Banana production ranks fifth behind cereals as a food crop and has potential, along with other major crops, to feed the world's increasing population. Globally, continuous efforts and techniques including the use of plant tissue culture (PTC) have been devised for increasing the production of several Musa species. The choice of cytokinin (CK) is one of the most critical factors in developing a successful PTC protocol. Since the discovery of topolins as naturally occurring aromatic CKs, they have emerged as genuine alternatives to the long serving CKs (benzyladenine = BA, zeatin = Z and kinetin = KIN) in PTC. Globally, the past 15 years has witnessed a surge in the use of topolins and their derivatives in research laboratories. Topolins have demonstrated great potential during culture initiation and protocol optimization as well as for counteracting various in vitro induced physiological disorders in some species. In terms of general physiology (growth, phytochemical and photosynthetic pigment contents as well as genetic fidelity), the topolins were compared with BA using 'Williams' bananas with minimal residual exogenous CK carry-over effects. The five topolins tested were meta-Topolin (mT); meta-Topolin riboside (mTR); meta-Methoxy topolin (MemT); meta-Methoxy topolin riboside (MemTR) and meta-Methoxy topolin 9-tetrahydropyran-2-yl (MemTTHP). Based on evidence of potential CK- and auxin-like activity of smoke-water (SW) and karrikinolide (KAR1) at low concentrations, a similar comparative study involving both compounds and mT was performed. For a further understanding of banana physiology in vitro, the effect of supplementing either mT- or BA-requiring cultures with roscovitine (a cyclin-dependent kinase and N-glucosylation inhibitor) and INCYDE (an inhibitor of CK degradation) on the endogenous CK profiles was investigated. In addition, greenhouse experiments geared towards improving the acclimatization competence of tissue-cultured banana plantlets via application of different concentrations of SW and vermicompost leachate was conducted. Sterile shoot-tip explants were cultured on modified Murashige and Skoog (MS) media supplemented with 10, 20 or 30 μM of the tested CKs for 42 days while rooting experiments involved the use of classic auxins as well as SW and KAR1. Apart from 10 μM BA and 30 μM MemTTHP treatments, the number of shoots produced with all the CK treatments were significantly higher than the control. Treatment with 30 μM mT resulted in the highest number of shoots (7.3±1.0) which is an indication of the requirement of exogenous CK for increased shoot proliferation in 'Williams' bananas The use of 10 μM MemTTHP had the least root inhibitory effect during the shoot proliferation phase. As an indication of the toxicity of applied CK, MemT- and MemTR-regenerants were the most deformed while mTR-regenerated plantlets demonstrated the best quality across all the CKs tested. In mT- and BA-derived shoots, SW and KAR1 significantly increased the number and length of roots compared to the control. During the rooting phase, topolin treatments produced more off-shoots than BA-treated ones which inevitably improved the overall number of regenerated shoots. Total phenolic levels were highest in 10 μM mT- and 30 μM MemTTHP-treated plantlets detected in the aerial and underground parts, respectively. It is interesting that in the underground parts, 10 μM mT resulted in the production of the highest amount of proanthocyanidins which was approximately five-fold higher than in the control plants. On the other hand, 10 μM MemTTHP-treated plantlets had significantly higher total flavonoids within the aerial parts. In view of the stimulation of secondary metabolites in the majority of the CK-treated plantlets, the current results indicate the role of the type and concentration of applied CK as potential elicitors in PTC. Generally, the maximum photosynthetic pigment content was attained between 40-50 days. The control plantlets had the highest pigment content (1150 μg/g FW) while 10 μM MemTTHP had the best pigment stimulatory effect among the tested CKs. Nevertheless, in vitro propagation of banana devoid of CKs is not a practical option due to low shoot proliferation rates. Scanning electron microscopy (SEM) of the foliar surface showed that the stomatal density was highest in 10 μM MemTTHP-treated and lowest in 10 μM MemTR-treated plantlets. Prolonging the culture duration as well as increasing CK concentrations reduced the pigment content. However, the drastic breakdown in chlorophyll pigments beyond 50 days was slightly inhibited by the presence of mT, mTR, MemTTHP and BA compared to the control. Current findings indicate the potential anti-senescence activity of the topolins such as mT, mTR and MemTTHP under in vitro conditions. This study articulates that the right choice and concentration of CKs applied during in vitro propagation may alleviate photomixotrophic-induced physiological stress that usually accompanies the transfer of plantlets to ex vitro conditions. Findings indicate that the effect of subculturing contributed significantly to the higher rate of variation in 'Williams' bananas in vitro. The presence of CK in the culture media apparently aggravated the stress on the explants as indicated in the relatively higher percentage polymorphic bands compared to the controls. Among the tested CKs, the use of mTR and MemTTHP caused the least detrimental effect on the regenerants while mT-treated plantlets had the most polymorphic bands. Hence, it is recommended that subculturing cycles from the initial explant establishment should be limited to a maximum of five. The use of SW and KAR1 improved the level of photosynthetic pigment and phenolic compounds in the micropropagated bananas. However, they had a negative effect on shoot proliferation; hence their inclusion is more desired when used at the rooting phase of micropropagation. Perhaps, these compounds could be used in conjunction with auxin to increase the number of roots prior to the acclimatization stage. The enhanced photosynthetic pigment level resulting from addition of SW and KAR1 would also play a vital role during acclimatization of the micropropagated plants. The present finding serves as an alternative approach, available to researchers for improving the quantity of secondary metabolites in micropropagated plants. The highest regeneration rate (93%) was observed in BA + roscovitine treatment while mT + INCYDE-treated plantlets produced most shoots. Treatment with BA + roscovitine had the highest shoot length and biomass. Although