of information on what effects estrogens may have on plants. In this study, the effects of mammalian estrogens (17-β-estradiol, estrone and estriol) on the growth and tuberization of potato plants (Solanum tuberosum L. cv 'Iwa') grown in liquid tissue culture medium are presented. It was found that at even 0.1 mg/L of estrogen, root growth of the plants was diminished and at 10 mg/L of estrogen, plant deformity was apparent and callus growth induced. Acid phosphatase activity of the plants was increased with the addition of 0.1 mg/L and 1 mg/L of estrogen but then decreased with the addition of 10 mg/L of estrogen. Tuber production was slightly reduced in plants treated with estrogen compared to the control.

Thesis (Ph.D.) -- University of Western Sydney, 2002. / "A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy" Bibliography: leaves 161-189.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Biotechnology is an important tool that is used to isolate and characterise genes. It is also used to produce clones that are genetically and phenotypically similar. Many Arabidopsis thaliana transcription factors have been isolated and characterised, but many have yet to be fully described. MYB proteins are members of a super-family of multifunctional transcription factors that can also interact with other transcription factors in the control of pathways. To date, more than 126 AtMYBs have been identified, but most have not been fully characterised, particularly in terms of the molecular role(s) they play in plants. Arabidopsis thaliana MYB3, MYB6, MYB7, MYB8 and MYB32 have been reported to be negative regulators of general phenylpropanoid metabolism. It has been reported that the five transcription factors mentioned above are likely to negatively regulate flavonoid biosynthesis, even though they may have different target genes. Studies on AtMYB13, AtMYB14 and AtMYB15 reported that they are likely regulators of general phenylpropanoid metabolism. The mentioned roles of the eight AtMYB transcription factors means that they can be manipulated in order to see what effect they have on primary and secondary metabolites in plants. The transcription factors ligated into the pUBI510-GRFCA vector were then used to transform sugarcane callus (Chapter 3). Sugarcane produces sucrose which makes up 70% of the sugar produced in the world, making sugarcane a commercially important and profitable plant. The sugarcane callus was transformed via particle bombardment. The transcription factors AtMYB3, AtMYB6, AtMYB7, AtMYB13 and AtMYB32 were successfully incorporated into the genomic DNA of the sugarcane callus. The data obtained for callus over-expressing AtMYB3, AtMYB13 and AtMYB32 on solid media and the callus in liquid media were contradictory (i.e callus on solid media producing more sucrose than the wildtype whereas the same transgenic line will poduce less sucrose that the wildtype in liquid media or vice versa). However, AtMYB13 transgenic lines produced more sucrose than the wildtype. Transgenic lines of AtMYB7 all produced less sucrose as compared to the wildtype both on solid and in liquid media. The transcription factors which resulted in increased production of starch when over-expressed were AtMYB7 and AtMYB13. The data obtained for AtMYB6 transgenic lines was highly inconsistent in lines grown on same media and across the two media. The effects of these transcription factors in the overall metabolism of the sugarcane callus, either on MSC3 solid or liquid media, could not be fully determined from the GC-MS analysis as there was no consistent