The establishment and germination in vitro can supply a high amount of explants to develop morphogenesis protocols necessary for flowering induction in vitro, haploid and double-haploid plant production, clonal propagation, somatic embryogenesis among other applications. The objective was to develop protocols for the establishment and germination in vitro of bottle gourd (Lagenaria siceraria (Mol.) Standl.) and squash (Cucurbita pepo L.) species to effort morphogenesis studies. Seed disinfection treatments were done with ethanol 70% and NaOCl. Seed germination in vitro was evaluated changing medium-osmotic pressure, light availability, tegument and auxin presence, imbibition s time and scarification methods. Cotyledonary explants were used to induce direct organogenesis. Apical and nodal segments were grown in MS medium without growth regulators. Bottle gourd seeds did not germinate in culture medium, but it occurred on four layers of germitest paper and distilled water in the proportion of 1:7.5 (w/w). Light is not necessary for bottle gourd seed germination. Osmotic pressure reduction did not increase bottle gourd and squash germination. Bottle gourd apical and nodal segments regenerate in culture medium without growth regulators. Cotyledonary explants of bottle gourd and squash induce aerial growth, but in a low proliferation rate / O estabelecimento e a germinação in vitro suprem explantes em grande quantidade para a organização de experimentos em morfogênese, a qual apresenta muitas aplicações, tais como a indução de flores in vitro, a obtenção de plantas haplóides e duplo-haplóides, a propagação clonal em massa, a embriogênese somática, entre tantas outras. O objetivo deste trabalho foi desenvolver protocolos que permitam o estabelecimento e a germinação in vitro das espécies de porongo (Lagenaria siceraria (Mol.) Standl.) e mogango (Cucurbita pepo L.) para subsidiar pesquisas em morfogênese. Foram realizados testes com álcool 70% e NaOCl (Hipoclorito de sódio) para a desinfestação de sementes e investigados vários fatores envolvidos na germinação in vitro, tais como pressão osmótica, fotoblastismo, presença do tegumento, adição de auxinas, tempo de embebição e escarificação. Explantes cotiledonares foram utilizados para a indução de organogênese direta. Ápices caulinares e segmentos nodais foram cultivados em meio MS sem a adição de reguladores de crescimento. Nenhum dos tratamentos utilizados foi eficiente para permitir a germinação de sementes inteiras de porongo em meio de cultura, porém houve germinação em papel germitest umidecido com 7,5 vezes a massa do papel. Não foi verificado fotoblastismo para o porongo. A redução da pressão osmótica não aumentou o percentual de germinação de porongo e mogango. Ápices caulinares e segmentos nodais de porongo regeneram sem a adição de reguladores de crescimento. Explantes cotiledonares podem ser utilizados para a indução de brotações adventícias, em porongo e mogango, porém a taxa de proliferação é baixa

In vitro micropropagation of saffron (Crocus sativus L.) by using direct and indirect organogenesis was the aim of this study. Also, the effect of plant growth regulators on growth parameters, such as corm production, sprouting time and germination ratio were investigated in ex vitro conditions. For in vitro regeneration of saffron, the effects of 2,4-D (2,4-dichlorophenoxyacetic acid) and BAP (6-benzylaminopurine) were tested initially. It was observed that 0,25 mg/L 2,4-D and 1 mg/L BAP combination was superior for indirect organogenesis while 1 mg/L 2,4-D and 1 mg/L BAP combination was favorable for direct organogenesis. During the improvement of direct organogenesis experiments, BAP (1 mg/L) without 2,4-D stimulated further shoot development. For adventitious corm and root induction, NAA (naphthaleneacetic acid) and BAP combinations were tested. Although a few corm formations were achieved, root development was not observed with these treatments. Further experiments with the culture medium supplemented with 1 mg/L IBA (indole-3-butyric acid) and 5% sucrose was effective on obtaining contractile root formation and increasing corm number. As a result, the overall efficiency was calculated as 59.26% for contractile root formation, 35.19% for corm formation and 100% for shoot development. In ex vitro studies, 50 mg/L IAA (indole-3-acetic acid) , 50 mg/L kinetin and 200 mg/L GA3 (gibberellic acid) were used. These applications were not as efficient as expected on assessed growth parameters.

