Application of molecular tools for detection of plant viruses

Grapevine leafroll disease (GLRD) is one of the most important virus diseases of grapevines worldwide, causing major economical impact. The disease has a complex aetiology and currently eleven phloem-limited viruses, termed in general Grapevine leafroll-associated virus (GLRaVs), have been identified. Two of the GLRaVs, GLRaV-1 and GLRaV-3, are included in the European certification scheme of propagation material. However, the flawed notion that GLRaV-3 is more frequent than GLRaV-1 and that all other GLRaVs are possibly not as relevant for GLRD, has until now precluded the development of specific serological and molecular detection assays and limited the scope of molecular characterization of the viruses known to be associated with the disease. Hence, few studies have addressed the phylodynamics of GLRaVs or even characterized the genetic structure of their natural populations. This generalized lack of molecular information, in turn underlie the deficient capacity to detect the viruses. The phylogenetic analyses were conducted on the basis of the heat shock protein 70 homologue (HSP70h) and the coat protein (CP) genes for GLRaV-1 and the HSP70h, the heat shock protein 90 homologue (HSP90h) and the CP genes for GLRaV-5. The data obtained for GLRaV-1 contributed 83 new CP sequences. This information was combined with previous analysis by other authors and used for the production of new polyclonal IgG, capable of detecting CP variants from all the phylogroups observed. Successful testing of this new tool included tissue print immunoblotting (TPIB) and in situ immunoassay (ISIA). The data obtained for GLRaV-5, contributed 61 new CP and 28 new HSP90h gene sequences. Eight phylogenetic groups were identified on the basis of the CP. Characterization of the genetic structure of the isolates revealed a higher diversity than previously reported and allowed the identification of dominant virus variants. For both GLRaV-1 and GLRaV-5, the effect of vegetative propagation on the virus transmission dynamics was addressed.

Etablierung neuer Methoden zur in vitro Kultivierung der Baumart Fraxinus excelsior L. und Entwicklung von Verfahren zur Kryokonservierung von in vitro Sprossspitzen

Die hier vorliegende Arbeit wurde im Rahmen eines europäischen Projektes mit dem Titel „Improving Fraxinus (Ash) productivity for European needs by testing, selection, propagation and promotion of improved genetic resources“ an der Niedersächsischen Forstlichen Versuchsanstalt, Abteilung Waldgenressourcen erstellt. Im Rahmen des Projektes wurden 62 Plusbäume aus einem 15 Jahre alten europäischen Herkunfts-/ Nachkommenschaftsversuch in den Niedersächsischen Forstämtern Bovenden und Dannenberg nach den Kriterien Stammform und Wuchsleistung für die vegetative Vermehrung ausgewählt. Ziel dieser Arbeit war die Optimierung bestehender in vitro Protokolle sowie die Entwicklung eines bisher noch nicht existierenden Kryokonservierungsprotokolls für in vitro Sprossspitzen. Im ersten Teil dieser Arbeit wird die Entwicklung des in vitro Protokolls für Fraxinus excelsior dargestellt. Die Optimierung der Methoden zur Etablierung, Vermehrung und Bewurzelung erfolgte durch Versuchsreihen mit unterschiedlichen Klonen, so dass insgesamt 26 der selektierten Plusbäume erfolgreich in vitro etabliert werden konnten. Achselknospen frischer Triebe der Pfropflinge der Mutterbäume stellten die beste Explantatquelle dar. Die Explantate wurden mit 0,2 % Quecksilberchlorid (HgCl2) oberflächensterilisiert bevor sie auf hormonfreies Woody Plant Medium (WPM) transferiert wurden. Nach zwei Wochen erfolgte ein Transfer auf WPM mit 4 mg/l 6-Benzylaminopurine (BAP) und 0,15 mg/l Indole-3-butyric acid (IBA). Die besten Vermehrungsraten wurden auf WPM mit 4 mg/l BAP, 0,15 mg/l IBA und 0,01 mg/l TDZ und 0,7 % Agar in Honiggläsern mit einem Plastikdeckel erzielt. Als Bewurzelungsmedium wurde 0,5 konzentriertes Murashige und Skoog (MS) Medium mit 2 mg/l IBA, 0,25 mg/l BAP und 0,8 % Agar verwandt. Im zweiten Teil der Arbeit werden die Versuchsreihen zur Entwicklung des Kryokonservierungsprotokolls von in vitro Sprossspitzen dargestellt. Zur Entwicklung der Methode wurden die Vorbehandlungsbedingungen verbessert und zwei Techniken, die Alginat- / Dehydrati-onsmethode und die Vitrifikationsmethode mit Hilfe der sogenannten PVS2-Lösung (Plant Vitrification solution number 2) getestet. Die optimierte PVS2-Methode erwies sich als die für Esche besser geeignete Technik und ließ sich erfolgreich zur Kryokonservierung juveniler und adulter Kulturen anwenden. Die Regenerationsraten lagen zwischen 50 und 100 % für juvenile bzw. 50 und 80 % für adulte Kulturen.