not significant, there was more proanthocyanidins in BA + roscovitine treatments compared to the treatment with BA alone. On the contrary, total phenolics were significantly higher in mT + roscovitine treatment than in the mT-treated regenerants. The presence of roscovitine and/or INCYDE had no significant effect on the photosynthetic pigments of the banana plantlets. Forty-seven aromatic and isoprenoid CKs categorized into nine CK-types were detected at varying concentrations. The presence of mT + roscovitine and/or INCYDE increased the levels of O-glucosides, while 9-glucosides remained the major derivative in the presence of BA. Generally, the underground parts had higher CK levels than the aerial parts; however the presence of INCYDE increased the level of CK quantified in the aerial parts of both CK treated plantlets. Apparently, the presence of INCYDE serves to enhance transportation of the CK towards the aerial regions. From a practical perspective, the use of roscovitine and INCYDE in PTC could be crucial in the alleviation of commonly observed in vitro-induced physiological abnormalities. Soil drenching with SW significantly increased the root length (1:1000 and 1:500 dilutions) as well as fresh and dry weight (1:1000; 1:500 and 1:250 dilutions) when compared to foliar application. Vermicompost leachate (1:10 and 1:5 dilutions) significantly enhanced the shoot length, root length, leaf area and dry weights. Vermicompost leachate (1:20; 1:10 and 1:5 dilutions) also significantly increased the number of off-shoots. The positive effect on rooting is beneficial for acclimatization and establishment of tissue-cultured banana plantlets in nurseries and subsequent transfer to the field. However, field trials will be necessary to substantiate the effects demonstrated by these compounds. In an attempt to contribute to improving banana micropropagation, the current findings provide additional evidence on the increasing advantage of topolins over BA. Nevertheless, some detrimental physiological effects observed with some of the topolins (for example, MemT and MemTR) are clear indication that they should not be taken as a panacea in PTC. Besides optimizing efficient PTC protocols through stringent choice of CKs, other associated physiological and metabolic events taking place in culture during the optimization process need more in-depth investigation. In addition to contributing towards the better understanding of the mode of action of these CKs, such an approach will help solve associated physiological and developmental problems in vitro. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Transport and post-harvest handling of flowers both cut and potted is one of the greatest challenges in the horticulture industry (REDMAN et al., 2002). Ethylene-induced flower abscission is responsible for the loss of crops (KIM et al., 2007). Flower abscission is greater when plants are transported (ABEBIE et al., 2005). This limits the sale of flowers and potted plants to areas close to the site of production and prevents export opportunities. South Africa is home to many spectacular species with great horticultural potential (RICE et al., 2011). Unfortunately however, development of a number of these species for export is difficult due to transport-induced flower abscission. Transport-induced flower abscission is a problem experienced by Dr Gert Brits, a breeder of Plectranthus in Stellenbosch in South Africa. In this study a number of Dr Brits’s Plectranthus varieties were used as model plants to understand the process of transport-induced flower abscission and develop a protocol for the prevention of such abscission. Flow cytometry was used to determine the ploidy levels of each of the varieties. It was important to be aware of this during the experiments as varieties with different ploidy levels have been reported to behave differently under stressful environmental conditions. Of the eight varieties examined, three were diploid (2n), one was triploid (3n), three were tetraploid (4n) and one was a mixopliod (2n/4n) variety. To determine the effects of packaging plants during transport and the effects of darkness on flower abscission, plants were packaged into perspex chambers and kept either in a 16 h photoperiod or in darkness for 96 h. Every 24 h the number of open and unopened flowers that had abscised was recorded. Both packaging and darkness increased flower abscission of open and unopened flowers in all eight varieties. Four varieties preferentially abscised open flowers; while the remaining four preferentially abscised unopened flowers. All eight varieties were exposed to different concentrations of ethylene (0, 0.1, 0.25 0.5, 1 and 2 μll-1) to determine their level of ethylene sensitivity. All of the Plectranthus varieties were determined to be extremely sensitive to ethylene. With 100% flower abscission occurring within 24 h at 1 and 2 μll-1 in all varieties. In order to determine what internal changes were causing this increase in flower abscission under these conditions, the changes in the expression of key ethylene biosynthetic enzymes, cytokinin content and carbohydrates in the flowers were examined. ACS and ACO are the two key enzymes in the ethylene biosynthetic pathway (JOHNSON & ECKER, 1998). Changes in the levels of mRNAs coding for these two enzymes were examined when plants were packaged and put into the dark. In general there was an upregulation of the ethylene biosynthetic pathway and in turn this may have increased ethylene production by the plants under simulated transport conditions. However, the changes were not large enough to be solely responsible for the increased flower abscission observed under simulated transport conditions. The concentrations of 43 cytokinins were measured in pedicle tissue from plants which had been kept in the dark for 0, 24, 48, 72 and 96 h. Of the 43 cytokinins measured 21 were below the level of detection. Concentrations for the remaining 22 cytokinins at each of the time points were examined and it was found that in general cytokinin concentrations increase when plants are packaged and put into the dark. DHZ-type cytokinins remained stable during the 96 h continuous dark monitoring period, with most of the changes observed in the tZ and iP types. Peaks in cytokinin concentrations are often followed by an increase in flower abscission, indicating that an increase in cytokinin concentrations may be one of the factors causing the increase in transport-induced flower abscission. Only glucose and fructose were detected in peduncle tissue. Changes in glucose and