Cowpea, Vigna unguiculata (L.) Walp is an excellent source of protein, vitamins and minerals and a major food crop many parts of Africa. Optimal production levels are hampered by insect pests and diseases. Biotechnological techniques such as tissue culture and genetic engineering can aid in the development of varieties with resistance to insect pests and diseases. The objective of this study was to investigate conditions necessary for the development of a reproducible tissue culture system that can be applied to regenerate transformed cells from culture. The in vitro manipulation of cowpea using Murashige and Skoog (MS) medium, auxins and cytokinins resulted in the formation of callus and rhizogenesis. Calli that were formed were separated into six classes based on color and texture. Yellowish friable callus, yellowish compact, soft yellowish callus and green and white were composed of largely vacuolated cells and were non-regenerative. Friable green callus was the most prevalent callus type and could form of roots in some hormone combinations. Green spots were formed on hard compact green callus. The green spots became nodular, forming root primordia and ultimately giving rise to roots. None of the six calli types gave rise to the formation of shoots. Embryogenic callus was induced from cowpea explants cultured on MS medium supplemented with dicamba and picloram. Embryogenic suspension cultures were initiated from callus induced on MS supplemented with 3.0 mg/L dicamba or picloram and conditions for maintenance of embryogenic suspension cultures were evaluated. Somatic embryos were formed in suspension cultures. Attempts to convert and germinate the somatic embryos resulted in the formation of callus or formation of appendages on the somatic embryos or in the death of the embryos. The appendages formed roots on prolonged culture. Further research is needed to determine appropriate optimal conditions for embryo conversion and germination and ultimately plant recovery from culture.

Diversos estudos elucidaram mecanismos envolvidos com a organogênese in vitro, porém pouco é conhecido a respeito da fase de aquisição de competência, fundamental para que a regeneração ocorra. Alguns genes já foram identificados por interferirem na fase de aquisição de competência em tomateiro (Solanum lycopersicum), mas ainda existem diversas lacunas a serem esclarecidas. Para investigar a expressão de genes e o controle hormonal na fase de aquisição de competência, foram utilizados os mutantes de tomateiro, sob o background genético da cultivar Micro-Tom (MT), MT-Rg1 e MT-pro (procera), os quais afetam positiva ou negativamente a organogênese in vitro, respectivamente. Embora a resposta constitutiva a giberelina no mutante MT-pro seja conhecida, a identidade molecular do gene RG1 permanece indefinida. O mutante MT-Rg1 apresenta aumento tanto na formação de gemas caulinares quanto de raízes e reduz o tempo necessário para a indução desses órgãos, devido à diminuição do período para a aquisição de competência. A partir do estabelecimento das fases de aquisição de competência e indução da organogênese in vitro para MT e MT-Rg1, foram identificados genes diferencialmente expressos entre estes genótipos. Entre estes genes, CELL DIVISION CYCLE ASSOCIATED 7 e LACCASE 1A estão regulados positivamente em MT-Rg1 e todos estão fortemente relacionados à fase de aquisição de competência, e a alterações na proliferação de células do protoxilema durante o início da organogênese. Por outro lado, a resposta constitutiva à giberelina no mutante MT-pro reduz a formação de gemas caulinares e raízes e aumenta a formação de calos in vitro, sem afetar o tempo requerido para a indução de gemas caulinares e raízes. De forma oposta a MT-Rg1, o gene CDCA7 apresenta expressão reduzida durante a fase de aquisição de competência em MT-pro, diminuindo o número de células do protoxilema em divisão. Outro fator importante para a divisão celular no mutante MT-pro é o aumento da expressão do gene WUS, causando um aumento da proliferação das stem cells, que são células indiferenciadas relacionadas à formação de novos órgãos. Esta proliferação celular inadequada, somada a uma alteração desfavorável na homeostase das citocininas, justifica o efeito negativo do alelo pro na formação de gemas caulinares, o que permitiu a criação de um novo modelo para organogênese in vitro / Several studies have enabled the discovery of mechanisms to achieve in vitro organogenesis; however, little is known about the phase of acquisition of competence, essential for regeneration. A few genes have been identified to interfere in the acquisition of the competence phase in tomato (Solanum lycopersicum), but there are still many gaps to be filled. We have used the mutants, under the genetic background of the Micro-Tom cultivar, MT-Rg1 and MT-pro (procera), which positively or negatively affect in vitro organogenesis, respectively, to investigate gene expression and the hormonal control in the phase of acquisition of competence. Despite the fact that the constitutive gibberellin response in the procera mutant is well-established, the molecular identity of RG1 gene remains unknown. The MT-Rg1 mutant presents an increase in the formation of both shoot and