Thesis (MSc)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.

Thesis (PhD) -- Stellenbosch University, 1984. / No abstract available

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Infection of potatoes by viral pathogens causes reduced crop yield and subsequent economic loss. In South Africa Potato virus Y (PVY) and Potato leafroll virus (PLRV) are the two most destructive viruses infecting potatoes. Several other viral pathogens exist, including Potato virus X (PVX), Potato virus M (PVM), Potato virus A (PVA), Potato virus S (PVS), Potato mop-top virus (PMTV), Tomato spotted wilt virus (TSWV) and Potato spindle tuber viroid (PSTVd). Although the aforementioned pathogens are found infecting potatoes around the world, there are no published information pertaining to the prevalence of these viral agents in South Africa. Currently, the occurrence of PLRV infection in potatoes of South Africa has reached epidemic proportions. A previous phylogenetic investigation undertaken in our laboratory of South African PLRV isolates, using coat protein (CP) gene sequences, found large variation between native South African PLRV isolates and most other isolates from elsewhere in the world; with their nearest relatives being single isolates from Australia and North America. In this study the incidence of PVX, PVM, PVA, PVS, PMTV, TSWV and PSTVd was investigated. A large number of potato plant and tuber samples was collected and infected samples were identified with reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the CP gene or the whole genome in the case of PSTVd. The amplified nucleic acid segments were sequenced, aligned with international reference sequences and analysed phylogenetically to determine their relative relationships with these reference sequences. The CP genes of PLRV isolates were sequenced and phylogenetically investigated to determine how these new isolates compared relative to the previous findings from our laboratory. In addition, the complete genomes of two PLRV isolates were sequenced and phylogenetically investigated as a preliminary study to investigate the apparent increase of pathogenicity of certain variants of South African PLRV. Results obtained showed that only PVX and PVS were present in the samples collected and the incidences of these viruses were very low (2.0 and 1.1% respectively). The phylogenetic analyses of the CP genes, indicated that the PVX and PVS variants isolated in this study, were part of the dominant types of variants found worldwide. From the analyses of the PLRV CP and whole genome sequences, it was determined that many of the PLRV variants found in South Africa, are genetically distinctly different from those around the world. This warrants further investigation into the increased pathogenicity experienced with South African PLRV. / AFRIKAANSE OPSOMMING: Infeksie van aartappels deur virale patogene veroorsaak verlaagde opbrengs en gevolglike ekonomiese verlies. In Suid-Afrika is Aartappelvirus Y (PVY) en Aartappelrolblad virus (PLRV) die twee mees vernietigende virusse wat aartappels infekteer. Verskeie ander virale patogene, insluitend Aartappelvirus X (PVX), Aartappelvirus M (PVM), Aartappelvirus A (PVA), Aartappelvirus S (PVS), Aartappel "moptop" virus (PMTV), Kromnekvirus (TSWV) en Aartappel "spindle tuber" viroïed (PSTVd) kom ook wêreldwyd in aartappels voor. Alhoewel hierdie virusse aartappels wêreldwyd besmet, is daar geen gepubliseerde inligting met betrekking tot die voorkoms van hierdie virusse of die viroïed in Suid-Afrika nie. Tans het die voorkoms van PLRV infeksie in aartappels in Suid-Afrika epidemiese proporsies bereik. In 'n vorige filogenetiese ondersoek van die mantelproteïen (MP) nukleotiedvolgordes van Suid Afrikaanse PLRV isolate in ons laboratorium, is groot variasie tussen hierdie inheemse isolate en die meeste ander isolate van elders in die wêreld bevind. Die Suid Afrikaanse PLRV variante betree 'n unieke intermediêre posisie tussen die internasionale isolate en enkele isolate van Australië en Amerika. In hierdie studie is die voorkoms van PVX, PVM, PVA, PVS, PMTV, TSWV en PSTVd ondersoek. Groot aantal aartappelplant