fructose over 24 h in the greenhouse and over 0, 24, 48, 72 and 96 h in simulated transport conditions were measured. During the day, glucose and fructose levels increased towards the afternoon and evening and decreased in the early morning. This is consistent with studies conducted on other species (ALONI et al., 1996). When plants were put into the dark, glucose and fructose levels increased slightly at 24 h and then decreased to levels similar to those measured in control plants. Although there were changes in glucose and fructose level in simulated transport conditions, they were very slight and it is unlikely that these changes are not responsible for the transport-induced flower abscission. These results suggest that the observed transport-induced flower abscission is the result of increased cytokinin concentrations and expression of ACO and ACS genes when plants are packaged and put into the dark. These changes in turn cause an increase in ethylene production by the plants, and the build-up of ethylene in the transport container causes flowers to abscise. Ethylene perception by the plant is the step which could be targeted to prevent flower abscission. A number of ethylene antagonists block the ethylene receptors in the plant and in so doing prevent the receptors from binding ethylene and transducing the abscission signal. 1-MCP isone such ethylene antagonist. To test whether 1-MCP could be used for the prevention of flower abscission in Plectranthus, plants were placed in sealed perspex chambers in the light and in the dark and treated with 100 nll-1 1-MCP for a single 6 h treatment, or for 6 h every day prior to continuous exposure to ethylene. 1-MCP treatment greatly reduced ethylene- and transport-induced flower abscission when plants were treated continuously, but reduced flower abscission for the first 24 h when pre-treated with a single 6 h exposure to 1-MCP.Transport-induced flower abscission in Plectranthus is the result of exposure to ethylene. The increase in ethylene production by the plants in transport conditions is likely due to an upregulation of the ethylene biosynthetic pathway and an increase in cytokinin concentrations or movement in the pedicle tissue. This transport-induced flower abscission can be prevented by continuous treatment with 100 nll-1 1-MCP during the transport period. By using 1-MCP plants can be transported for up to 4 d and the opportunity for export is made possible. / Thesis (Ph.D.)-University of KwaZulu-Natal, Piertermaritzburg, 2013.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Estudos anteriores realizados no Laboratório de Fisiologia Vegetal do IBUSP com Guzmania monostachia demonstraram que quando essas plantas são submetidas ao déficit hídrico ocorre a indução do CAM, com maior expressão desse metabolismo na porção foliar apical. Para outra espécie (Vriesea gigantea), foi verificada a maior atividade da enzima nitrato redutase (NR) na porção basal durante o período diurno. Em uma bromélia terrestre (Ananas comosus) foi observada a sinalização por citocininas tanto na indução da expressão gênica, quanto na ativação da NR. Outros laboratórios evidenciaram que plantas de Mesembryanthemum crystallinum induzidas ao CAM apresentaram uma provável regulação negativa da fosfoenolpiruvato carboxilase (PEPC) por citocininas. Em decorrência desses conhecimentos acumulados, surgiram novos questionamentos: haveria variações diuturnas da atividade das enzimas PEPC e NR nas diferentes porções das folhas de G. monostachia induzidas ao CAM? A maior disponibilidade de esqueletos carbônicos à noite (acúmulo de acidez) influenciaria positivamente a atividade da NR, deslocando seu pico de atividade para o período noturno? As variações dos teores endógenos de citocininas acompanhariam as possíveis mudanças da atividade da PEPC e da NR, indicando, assim, a participação dessa classe hormonal na regulação dessas enzimas? O presente trabalho teve por objetivo principal investigar uma possível regulação da atividade das enzimas PEPC e NR por citocininas em folhas destacadas da bromélia epífita com tanque, Guzmania monostachia (Bromeliaceae) induzidas ao CAM. Foi esperado com esta pesquisa aprofundar os estudos sobre a inter-relação entre o comportamento fotossintético, a capacidade de assimilação de nitrogênio e a possível regulação das atividades da PEPC e da NR por citocininas endógenas. Análises de acidez titulável, ácidos orgânicos, amido endógeno e da atividade da enzima malato desidrogenase (MDH) foram realizadas, confirmando a indução do CAM nas folhas isoladas de G. monostachia mantidas em polietilenoglicol (PEG) a uma concentração de 30%. O uso desse composto foi eficiente na redução do conteúdo relativo de água e na imposição da deficiência hídrica foliar. Além disso, pôde-se verificar a maior expressão do CAM na porção apical das folhas mantidas em PEG 30%, quando comparada à porção basal. Análises da atividade da PEPC e da NR permitiram verificar a separação espacial dessas enzimas. A primeira apresentou maior atividade no ápice foliar, enquanto a segunda mostrou a maior atividade na porção basal. Apesar disso, não foi observada a separação temporal dessas enzimas, uma vez que ambas apresentaram picos de atividade noturna. A maior atividade da NR durante o período escuro (01 hora) foi verificada nas folhas-controle ou sob deficiência hídrica. Esse resultado sugere que outros fatores, diferentes do metabolismo CAM, influenciaram para a ocorrência da maior atividade dessa enzima durante o período noturno. Os resultados obtidos ainda sugerem que as citocininas possivelmente atuaram como um regulador negativo para a atividade da PEPC durante o dia, uma vez que os maiores níveis endógenos desse hormônio foram observados durante esse período, enquanto a maior atividade dessa enzima foi verificada durante a noite, quando os teores de Z+iP decaíram significativamente. A aplicação de Z ou iP resultou também num decréscimo da atividade dessa enzima. Por outro lado, as citocininas atuaram como um provável regulador positivo para a atividade da NR, uma vez que a maior atividade noturna dessa enzima foi antecedida em 3 ou 6 horas pelos maiores níveis endógenos de citocininas na porção basal das folhas mantidas em água ou PEG 30%, respectivamente. A aplicação de citocininas-livres aumentou significativamente a atividade da NR na base das folhas destacadas mantidas em água ou PEG 30% / Prior studies undertaken in the Laboratory of Plant Physiology on IBUSP with Guzmania monostachia have shown that during water shortage, CAM induction occurs with greater expression in the