Agrobacterium-mediated transformation of Mycosphaerella fijiensis, the devastating Black Sigatoka pathogen of bananas
Díaz-Trujillo, C. ; Adibon, H. ; Kobayashi, K. ; Zwiers, L.H. ; Souza, M.T. ; Kema, G.H.J. - \ 2010
Gewasbescherming 41 (2010)3. - ISSN 0166-6495 - p. 151 - 151.
mycosphaerella fijiensis - fungiciden - bananen - genotypen - fenotypen - rhizobium - genetische transformatie - genoomanalyse - mycosphaerella fijiensis - fungicides - bananas - genotypes - phenotypes - rhizobium - genetic transformation - genome analysis
Mycosphaerella fijiensis, M. musicola en M. eumusae veroorzaken de Sigatoka-ziekte in banaan. Op dit moment is de toepassing van fungiciden de enige optie om deze ziekte te bestrijden. Het PRPB (Pesticide Reduction Program for Banana) investeert in de ontwikkeling van technieken voor de genotype- en fenotypebepaling van M. fijiensis. Hierbij wordt gebruikt gemaakt van ATMT (Agrobacterium tumefaciens-mediated transformation).
On the introduction of genetically modified bananas in Uganda: social benefits, costs, and consumer preferences
Kikulwe, E.M. - \ 2010
Wageningen University. Promotor(en): Ekko van Ierland, co-promotor(en): Justus Wesseler; J.B. Falck-Zepeda. - [S.l. : S.n. - ISBN 9789085856108 - 198
bananen - musa - bioveiligheid - genetische modificatie - genetische transformatie - sociale uitkeringen - houding van consumenten - consumenten - consumentenvoorkeuren - kosten - gewassen - uganda - bananas - musa - biosafety - genetic engineering - genetic transformation - social benefits - consumer attitudes - consumers - consumer preferences - costs - crops - uganda
Agriculture is the mainstay for the great majority of rural people in most African countries
and is essential for poverty reduction and food security. The role of agriculture towards
poverty reduction, however, has not been realized in Africa, despite advances in development
of technologies such as improved varieties suitable to local conditions and resistant to pests,
diseases and droughts stresses. Plant breeding using modern biotechnology and genetic
modification in particular has the potential of speeding-up crop improvement. However, the
central issue in agricultural biotechnology particularly in Africa is to achieve a functional
biosafety system to ensure that a country has the capacity to assess risks that may be associated
with modern biotechnology. Several countries have designed and implemented policies to address
the safety concerns of consumers and producers, including environment and food safety. One of
the requirements, as proposed in Article 2 of the Cartagena Protocol, is the inclusion of
socioeconomic considerations in the biosafety assessment process. Many developing countries,
including Uganda, have not determined whether and how to include socioeconomic
considerations. Specifically, at what stage of the regulatory process should they be included, the
involved scope, as well as the nature of the decision-making process within the biosafety
regulations. The aim of my thesis is to examine potential social welfare impacts of introducing a
GM banana in order to illustrate the relevance of socioeconomic analyses for supporting
biotechnology decision-making and in particular the importance of consumer perceptions but
also for contributing to the development and implementation of biosafety regulations. I
present a general approach using GM banana as an example, while assuming the GM banana
has passed standard food and biosafety safety assessments, i.e. can be considered to be safe. I
explore the benefit-cost trade-offs of its introduction and the farmers’ and consumers’
willingness to pay for the technology and the end product. In the study I present a framework
for considering concerns about genetically modified crops within a socioeconomic analysis of GM
crops, using real options and choice experiment approaches. The approaches relate the economic
benefits to consumers’ concerns. The results show that the introduction of GM bananas would be
desirable for the Ugandan society as a whole, mainly benefit poor rural households and would
merit policy support. Nevertheless, if such a GM banana is introduced its introduction may
result in strong opposition from the opponent segment of the population, which is composed
of mainly urban consumers with an on average higher education and income. Interestingly
and in contradiction to common wisdom only providing additional information about the
technology and its safety will not result in higher acceptance. Based on this case study
biosafety regulators would need to consider these socioeconomic effects before a decision to
introduce a GM banana is made. However, the decision to consider socioeconomic impacts
for other GM crops elsewhere depends on the crop and the country. The research
methodology in this thesis provides the basis for assessing other GM crops as well.
Genetic analysis of breeding-related traits in Brassica rapa
Bagheri, H. - \ 2009
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - [S.l. : S.n. - ISBN 9789085855491 - 130
brassica campestris - brassica - genetische analyse - loci voor kwantitatief kenmerk - kwantitatieve kenmerken - kenmerken - rhizobium - zaadkenmerken - zaden - genetische transformatie - plantenveredeling - brassica campestris - brassica - genetic analysis - quantitative trait loci - quantitative traits - traits - rhizobium - seed characteristics - seeds - genetic transformation - plant breeding
Brassica rapa is an important crop with a variety of forms, and a wide distribution in the world. It is used as oil seed and vegetable crop and a valuable source of diverse health-promoting metabolites. It also can serve as a model for genetic and molecular analysis in the Brassica genus, to which all rapes, kales and cabbages belong, as it has the smallest genome size and some genotypes with a rapid life cycle.