phenotypic effect between different transgenic lines for any of the MYB transcription factors used. In Chapter 4, a micropropagation strategy was developed and phytochemicals and their biological activities were determined for the medicinal plant Salvia repens. Salvia plants have been found to be medicinally important due to the secondary metabolites, particularly the essential oils that they produce. The plant extracts have been found to have many biological activities such as antibacterial, anti-inflammatory, antioxidant and anticancer activities. Salvia repens was successfully germinated in vitro,with 60% germination being achieved in MS media containing 1x10-5 times diluted smoke water following scarification for 12 min in 75% (v/v) H2SO4. Success rates of 100% were achieved in the hardening off process when the seedlings were moved into the greenhouse. Germination of S. repens ex vitro was 100% in an autoclaved soil mixture of 1:1 (v/v) sand and vermiculite. Importantly the medicinal value of S. repens produced in vitro or ex vitro was not lost as the GC-MS metabolite analysis showed that the plants produced the chemicals that are medicinally important. Metabolite extracts of S. repens were for the first time reported to be active against fungi with MIC values lower than 1 mg/ml over 4-5 d period against four Fusarium spp. tested. Lastly (Chapter 5), transcription factors AtMYB6 and AtMYB13 were used to trasnform Salvia stenophylla via Agrobacterium-mediated transformation, in order to determine whether the over-expression of these transcription factors could up-regulate the production of medicinally and commercially important secondary metabolites in S. stenophylla. Whilst both A. tumefaciens and A. rhizogenes strains were utilised for the transformation procedure, transformation was only achieved using A. rhizogenes and no transformants could be generated from the A. tumefaciens-treated material. Transgenic hairy roots did not produce any of the medicinally important metabolites. The GC-MS analysis of the transgenic root material identified mainly sugars and other primary metabolites. / AFRIKAANSE OPSOMMING: Biotegnologie is 'n belangrike instrument wat gebruik kan word om gene te isoleer en te karakteriseer. Dit word ook gebruik om klone wat geneties en fenotipies identies is te produseer. Baie Arabidopsis thaliana transkripsiefaktore is al geïsoleer en gekarakteriseer, maar baie moet nog volledig beskryf word. MYB proteïene is lede van 'n super-familie van multifunksionele transkripsiefaktore wat ook interaksie het met ander transkripsiefaktore tydens die beheer van metaboliese weë. Tot op hede is meer as 126 AtMYBs geïdentifiseer, maar die meeste is nie volledig gekarakteriseer nie, veral nie ten opsigtigte van die molekulêre rol(le) wat hulle in plante speel nie. Arabidopsis thaliana MYB3, MYB6, MYB7, MYB8 en MYB32 is gevind om negatiewe reguleerders van algemene fenielpropanoied-metabolisme te wees. Daar is ook berig dat dié vyf transkripsiefaktore moontlik flavenoied-biosintese negatief kan reguleer, selfs al kan hulle verskillende teikengene hê. Studies op AtMYB13, AtMYB14 en AtMYB15 het berig dat hulle waarskynlik reguleerders van algemene fenielpropanoied-metabolisme is. Die genoemde rolle van die agt AtMYB transkripsiefaktore beteken dat hulle gemanipuleer kan word om te bepaal watter effek hulle op primêre en sekondêre metaboliete in plante het. Die transkripsiefaktore, wat in die pUBI510-GRFCA vektor geligeer was, is toe gebruik om suikerriet-kallus te transformeer (Hoofstuk 3). Suikerriet vervaardig sukrose wat tot 70% van