apical portion of the leaf. In the case of another species (Vriesea gigantean), more intense nitrate reductase (NR) enzyme activity was observed in the basal portion during the daytime. In a certain terrestrial bromeliad (Ananas comosus), signaling by cytokinins, both in the induction of gene expression as well as NR activation, was observed. According to other laboratories, the cytokinins seem to play a negative regulation of phosphoenolpyruvate carboxylase (PEPC) in CAM induced Mesembryanthemum crystallinum plants. As a result of accumulated knowledge, new questions have arisen, such as: Are there daily variations in PEPC and NR enzymes activity in the different portions of CAM induced leaves of G. monostachia? Would the more pronounced nocturnal availability of carbon skeletons (accumulation of acidity) positively influence NR activity, with consequential displacement of its peak of activity to this period? Would variations in endogenous cytokinins concentration accompany possible changes in PEPC and NR activity, thereby indicating the participation of this hormonal class in their regulation? The main aim in the present study was to investigate the possible regulation of PEPC and NR activity by cytokinins in detached CAM-induced leaves of the epiphyte tank bromeliad Guzmania monostachia (Bromeliaceae). The expectations with this research were to study more deeply the inter-relationship between photosynthetic behavior, the capacity for nitrogen assimilation and the possible regulation of PEPC and NR activity by endogenous cytokinins. Analyses of titratable acidity, organic acids, endogenous starch and malate dehydrogenase (MDH) enzyme activity confirmed CAM induction in isolated leaves of G. monostachia kept in polyethylene glycol (PEG) at a concentration of 30%. The use of this compound was efficient in reducing relative water content and imposing leaf water deficiency. Furthermore, compared to the basal portion, greater CAM expression could be observed in the apical portion of leaves kept in PEG 30%. Analyses of PEPC and NR activity allowed detecting their mutual spatial separation, seeing that, in the first greater activity was concentrated in the leaf apex, while in the second this was more pronounced in the basal portion. Even so, no temporal separation could be observed, since peak of activity for both occurred at night. The peak of nocturnal NR activity (1 hour) was observed in control leaves or those undergoing water deficiency, thereby implying that factors, other than CAM metabolism, exerted an influence on the occurrence of more intense activity of this enzyme at this time. Furthermore, there were indications that cytokinins possibly act as a negative regulator of PEPC activity during the daytime, when the highest endogenous levels of this hormone were observed, whereas it was apparent that the most intense activity of this enzyme actually occurred at night, when Z+iP rates decreased significantly. Z or iP application also induced a decrease in the activity of this enzyme. On the other hand, the cytokinins acted as a positive regulator of NR activity, since the nocturnal peak of activity of this enzyme was preceded by 3 or 6 hours by higher endogenous levels of cytokinins in the basal portion of leaves maintained in water or PEG 30%, respectively. The application of free cytokinins induced a significant increase in NR activity in the base of detached leaves kept in water or PEG 30%

En réponse à une carence en azote dans le sol, les légumineuses sont capables d’interagir avec une bactérie du sol, Rhizobium, pour former un nouvel organe spécifique: la nodosité fixatrice d’azote atmosphérique. Chez la plante modèle des légumineuses Medicago truncatula, le récepteur aux cytokinines MtCRE1 est essentiel pour cette interaction symbiotique. Cependant, trois autres récepteurs aux cytokinines CHASE Histidine Kinase (CHK) existent chez M. truncatula. Les quatre CHKs ont des profils d’expression redondants dans les étapes précoces de la formation des nodosités, et plus divergeant dans les nodosités différenciées, même si MtCRE1 est le plus exprimé. Le locus génomique du plus proche homologue de MtCRE1 chez la plante non-symbiotique Arabidopsis, AHK4, complémente l’initiation des nodosités, mais seulement partiellement les phénotypes de croissance des nodosités et de fixation d’azote. Parmi les Régulateurs de Réponse de type B agissant en aval des CHKs, RRB3 a été sélectionné pour réaliser une stratégie de délétion de domaines protéiques, révélant son rôle positif dans la nodulation. Des données transcriptomiques indiquant une régulation de MtCRE1 dans l’épiderme en réponse à un « traitement symiotique », des approches fonctionnelles ont été réalisées et ont permis d’identifier un rôle négatif des cytokinines et de la voie MtCRE1 dans l’épiderme en réponse à ces « conditions symbiotiques ».En parallèle de ces travaux, des mutants des CHKs chez le pois ont été générés. A terme, ces recherches permettront de sélectionner un génotype tolérant à différents stress biotiques et abiotiques sans affecter les symbioses bénéfiques chez une espèce d’intérêt agronomique. / Legume plants adapt to low nitrogen by developing an endosymbiosis with nitrogen-fixing soil bacteria to form a new specific organ: the nitrogen-fixing nodule. In the Medicago truncatula model legume, the MtCRE1 cytokinin receptor is essential for this symbiotic interaction. As three other CHASE Histidine Kinase (CHK) cytokinin receptors exist in M. truncatula, we determined their potential contribution to this symbiotic interaction. The four CHKs have extensive redundant expression patterns at early nodulation stages but diverge in differentiated nodules, even though MtCRE1 has the strongest expression. Interestingly, a genomic locus of the MtCRE1 homolog from the aposymbiotic Arabidopsis plant, AHK4, rescues noduleinitiation, but only partially the nodule growth phenotype, and not the nitrogen fixation capacity. Among type-B Response Regulators acting downstream of CHKs, RRB3 has been selected to perform protein deletions, revealing a positive role RRB3 in nodulation. Transcriptomic data indicating an epidermis MtCRE1 regulation during a « symbiotic traitment », functional approaches showed a negative role of cytokinins and MtCRE1 pathway in epidermis in response to « symbiotic conditions ». chk mutants have been generated in Pisum sativum. Ultimately, the aim of these researches is to set the bases to select in legume species of agronomic interest a genotype tolerant to biotic and abiotic stresses without affecting beneficial symbioses.