en -knol monsters is versamel en infeksie is getoets met tru-transkripsie polimerase kettingreaksie (RT-PCR) amplifisering van die MP geen, of die hele genoom in die geval van PSTVd. Die nukleïensuurvolgordes is bepaal en vergelyk met internasionale verwysingsvolgordes. Die relatiewe verhoudings tussen die bepaalde volgordes en die verwysingsvolgordes is geanaliseer met filogeneties ontledings. Die MP gene van PLRV isolate se volgordes is bepaal en filogeneties ontleed om hierdie nuwe isolate te vergelyk relatief tot vorige bevindinge in ons laboratorium. Die volledige genome van twee PLRV isolate se volgordes is bepaal en filogeneties ontleed as 'n voorlopige studie om die oënskynlike toename in patogenisiteit van Suid-Afrikaanse PLRV te ondersoek. Resultate het getoon dat slegs PVX en PVS teenwoordig was in die monsters wat versamel is en dat die voorkoms van hierdie virusse baie laag was (2.0% en 1.1% onderskeidelik). Die filogenetiese ontleding van die MP gene het aangedui dat die Suid Afrikaanse variante van PVX en PVS, geisoleer in hierdie studie, van die dominante tipes is wat mees gereeld internationaal voorkom. Uit die ontleding van die PLRV MP en heelgenoom volgordes, is vasgestel dat baie van die PLRV variante wat in Suid-Afrika aangetref word, geneties meer verskillend is as die van regoor die wêreld. Dus, regverdig dit, verdere ondersoek van die verhoogde patogenisiteit van Suid Afrikaanse PLRV variante.

Thesis (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Grapevine leafroll-associated virus-3 is one of ten members of the C/osteroviridae that are known to infect grapevine. Nine of these viruses are associated with grapevine leafroll disease, of which GLRaV-1 and GLRaV-3 are the most important and widespread. Members of the C/osteroviridae are unique amongst the viruses, as it is the only known family whose members encode a heat shock protein 70 kOa homolog (Hsp70h). The Hsp70h is a movement protein (MP) that is required for the active translocation of the virion structure through the plasmodesmata into adjacent cells. Broad-spectrum resistance to unrelated viruses can be obtained by a pathogen-derived resistance (POR) strategy that is based on the expression of a dysfunctional MP in plants. The Hsp70h has two distinct domains. The N-terminal two thirds of the protein is an ATPase domain and shares high homology with the ATPase domains of all Hsp70h proteins from the C/osteroviridae and Hsp70 proteins from the prokaryote and eukaryote kingdoms. Conserved amino acids are found in the ATPase domain and are required for the positioning of the ATP at the catalytic site for ATP hydrolysis. The C-terminal domain is variable and the function of this domain in the Closteroviridae is not known. In prokaryote and eukaryote Hsp70 proteins, the C-terminal domain is required for protein-protein interactions. The American NY-1 isolate of GLRaV-3 has been sequenced and POR strategies have been attempted with the coat protein, divergent coat protein and replicase genes, but not with a dysfunctional form of the hsp70h gene. In this study, double-stranded RNA was isolated from a commercial vineyard with unknown virus status, but with distinct grapevine leafroll symptoms, and from two grapevine sources of known virus status, one with mild and one with severe symptoms. The GLRaV-3 hsp70h gene was amplified by RT-PCR from the dsRNA and the gene sequence was analysed. The hsp70h gene from the three virus sources contained more than 94% nucleotide sequence homology to the NY-1 isolate and the conserved amino acids required for ATPase activity were present. The hsp70h gene isolated from GLRaV-3 from a commercial Stellenbosch vineyard showing clear leafroll symptoms was selected for further work and was subjected to site-directed mutagenesis to engineer four point mutations in the gene. These four mutations resulted in the substitution of Asn for Asp", Gly for Thr1O, Lys for Glu 174 and Asn for Asp 197. The wild type (WT) and mutated (Mut) forms of the hsp 70h genes were cloned into a bacterial expression vector. Expression of both the WT- and Mut-Hsp proteins was achieved, and the protein