die suiker wat in die wêreld geproduseer word opmaak. Dít maak suikerriet 'n kommersieel belangrike en winsgewende plant. Die suikerriet-kallus is getransformeer deur middel van partikel-bombardering. Die transkripsiefaktore AtMYB3, AtMYB6, AtMYB7, AtMYB13 en AtMYB32 was suksesvol in die DNA van die suikerriet-kallus opgeneem. Data wat verkry was vir kallus wat AtMYB3, AtMYB13 en AtMYB32 ooruitgedruk het op soliede media en kallus in vloeibare medium was teenstrydig (m.a.w. kallus op soilede media wat meer sukrose as die wildetipe op soliede media geproduseer het, terwyl dieselfde transgeniese lyn minder sukrose as die wildetipe geproduseer het in vloeibare medium, en anders om). Nietemin, het AtMYB13 transgeniese lyne meer sukrose geproduseer as die wildetipe. Transgeniese lyne van AtMYB7 het almal minder sukrose geproduseer as die wildetipem op beide soliede en vloiebare media. Die transkriopsiefaktore wat gelei het tot 'n styging in stysel produksie wanneer hulle ooruitgedruk was was AtMYB7 en AtMYB13. Data wat verkry is van die AtMYB6 transgeniese lyne was hoogs veranderlik in lyne wat op dieselfde medium gegroie was en oor die twee media. Die effek van hierdie transkripsiefaktore op die algehele metabolisme van die suikerriet-kallus, hetsy op MSC3 soliede of vloeibaremedia, kon egter nie van die GC-MS analise ten volle bepaal word aangesien daar geen konsekwente fenotipiese effek tussen die verskillende transgeniese lyne vir enige van die gebruikte MYB transkripsiefaktore was nie. In Hoofstuk 4 was ‘n mikropropagerings strategie ontwikkel. Fitochemikalieë en hul biologiese aktiwiteite was ook bepaal vir die medisinale plant Salvia repens. Salvia plante is gevind om medisinaal belangrik te wees as gevolg van die sekondêre metaboliete, veral die essensiële olies, wat hulle produseer. Dit is ook bevind dat die plant-ekstrakte baie biologiese aktiwiteite soos anti-bakteriese, anti-inflammatoriese, anti-oksidant en anti-kanker aktiwiteite het. Salvia repens is suksesvol ontkiem in vitro, met 60% ontkieming wat bereik is in MS media met 1x10-5 maal verdunde rook-water na insnyding vir 12 min in 75% (v/v) H2SO4. Suksessyfers van 100% was behaal in die afhardingsproses wanneer die saailinge na die glashuis verskuif was. Ontkieming van S. repens ex vitro was 100% in 'n geoutoklaveerde grondmengsel van 1:1 (v/v) sand en vermikuliet. Gewigtig het die medisinale waarde van S. repens wat in vitro of ex vitro geproduseer was nie verlore gegaan nie. Die GC-MS data metaboliete analise het aangetoon dat die plante die medisinaal belangrike chemikalieë geproduseer het. Metaboliet-ekstrakte van S. repens was vir die eerste keer na berig aktief teen swamme, met MIK waardes laer as 1mg/ml oor ‘n tydperk van 4-5 d, teen vier Fusarium spp wat getoets was. Laastens (Hoofstuk 5), transkripsiefaktore AtMYB6 en AtMYB13 was gebruik om Salvia stenophylla te transformeer deur Agrobacterium-bemiddelde transformasie, om sodoende te bepaal of die ooruitdrukking van hierdie transkripsiefaktore die produksie van medisinale en kommersieël-belangrike sekondêre metaboliete in S. stenophylla kan verhoog. Alhoewel beide A. tumefaciens en A. rhizogenes stamme gebruik was vir die transformasie proses, kon transformasie slegs deur die gebruik van A. rhizogenes bereik word. Geen transformante kon gegenereer word vanuit die A. tumefaciens behandelde materiaal nie. Transgeniese harigewortels het geen van die medisinaal belangrike metaboliete vervaardig nie. Die GC-MS analise van die transgeniese wortel materiaal het hoofsaaklik suikers en ander primêre metaboliete geïdentifiseer.