roots and a reduced period for the induction of these organs, because of the reduced time required for acquisition of competence.We searched for the identity of differentially expressed genes between MT and MT-Rg1 after the establishment of the competence acquisition phase and organogenesis induction stages. Among those genes, CDCA7 and LAC1A are upregulated in MT-Rg1 and these genes appear to be strongly related with the acquisition of competence phase and changes in proliferation of protoxylem cells during early organogenesis. The constitutive response to gibberellin in the MT-pro mutant decreases the formation of shoot and roots and increase in vitro calli formation, without reducing the induction phase of shoots and roots. Unlike MT-Rg1, MT-pro reduces the CDCA7L expression during the acquisition of competence phase, causing a reduction of the protoxylem dividing cells. Another important factor for cell division in MT-pro mutant is the increased expression of the WUS gene, leading to an abnormal proliferation of stem cells. Thereby, this abnormal cell proliferation, in addition to an unfavorable change in the cytokinin homeostasis, justify the negative effect of the pro allele in the shoot formation, which enabled the proposal of a new model for in vitro organogenesis

Diversos estudos têm demonstrado o envolvimento benéfico da utilização do aminoácido glutamina em meios de cultura, favorecendo a organogênese dos tecidos vegetais cultivados. Sabe-se que as fontes de nitrogênio podem influenciar na produção endógena de fitormônios, entretanto o papel exato da glutamina ainda não está bem estabelecido. Em Ananas comosus (L.) Merr., a adição de glutamina ao meio de cultura exerceu efeito promotor sobre a taxa de organogênese e o vigor do crescimento das gemas caulinares a partir de bases foliares. Além da glutamina, discute-se se o estresse resultante da explantação também poderia estar envolvido com a indução do processo organogenético, acarretando na produção de espécies reativas de oxigênio e na alteração do estado redox endógeno. Esse estresse para ser benéfico, entretanto, deveria estar restrito a certo limite. O presente trabalho visou compreender o efeito favorável da glutamina na organogênese adventícia em bases foliares de abacaxizeiro cultivadas in vitro. O envolvimento da glutamina com uma possível diminuição do estresse oxidativo durante o período de indução da organogênese também foi abordado. Para tanto, buscou-se correlacionar a influência do suprimento de glutamina no meio de cultura com os teores endógenos de peróxido de hidrogênio, glutationa e ascorbato. O estado redox da glutationa e do ascorbato durante o período de indução da organogênese adventícia também foi analisado. Além disso, foram analisadas as atividades de duas enzimas antioxidantes nesses explantes foliares, a superóxido dismutase e a catalase. Tentativamente, a glutationa foi adicionada ao meio de cultura, contendo ou não glutamina, visando conhecer o efeito desse antioxidante no processo organogenético. Os resultados mostraram que a glutationa substituiu, mas não intensificou, o efeito benéfico da glutamina sobre a taxa de organogênese das bases foliares de abacaxizeiro. Esse antioxidante não substituiu o efeito positivo do aminoácido no ganho de massas fresca e seca dos eixos caulinares formados, no entanto atuou favoravelmente na formação de um maior número de gemas adventícias por explante inoculado. Ao que parece, o cultivo in vitro das bases foliares gerou um estresse oxidativo nesses tecidos logo no início do período de cultivo, a julgar pela alta concentração de H2O2 detectada nas primeiras 24 horas. Entretanto, essa possível condição estressante foi controlada ao longo do período de cultivo, retornando a uma homeostase do tecido e conferindo condição para que as células se reprogramassem para seguir a uma rota de organogênese caulinar. A glutamina pareceu favorecer a manutenção de um estado redox reduzido tanto de ASC quanto de GSH durante o período em que houve o possível estresse oxidativo. Os resultados das atividades das enzimas antioxidantes sugeriram que a CAT pode ter sido responsável pela regulação do conteúdo endógeno de H2O2, já que a SOD não apresentou alterações expressivas ao longo do período de indução da organogênese tanto em SIM quanto em SIMGln. Em conjunto, os resultados sugerem que o estresse oxidativo causado pelo cultivo in vitro pode ter gerado uma sinalização importante para que a organogênese se inicie, sendo que a glutamina exerceria um papel de manter o estado redox dos tecidos foliares reduzido no momento da maior concentração de H2O2 endógeno. / A positive influence of glutamine on organogenesis of in vitro cultured plant tissues has been demonstrated by several studies. It is well known that the endogenous synthesis of phytohormones can be influenced by nitrogen sources, although it is not completely established in which way glutamine acts in this process. The addition of this amino acid to the culture medium has enhanced the organogenesis rate and resulted in a better vigor of the shoots that were originated from the leaf bases of Ananas comosus (L.) Merr. cultured in in vitro conditions. It is also suggested that the tissue excision may result in a