was expressed in the insoluble inclusion bodies. All attempts to refold and isolate active proteins from the inclusion bodies were unsuccessful. Attempts to increase the concentration of soluble protein within the expressing bacteria were unsuccessful. Due to the lack of active protein, biochemical tests on the ATPase activity of the WT- and Mut-Hsp proteins could not be conducted. The wt- and mut-hsp genes were cloned into a plant expression vector for transformation into tobacco plants. These transformations were successful and gave rise to 22 Km' and 18 Km' plants from the WT- and Mut-Hsp constructs respectively. Two plant lines, M5 and M10, transformed with the mut-hsp transgene construct, appeared to have a high level of resistance to the challenging potato X potexvirus, whereas all the other tested plants were susceptible to the challenging virus. It was thus shown that a dysfunctional form of the GLRaV-3 Hsp70h could provide resistance to an unrelated virus in tobacco. / AFRIKAANSE OPSOMMING: Wingerdrolblaar-geassosieerde virus 3 (GLRaV-3) is een van 10 lede van die Closteroviridae wat wingerd kan infekteer. Nege van die virusse is met wingerdrolblaar geassosieer. Die GLRaV-1 en GLRaV-3 is die belangrikste en mees wyd verspreide lede van die rolblaar-geassosieerde Closteroviridae. Lede van die Closteroviridae is uniek in die opsig dat die virusse vir 'n 70 kDa-homoloë hitteresponsproteïen (Hsp70h) kodeer. Die Hsp70 is 'n bewegingsproteïen (MP) wat belangrik is vir die translokasie van die virus deur die plasmodesmata na die naasliggende sel. Breë-spektrum weerstand teen onverwante virusse kan behaal word deur 'n patogeen-afgeleide weerstandstrategie (POR), wat op die uitdrukking van 'n disfunksionele MP wat in plante uitgedruk word, gebaseer is. Die Hsp70hproteïen het twee gebiede. Die N-terminale gebied is In ATPase-gebied en toon hoë homologie met ander ATPase-gebiede van Hsp70h-proteïene van die Closteroviridae, asook die prokariotiese en eukariotiese koninkryke. Gekonserveerde aminosure wat belangrik is vir die posisionering van ATP in die katalitiese domein vir ATP-hidrolise is in die ATPase-gebied gevind. Die C-terminale gebied is variërend en die funksie van die gebied in die Closteroviridae is onbekend. In prokariotiese en eukariotiese Hsp70h-proteïene is die C-terminale gebied belangrik vir proteïenproteïen interaksies. Die nukleotiedvolgorde van die Amerikaanse NY-1-isolaat van GLRaV-3 is al bepaal en POR-strategieë is ook op die kapsiedproteïen, uiteenlopende kapsiedproteïen en die replikasie-proteïen uitgevoer, maar nog nie op 'n disfunksionele vorm van die Hsp70h-geen nie. In hierdie studie is dubbelstring-RNA (dsRNA) van 'n kommersiële wingerd met onbekende virusstatus wat rolblaarsimptome toon, geïsoleer, asook van twee wingerde met 'n bekende virusstatus, een met ligte en een met strawwe simptome. Die GLRaV-3 hsp70h-geen is met hulp van die polimerasekettingreaksie-metode (PKR) vanaf die dsRNA geamplifiseer en die geen se nukleotiedvolgorde is bepaal. Die hsp 70-gene van drie verskillende wingerde het meer as 94% homologie met die NY-1-isolaat getoon. Die gekonserveerde aminosure wat vir ATPase-aktiwiteit belangrik is, was teenwoordig. Die hsp70h-geen van GLRaV-3, wat uit 'n kommersiële wingerd met duidelike rolblaarsimptome in die Stellenbosch-gebied geïsoleer is, is vir verdere navorsing gekies en dit is aan setel-gerigte mutagenese blootgestelom vier mutasies van die geen te bewerkstellig. Die gevolg van hierdie vier mutasies was die verandering van Asn na Asp", Gly na Thr1o, Lys na Glu174 en Asn na Asp197. Die wilde (WT) en veranderde (Mut) vorms van die hsp-gene is in 'n bakteriese uitdrukkingsvektor gekloneer. Uitdrukking van beide die WT- en die Mut-Hspproteïene is behaal, maar die proteïene was in die onoplosbare fraksie geleë. Pogings om die onoplosbare proteïene te isoleer en in 'n aktiewe oplosbare vorm te verkry, was onsuksesvol. Verdere pogings om die proteïene in die oplosbare fraksie van die bakteriese ekspressiesisteem uit te druk, was ook onsuksesvol. As gevolg van die gebrek aan aktiewe proteïen kon biochemiese toetse nie op die ATPaseaktiwiteit van