L’interaction symbiotique légumineuses-rhizobium nécessite l'infection des racines de la plante par les bactéries et l’initiation de divisions cellulaires dans le cortex racinaire.Les cytokinines sont des hormones végétales agissant via une signalisation par phosphotransfert qui conduit à l’activation de Régulateurs de Réponse de type B (RRBs), des facteurs de transcription régulant l'expression des gènes de réponse primaire aux cytokinines. Une étude phylogénétique menée sur plusieurs espèces de légumineuses a révélé une expansion génique de la famille des RRBs et l’apparition de formes non-canoniques de ces facteurs de transcription. Chez Medicago truncatula,MtRRB3 est le RRB le plus fortement exprimé dans les racines et les nodosités et est impliqué dans la nodulation. En effet, les plantes dont l’expression de MtRRB3 a été réduite par ARNi ainsi que des mutants rrb3 présentent une diminution significative du nombre de nodosités formées. De plus, l’expression de gènes associés à la nodulation, tels que "Nodulation Signalling Pathway 2" (MtNSP2) et "Cell Cycle Switch 52A"(MtCCS52A), est réduite en réponse aux cytokinines dans ces mutants. Des fusions transcriptionnelles avec le rapporteur GUS montrent que MtRRB3, MtNSP2 et MtCCS52Aprésentent un profil d’expression spatiale largement chevauchant dans les racines et lesnodosités. Des expériences de ChIP-qPCR et de trans-activation en protoplastes indiquent par ailleurs que MtRRB3 peut respectivement interagir avec et activer les promoteurs des gènesMtNSP2 et MtCCS52A. Cette thèse a donc permis d’établir des mécanismes moléculaires impliqués dans les régulations transcriptionnelles médiées par les cytokinines lors de la mise en place des nodosités symbiotiques fixatrices d’azote. / The legume-rhizobium interaction requires the infection of plant roots by rhizobia and the initiation of cell divisions in the root cortex. Cytokinins, a class of plant hormones acts trough a phosphotranfert signalling leading to the activation of Type-B Response Regulators(RRBs) which are transcription factors regulating the expression of cytokinins primary response genes. Phylogenetic analyses carried out indifferent legume species genomes showed anexpansion of the RRB genes family associated toan increase in non-canonical RRBs. In Medicago truncatula nodules, MtRRB3 is the most expressed RRB in roots and nodules. MtRRB3 islinked to nodulation as MtRRB3 RNAi silencedplants as well as rrb3 mutants display asignificant decrease of nodule number. Inaddition, the expression of the nodulation related genes Nodulation Signalling Pathway 2(MtNSP2) and Cell Cycle Switch 52A(MtCCS52A) is reduced in response to cytokininsin rrb3 mutants. The expression pattern of apMtRRB3-GUS fusion overlaps with thepMtNSP2-GUS and pMtCCS52A-GUS fusions in roots and nodules. Finally, ChIP-qPCR and protoplast trans-activation experiments showed that MtRRB3 can respectively interacts with and activate MtNSP2 and MtCCS52A promoters. This thesis have thus established molecular mechanisms associated to transcriptional regulations mediated by cytokinins during the legume symbiotic nitrogen-fixing nodulation.