Reproductive biology and population studies in two annual shuttle mosses

Observations on clumps of Phascum cuspidatum during the summer and autumn indicated that this species is at least a short-lived perennial, as young shoots develop from old, brown shoots persisting from the previous winter. No young shoots arising by vegetative propagation were recorded in Pottia truncata. Rhizoid tubers were observed in this species, but only in one of the many clumps examined. Spores of both species germinated freely in culture, but when spores were planted in the field young gametophytes developed inconsistently in P. truncata and never in P. cuspidatum. An investigation of spore deposition around an isolated clump of P. truncata suggested that 67% of the spores released were deposited within the clump, and 70% within 2m. Electrophoretic studies indicated limited genetic variation within two populations of each species, with no genotypes in common between the populations. No genetic variation was recorded between gametophytes within individual clumps of either species, nor between sporophytes and their maternal gametophytes, suggesting a high incidence of inbreeding in these monoecious mosses. (author abst.)

Technologies for biological containment of GM and Non-GM crops

International Perspective The development of GM technology continues to expand into increasing numbers of crops and conferred traits. Inevitably, the focus remains on the major field crops of soybean, maize, cotton, oilseed rape and potato with introduced genes conferring herbicide tolerance and/or pest resistance. Although there are comparatively few GM crops that have been commercialised to date, GM versions of 172 plant species have been grown in field trials in 31 countries. European Crops with Containment Issues Of the 20 main crops in the EU there are four for which GM varieties are commercially available (cotton, maize for animal feed and forage, and oilseed rape). Fourteen have GM varieties in field trials (bread wheat, barley, durum wheat, sunflower, oats, potatoes, sugar beet, grapes, alfalfa, olives, field peas, clover, apples, rice) and two have GM varieties still in development (rye, triticale). Many of these crops have hybridisation potential with wild and weedy relatives in the European flora (bread wheat, barley, oilseed rape, durum wheat, oats, sugar beet and grapes), with escapes (sunflower); and all have potential to cross-pollinate fields non-GM crops. Several fodder crops, forestry trees, grasses and ornamentals have varieties in field trials and these too may hybridise with wild relatives in the European flora (alfalfa, clover, lupin, silver birch, sweet chestnut, Norway spruce, Scots pine, poplar, elm, Agrostis canina, A. stolonifera, Festuca arundinacea, Lolium perenne, L. multiflorum, statice and rose). All these crops will require containment strategies to be in place if it is deemed necessary to prevent transgene movement to wild relatives and non-GM crops. Current Containment Strategies A wide variety of GM containment strategies are currently under development, with a particular focus on crops expressing pharmaceutical products. Physical containment in greenhouses and growth rooms is suitable for some crops (tomatoes, lettuce) and for research purposes. Aquatic bioreactors of some non-crop species (algae, moss, and duckweed) expressing pharmaceutical products have been adopted by some biotechnology companies. There are obvious limitations of the scale of physical containment strategies, addressed in part by the development of large underground facilities in the US and Canada. The additional resources required to grow plants underground incurs high costs that in the long term may negate any advantage of GM for commercial productioNatural genetic containment has been adopted by some companies through the selection of either non-food/feed crops (algae, moss, duckweed) as bio-pharming platforms or organisms with no wild relatives present in the local flora (safflower in the Americas). The expression of pharmaceutical products in leafy crops (tobacco, alfalfa, lettuce, spinach) enables growth and harvesting prior to and in the absence of flowering. Transgenically controlled containment strategies range in their approach and degree of development. Plastid transformation is relatively well developed but is not suited to all traits or crops and does not offer complete containment. Male sterility is well developed across a range of plants but has limitations in its application for fruit/seed bearing crops. It has been adopted in some commercial lines of oilseed rape despite not preventing escape via seed. Conditional lethality can be used to prevent flowering or seed development following the application of a chemical