die WT- en Mut-Hsp proteïne gedoen word nie. Die wt- en mut-hsp-gene is ook in In plantekspressievektor gekloneer vir transformasie in tabakplante. Hierdie transformasies was suksesvol en het aanleiding gegee tot 22 kanamisienbestande (Km') en 18 Km' plante vanaf die WT- en Mut-Hspkonstrukte onderskeidelik. Twee plantlyne, M5 en M10, wat met die mut-hsptransgene getransformeer is, het 'n hoë vlak van weerstand teen die infekterende aartappelvirus X getoon in vergelyking met ander plante wat met die virus geïnfekteer is. Daar is dus bewys gelewer dat 'n disfunksionele vorm van die GLRaV-3 Hsp70h weerstand kan bied teen 'n onverwante virus in tabak.

No description available.

Orientador: Antonio Carlos Maringoni / Banca: Maria José de Marchi Garcia / Banca: Margarida Fumiko Ito / Banca: Marcelo Agenor Pavan / Banca: Tadeu Antonio Fernandes da Silva Júnior / Resumo: A cultura do pimentão (Capsicum annuum L.) tem elevada importância econômica e social para o Equador. Vários fatores têm contribuído para a baixa produtividade dessa cultura no país e dentre eles destacam-se as doenças de diversas etiologias. Poucas informações sobre a ocorrência de doenças nessa cultura estão disponíveis nas para as condições equatorianas. Neste trabalho foi realizado levantamento da ocorrência da doença "pata seca" e de viroses na cultura do pimentão, em plantios comerciais, durante anos de 2013 e 2014; a identificação dos agente(s) causal(ais) da "pata seca"; avaliações do eficácia da pulverização da aplicação de produtos fitossanitários (sulfato de cobre + fosetil alumínio; iprodione + clorotalonil; Trichoderma harzianum + T. koingii) em condições de campo, no controle da doença nos híbridos Nathalie e Quetzal e a sensibilidade in vitro do(s) patógeno(s) dessa doença a fungicidas (tebuconazole, benomil, clorotalonil, fosetil alumínio, iprodione, metalaxil + propanocarb e tebuconazole). A "pata seca" foi constatada em um 79,2% das propriedades, com incidência entre 5% a 53,6%, em quanto as viroses, foram detectada em 62,5% das propriedades, com incidência entre 9,6% a 61,2%. Os fungos S. clerotium rolfsii e Fusarium spp. foram isolados com maior freqüência das plantas com sintomas de "pata seca". Entretanto, S.rolfsii foi mais virulento, sendo considerado o principal agente causal da doença "pata seca". Não foi observada diferença dos produtos fitossanitários, aplicados no colo das plantas, na incidência de "pata seca", nos dois ensaios realizados em campo. A aplicação de iprodione + clorotalonil propiciou uma maior quantidade de frutos comerciais nos dois ensaios e uma maior produção em um dos ensaios. Os Isolados de S. rolfsii apresentaram maior sensibilidade aos fungicidas tebuconazole, benomil e iprodione, ... / Abstract: Sweet pepper (Capsicum annuum L.) has high economic and social importance for Ecuador. Several factors have contributed to the low productivity of this crop in the country and among them there are the diseases of different etiologies. Little information on the occurrence of diseases in this culture is available to the Ecuadorian conditions. This study was carried out survey of the occurrence of the disease "pata seca" and viruses in sweet pepper in commercial plantations, during the years 2013 and 2014; the identification of the agent (s) causal (es) of "pata seca"; evaluations of the effectiveness of spray application of pesticides (copper sulfate + fosetyl aluminum, iprodione + chlorothalonil; Trichoderma harzianum + T. koingii) in field conditions in controlling the disease in hybrid Nathalie and Quetzal and in vitro sensitivity ( s) pathogen (s) of the disease to fungicides (tebuconazole, benomyl, chlorothalonil, fosetyl aluminum, iprodione, metalaxyl + propanocarb and tebuconazole). The "pata seca" was found in a 79.2% of the properties, with an incidence of 5% to 53.6%, as in the viruses were detected in 62.5% of the properties, with an incidence of 9.6% to 61.2%. The fungus Sclerotium rolfsii and Fusarium spp. were isolated more frequently plants with symptoms of "pata seca". However, S. rolfsii was more virulent, being considered the main causative agent of the disease "pata seca". There was no difference of pesticides applied in the base of the plants, the incidence of "pata seca" in the two trial carried out in the field. The application of iprodione + chlorothalonil provided a larger amount of fruits in the two ... / Doutor