Insertional mutagenesis using heterologous maize transposons has been a valuable tool for the identification and isolation of genes in Arabidopsis. Transposon-based activation tagging systems use a construct with constitutive enhancer elements that can cause transcriptional activation of flanking plant genes, which can result in dominant mutant phenotypes and subsequent isolation of the genes involved. Chapter 2 describes the action of an En/I activation tagging construct in B. rapa through Agrobacterium rhizogenes–mediated hairy root transformation. Successful transformation of this construct to B. rapa ssp. by A. tumefaciens was not achieved, probably due to the combination of an inefficient plant transformation and regeneration system, the length of the construct and most importantly the presence of the SU1 gene in the construct that appears to inhibit the regeneration of transformed shoots.
As an alternative to the insertional mutagenesis approach to identify genetic loci that impact traits, there is a genetic approach based on quantitative trait locus (QTL) analysis. Segregating populations are needed to map QTLs for traits of interest. Chapter 3 describes the analysis of an F2 population derived from a cross between two distinct, but early flowering and self compatible, B. rapa genotypes, L58 and R-o-18. Amplified fragment length polymorphism (AFLP) markers together with simple sequence repeat (SSR) markers were used to genotype this F2 population and anchor the linkage map to the reference genetic map of B. rapa. Highly significant QTLs associated with the production of adventitious roots and the transformation competence to A. rhizogenes were detected, which will allow the selection of lines that are more efficient in transformation experiments. The analysis detected a strong QTL associated with seed coat color as well as QTLs for various morphological traits.
To fix the recombination events as much as possible and to obtain an “immortal” mapping population, a recombinant inbred line (RIL) population was developed from this F2 population. Chapter 4 describes development of this RIL population, for which a genetic linkage map was constructed using the Illumina® BeadXpressTM genotyping platform of Keygene NV and additional SSR markers. Analysis revealed an additional QTL for seed coat colour as well QTL for pod shattering, carpel number, cuticular wax and seed vivipary. Chapter 5 describes the detection of QTLs related to primary and secondary metabolites in this RIL population. The two parental lines show clear differences in metabolite profile, which allowed the finding of QTLs for glucosinolates, phenylpropanoids, glucose, glutamate and amino acids after analysis with H1- NMR. HPLC analysis of tocopherols revealed four QTLs controlling the levels of this important antioxidant.
The information on the genetic control of health related compounds indicates the potential to improve nutritional quality in classical crop breeding programs.
The institutional and legal environment for GM soy in Brazil
Franke, A.C. ; Greco, F.M. ; Kleter, G.A. ; Noordam, M.Y. ; Roza, P. ; Eaton, D.J.F. ; Bindraban, P.S. ; Lotz, L.A.P. - \ 2009
Wageningen : Plant Research International (Report / Plant Research International 298) - 42
glycine - sojabonen - gewasproductie - genetische transformatie - wetgeving - handel - brazilië - glycine - soyabeans - crop production - genetic transformation - legislation - trade - brazil
Potential environmental introduction of unapproved GM crop species in the Netherlands
Prins, T.W. ; Wiel, C.C.M. van de; Kleter, G.A. ; Dolstra, O. ; Kok, E.J. - \ 2009
Wageningen : RIKILT Wageningen UR (RIKILT / PRI report 2009.007) - 50
gewassen - genetische modificatie - genetische transformatie - biotechnologie - transgene planten - risicoschatting - plant introduction - monitoring - nederland - crops - genetic engineering - genetic transformation - biotechnology - transgenic plants - risk assessment - plant introduction - monitoring - netherlands
Designing experimental protocols to investigate the impact of GM crops on non-target arthropods
Charleston, D.S. ; Dicke, M. - \ 2008
Bilthoven : Cogem (COGEM onderzoeksrapport CGM-2008-01) - 61
gewassen - geleedpotigen - transgene planten - genetische modificatie - biotechnologie - genetische transformatie - insecten - laboratoriumproeven - populatiegroei - proefopzet - insect-plant relaties - ecologische risicoschatting - protocollen - crops - arthropods - transgenic plants - genetic engineering - biotechnology - genetic transformation - insects - laboratory tests - population growth - experimental design - insect plant relations - ecological risk assessment - protocols
Het waarom en hoe van DuRPh: duurzame resistentie tegen Phytophthora in aardappel door cisgene merkervrije modificatie
Haverkort, A.J. ; Boonekamp, P.M. ; Jacobsen, E. ; Lotz, L.A.P. ; Visser, R.G.F. - \ 2007
Gewasbescherming 38 (2007)5. - ISSN 0166-6495 - p. 238 - 241.
