Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this research was to determine number, crown diameter and root nodules of runner tips produced by micropropagated and multiplied strawberry stock plants at different planting times. The experiment was conducted between October, 2008 and April, 2009, with cultivars INIA-Arazá, INIA-Guenoa and INIA-Yvapitá and ten planting times, at 37; 40; 46; 51; 58; 64; 67; 75 and 87 days after planting the micropropagated stock plants, respectively. Number of runner tips decreased linearly by delaying planting time of stock plants, while crown diameter and number of root nodules were not affected. Higher number of runner tips was produced by micropropagated plants and INIA-Arazá was the most prolific cultivar. Multiplying stock plants may be a new propagation method than can be used for reducing the number of micropropagated stock plants required for the production of strawberry commercial plug transplants. / O objetivo desta pesquisa foi determinar o número de pontas de estolões, o diâmetro de coroa e o número de primórdios radiculares de pontas emitidas por plantas matrizes micropropagadas e por plantas matrizes multiplicadas em diferentes épocas de plantio. O experimento foi conduzido entre outubro de 2008 e abril de 2009, com as cultivares INIAArazá, INIA-Guenoa e INIA-Yvapitá em dez épocas de plantio, aos 37, 40, 46, 51, 58, 64, 67, 75 e 87 dias após o plantio da plantas micropropagadas, respectivamente. O número de pontas de estolão diminuiu linearmente com a época de plantio enquanto o diâmetro da coroa e o número de nódulos radiculares não foi afetado. O número mais elevado de pontas foi obtido com as plantas micropropagadas e a cultivar INIA-Arazá foi a mais prolífica. A multiplicação de plantas matrizes pode ser um novo método de propagação a ser empregado para reduzir o número de plantas matrizes micropropagadas na produção de mudas de morangueiro em bandejas.

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Horticultural Sciences in the Faculty of Applied Sciences at the CAPE PENINSULA UNIVERSITY OF TECHNOLOGY Supervisor: Dr L Kambizi Co-supervisor: Dr NP Makunga Cape Town December 2013 / Agathosma betulina (Berg.) Pillans, previously known as Barosma betulina, is a member of the Rutaceae family, and indigenous to the fynbos botanical biome of the Western Cape of South Africa. It is commonly known as buchu. Extracts as well as powdered leaves have traditionally been used for the treatment of various ailments. The increase in the international demand for A. betulina for health as well as food and beverage benefits, have raised concerns over exploitation of wild populations and the lack of horticultural information necessitates this study to evaluate the propagation of this economical important species. The main objective of this study was to establish a simple and highly productive micropropagation protocol for A. betulina through experimenting with nodal explants. Testing of the effect of various treatments (physical scarification, chemical scarification, GA, stratification, smoke and combinations thereof) on the in vitro germination of A. betulina seeds was done to elucidate the factors which control seed germination. The study revealed that the physical scarification and smoke-induced germination had a significant effect on germination percentages. In terms of germination rate, the radical generally started to appear after approximately 10 days in the physical scarification with smoke treatment. Initial decontamination methods with the exposure of various concentrations of NaOCl gave fatal results, however 1.5% NaOCl had more phenolic reactions rather than fungal or bacterial contamination. Interestingly, contamination rates of explants were influenced by the stage of maturity of the explant material. This plant material was used to test different strengths of regeneration media, to ensure that the explants receive ample nutrients. Results made exhibited that ½ MS was the best strength for growing A. betulina nodal explants. Compared comparison between in vitro derived explants and ex vitro collected explants showed that the ex vitro derived explants had significant results, but the explants lost vigour soon after the initial exponential growth leading to the explants dying off. Furthermore, ex vitro decontaminated plant material was not economically viable to continue with. Seedlings derived from germinated seeds appeared to be the preferred method of propagation as this spent the least time in culture and produced a stable plant with an established root system, which is essential during the hardening off process after in vitro growth. When exposing nodal explants to phytohormone 2,4-D it responds best to dosages 0.5mg Lˉ¹ and 1mg Lˉ¹. Phytohormone BA was very effective in producing soft friable callus. The best results were shown when 0.5mg Lˉ¹ BA was applied to ½ MS media. For both shoot length and multiple shoot production, a combination of phytohormones BA-NAA (1: 0.5mgLˉ¹) had the most significant results. Interestingly, a higher phytohormone concentration of NAA is necessary to develop multiple adventitious roots. The effect of 3mg Lˉ¹ was significant in that it resulted in multiple adventitious roots, but fewer calli was observed in this treatment. Micropropagation becomes valuable as little attention between subcultures is needed; making it less labour intensive compared to conventional nursery propagation systems where weeding watering and spraying of plants are labour intensive. In the traditional world of medicine, more so in Southern Africa, extracts are prepared by adding boiling water to the plant material; however commercial ethanol is used as an extractant. Establishment of the essential oil quality of the in vitro cultures post exposure to various treatments was done. Analysis of essential oils from A. betulina resulted in the identification of twenty one compounds. The results showed qualitative as well as quantitative differences amongst the samples used in the study. The highest relative concentration of limonene was observed in the callus of nodal explants after it was exposed to 0.5mg lˉ¹ NAA. No pulegone was found in this treatment making it ideal for limonene production. This suggests that liquid culture with the same treatment may produce more calli making it ideal for the production of limonene.