Orientador: Marcelo Agenor Pavan / Coorientador: Renate Krause Sakate / Banca: Marcio Martinello Sanches / Banca: Valdir Atsushi Yuki / Banca: Julio Massaharu Marubayashi / Banca: Antonio Carlos Maringoni / Resumo: Espécies de vírus dos gêneros Potyvirus, Carlavirus e Allexivirus são comumente encontradas na cultura do alho (Allium sativum L.) e em decorrência da sua propagação vegetativa, há o acúmulo de vírus de um ciclo para outro, formando um complexo entre espécies dos gêneros citados. Uma das principais causas da redução da produtividade ocorre devido à infecção por vírus, e a termoterapia associada à cultura de tecido pode ser um método eficiente para limpeza clonal e obtenção de alho semente livre de vírus. O estudo da degenerescência do alho livre de vírus é, portanto, importante para se conhecer o número de ciclos em que o alho semente, inicialmente sadio, pode ser multiplicado no campo, sem que ocorra a redução da produtividade. Neste estudo foi verificado que após três anos a produtividade foi 13,75% superior ao alho 100% infectado por vírus. Os bulbilhos aéreos também podem ser utilizados para a multiplicação de sementes e tem como vantagem o baixo custo quando comparado à utilização de bulbos provenientes da cultura de tecido. Entretanto, essa técnica só se torna viável com a utilização de matrizes isentas de vírus, pois a transmissão de vírus para os bulbilhos aéreos, provenientes de matrizes infectadas, podem chegar a 83,33% no caso dos potyvirus, 20% para carlavirus e 70% para allexivirus, segundo dados obtidos neste trabalho. Realizar uma diagnose precisa dos vírus que infectam o alho torna-se essencial para que não ocorram falhas durante a indexação. Pôde-se observar que para a detecção dos potyvirus, utilizando o teste de ELISA indireto, deve-se utilizar folhas novas, entre 21 e 63 dias após o plantio, pois a concentração viral nesse período é a mais elevada. Diferentes técnicas como o DIBA colorimétrico, DIBA quimioluminescente, ELISA e PCR em tempo real foram avaliadas para detecção do LYSV, aos 21 dias após a inoculação ... / Abstract: Virus species of genera Potyvirus, Carlavirus and Allexivirus are commonly infecting garlic plants (Allium sativum L.). Due to the vegetative propagation, the accumulation of viruses might occur from one cycle to another. The infection of viruses might induce yield losses, and the technique of thermotherapy associated with meristem tip culture is an efficient method to obtain virus-free seeds. The study of degeneration of seeds free of virus is important to know the number of cycles in which garlic may be multiplied in the field without reduction of productivity. In this study it was observed that after three years, the productivity increased 13,75% compared to 100% infected seeds. Currently, aerial bulbils may be used for multiplication of seeds with low cost, compared to meristem tip culture. However, this is a viable technique only if the matrix is free of viruses, because the transmission to aerial bulbils, from infected plants can reach 83.33 % for potyviruses, 20% for carlavirus and 65% for allexivirus, data obtained in this work. An accurate diagnosis in viruses that infect garlic is important to avoid mistakes during indexing material. It was 4 observed that the best time to detect potyvirus, through ELISA test is between 21 and 63 days after planting on young leaves, as the virus concentration is higher in this period. Whem using different techniques, such as the colorimetric DIBA, chemiluminescent DIBA, ELISA, and real-time PCR, it was possible to perform the detection of LYSV 21 days after inoculation. ELISA test detected LYSV only at 21 days after inoculation, whereas using real time PCR, in 5 days after inoculation it was possible to detect the virus due to its high sensitivity. The other tests also detected the virus 21 days after infection / Doutor