In this study, optimization of a transformation and regeneration system via indirect and direct organogenesis in cotyledon, hypocotyl, petiole, leaf and shoot base tissues of sugar beet (Beta vulgaris L. cv. ELK 345 and 1195) was investigated. Two different germination, three different callus induction and shoot induction medium was used for indirect organogenesis of sugar beet cultivar ELK 345. Except cotyledon, other explants (hypocotyl, petiole and leaf) produced callus. However no shoot development was observed from callus of these explants. Shoot base tissue of sugar beet cultivar 1195 was employed for direct organogenesis. Shoot development was achieved via direct organogenesis using 0.1 mg/L IBA and 0.25 mg/L BA. Root development and high acclimatization rate were accomplished from shoot base tissue. Different concentrations of kanamycin and PPT were applied to leaf blade explants to find out optimum dose for selection of transformants. Kanamycin at 150 mg/L and PPT at 3 mg/L totally inhibited shoot development from leaf blades. Moreover, an Agrobacterium mediated transformation procedure for leaf explants of ELK 345 was also optimized by monitoring transient uidA expression 3rd days after transformation. Effects of different parameters (vacuum infiltration, bacterial growth medium, inoculation time with bacteria, Agrobacterium strains and L-cysteine application in co-cultivation medium) were investigated to improve transformation procedure. Vacuum infiltration and Agrobacterium strains were significantly improved transformation procedure. Percentage of GUS expressing areas on leaves increased three folds from the beginning of the study.

Patchouli is an aromatic species of Asian origin that has been cultivated in various parts of the world for the extraction of essential oil of its leaves. The essential oil is used by the perfume, cosmetic and food industries. Patchouli is an economically important species need to have its genetic material preserved. In this context, the aim of this study was to develop protocols for micropropagation and in vitro conservation of three accessions of patchouli. For the experiments of micropropagation MS medium supplemented with different concentrations of kinetin and IAA or NAA were tested. For the acclimatization assays different mixtures of coconut coir and vermiculite supplemented with NPK fertilizer and limestone were tested. For callus induction, different doses of 2,4-D were combined with BAP and kinetin. For the in vitro conservation assays different growth temperatures, osmotic regulators and/or inhibitors of growth were tested. The different accessions showed shoot regeneration via direct organogenesis. Accession POG002 can be propagated using 0.5 mg.L-1 kinetin and 0.1 mg.L-1 NAA, accession POG014 using 2.0 mg L-1 kinetin and 0.1 mg.L-1 NAA, and accession POG021 using 1.0 mg.L-1 kinetin and 0.5 mg.L-1 IAA. The acclimatization of accessions POG002, POG014 and POG021 can be performed with the substrate coconut coir + NPK (3-12-6) fertilizer (12 g.L-1) + limestone (1 g.L-1). The larger sizes of calluses were obtained using: 0.022 mg.L-1 2,4-D and 0.113 mg.L-1 BAP, 0.022 mg.L-1 2,4-D and 0.225 mg.L-1 BAP and 0.110 mg.L-1 2,4-D and 0.113 mg.L-1 BAP for accession POG014; 0.022 mg.L-1 2,4-D and 0.113 mg.L -1 BAP, 0.022 mg.L-1 2,4-D and 0.225 mg.L-1 BAP for POG021; and 0.022 mg.L-1 2,4-D and 0.225 mg.L-1 BAP for POG002. The conservation of accession POG002 can be realized using 0.5 mg.L-1 abscisic acid for three months at 18°C; accession POG014 can be conservated for three months using 0.5 mg.L-1 of abscisic acid at 18°C; and POG021 can be maintained for six months using sucrose 10 g.L-1 and sorbitol 5 g.L-1 at 