The objectives of this research were to select new strawberry clones for the estate of Rio Grande do Sul, Brazil, and to develop methods of multiplication to obtain disease-free transplants with high physiological quality. Five advanced strawberry clones from the Breeding Program and two controls were evaluated in an annual hill system in low tunnels from April to December, 2006. A closed soilless system was developed, based on a growing bed with substrate over a cement tile. A nutrient solution was delivered from a reservoir to the upper end of the tile and drained off back by gravity. An inert substrate (sand) and an organic substrate (Plantmax®) and two advanced strawberry clones were tested. In another experiment fruit yield of plug transplants of different sizes was compared to that of bare-root transplants. Plug transplants were produced rooting runner tips in plastic trays with different volumes of organic substrate. Bare-root transplants were produced in the closed soilless growing system described above. Clones LBD 15.1, LBH 27.2, LBD 35.2 and LBG 121.4 were identified as having potential to be used in the estate of RS. These clones combine earliness, high yield and fruit quality, high content of bioactive compounds and resistance to diseases. A high number of healthy bare-root and runner tips with high quality were obtained with both substrates and both clones. A higher early fruit yield during fall and winter was obtained with plug transplants. Both plug and bare-root transplants reached a high total yield. It was concluded that selected strawberry clones of this research can be recommended to substitute commercial cultivars now planted in the RS or used in combination with them and that disease-free bare-root transplants and runner tips for plug transplants, both with high physiological quality can be produced in the closed soilless system, providing a sustainable alternative for nurseries. / Os objetivos deste trabalho foram selecionar novos clones de morangueiro para o RS e desenvolver um sistema de multiplicação para a obtenção de mudas com alta qualidade fisiológica e sanitária. Foram avaliados cinco clones avançados do Programa de Melhoramento e duas testemunhas, em túneis baixos, durante os meses de abril e dezembro de 2006. Para a multiplicação foi utilizado um sistema fechado sem solo, baseado em um leito de cultivo com substrato sobre telhas de fibrocimento. A circulação da solução nutritiva foi feita a partir de um reservatório até a extremidade mais alta da telha, drenando por gravidade. Como substrato, testou-se a areia na categoria inerte e o Plantmax® na categoria orgânica, com dois clones. Em outro experimento foi comparada a produtividade de mudas com torrão de diferentes tamanhos e com raízes nuas. As mudas com torrão foram produzidas a partir de pontas de estolão enraizadas em bandejas com diferentes volumes de substrato orgânico. As mudas com raízes nuas foram provenientes do sistema fechado sem solo. Foram identificados os clones LBD 15.1, LBH 27.2, LBD 35.2 e LBG 121.4 com potencial para serem cultivados no RS. Esses clones combinam alta produtividade precoce e total, qualidade de fruta, conteúdo de componentes bioativos na fruta e resistência às doenças. Um alto número de mudas e pontas de estolão sadias e de alta qualidade foi obtido no sistema fechado sem solo com ambos os substratos e clones. As mudas com torrão apresentaram maior produtividade precoce no outono e inverno. Tanto as mudas com torrão como as de raízes nuas alcançaram elevada produtividade total. Concluiu-se que os novos clones selecionados podem ser indicados em substituição ou em combinação com as cultivares atualmente em uso no RS e que o sistema fechado sem solo é uma alternativa sustentável para ser empregada na produção de mudas com raízes nuas e de pontas de estolão para mudas com torrão.