Orientador: Marcelo Agenor Pavan / Coorientador: Renate Krause Sakate / Banca: Julio Massaharu Marubayashi / Banca: Rumy Goto / Resumo: A mandioquinha-salsa (Arracacia xanthorrhiza Bancroft) é uma hortaliça originária da região andina, Venezuela, Colômbia, Equador, Peru e Bolívia. A família das apiáceas compreende também outras hortaliças importantes, como cenoura, salsa, coentro, aipo, dentre outras. A mandioquinha-salsa é propagada de forma vegetativa através dos propágulos, e isso pode favorecer a ocorrência de um número considerável de patógenos que causam degenerescência, principalmente os vírus. Está comprovado que alguns vírus são fator limitante em culturas de propagação vegetativa. Portanto, o presente trabalho teve como objetivos avaliar por meio de métodos sorológico e molecular 241 amostras oriundas de diferentes localidades, a fim de fazer um estudo de ocorrência e predominância das espécies virais e verificar a variabilidade biológica dos isolados encontrados por meio de transmissão por extratos vegetais. A detecção foi realizada através de Enzyme linked immunossorbent assay (ELISA) utilizando antissoro comercial anti-poty para o gênero Potyvirus. Para a análise da variabilidade, foram desenhados 2 oligonucleotídeos para amplificar a região codificadora da proteína capsidial para o gênero Cheravirus, e para a detecção de potyvirus e nepovirus, oligonucleotídeos foram obtidos da literatura. No estudo foi possível detectar a espécie Arracacha motlle virus do gênero Potyvirus, coletadas nos estados de Goiás, Minas Gerais, Brasília e Paraná. Os demais vírus testados não foram identificados em nenhuma das amostras analisadas. Além desses testes de identificação, ainda foi realizada microscopia eletrônica de transmissão em seis amostras, das quais duas foi possível a identificação de potyvirus. O teste de gama de hospedeiras foi realizado e mostrou que o AMoV tem gama de hospedeiras bastante restrita, sendo possível infectar somente três de nove espécies testadas ... / Abstract: Arracacha (Arracacia xanthorrhiza Bancroft) is a vegetable crop of the Andean region, formed by Venezuela, Colombia, Ecuador, Peru and Bolivia. The Apiacea family also have other important vegetable crops such as carrots, parsley, cilantro, celery, among others. Arracacha is propagated vegetatively and this can favor the occurrence of a considerable number of pathogens that cause degeneration, especially viruses. Some viruses are a limiting factor in vegetatively propagated crops. Therefore, the present study aimed to evaluate by serological and molecular methods 241 samples from different localities in order to verify the occurrence and predominance of the viruses species and verify the biological variability by sap inoculation. The detection was performed using enzyme-linked immunossorbent assay (ELISA) technique, using commercial antiserum against-potyvirus. For variability analysis, primers were designed to amplify the coding region of the coat protein for the 4 genus Cheravirus, and oligonucleotides were obtained from literature for the detection of the genus potyvirus and nepovirus, . It was possible to detect Arracacha mottle virus, from genus Potyvirus, in the states of Goiás, Minas Gerais, Brasilia and Paraná. The others viruses tested were not identified in the collected samples. Six samples were analysed by electron microscopy, and the potyvirus were identified only in two samples. The host range was restricted, infecting only three species of nine tested. The nucleotide identity ranged from 96% to 99%, between collected samples and sequence from GenBank (acess DQ925486), showing low genetic variability among them / Mestre