inducer, but requires 100% induction of the trait and sufficient application of the inducer to all plants. Equally, inducible expression of the GM trait requires equally stringent application conditions. Such a method will contain the trait but will allow the escape of a non-functioning transgene. Seed lethality (‘terminator’ technology) is the only strategy at present that prevents transgene movement via seed, but due to public opinion against the concept it has never been trialled in the field and is no longer under commercial development. Methods to control flowering and fruit development such as apomixis and cleistogamy will prevent crop-to-wild and wild-to-crop pollination, but in nature both of these strategies are complex and leaky. None of the genes controlling these traits have as yet been identified or characterised and therefore have not been transgenically introduced into crop species. Neither of these strategies will prevent transgene escape via seed and any feral apomicts that form are arguably more likely to become invasives. Transgene mitigation reduces the fitness of initial hybrids and so prevents stable introgression of transgenes into wild populations. However, it does not prevent initial formation of hybrids or spread to non-GM crops. Such strategies could be detrimental to wild populations and have not yet been demonstrated in the field. Similarly, auxotrophy prevents persistence of escapes and hybrids containing the transgene in an uncontrolled environment, but does not prevent transgene movement from the crop. Recoverable block of function, intein trans-splicing and transgene excision all use recombinases to modify the transgene in planta either to induce expression or to prevent it. All require optimal conditions and 100% accuracy to function and none have been tested under field conditions as yet. All will contain the GM trait but all will allow some non-native DNA to escape to wild populations or to non-GM crops. There are particular issues with GM trees and grasses as both are largely undomesticated, wind pollinated and perennial, thus providing many opportunities for hybridisation. Some species of both trees and grass are also capable of vegetative propagation without sexual reproduction. There are additional concerns regarding the weedy nature of many grass species and the long-term stability of GM traits across the life span of trees. Transgene stability and conferred sterility are difficult to trial in trees as most field trials are only conducted during the juvenile phase of tree growth. Bio-pharming of pharmaceutical and industrial compounds in plants Bio-pharming of pharmaceutical and industrial compounds in plants offers an attractive alternative to mammalian-based pharmaceutical and vaccine production. Several plantbased products are already on the market (Prodigene’s avidin, β-glucuronidase, trypsin generated in GM maize; Ventria’s lactoferrin generated in GM rice). Numerous products are in clinical trials (collagen, antibodies against tooth decay and non-Hodgkin’s lymphoma from tobacco; human gastric lipase, therapeutic enzymes, dietary supplements from maize; Hepatitis B and Norwalk virus vaccines from potato; rabies vaccines from spinach; dietary supplements from Arabidopsis). The initial production platforms for plant-based pharmaceuticals were selected from conventional crops, largely because an established knowledge base already existed. Tobacco and other leafy crops such as alfalfa, lettuce and spinach are widely used as leaves can be harvested and no flowering is required. Many of these crops can be grown in contained greenhouses. Potato is also widely used and can also be grown in contained conditions. The introduction of morphological markers may aid in the recognition and traceability of crops expressing pharmaceutical products. Plant cells or plant parts may be transformed and maintained in culture to produce recombinant products in a contained environment. Plant cells in suspension or in vitro, roots, root cells and guttation fluid from leaves may be engineered to secrete proteins that may be harvested in a continuous, non-destructive manner. Most strategies in this category remain developmental and have not been commercially adopted at present. Transient expression produces GM compounds from non-GM plants via the utilisation of bacterial or viral vectors. These vectors introduce the trait into specific tissues of whole plants or plant parts, but do not insert them into the heritable genome. There are some limitations of scale and the field release of such crops will require the regulation of the vector. However, several companies have several transiently expressed products in clinical and pre-clinical trials from crops raised in physical containment.