Abstract Ficus carica L. is considered one of the oldest fruit trees in the Mediterranean basin and is widely grown and harvested for the consumption of its fruits dry and fresh. This species is affected by different virus diseases, especially by Fig mosaic disease (FMD), for which Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV-2), Fig mild mottling-associated virus (FMMaV), Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig badnavirus 1 (FBV-1) and Fig fleck-associated virus (FFkaV) are associated. FMD is the most widespread disorder of this species, which represents a threat and a constraint for healthy fig production and germplasm exchange. Thus, the objective of the present doctoral research was the establishment of an efficient and rapid in vitro F. carica propagation, sanitation and conservation of free-FMD plant material for future large-scale commercialization. Initially, FMD-related viruses distribution was screened within the different fig plant organs (buds, leaves, syconia and seeds) of 14 Mediterranean genotypes (Palazzo, Severoni precoce, Bianca, Pilusedda, Dottato bianco, Bifera, Zidi, Baiyadi, Biancu, Brogiotto nero, Catalanisca, Houmairi, Triboiti and Turca 'Serilop') which were utilized afterward as in vitro plant source material. RT-PCR assays revealed that all the aforementioned viruses were present without any exception in seeds, whereas only 4 viruses (FBV, FFkaV, FLMaV-1 and FMV) were detected in buds, leaves and syconia with highly variable infection rates. Moreover, encapsulation technology proved to be a powerful multiplication technique to sustain standard fig tissue culture protocol for three cultivars (Catalanisca, Palazzo and Bifera) and it gave high, almost similar, viability, regrowth and conversion rates. Microcutting rooting in one-step was achieved and conversion rate was comparable for the three cultivars. Furthermore, in order to eliminate FMD associated viruses, with the exception of FBV-1 which resisted to all the sanitation attempts, Caulogenesis and Meristem Tip Culture Protected by the Synthetic Seeds technique (MTC-SS) gave the best sanitation rates. Finally, F. carica (cv. Houmairi) artificial seeds conservation, for final delivery, was achieved. A high viability and moderate regrowth rates were registered with a lesser conversion rate strictly related to the plant growth regulators (PGRs) used. Keywords: Fig, mosaic, RT-PCR, virus distribution, cytokinins, encapsulation, micropropagation, synthetic seed. / Resumen La higuera (Ficus carica L.) es considerada como uno de de los árboles frutales más antiguos de la cuenca mediterránea y es ampliamente cultivado y cosechado para el consumo de sus frutos tanto secos como en fresco. Esta especie se ve afectada por diversas enfermedades virales, especialmente por la denominada "Fig mosaic disease" (FMD) asociada actualemnte a los virus: Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV-2), Fig mild mottling-associated virus (FMMaV), Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig badnavirus 1 (FBV-1) y Fig fleck-associated virus (FFkaV). Esta enfermedad representa una amenaza y un obstáculo para la producción de higos y el intercambio de germoplasma. El principal objetivo del presente trabajo fue establecer un método de propagación de higuera in vitro para el saneamiento y la conservación de material vegetal libre de FMD para su posterior comercialización. Inicialmente, se estudió la distribución de los virus implicados en la enfermedad en diversos órganos de 14 genotipos de F. carica (Palazzo, Severoni precoce, Bianca, Pilusedda, Dottato bianco, Bifera, Zidi, Baiyadi, Biancu, Brogiotto nero, Catalanisca, Houmairi, Triboiti y Turca 'Serilop'), los cuales fueron utilizados posteriormente como fuente material vegetal in vitro. Los resultados obtenidos mediante RT-PCR revelaron que todos los virus mencionados estaban presentes sin excepción en las semillas, mientras que sólo cuatro de ellos (FBV, FFkaV, FLMaV-1 y FMV) fueron en brotes, hojas y siconios con tasas de infección variables. Además, la tecnología de encapsulación demostró ser una técnica de multiplicación eficaz para poder aplicar el protocolo estándar de cultivo de tejidos de higo para tres cultivares (Catalanisca, Palazzo y Bifera) dando altas tasas de viabilidad, rebrote y conversión. Se logró el enraizamiento de microcortes en un solo paso y el índice de conversión fue comparable para los tres cultivares. La callogénesis y el culñtivo de meristemos con la técnica de la semilla sintética (MTC-SS) fueron las técnicas que proporcionaron mayores tasas de desinfección para los virus estudiados a excepción de con FBV-1, entidad viral que no fue eliminada con ninguna de las técnicas ensayadas. Por último, se logró la conservación de las semillas artificiales de higuera (cv Houmairi), registrándose una alta viabilidad y tasas de rebrote moderadas con un menor grado de conversión estrictamente relacionado con hormonas utilizadas. Palabras clave: Higuera, mosaico, RT-PCR, la distribución de los virus, hormonas, encapsulación, micropropagación, y la semilla sintética. / Resum La figuera (Ficus carica L.) és considerada un dels arbres fruiters més antics de la conca mediterrània i és àmpliament conreat i collit per al seu consum fresc i sec. Les malalties virals, especialment "Fig mosaic disease" (FMD), associada amb els viruses: Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV-2), Fig mild mottling-associated virus (FMMaV), Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig badnavirus 1 (FBV-1) i Fig fleck-associated virus (FFkaV). Esta malaltia representa una amenaça per a la producció de figues i l'intercanvi de germoplasma. El principal objectiu d'aquest treball va ser estableixerun mètode de propagació de figuera in vitro per al sanejament i la conservació de material lliure de FMD per a su posterior commercialització. Inicialment, es va estudiar la distribució dels virus associats a FMD en diversos òrgans en 14 genotips de F. carica (Palazzo, Severoni Precoce, Bianca, Pilusedda, Dottato bianco, Bifera, Zidi, Baiyadi, Biancu, Brogiotto diners, Catalanisca, Houmairi, Triboiti i Turca 'Serilop'), els quals van ser utilitzats posteriorment com a font de material vegetal in vitro. Els resultats obtinguts del anàlisis realitzats per RT-PCR van revelar que tots els virus eren presents sense excepció en les llavors, mentre que només quatre virus (FBV, FFkaV, FLMaV-1 i FMV) van ser detectats en brots, fulles i siconis amb taxes d'infecció variables. A més, la tecnologia d'encapsulació va demostrar ser una tècnica de multiplicació eficaç per poder aplicar el protocol estàndard de cultiu de teixits de figa per a tres cultivars (Catalanisca, Palazzo i Bifera) donant taxesadequades de viabilitat, rebrot i conversió. Es va aconseguir l'arrelament de microtalls en un sol pas i l'índex de conversió va ser comparable per als tres cultivars. La calogènesi i el cultiu de meristems protegits per llavors sintètiques (MTC-SS)van ser les tècniques que proporcionarem millores tases de desinfecció per als virus estudiats amb l'excepció de FBV-1 que es va resistir a tots els mètodes de sanejament. Finalment, es va aconseguir la conservació de la llavors artificials de figuera (cv. Houmairi), registrant-ne una alta viabilitat i taxes de rebrot moderades amb un menor grau de conversió estrictament relacionat amb hormones utilitzades. Paraules clau: Figuera, mosaic, RT-PCR, la distribució dels virus, hormones, encapsulació, micropropagació, i la llavor sintètica. / Yahyaoui, E. (2017). Use of standard and setup of non conventional techniques for the elimination of viruses associated with Fig Mosaic Disease (FMD) in fig germplasm (Ficus carica L.) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/79876