25°C. / O patchouli é uma espécie aromática de origem asiática que tem sido cultivada em diversas partes do mundo para extração de óleo essencial de suas folhas. Este óleo essencial é empregado nas indústrias de perfumes, cosmética e alimentícia. Por se tratar de uma espécie de importância econômica local e mundial o patchouli necessita ter seu material genético preservado. Neste contexto, o objetivo geral deste trabalho foi desenvolver protocolos de micropropagação e conservação in vitro de três acessos de patchouli. Para os experimentos de micropropagação foi testado meio MS suplementado com diferentes concentrações de cinetina e as auxinas AIA ou ANA. Nos ensaios de aclimatização testou-se diferentes misturas de pó de coco e vermiculita suplementado com adubo NPK formulado e calcário. Para indução de calos, doses distintas de 2,4-D foram combinadas com as citocininas BAP e cinetina. Nos ensaios de conservação in vitro testou-se diferentes temperaturas de cultivo, reguladores osmóticos e/ou inibidores de crescimento. Os diferentes acessos apresentaram regeneração de brotos via organogênese direta. O acesso POG002 pode ser propagado na presença de 0,5 mg.L-1 de cinetina e 0,1 mg.L-1 de ANA; o acesso POG014 com 2,0 mg.L-1 de cinetina e 0,1 mg.L-1 de ANA; e o acesso POG021 com o emprego de 1,0 mg.L-1 de cinetina e 0,5 mg.L-1 de AIA. A aclimatização dos acessos POG002, POG014 e POG021 pode ser realizada com o substrato pó de coco + NPK (3-12-6) (12 g.L-1) + calcário (1 g.L-1). Os maiores tamanhos de calos foram obtidos nos seguintes meios: 0,022 mg.L-1 de 2,4-D e 0,113 mg.L-1 de BAP, 0,022 mg.L-1 de 2,4-D e 0,225 mg.L-1 de BAP, e 0,110 mg.L-1 de 2,4-D e 0,113 mg.L-1 de BAP para o acesso POG014; 0,022 mg.L-1 de 2,4-D e 0,113 mg.L-1 de BAP, 0,022 mg.L-1 de 2,4-D e 0,225 mg.L-1 de BAP para o POG021; e 0,022 mg.L-1 de 2,4-D e 0,225 mg.L-1 de BAP para o POG002. A conservação do acesso POG002 pode ser realizada com 0,5 mg.L-1 ácido abscísico por três meses a 18°C; o acesso POG014 pode ser conservado por três meses empregando 0,5 mg.L-1 de ácido abscísico a 18°C; e o acesso POG021 pode ser mantido por seis meses com uso de sacarose 10 g.L-1 e sorbitol 5 g.L-1 a 25°C.

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2016-08-24T20:52:09Z No. of bitstreams: 1 Disserta??o Final_Fl?via_PPGRGV (2).pdf: 1857571 bytes, checksum: 967f2b7d728622a11a98a803f13ff0de (MD5) / Made available in DSpace on 2016-08-24T20:52:09Z (GMT). No. of bitstreams: 1 Disserta??o Final_Fl?via_PPGRGV (2).pdf: 1857571 bytes, checksum: 967f2b7d728622a11a98a803f13ff0de (MD5) Previous issue date: 2015-03-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Hyptis ramosa Pohl ex Benth (Lamiaceae) is native and endemic to the semi-arid northeast, with its unknown phytochemical constitution so far. Considering the pharmacological importance of the species of this family, the development of forms of propagation and in vitro culture can contribute to the inclusion of these species in sustainable production systems and the conservation of the same. Thus, the objective of this work was to study the in vitro propagation of the species H. ramosa, through direct organogenesis and callus formation, and the biochemical characterization of the obtained calluses, thus allowing the establishment of strategies for their conservation and sustainable use. To this, H. ramosa seeds were disinfected and established in medium MS / 2 culture. In the multiplication phase was tested the influence of cytokinins BAP, CIN and TDZ on different explants. The obtained shoots were individualized and transferred to MS / 2 medium containing different concentrations of auxin IBA and activated carbon for rooting them. Regenerated