Propagação de espécies nativas com potencial ornamental : Kelissa brasiliensis (Baker) Ravenna e Sinningia lineata (Hjelmq.) Chautems

A pesquisa em tecnologia de sementes contribui para a manutenção de bancos de germoplasma. Kelissa brasiliensis (Baker) Ravenna e Sinningia lineata (Hjelmq.) Chautems são espécies nativas com potencial ornamental. Os objetivos deste trabalho foram desenvolver testes para a germinação de sementes, conhecer a qualidade fisiológica de lotes coletados in situ, desenvolver protocolos de propagação vegetativa e conhecer estratégias reprodutivas no ambiente. Avaliaram-se o vigor, bem como o comportamento germinativo das sementes em diferentes substratos, temperaturas e condições de luz. Foram realizados experimentos com propagação vegetativa de S. lineata. O trabalho foi conduzido no Laboratório de Sementes da Fepagro, no Jardim Botânico de Porto Alegre e na Faculdade de Agronomia da UFRGS. Durante as coletas, foi feita a observação dos nichos das espécies. O delineamento experimental foi completamente casualizado com quatro repetições de 10 e 20 sementes para K. brasiliensis e S. lineata, respectivamente; para S. lineata, três tratamentos com 17 repetições foram utilizados no experimento com estaquia, e nove tratamentos com quatro repetições para o teste de divisão dos tubérculos. A comparação das médias foi realizada através do teste de Duncan (P<0,05). A temperatura mais adequada para a germinação de sementes de K. brasiliensis foi 10ºC, o que explica a sua restrita distribuição ao bioma Pampa; a combinação 24h/25ºC levou a uma liberação maior de lixiviados na condutividade elétrica; o envelhecimento acelerado (72 h em 41ºC e 100% de umidade) não provocou redução significativa no percentual de germinação das sementes de K. brasiliensis. Já as sementes de S. lineata não germinaram após serem expostas ao estresse. A temperatura mais adequada para o teste de germinação de sementes de S. lineata foi 20ºC; para ambas espécies, o substrato papel e a presença de luz foram as condições mais adequadas para a germinação. S. lineata demonstrou ser facilmente propagada por sementes e via assexuada, o que revelou a rusticidade característica das espécies rupícolas.

Enraizamento de estacas de Camellia sinensis L. em função da época de coleta de ramos, genótipos e ácido indolbutírico

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Distribuição do sistema radicular de porta-enxertos de umezeiro enxertados com o pessegueiro 'Aurora-1'

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Produção de mudas de maracujazeiro-amarelo por enxertia hipocotiledonar sobre sete espécies de passifloras

The yellow passion fruit (Passiflora edulis Sims f. flavicarpa Deg.) Passifloraceae is the main crop in Brazil, and the crop has found some difficulties, particularly concerned to the orchards longevity, which has been reduced due to diseases and nematodes that attack the root system. This study was carried out to establish technologies for the production of seedlings of yellow passion fruit by hypocotyledonary grafting on seven rootstocks. The treatments used as rootstocks were: P. edulis f. flavicarpa, P. caerulea, P. alata, P. giberti, P. coccinea, P. cincinnata and P. setacea. The experimental design was completely randomized, with 36 replications for each rootstock. The percentage of graft survival, plant height, number of leaves and rootstock diameter were the characteristics evaluated. The hypocotyledonary grafting methodology was successful for most species tested. P. caerulea, P. giberti, P. cincinnata and P. flavicarpa were the stood out rootstocks by their high conditions of plants growth, and by their early plants production.

Propagação de jambeiro vermelho (Syzygium malaccense L.) por estaquia de ramos herbáceos

The consumption of exotic fruits has been showing accentuated increase and the cultivated area is in expansion, generating demand for adequate culturing techniques. The Malay apple (Syzygium malaccense), with probable origin in India, has a fruit widely known and appreciated in the North and Northeasthern Brazilian states. The Malay apple tree is extremely tall and has a long juvenile period when propagated by seed, making its vegetative multiplication is desirable, to anticipate the productive period and decrease its size, and also to obtain uniform orchards. The experiment was conducted at UNESP/FCAV, Jaboticabal Campus, using Malay apple herbaceous cuttings subjected to treatments with indol butyric acid (IBA) (0; 1,000; 3,000 and 5,000 mgL(-1)) and cuttings with and without basal incision. The variables analyzed were percentage of survival and rooting of the cuttings, number and mean length of roots per cutting. The experiment was conducted under CRB on a factorial scheme (4 X 2) with 4 replicates constituted by 10 cuttings each. Data were analyzed by Tukey's mean test at 5% probability. The vegetative propagation by rooting of herbaceous cuttings of the Malay apple is possible, however, both IBA treatments and basal incision have not shown significant effect on the analyzed variables.