Bermudagrass (<i>Cynodon</i> spp.), when cultured at the northern limit of adaptation for semitropical grasses, is exposed seasonally to temperatures low enough to limit growth and turf quality. Research was conducted to investigate the influence of foliar iron and cytokinin applications on bermudagrass growth during fall and spring. The relationship of photosynthesis, respiration, and nonstructural carbohydrate composition to chilling temperatures was also studied. Foliar applications of Fe in late-summer and fall extended bermudagrass performance during low temperature periods of fall. Frequent Fe applications aided the retention of green bermudagrass turf during prolonged exposure to chilling temperatures. Iron applied the previous season stimulated post-dormancy recovery. Benzyladenine (BA) applied alone was not as effective as Fe for promoting green bermudagrass color retention during fall and BA had few effects on spring growth when applied the previous season. Applications of BA in conjunction with Fe were beneficial for retention of green bermudagrass color during late fall when clear plastic covers were used to prevent frost injury. A 6- to 8-week longer bermudagrass growing season occurred when clear plastic covers were used to prevent frost injury. Iron and BA did not significantly affect the total nonstructural carbohydrate (TNC) levels in Midiron bermudagrass rhizomes and stolons at the onset of dormancy in field studies. Midiron bermudagrass had higher photosynthetic and respiration rates than Tifgreen bermudagrass after 4 days exposure to chilling (10/7°C day/night) temperatures in controlled environment studies. Midiron recovered higher photosynthetic rates than Tifgreen when returned to a warm (30°C) environment after exposure to chilling temperatures. The TNC in leaves of Midiron and Tifgreen increased 88 and 160%, respectively, during 5 days at chilling temperatures. The inability to transport carbohydrate from and the subsequent accumulation of high photoassimilate levels in leaves was associated with the inability of bermudagrass to fully recover high photosynthetic rates following chilling. Reduced respiratory activity was apparently responsible for the accumulation of high TNC levels in leaves. In contrast to photosynthesis, respiration was reversibly inhibited by short term exposure of bermudagrass to chilling temperatures. Rapid recovery of high respiratory activity may be important for maintenance of aesthetically pleasing bermudagrass turf following chilling. Foliar applied Fe or BA generally caused darker green Midiron and Tifgreen turf after exposure to chilling temperatures in a controlled environment, although the enhancement of physiological activity differed with chemical applied and cultivar. Iron stimulated recovery of photosynthetic and respiratory activity in both cultivars after exposure to chilling temperatures. However, during chilling Midiron CO₂ exchange was more responsive to Fe applications. Benzyladenine increased photosynthesis in Tifgreen but not in Midiron and did not significantly affect respiration in either cultivar. Neither Fe nor BA had a consistent effect on TNC levels in bermudagrass leaves, rhizomes, or stolons. These investigations indicate that cultivar selection may play a major role in determining turf quality at chilling temperatures. Iron may modify bermudagrass physiology and enhance performance of bermudagrass exposed to chilling temperatures. / Ph. D. / incomplete_metadata

Autumn senescence is a visually spectacular phenomenon in which trees prepare for the oncoming winter. The mechanism for regulation of autumn senescence in trees has been very hard to pinpoint. In this thesis the main focus is to investigate how autumn senescence is regulated in aspens (Populus tremula). Previous work has established that autumn senescence in aspens is under daylight control, in this thesis the metabolic status and the effect on autumn senescence was investigated. The metabolic status was altered by girdling which leads to accumulation of photosynthates in the canopy. This resulted in an earlier onset of senescence but also the speed of senescence was changed. At the onset of senescence the girdled trees also accumulated or retained anthocyanins. The nitrogen status of aspens during autumn senescence was also investigated, we found that high doses of fertilization could significantly delay the onset of senescence. The effects of various nitrogen forms was investigated by delivering organic and inorganic nitrogen through a precision fertilization delivery system that could inject solutes directly into the xylem of the mature aspens. The study showed that addition of nitrate delayed senescence, addition of arginine did not have any effect on the autumn senescence in aspens, and furthermore the nitrate altered the trees leaf metabolism that was more profound in high dosages of supplied nitrate.  Cytokinins are plant hormones believed to delay or block senescence, studies have suggested that the decrease of cytokinins and/or cytokinin signalling may precede senescence in some plants. To investigate how cytokinin regulates autumn senescence in aspens we profiled 34 cytokinin types in a free growing mature aspen. The study begun before autumn senescence was initiated and ended with the shedding of the leaves, and spanned three consecutive years. The study showed that the individual cytokinin profiles varied significantly between the years, this despite that senescence was initiated at the same time each year. Senescence was furthermore not connected to the depletion of either active or total cytokinins levels. The gene pattern of genes known to be associated with cytokinin was also studied, but no gene expression pattern that the profile generated could explain the onset of senescence. These results suggest that the depletion of cytokinins is unlikely to explain the tightly regulated onset of autumn leaf senescence in aspen.