Orientador: Giuseppina Pace Pereira Lima / Banca: Eny Iochevet Segal Floh / Banca: João Domingos Rodrigues / Resumo: O presente trabalho teve como objetivo estabelecer o protocolo de desinfestação de estacas e sernentes e germinação de Pothomorphe umbeilata (L) e adaptar o protocolo de micropropagação de Pothomorphe umbeilata (L), incluindo a produção de calos e organogênese, além de comparar o teor de carotenóides entre calos e plântulas. O experimento foi conduzido no Laboratório de Biotecnologia Vegetal do Departamento de Química e Bioquímica, do Instituto de Biociências da UNESP - Botucatu, SP e o material vegetal (sementes e estacas) foi obtido no município de Adrianópolis-PR. Semente germinadas de P. umbeilata foram inoculadas em diferentes concentrações de BAP (0,5 mg.L’; 1,0 mgL’; 1,5 mg.L) e NAA (0,4 mg.L 0,6 mg.L’; 0,6 mg.L’) respectivamente, visando estimular a produção de calos e dosar o carotenóides. Após 60 dias do cultivo, os calos contendo algumas brotações, foram transferidos para meio de diferenciação das plântulas (GA3 0,1 mg.L’, BAP 0,5 mgL’) por um período de 40 dias, para logo serem transferidos para meio de diferenciação de plântulas. Calos (coletados aos 60 dias) e plântulas (coletadas aos 140 dias) foram congelados em nitrogênio líquido e mantidos em freezer a 80°C para posteriores análises de carotenóides. O melhor tratamento para a produção de calos e formação de gemas foi NAA 0.6 mgL’ em combinação com BAP 10 mg.L’. Nas plântulas sem adição de reguladores vegetais foi encontrada uma maior concentração de carotenõides, em comparação aos calos. / Abstract: The present research aimed at establishing a protocol of stalks and seed desinfectation, and seed germination of Pothomorphe umbeilata (L.). A protocol of micropropagation including the induction of callus formation and organogenesis was adapted. Besides, it was compared the carotenoids quantity between Porhomorphe umbeliata calius and plantlets. This experiment was carried out iii Biotechnology Vegetal Laboratory of the Chernistr and Biochemistry Department at the Instituto de Biociências of Universidade Estadual Paulista, Botucatu campus. The vegetal matenal (seed and stalks) was obtained from Adranópolis - PR. Pothomorphe umbeilata germinated seeds were inoculated in different concentrations of BAP (0,5 mg.L-l; 1,0 mg.L.-l; 1,5 mg.L-l) and NAA (0,4 mg.L-l; 0,6 mg.L-1; 0,6 mg.L-l) respectively, in order to stimulate calius induction and increase quantity of earotenoids. Afler 60 days, calius which contained shoots were inoculated in plantlets diferenciation medium GA3 0,1 mg.L4, BAP 0,5 mg.U’) during 40 days and transferred to plantlets growth medium. Plantlets were acclimatized Calius collected after 60 days and plantlets were collected afier 140 days. There were frozen in liquid nitrogen and maintained in freezer 80°C to be used in further carotenoids test. The best treatment for callus production and shoots elongation was NAA 0.6 mg.L’ in association with BAP 1.0 mg.L’. The higher carotenoids coneentration was in plantlets without growth regulators, compared with calius. / Doutor

Atualmente a procura por produtos sustentáveis têm-se mostrado cada vez mais frequente e promissora. Em espécies de importância comercial, procura-se obter a maior produtividade possível dentro de um curto espaço de tempo aliado à preservação do meio ambiente. Dentro disso, a transformação genética de plantas se mostra uma alternativa atrativa para a geração de variedades de cana-de-açúcar que gerem produtos de maneira mais eficaz. O sucesso da transformação genética está diretamente associada a cultura de tecidos de plantas que precisa ser adequada a cada genótipo e situação de cultivo, sendo a luminosidade um dos principais fatores para a produção de plantas vigorosas. Outro fator importante é a seleção das plantas transgênicas, que precisam ser submetidas a uma quantidade de agente seletivo suficiente para identificar as plantas modificadas geneticamente. Em cana-de-açúcar, a identificação de plantas transgênicas por PCR e a definição do número de cópias é um procedimento de difícil execução e muito oneroso. Isto se dá pois no processo transformação via biolística, a inserção de genes é aleatória, produzindo plantas com variados números de cópias. Em consideração a estes fatores envolvidos na eficiência de obtenção de plantas transgênicas de cana-de-açúcar, os objetivos