stressful condition by increasing the production of reactive oxygen species and changing the endogenous redox state, which might be involved in the induction of organogenic process. However, this stress should be beneficial only if restricted. The aim of this work was to comprehend the positive influence of glutamine on the in vitro adventitious organogenesis of pineapple leaf bases. It was also attempted to determine whether the glutamine would be involved on a possible oxidative stress decrease during the organogenesis induction. In order to answer these questions, we tried to correlate the presence of glutamine in the culture medium and the endogenous hydrogen peroxide, glutathione, ascorbate levels. The redox state of these antioxidants is also analyzed during the induction of adventitious shoot organogenesis. Moreover, two antioxidants enzymes activities are quantified in the leaf explants: catalase and superoxide dismutase. The glutathione influence on the process was also investigated, considering the glutamine presence or not. It was done in order to establish the effect of this antioxidant in the organogenic process. The results showed that glutathione could replace, but not enhance, the positive effect of glutamine on the organogenesis rate of pineapple leaf bases. This antioxidant did not substitute the positive effect presented by the glutamine on the acquisition of fresh and dry masses by the new shoots. On the other hand, glutathione enhanced the number of adventitious buds per explant. Apparently, the excision of the leaf bases and its subsequent cultivation in the induction culture medium resulted in the tissue oxidative stress early in the first 24 hours of incubation. This could be inferred by the high H2O2 concentrations detected during this period. However, this possible stressful condition was controlled during the culture period, leading to the return of the homeostasis of the tissue and allowing the cells to become determined to shoot organogenesis. During the probable period of oxidative stress, glutamine seemed to maintain the reduced redox state on both ASC and GSH. The results of the antioxidant enzymes activities suggested that CAT may have been responsible for the regulation of the endogenous H2O2 levels, while SOD did not showed significant changes during the induction of organogenesis of leaf bases cultivated either in SIM or SIMGln. Taken together, the results obtained in this work suggest that the oxidative stress caused by the excision of the leaf tissues and its in vitro cultivation may be an important signal to the induction of the leaf organogenesis. Furthermore, the glutamine may have a role in the maintenance of the reduced redox state when higher levels of endogenous H2O2 are present in the tissues.

Cell dedifferentiation is a cell fate regression process in which the cell fate memory of a differentiated cell is erased, leading to regain stem cell characteristics. Auxin regulates both cell dedifferentiation and differentiation in plants. It is unknown how auxin controls the two opposite processes. Here the minimal auxin requirements for cell dedifferentiation were found, molecular markers associated with the cell dedifferentiation event were identified. When cellular auxin concentration exceeds the level of meristem cell, most differentiated cells undergo dedifferentiation. In differentiated cells, the polar auxin efflux system prevents cell dedifferentiation by reducing auxin accumulation, particularly in the presence of exogenous auxin. Classic plant tissue culture experiments have shown that exposure of cell culture to a high auxin to cytokinin ratio promotes root formation and a low auxin to cytokinin ratio leads to shoot regeneration. Since the auxin level is highly elevated in the shoot meristem tissues, it is unclear how a low auxin to cytokinin ratio promotes the regeneration of shoots. To identify genes mediating the cytokinin and auxin interaction during organogenesis in vitro, three allelic mutants that display root instead of shoot regeneration in response to a low auxin to cytokinin ratio are identified using a forward genetic approach in Arabidopsis. Molecular characterization shows that the mutations disrupt the AUX1 gene, which has been reported to regulate auxin influx in plants. Meanwhile, it was found that cytokinin substantially stimulates auxin accumulation and redistribution in calli and some specific tissues of Arabidopsis seedlings. In the aux1 mutants, the cytokinin regulated auxin accumulation and redistribution is substantially reduced. These results suggest that auxin elevation and other changes stimulated by cytokinin, instead of low auxin or exogenous auxin directly applied, is essential for shoot regeneration. In this study, as a part of interaction between auxin and cytokinin it was identified that the induction of ARR5 and ARR6 expression by cytokinin is subjected to the regulation of auxin. The expression of ARR5 and ARR6 follows a mutual exclusive pattern in response to the induction of exogenous auxin in Arabidopsis seedlings and calli. The results suggest that auxin interacts with the cytokinin via a gene and tissue specific induction of the negative regulators in the cytokinin signaling pathway.