This work was aimed to investigate the basic viral infection protocols mainly focusing on Nicotiana benthamiana hairy root cultures and wild-type tobacco mosaic virus (TMV). The application of transgenic virus containing the gene for green fluorescent protein (GFP) for foreign protein production in plant tissue cultures and whole plants was also studied. The effect on viral accumulation of the form of plant tissue culture used, such as hairy roots, shooty teratomas and suspended cells, was investigated. Viral infection was shown to have no effect on culture growth and morphology. Hairy root cultures are a superior host for viral propagation and production in vitro. The maximum specific rate of viral accumulation occurred mainly during the root growth phase. The average maximum virus concentration in the hairy roots was 0.82 ?? 0.14 mg g-1 dry weight and virus protein represented a maximum of approximately 6% of total soluble protein in the root biomass. Proportional scale-up of TMVinfected hairy roots in shake flasks and bioreactors can be achieved without changing the average virus concentration accumulated in the hairy roots. The level of viral accumulation was much lower in N. benthamiana hairy roots infected with transgenic virus containing GFP (TMVGFPC3) compared with TMV and low levels or no GFP was detected. Viral accumulation and GFP production in whole plants was studied using different generations of transgenic TMV-GFPC3 virus. Hybrid viruses with the foreign gene GFPC3 deleted may have been formed in successive TMV-GFPC3 generations, resulting in the loss of GFP production and enhanced viral infectivity. In vitro generated RNA transcript and first generation TMV-GFPC3 were found to be more suitable for infection than the second generation TMV-GFPC3. However, the accumulation of GFP and virus concentration did not occur at the same ratio. Provided a more genetically stable transgenic viral vector is used for infection, transient viral infection of hairy roots can be a potential alternative system for foreign protein production than plants grown in the field as the containment or safety issues can be addressed.

Lily hybrids vary in their ability to produce virus-free (VF) bulblets when grown from virus-infected scales in tissue culture. Asiatic hybrids typically produce a higher percentage of in vitro VF scale bulblets than do Lilium longiflorum cultivars. Three hypotheses concerning the cause of this variation are tested on five lily hybrids: an Asiatic hybrid, two L. longiflorum cultivars, an Oriental hybrid and L. candidum. The first hypothesis states that VF scale bulblets originate from wound tissue that is naturally low in virus concentration and blocks the passage of virus particles from one cell to the next. The second hypothesis says that scale-to-bulblet vascular connections, which serve as virus pathways, occur in hybrids showing high percentages of virus-infected scale bulblets, while connections are absent in those hybrids with low numbers of virus-infected bulblets. The third hypothesis concerns the virus concentration in the scale at the site of bulblet origin: bulblets of hybrids producing large numbers of VF bulblets originate from scale tissues low in virus concentration; bulblets of low percentage VF bulblet hybrids originate from scale tissues high in virus concentration. The first two hypotheses are not supported by the results of this study. First, lily bulblets do not originate from wound tissue. Second, scale-to-bulblet vascular connections consistently occur in 'Enchantment,' an Asiatic hybrid, and occasionally occur in L. candidum. Vascular connections are not detected in the low VF bulblet producers, L. longiflorum cultivars 'Ace' and 'Nellie White,' nor are they seen in the Oriental hybrid 'Stargazer.' Speculative support exists for the third hypothesis concerning uneven virus concentration in the scale. Distinct virus particles are observed with the electron microscope in the double virus-infected L. longiflorum cultivars and not in the other singly-infected lilies. The doubly-infected lilies produce a continuous layer of divided cells in the adaxial subepidermis of the scale where bulblets originate, whereas the singly-infected lilies produce cell division masses in the same area but only beneath forming bulblets. This study suggests that virus particles in L. longiflorum cultivars are more uniformly distributed than particles in the other lilies examined. This occurs not only at the site of bulblet origin but also throughout the scale mesophyll. Whether this is due to concurrent viral infection or to hybrid variation is unknown. / Graduation date: 1992