deste trabalho foram o aperfeiçoamento do protocolo de cultura de tecidos, transformação genética da variedade SP803280 com os genes xth, AtDdm1, como também, definir genes de referência para a quantificação do número de cópias dos genes xth e AtDdm1 inseridos na variedade SP803280 e do gene neo na RB835089, análise de ploidia e tamanho de genoma dos eventos transgênicos comparado com as plantas controle. No estudo a respeito da melhor qualidade de luz durante o cultivo in vitro na fase de regeneração de plantas, tem-se que a luz branca e a junção das luzes LED e branca se mostraram melhores para regeneração e desenvolvimento das plantas enquanto que para plântulas, as luzes LED e branca separadamente foram mais efetivas no crescimento. Para a seleção das plantas uma concentração de geneticina entre 40 e 50 mgL-1 é recomendada. As taxas de sucesso nas transformações genéticas para o gene xth variaram entre 2,5 a 18,3% dependendo do experimento e para AtDdm1 foi de 2,2% em um bombardeamento.Não houveram alterações de ploidia e tamanho do genoma nos transgênicos das duas variedades em relação à planta selvagem. Os genes p4h e prr foram identificados como os melhores para a quantificação relativa de genes inseridos por PCR em tempo real na variedade SP803280 enquanto que para a RB835089 aprt e prr se mostraram mais eficazes. A análise do número de cópias inseridas em eventos transgênicos por PCR em tempo real foi possível através das duas metodologias de cálculo testados por este trabalho, com resultados que concordam com uma tendência nesta determinação de maneira simples e rápida. / Currently the demand for sustainable products has been shown to be frequent and promising. In species of commercial importance, there is an effort to obtain the highest possible productivity in a short time, along with the environment preservation. In this context, genetic transformation of plants appears as an attractive alternative for the development of sugarcane varieties able to generate products in a more effective way. The genetic transformation success is directly associated to plant tissue culture that requires specific condition for each genotype and cultivation process, in which, luminosity is one of the main factors that determines the production of vigorous plants. Another important factor is the selection of transgenic plants, that occur by exposing plants to a sufficient amount of selective agent in order to identify only genetic modified plants. In sugarcane, identification of transgenic plants by PCR and the definition of copy numbers is a difficult procedure to implement and usually is very costly. It is because in the process of genetic transformation by biolistic, the insertion of genes occurs randomly and also produce plants with varied copy numbers. In consideration of these factors directly involved in the efficiency to obtain sugarcane transgenic plants the objectives of this study were the improvement of a tissue culture protocol, the genetic transformation of the variety SP803280 with xth and AtDdm1 genes. Also, the studies include the definition of reference genes for determining the number of copies inserted of xth and AtDdm1 genes into the variety SP803280 and neo gene in RB835089, ploidy analysis and genome size of the transgenic events compared to control plants. In the study related to the best light quality for in vitro plant regeneration, white light and the combination of LED and white lights proved to be better for plants regeneration and development while for seedlings, LED and white light separately were more effective for growth. In order to obtain selection of transgenic plants, geneticin concentration between 40 and 50 mg L-1 is recommended. Success rates in xth genetic transformation ranged from 2.5 to 18.3% depending on the experiment, and for AtDdm1 was only 2.2% in just one biolistic bombardment. There were no changes in ploidy and genome size in transgenic events related to their wild type plant. The genes p4h and prr were defined to be the best for determining the copy number of transgenic events by real time PCR in SP803280 variety, while for RB835089, the genes aprt and prr were the most effective. The analysis of the number of inserted copies was possible using the two calculation methodologies tested by this work, with results that agree with a tendency in a simple and fast quantification methodology.