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.

Caenorhabditis elegans is a powerful tool to study cellular migration and morphogenesis during organ development. During pharynx development, the dorsal gland cell, g1p, is born in the anterior aspect of the pharyngeal primordium and undergoes a form of morphogenesis called retrograde extension. egl-15, the single Fibroblast Growth Factor Receptor (FGFR) in C. elegans and ina-1, one of two α-integrin receptors, are both required for the proper extension or migration of g1p cell. Mutations in either egl-15 or ina-1 show similar gland cell over-migration defects where the gland cell body migrates past the terminal bulb and is located in proximity of the intestine. The kinase domain of EGL-15 was found to be required for migration and transgenic rescue strategies were used to determine the tissue of EGL-15 function. RNA interference was used to determine if egl-15 and ina-1 are functioning in the same pathway to regulate gland cell migration. / February 2015

Le tégument est formé de deux tissus, un épithélium et un mésenchyme. Il comprend la peau et la cornée. Au niveau de la face, ces derniers ont une origine embryonnaire commune: ectoderme et cellules des crêtes neurales. J'ai tout d'abord contribué à la mise en évidence de l'acquisition par le derme embryonnaire de ses capacités inductrices sur l'épiderme, et de l'indépendance de la différenciation de l'épithélium cornéen vis-à-vis de son mésenchyme, le stroma. Mes travaux principaux ont été d'établir quelle était la signature moléculaire du programme cornéen, et à quel moment et comment ce programme est mis en place chez l'embryon. La comparaison des transcriptomes des cellules souches de la cornée à celui des cellules souches épidermiques chez la souris montre 3621 gènes communs et 1768 gènes cornéens propres, en plus de Pax6, le gène clé de l'oeil, et de K12, la kératine type de la différenciation terminale de l'épithélium cornéen. La cornée résultant, selon un dogme ancien, d'une induction par le cristallin, j'ai effectué des expériences chez l'embryon de poulet de 2 jours afin de le vérifier. L'ablation chirurgicale de la placode cristallinienne ayant mis en évidence sa régénération, j'ai prévenu sa formation en électroporant Gremlin, un inhibiteur de BMP4, requis lors la spécification du cristallin par la vésicule optique. L'obtention d'oeil sans cristallin mais avec un épithélium cornéen exprimant K12 montre que le programme cornéen s'effectue par défaut lorsque le programme cristallinien est interrompu. Cet arrêt se produit normalement à la périphérie de la placode cristallinienne qui s'invagine, lors de la migration des cellules des crêtes neurales, productrices de Gremlin. Les précurseurs de l'épithélium cornéen sont donc communs avec ceux du cristallin, qui sont connus pour se ségréger chez l'embryon lors de la formation du domaine préplacodal au stade neurula.

The Nance-Horan syndrome gene (NHS) plays a role in lens, eye and brain development. To date, the function of NHS remains unclear. Recent evidence showed that p53 isoform, Δ113p53, inhibits abnormal cell growth during organogenesis. We show that NHS is expressed in the retinas of Danio rerio and Xenopus tropicalis during key stages of retinogenesis, and that knockdown of the gene resulted in a small eye phenotype in both species. Initially, knockdown of nhsb in zebrafish had no visible defects at 24hpf. But examination of the retina at 48hpf, we see a marked difference in size compared to control embryos. Cell proliferation is a major feature of the developing retina from 24 hpf to 48 hpf. Differentiation of neurons was delayed, while the total number of cells that makes up the volume of the retina was markedly reduced. Here we show that the small retina in nhsb knockdown embryos are due to p53-dependent cell cycle arrest with specific induction of p53 target gene, Δ113p53 and p21. Δ113p53 protects nhsb- knockdown cells from p53-mediated apoptosis. We hypothesize that nhsb overcomes a proliferation restriction in retina progenitor cells during retinogenesis, while knockdown of nhsb increases expression of Δ113p53 and p21, lengthening the cell cycle.