Light and phytohormones, such as cytokinins, have been known to play a pivotal role in numerous physiological processes in plant cells. Previous work in our laboratory has revealed the light- and cytokinin- modulated changes both in the levels of specific tRNA species and their modified nucleotide contents, in addition to the characterization of specific tRNAs and tRNA genes from higher plants. The plant hormone cytokinin, which is of particular interest to us has been implicated to be involved in processes such as induction of cell division, plastid biogenesis and delay of senescence. Ongoing work in our laboratory also points towards the role of Ca2+ as a second messenger in cytokinin mediated gene expression. With the objective of isolation of specific tRNA genes which could then be used as probes to study the light- and phytohormone- induced changes in the levels of respective functional mature tRNAs, a previously isolated clone containing a 6.6kb insert that hybridized with 3 end labeled cucumber total cellular tRNA was sequenced by the dideoxy chain termination method. Sequence analysis of the 6.6 kb DNA fragment has revealed a chloroplast genome DNA fragment containing the trnNGUU and trnRACG genes in addition to the genes coding for the ribosomal RNAs 4.5S, 5S and 23S as well as the protein coding genes ccsA (cytochrome c-synthesis) and ndhD(NADH plastoquinone oxidoreductase).These genes were found to be arranged in the order-23S-4.5S-5S-trnRACG-trnNGUU-ccsA-ndhD. This shows a divergence from the gene organization in the completely sequenced chloroplast genomes of other higher plant species such as tobacco, maize, rice and Arabidopsis, especially with regard to the absence of a highly conserved trnLUAG gene that has been shown to be present in the trnNGUU-ndhD intergenic region. The cucumber chloroplast trnNGUU and trnRACG genes have shown very high homology (>90%) whereas ccsA and ndhD show 50-61% similarity to corresponding genes from chloroplast genomes of other plant species. The relative levels of tRNAArg and tRNAAsn were determined by Northern analysis using the tRNA gene probes, in etiolated excised cucumber cotyledons treated with light or phytohormones, such as cytokinin (BA) and auxin (2,4-D). Light and phytohormones were found to significantly increase the levels of tRNAArg unlike in the case tRNAAsn where no significant changes in the levels were observed. This result points towards the regulation of relative levels of specific tRNA species by light and cytokinin so as to match the codon usage of the mRNA population during light- and cytokinin- induced plant development in cucumber. Northern analyses were also performed to monitor the relative transcript levels of the plastid encoded ccsA and ndhD in etiolated excised cucumber cotyledons treated with light or phytohormones. ccsA transcript levels were found to be significantly reduced in auxin treated cucumber cotyledons where as exogenous application of cytokinin to either dark-grown or light exposed cotyledons did not seem to have any pronounced effect. ndhD transcripts were found to be up-regulated by cytokinin treatment or light exposure in comparison to un-treated controls probably indicating a point of overlap in the light/ cytokinin mediated signal transduction pathways. Auxin treatment on the other hand was found to down-regulate ndhD transcript levels also. Recent studies from our laboratory have demonstrated the involvement of a calcium-dependent protein kinase(CDPK) in the cytokinin-signal transduction pathway associated with the induction of pathogenesis-related proteins (chitinase and β 1-3 modulation of nuclear-encoded CDPK transcripts in response to light and exogenously added phytohormones such as cytokinins and auxin. Towards this end, partial CDPK cDNAs were generated from Cucumis Sativus by RT-PCR using degenerate primers designed based on the conserved regions of the known CDPK proteins available in the database, cloned in pGEM-T and sequenced. Sequence analysis of twenty partial cDNA clones revealed the presence of at least four CDPK isoforms in Cucumis sativus (CuCDPK 1-4). Of the four partial CDPK cDNAs, the tissue-specific expression level of CuCDPK3 was studied using the highly sensitive Taqman Analysis (Quantitative RT-PCR). The results obtained indicate that, in excised dark-grown cucumber cotyledons light and cytokinin were found to up-regulate the levels of CuCDPK3 unlike auxin, which was found to have no significant effect. In cucumber hypocotyls, which had the highest levels of CuCDPK3, light was found to have a down-regulatory effect whereas cytokinin and auxin did not bring about any significant changes in the levels of CuCDPK3. In cucumber root tissue, both light and cytokinin were found to have a down-regulatory effect on the levels of CuCDPK3, unlike auxin. The southern analysis of cucumber genomic DNA revealed a CDPK multi-gene family in cucumber. Since cytokinins have been known to play a role in both etioplast and chloroplast biogenesis and since various groups have recently reported the presence of higher plant homologues of bacterial cell-division protein FtsZ and the requirement of plant nuclear-encoded FtsZs for plastid division, efforts were also made to isolate and to study the expression of cucumber FtsZ in dark-grown cucumber cotyledon tissue treated exogenously with light/phytohormones. Towards this end, a partial FtsZ cDNA was generated from cucumber by RT-PCR using degenerate primers designed based on conserved regions of known plant FtsZ proteins. Results of the Taqman Analysis indicate that cytokinin, unlike auxin, mimics the action of light by increasing the levels of CuFtsZ transcripts in dark-grown cotyledon tissue suggesting the involvement of FtsZ in cytokinin-induced plastid-biogenesis.