Made available in DSpace on 2014-08-20T14:22:04Z (GMT). No. of bitstreams: 1 Dissertacao_Luciane_Leitzke.pdf: 527181 bytes, checksum: 1bd073c7e15cf594e5e31a8d64217660 (MD5) Previous issue date: 2006-03-05 / The success of mass micropropagation of fruit trees may be reached by using plant tissues culture techniques, since this has showed efficient results on seedlings production with high quality and health. However, for the commercial viability of micropropagation application in the field of horticulture and how this might compete with traditional methods of propagation (cuttings, etc.), it is necessary to decrease production costs. Therefore, the development of photo-autotrophic micropropagation systems (production of micropropagules in sugar-free medium under environmental conditions that promote photosynthesis of the culture) with natural light appear as a possibility to improve the efficiency of micropropagation and to reduce costs. This research aimed the photo-autotrophic multiplication of blackberry (Rubus spp.) cultivar Xavante and raspberry (Rubus idaeus L.) cultivars Batum and Heritage. Preliminary experiments was carried out to define the constitution of culture medium that provides better results, as on multiplication as on in vitro rooting of blackberry and raspberry, under conventional conditions of micropropagation. Then, using the best constitution of culture medium, it was done the study of the photo-autotrophic multiplication by using natural light. The MS medium enriched with BAP at 13 µM was the more efficient treatment on in vitro multiplication of leaves of blackberry Xavante and raspberries Batum and Heritage , inducing a higher number of leaves, shoots and buds. The best rooting condition for explants of the blackberry Xavante was reached by keeping the explants in WPM enriched with 2,5 µM AIB for a week followed by a regulator-free medium growth. Nevertheless, for raspberry Batum rooting, it is necessary the addition of 6,5 µM AIB. Under photo- autotrophic conditions, the aluminum foil was the best sealing material for the flasks. Regarding to in vitro multiplication for blackberry cultivar Xavante the best growth local was in greenhouse with the addition of 22 g L -1 sucrose to the medium; and for raspberry Batum was at 11,5 g L-1 sucrose but kept in growth room. For in vitro rooting of raspberry Batum cotton was the best sealing material, growth room and sugar-free medium were the best condition having a higher rooting percentage and root numbers per explants. / A cultura de tecidos é uma técnica que proporciona com sucesso a micropropagação massal de frutíferas e que já vem sendo utilizada com eficientes resultados para a produção de mudas sadias com alta qualidade. Entretanto, para que a aplicação da micropropagação na fruticultura torne-se viável comercialmente e possa competir com métodos tradicionais de propagação (estaquia, etc), é necessária a redução do custo de produção. Diante disso, o desenvolvimento de sistemas de micropropagação fotoautotrófica (produção de micropropágulos sem adição de sacarose no meio de cultura e sob condições ambientais que promovam a fotossíntese na planta) com o uso de luz natural surge como possibilidade que apresenta potencial para aumentar a eficiência da micropropagação e auxiliar na redução de seu custo. Assim, este trabalho teve como objetivo a multiplicação fotoautotrófica de amoreira-preta (Rubus spp.) cv. Xavante e de framboeseira (Rubus idaeus L.) cvs. Batum e Heritage. Dessa forma, foram realizados os estudos preliminares a fim de definir a constituição do meio de cultura que propicie os melhores resultados, tanto na multiplicação como no enraizamento in vitro de amorapreta e framboesa, sob condições convencionais de micropropagação. A partir daí, foi realizado o estudo da micropropagação fotoautotrófico com o uso da luz natural, utilizando a constituição do meio de cultura que propiciou os melhores resultados. Pelos resultados obtidos, conclui-se que o meio MS adicionado de BAP na concentração de 13µM é o tratamento mais eficiente na multiplicação in vitro de explantes com folhas de amoreira-preta Xavante e framboeseira Batum e Heritage , induzindo maior número de folhas, brotações e gemas. Para o enraizamento in vitro de amoreira-preta Xavante , o meio WPM adicionado de 2,5µM AIB e mantido por uma semana, seguido do cultivo em meio livre de regulador é o melhor meio de enraizamento; para framboeseira Batum , é necessária a adição de 6,5µM de AIB. Em condições fotoautotróficas o alumínio é o melhor modo de vedação dos frascos de cultivo. Para a multiplicação in vitro de amoreira-preta cv. Xavante , o melhor local de cultivo é a casa de vegetação e a adição de 22 g L -1 de sacarose no meio de cultura e de 11,5g L-1 para framboeseira Batum , mantida na sala de crescimento. Para o enraizamento in vitro de framboeseira cv. Batum, o algodão é o melhor modo de vedação dos frascos de cultivo; o melhor local de cultivo é a sala de crescimento, sem a adição de sacarose no meio de cultura, obtendo-se maior porcentagem de enraizamento, número de raízes por explante.