O óxido nítrico (NO) tem se mostrado um sinalizador presente em muitas fases do desenvolvimento vegetal e elemento essencial nas respostas aos estresses bióticos e abióticos em plantas. Entretanto, poucos estudos correlacionam esse radical à organogênese vegetal. Folhas de Ananas comosus (Bromeliaceae) podem ser consideradas um modelo interessante para o estudo da organogênese, pois sua região basal possui um conjunto de células que, sob cultivo in vitro e balanço adequado de certos reguladores de crescimento, dá início à proliferação celular e formação de meristemas adventícios. Primeiramente, formam-se estruturas denominadas protuberâncias que, depois, se desenvolvem em novos eixos caulinares, sem formação intermediária de calos. A relação ótima dos reguladores de crescimento para a indução da organogênese adventícia é 0,53 uM de ácido naftalenoacético e 8,87 uM de benziladeninapurina e sabe-se que o balanço hormonal entre auxinas e citocininas endógenas é alterado nesses explantes, encontrando-se um pico desses hormônios no terceiro dia da indução. Esse trabalho teve como objetivo verificar se o NO participaria da cascata de sinalizações que leva à organogênese adventícia, utilizando bases foliares de abacaxizeiro cultivadas in vitro. A quantificação do NO foi determinada pela técnica de quimiluminescência durante um período de 7 dias a partir da obtenção dos explantes. Essa produção foi dosada em fluxo contínuo com medições feitas a cada 10 segundos. O tratamento indutor de organogênese mostrou um aumento significativo na produção de NO em comparação aos explantes incubados em meio de cultura sem reguladores de crescimento, indicando uma possível participação do óxido nítrico no processo da organogênese. Houve um pico de NO no primeiro dia logo nas primeiras horas de cultivo em meio indutor. Além disso, foi realizada a dosagem de peróxido de hidrogênio (H2O2) e foi encontrada uma correlação com a produção de óxido nítrico durante a fase clara do fotoperíodo. A produção de H2O2 aumentou em seguida ao incremento de NO no primeiro dia de cultivo no meio de cultura com reguladores de crescimento. Finalmente, o tratamento com L-NAME mostrou ser a enzima NOS uma possível rota de síntese de NO, já que reduziu significativamente o número de novos meristemas adventícios e, conseqüentemente, de novos eixos caulinares formados após 60 dias de cultivo em meio não indutor da organogênese. A localização in situ do NO pareceu indicar que o aumento da produção estaria mais relacionado com células do parênquima foliar e dos feixes vasculares. Assim, esta pesquisa permitiu incluir temporalmente dois novos possíveis sinalizadores (NO e H2O2) à cascata de sinalização da organogênese adventícia em folhas de abacaxizeiro. A sinalização parece iniciar-se com o NO e logo em seguida, ainda no primeiro dia, viria o aumento de H2O2 e dos hormônios auxinas e citocininas no terceiro dia (resultados anteriores do grupo) do período de indução. / The nitric oxide (NO) was shown to be an ubiquitous molecule for many phases of the development of a plant and an important element for biotic and abiotic stress responses. However, few studies correlate this radical with the organogenesis in plants. Ananas comosus> (Bromeliaceae) leaves can be considered an interesting model for the organogenesis studies, because the basal region have a set of cell which, under in vitro cultivation and fine growth regulators balance, start cell division and form adventitious meristems. Firstly, structures named protuberances are formed, which develop forming new buds, without the intermediary formation of callus. The optimum rate of the growth regulators for the induction to organogenesis is 0.53 uM of naphthalene acetic acid and 8.87 uM of benziladeninepurine. It is known that the endogenous hormonal balance between auxins and cytokinins is altered in these explants, and a peak of these hormones is found at the third day of induction. This work intended to verify whether the NO would participate on the signaling cascade that led to the adventitious organogenesis, using basal leaves of pineapple cultivated in vitro. The nitric oxide quantification was made by chemiluminescence for a period of 7 days after the explants obtainment. The production was dosed in continuous flow air with measurements made every 10 seconds. The inductive treatment to organogenesis showed a significant rise in NO production in comparison to the explants incubated in medium culture without growth regulators, indicating a possible participation of NO at the organogenesis process. There was a NO peak at the first day, soon at the first hours of cultivation at inductive medium. Besides, the dosage of hydrogen peroxide (H2O2) was done and a correlation was found to the NO production at the light phase of the photoperiod. The H2O2 production rose after the increase of NO at the first day of cultivation in medium containing growth regulators. Finally, the treatment with L-NAME showed the NOS enzyme as a possible pathway involved with the NO production, since the number of new adventitious meristems reduced significantly and subsequently, of new shoots after 60 days of cultivation in inductive to organogenesis medium. The in situ localization of NO seemed to indicate that the rise in production could be related to the cells of the leaf parenchyma and vascular tissues. Thus, this research permitted to include temporally two new possible signals (NO and H2O2) to the signalization cascade of the adventitious organogenesis in pineapple leaves. The signalization seems to initiate with NO followed by H2O2 at the first day and by the auxins and cytokinins hormones at the third day (previous results of the group) of the induction period.