Effect of wounds applied to the bases of herbaceous cuttings on the rooting of four Japanese apricot clones (Prunus mume Sieb. et Zucc.) in an intermittent mist system

The Japanese apricot (Prunus mume Sieb. et Zucc.) is a fruit tree of the Rosaceae family which produces very acid and bitter fruits, highly appreciated by Orientals. In Brazil, this species has been studied as a rootstock for peach and nectarine trees, its main advantage being the reduction in plant vigour, which can favour the production of compact trees and orchard cultural treatments. This study was conducted in the Vegetable Production Department of FCAV/UNESP, Jaboticabal Campus, São Paulo State, Brazil, and the objective was to examine the effect of wounding the herbaceous cutting bases on the rooting of four Japanese apricot clones. The clones were obtained from plants under cultivation in the Instituto Agronomico de Campinas, Brazil, and were identified as Clones 02, 05, 10 and 15. The stock plants, obtained through herbaceous cuttings, were maintained under lath house conditions (50% of natural light). Cuttings 12 cm long with 3 to 5 leaves were collected from these clones and prepared. The experiment was carried out in a completely randomised design with 4 repetitions of 20 cuttings per replication, in a factorial 4 x 2 design, the clone factor having 4 levels (Clones 02, 05, 10 and 15) and the wounding factor at 2 levels of incisions into the cutting base (with and without). All the cuttings were treated with 2000 mg.L-1 of IBA for five seconds. Differences between the clones were observed concerning the rooting percentage, dead cuttings, number and length of roots. The incision (wound) at the base of the herbaceous cuttings of the Japanese apricot increased the number of roots and improved the distribution of these in the damaged tissue but the results were not considered sufficiently beneficial to make the treatment worthwhile.

Influência da fonte material e do tempo de cura na propagação vegetativa da pitaya vermelha (Hylocereus undatus Haw)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Propagação vegetativa de jambeiro vermelho [Syzygium malaccense (L.) Merr. & L.M. Perry]

The Malay Apple [Syzygium malaccense (L.) Merr. & L. M. Perry] is an option for the producer as its fruits are attractive and well accepted by the consumer. The difficulty of culture of this species is that the plant is very tall and has a long juvenile period when propagated by seed, making its vegetative propagation necessary to anticipate the productive period and decrease its size. The possibility of vegetative propagation of this species was studied by cutting, layering, and grafting. In the cutting experiment, three doses of indolbutyric acid (IBA) (0, 1,000, 3,000, and 5,000 mg L(-1)) were tested in two lengths of herbaceous cuttings (15 and 25 cm). In the layering experiment, two periods of layering (summer and autumn) and four doses of IBA (0, 1,000, 4,000, 7,000, and 10,000 mg kg(-1)) were evaluated. For grafting, the compatibility between S. malaccense and S. jambos (L.) Alston as rootstock was studied with two diameters (0.5 and 0.8 cm) and in two periods (winter and summer), by method of full graft. For cuttings, the percentage of rooting was 20%, independently of the IBA doses, except for 5,000 mg L(-1) that showed negative effect on 15 cm cuttings; layering and grafting were not successful in the studied conditions.