Le retrait des nématicides rend urgent la mise au point de méthodes alternatives de lutte contre les nématodes parasites des cultures et la création de variétés résistantes est une voie prometteuse. En vignoble, le nématode Xiphinema index a un impact économique élevé en transmettant le Grapevine fanleaf virus (GFLV), principal virus du court-noué de la vigne et première virose de la vigne à l’échelle mondiale. Des porte-greffe résistants vis-à-vis du vecteur X. index basés sur la source de résistance muscadine (Muscadinia rotundifolia) sont en cours de sélection chez la vigne afin de stopper ou retarder l’infection. Sur cette culture, une étude antérieure avait montré que ce nématode parthénogénétique méiotique est aussi capable de se reproduire (rarement) de façon sexuée. Un travail préliminaire de phylogéographie avait permis de révéler les groupes prédominants de diversité et de sélectionner des populations représentatives pour la création de lignées monofemelles. La durabilité de la résistance doit prendre en compte la diversité du nématode. Dans ce contexte, la thèse a d’abord complété et approfondi l’approche phylogéographique en utilisant une très large gamme d’échantillons originaires de l’aire mondiale de répartition de la vigne. Nos résultats permettent de proposer des hypothèses fortes afin de localiser l’aire native du nématode X. index au Moyen-Orient et de retracer ses itinéraires de dissémination à partir de l’Antiquité. IIs illustrent également le lien étroit depuis cette époque entre la dissémination du nématode et celle de la vigne domestiquée par l’homme. La deuxième partie de la thèse a évalué la durabilité de la résistance de matériel porte-greffe issu de la muscadine en serre (nématodes non virulifères sur plants entre 3 et 6 ans) et en vignoble (nématodes virulifères sur plants âgés de 16 ans). En serre, des accessions résistantes F1 ou BC1, préalablement obtenues à partir d’in vitro ou de boutures ligneuses, ont été inoculées avec un mélange de 4 lignées représentatives, chaque lignée étant traçable avec des marqueurs microsatellites. Nous avons montré que les nématodes issus de plants obtenus par multiplication in vitro surmontent progressivement la résistance tandis que le matériel issu de boutures exprime une résistance durable. La multiplication progressive des nématodes sur le matériel résistant uniquement dans le cas où il est issu d’in vitro écarte a priori l’hypothèse d’une adaptation génétique du nématode. Elle apparaît liée à une architecture différente du système racinaire chez les plants issus de ce type de multiplication, multiplication qui pourrait induire des changements physiologiques discrets mais durables dans les tissus racinaires apicaux à partir desquels les nématodes se nourrissent. Le génotypage des nématodes par microsatellites a permis de détecter un taux bas mais croissant d’individus hybrides entre lignées sur les plants âgés de 4 à 6 ans, ce qui confirme l’aptitude de multiplication sexuée précédemment observée en vignoble. Du fait que l’observation d’individus hybrides apparaît indépendante du type de propagation et du statut de résistance de la plante, nos résultats écartent l’hybridation comme mode d’adaptation du nématode qui serait à même d’expliquer le contournement de la résistance chez les plants issus d’in vitro. En vignoble, après 16 années, les nématodes ont été quasi-impossibles à détecter sur l’accession résistante BC1 qui est également peu affectée par les attaques virales, tandis que des effectifs de nématodes plus élevés ont été retrouvés sur une accession témoin sensible dont les plants sont par contre très majoritairement morts ou en dépérissement. Considérés globalement, nos résultats montrent que la stratégie de résistance basée sur la muscadine apparaît durable. Cette stratégie ciblée sur le nématode vecteur contribuera à réduire significativement l’impact du GFLV transmis par X. index. / The ban of most nematicides renders urgent control alternatives against plant-parasitic nematodes and breeding for resistant plant varieties is promising. In vineyards, the nematode Xiphinema index has a high economical impact by transmitting Grapevine fanleaf virus (GFLV), the main virus of ‘Court-noué’ disease and the first grapevine viral disease worldwide. Resistant rootstocks are being selected in grapevine, using Muscadinia rotundifolia (muscadine) as a resistance source to the vector, in order to arrest or delay GFLV transmission. In this crop, a previous study had shown that this meiotic parthenogenetic nematode is able to reproduce sexually (rarely) in the field. A preliminary phylogenetic work had allowed to reveal the predominant diversity groups and to select representative populations for the creation of single-female lines. Resistance durability is a real challenge that must consider the key information of the nematode diversity. In this context, the PhD project first completed and deepened our phylogeographical approach using an extended geographic coverage of the worldwide nematode distribution. Our results allow proposing strong hypotheses to locate the native area of X. index in the Middle-East and trace its dissemination routes from the Antiquity. They also highlight the close link since this epoch between dissemination of the nematode and domesticated grapevine by man. The second part of the PhD project has then evaluated the durability of muscadine-derived rootstock material in greenhouse (non viruliferous nematodes on plants aged 3 to 6 years) and field (viruliferous nematodes on plants aged 16 years) conditions. In the greenhouse, F1 and BC1 resistant accessions, previously obtained from both in vitro and hardwood-cutting propagation, were inoculated with 4 mixed representative X. index lines, traceable each with microsatellite markers. We showed that nematodes from plants obtained from in vitro progressively overcame the resistance while the material obtained from cuttings displayed a durable resistance. Nematode progressive multiplication in resistant accessions obtained only from in vitro removes a priori the hypothesis of a nematode genetic adaptation and appears linked to a different architecture of the root system in this propagation type. This type may have induced discrete but durable physiological changes in apical root tissues from where nematodes feed. Nematode microsatellite genotyping allowed detecting a low but increasing rate of hybrid individuals from 4 to 6 years, which confirms data from the vineyard. As the hybrid occurrence appears independent from the propagation type and the resistance status of the plant, our data discard hybridization as the mode of adaptation of the nematode underlying resistance breakdown from in vitro plants. In field conditions, after 16 years, nematodes were almost undetectable on the resistant BC1 accession, also almost unaffected by the viral attacks, while higher numbers were detected on a susceptible control accession, whose plants were by contrast in high majority dead or poorly vigorous. Taken all together, our results show that the muscadine-derived resistance strategy appears durable. This strategy focused on vector control will significantly contribute to reduce the impact of GFLV transmitted by X. index.

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