and rooted in vitro microplants were subjected to pre-acclimatization in different cultivation container closure and then were transferred to ex vitro conditions in commercial substrate Plantmax? being quantified plant survival rate at 30 days after transfer. For callus induction, we used explants and different concentrations of 2,4-D and BAP, determining the growth curve from the fresh weight of callus until the 28th day of cultivation, at intervals of seven days. Concurrent with obtaining the growth curve it is quantified in calluses obtained the total soluble sugar content, reducing sugar and crude protein. The in vitro propagation of H. ramosa is possible using the nodal segment as a source of explants in MS medium supplemented with BAP. In vitro rooting occurs, even in free auxin. The species showed survival of 100%, regardless of the day of pre-acclimatization phase. For callus induction the best explant is the nodal segment, and the combination of 2.4-D and BAP favor the formation of the same. The callus growth curve showed quadratic behavior with two different phases and biochemical analysis showed the maximum level of total soluble sugars, reducing sugars and crude protein at 14 ?, 21 ? and 14 ? days, respectively. / Hyptis ramosa Pohl ex Benth (Lamiaceae) ? uma esp?cie nativa e end?mica do semi?rido nordestino, sendo sua constitui??o fitoqu?mica desconhecida at? o momento. Considerando a import?ncia farmacol?gica das esp?cies dessa fam?lia, o desenvolvimento de formas de propaga??o e cultivo in vitro poder? contribuir para a inser??o dessas esp?cies em sistemas de produ??o sustent?veis e a conserva??o das mesmas. Diante disso, o objetivo deste trabalho foi estudar a propaga??o in vitro da esp?cie H. ramosa, atrav?s de organog?nese direta e calog?nese, bem como a caracteriza??o bioqu?mica dos calos obtidos, permitindo assim o estabelecimento de estrat?gias para a sua conserva??o e explora??o sustent?vel. Para isso, sementes de H. ramosa foram desinfestadas e estabelecidas em meio de cultura MS/2. Na fase de multiplica??o foi testada a influ?ncia das citocininas BAP, CIN e TDZ sobre diferentes explantes. As brota??es obtidas foram individualizadas e transferidas para meio MS/2 contendo diferentes concentra??es da auxina AIB e de carv?o ativo para o enraizamento das mesmas. As microplantas regeneradas e enraizadas in vitro foram submetidas ? pr?-aclimatiza??o em diferentes tipos de fechamento do recipiente de cultivo e, posteriormente, foram transferidas para a condi??o ex vitro em substrato comercial Plantmax?, sendo quantificada a taxa de sobreviv?ncia das plantas aos 30 dias ap?s a transfer?ncia. Para a indu??o de calos utilizou-se diferentes explantes e concentra??es de 2,4-D e BAP, determinando-se a curva de crescimento a partir da mat?ria fresca dos calos at? o 28o dia de cultivo, em intervalos de sete dias. Concomitante com a obten??o da curva de crescimento quantificou-se nos calos obtidos o teor de a??cares sol?veis totais, a??cares redutores e prote?na bruta. A propaga??o in vitro de H. ramosa ? poss?vel utilizando-se o segmento nodal como fonte de explante, em meio de cultura MS suplementado com BAP. O enraizamento in vitro ocorre, mesmo em meio isento de auxina. A esp?cie apresentou sobreviv?ncia de 100%, independentemente da realiza??o da fase de pr?-aclimatiza??o. Para indu??o de calos o melhor explante ? o segmento nodal, sendo a combina??o de 2.4-D e BAP favor?vel a forma??o dos mesmos. A curva de crescimento de calos mostrou comportamento quadr?tico com duas fases distintas e a an?lise bioqu?mica evidenciou o teor m?ximo de a??cares sol?veis totais, a??cares redutores e prote?na bruta aos 14?, 21? e 14? dias, respectivamente.