Propagação de três genótipos de abieiro (Pouteria caimito) por estaquia de ramos herbáceos

Foram selecionadas três plantas de um pomar comercial, com características agronômicas desejáveis, proveniente de propagação seminífera, que forneceram estacas herbáceas, contando com cerca de 15 cm de comprimento, um par de folhas no nó superior, corte em bisel na base e foram tratadas com 4 doses de IBA (0, 1.000, 3.000 e 5.000 mg.l-1) e, posteriormente, plantadas em caixas plásticas perfuradas contendo como substrato, vermiculita textura média. A condução do experimento foi em ambiente de 50 % de sombreamento, sob nebulização intermitente. A análise estatística foi em esquema fatorial 3X4, (genótipos X doses de IBA), com 4 repetições de 10 estacas cada. Após 90 dias foram avaliados a percentagem de enraizamento e sobrevivência, o comprimento e número médio de raízes por estaca. O IBA não influenciou as variáveis analisadas. O genótipo somente induziu mais raízes na seleção 3 e maiores raízes nas seleções 1 e 3.

CLONAGEM do JAMBEIRO-ROSA (Syzygium malacensis) POR ESTAQUIA DE RAMOS ENFOLHADOS

O jambeiro-rosa é uma fruteira exótica que representa uma alternativa aos fruticultores, devido às características organolépticas de seus frutos. em virtude da segregação genética e ausência de sementes em vários clones, procurou-se, neste trabalho, estudar a propagação vegetativa, utilizando-se de estacas com folhas e a influência do tratamento com AIB. O trabalho foi realizado na Área experimental de fruticultura da Faculdade de Ciências Agrárias e Veterinárias -- UNESP, Câmpus de Jaboticabal, São Paulo, no período de outubro de 1998 a março de 1999, tendo como objetivo avaliar a capacidade de enraizamento de estacas com folhas apicais e subapicais de jambeiro-rosa com a utilização de diferentes concentrações de ácido indolbutírico (AIB). O delineamento experimental utilizado foi o inteiramente casualizado, em um esquema fatorial 2 x 4 (2 tipos de estacas e 4 concentrações de AIB), com 4 repetições e 10 estacas por parcela, num total de 320 estacas. As concentrações de AIB utilizadas foram: 0; 100; 200 e 400 mg.L-1, e as estacas foram colocadas em imersão lenta por 14 horas, no escuro. Foram avaliados os percentuais de sobrevivência das estacas e enraizamento das estacas, número médio de raízes e brotos por estaca, e comprimento médio dos brotos. Observou-se que o jambeiro-rosa pode ser propagado por estacas com folhas apicais sem a utilização de AIB.

PROPAGAÇÃO do UMEZEIRO (Prunus mume Sieb & Zucc.) POR ESTAQUIA HERBÁCEA

A utilização do umezeiro ou damasqueiro-japonês (Prunus mume Sieb & Zucc.) como porta-enxerto de Prunus sp. vem despertando grande interesse em função de sua rusticidade, resistência a pragas e doenças, adaptação e, principalmente, por reduzir o porte de pessegueiros e nectarineiras. Este trabalho foi conduzido no Departamento de Produção Vegetal da FCAV/UNESP, Câmpus de Jaboticabal-SP, e teve por objetivo estudar a propagação vegetativa desta espécie. Para tanto, utilizaram-se estacas herbáceas com 12cm de comprimento dos Clones 02; 05; 10 e 15, provenientes do Programa de Melhoramento Genético do Instituto Agronômico de Campinas, submetidas às concentrações de 0 e 2000mg.L-1 de AIB, por cinco segundos. O experimento foi conduzido em delineamento experimental inteiramente casualizado, com 4 repetições de 20 estacas por parcela, esquema fatorial 4 x 2, sendo o fator clone em 4 níveis (Clones 02; 05; 10 e 15) e AIB em 2 níveis (0 e 2000mg.L-1). de acordo com os resultados, verificou-se diferença entre os clones quanto à porcentagem de enraizamento, sendo o Clone 15 significativamente superior ao Clone 02 (93,75% e 78,13%, respectivamente). Os Clones 05 (85,0%) e 10 (83,13%) comportaram-se como intermediários, não diferindo dos demais. Não houve diferença entre os clones testados quanto à formação de calo, raízes por estaca, comprimento de raízes e porcentagem de estacas brotadas. O ácido indolbutírico na concentração de 2000mg.L-1 favoreceu a emissão de raízes adventícias e aumentou o comprimento das raízes, mas não teve influência na brotação das estacas. Não houve efeito da interação entre os fatores testados para as variáveis analisadas.