aardappelen - vermeerderingsmateriaal - rassen (planten) - phytophthora infestans - plantenziekteverwekkende schimmels - pesticiden - genetische modificatie - milieueffect - plaagresistentie - innovaties - duurzaamheid (sustainability) - celkloneren - veredelingsmethoden - genetische transformatie - genetische gewasbescherming - economie - potatoes - propagation materials - varieties - phytophthora infestans - plant pathogenic fungi - pesticides - genetic engineering - environmental impact - pest resistance - innovations - sustainability - cell cloning - breeding methods - genetic transformation - genetic control - economics
Begin 2005 verzocht de interdepartementale Commissie Biotechnologie Wageningen UR een onderzoeksprogramma op te stellen voor een perspectiefvol GMO-project. Dit met het oog op de Nederlandse innovatieagenda en behoud of vergroting van de concurrentiekracht van de Nederlandse economie. Uitgangspunt daarbij was de Integrale Nota Biotechnologie (2000) met als centraal motto het verantwoord en zorgvuldig benutten van kansen ten behoeve van economie en duurzame landbouw. De keuze viel op een 'proof of principle' project van een duurzaam Phytophthoraresistente aardappel vanwege de economische aspecten zoals de kosten van beheersing van de ziekte en het veiligstellen van de winstgevendheid van de pootgoedsector. Milieuaspecten zoals reductie van de emissie van chemicaliën en een verminderd energiegebruik voor de toediening telden eveneens mee alsook het vergroten van de wetenschappelijk innovatiekracht
Exploring variation in pea protein composition by natural selection and genetic transformation
Tzitzikas, E. - \ 2005
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Jean-Paul Vincken; C.J.J.M. Raemakers. - s.n. - ISBN 9789085042938 - 109
pisum sativum - erwten - eiwitten - globulinen - genetische variatie - genetische transformatie - rhizobium radiobacter - nieuwe voedingsmiddelen - plantenveredeling - luciferasen - rassen (planten) - cultivars - pisum sativum - peas - proteins - globulins - genetic variation - genetic transformation - rhizobium radiobacter - novel foods - plant breeding - luciferases - varieties - cultivars
Pea (Pisumsativum L.) seeds are a rich and valuable source of proteins, which can have potential for food industrial applications. Pea storage proteins are classified into two major classes: the salt-soluble globulins, and the water-soluble albumins. The globulins are subdivided into two major groups based on their sedimentation coefficient: the 11S fraction (comprising the class ofleguminwith variousisoforms) and the 7S fraction (comprising the classes ofvicilinandconvicilin, each with variousisoforms).Pea cultivars with extreme variation in globulin composition (i.e. lacking a particular class of proteins) might become important for the food industry, because they could provide new raw materials for specific applications, like the production of Novel Protein Foods (NPFs), which receive attention as possible meatreplacers.This thesis aimed at (i) to determine the existing natural variation in pea's globulin content and composition, in order to identify suitable cultivars for the production ofNPFs, (ii) to develop a more efficient protocol for genetic modification of pea, and (iii) to modulate pea protein composition, based ondsRNAdirected silencing.An inventory of protein content and composition of pea was performed to characterize the genetic variation for these traits (Chapter 2). To include a wide range of natural genetic variation, cultivars from a wide geographic distribution, with differences in leaf and seed characteristics, were selected and characterized. Large variation was observed between the various lines. Results on protein content showed a variation from 16.3% to 36.6% of dry matter (DM), with an overall average content of 26.6%. Globulins content varied between 49.2% and 81.8% of the proteins of the total pea protein extract (TPPE). On individual globulins level,legumincontent varied between 5.9% and 24.5%.Vicilinwas the most abundant protein of pea, and its content varied between 26.3% and 52.0% of the TPPE. The processedvicilinwas the predominant of the two, with values between 17.8% and 40.8%, whereas the non-processed ones constituted between 3.1% and 13.5% of the TPPE.Convicilinwas the least abundant globulin having an average content of 6.1%. Its content ranged from 3.9% to 8.3%. Finally, the globulin-related proteins were present in amounts ranging from 2.8% to 17.3% of the TPPE. The globulins showed the largest relative variation of the four globulin classes.It is known that a lowvicilin/convicilinratio can result in poorgelation. Based on our data (Chapter 2) and the literature, it is concluded that pea isolates have a morefavourableprotein composition for gelling applications as compared to those from soybean. Moreover, the genetic variation for this trait appears to be larger in pea than in soybean, which might offer opportunities to reduce theconvicilincontent further.Our inventory did not show cultivars lacking a specific globulin. Such cultivars might be important, because they could have morefavourablephysical properties for the production ofNPFs. To produce such lines genetic modification approaches were employed. To carry out genetic modification a reliable protocol is needed. At the time this study started, protocols for the production of genetically modified peas were available, but particularly the regeneration of plants from transgenic cells was very inefficient. Most of the plants obtained were either escapes orchimeric(not all cells of a plant are genetically modified). Therefore, our study focused on obtaining a novel regeneration protocol, which in combination with the transformation procedure would result in an improved method for obtaining transgenic pea lines.The novel regeneration protocol started with subculture of stem tissue with one node (Chapter 3) or whole seeds (Chapter 4) on TDZ supplemented medium. Repeated subculture of stem tissue with one node resulted in a greenhyperhydrictissue in the swollen bases of the multiple shoots, which is fully covered with small buds [bud-containing tissue (BCT)] was formed. BCT fragments were isolated andsubculturedin the same medium and, as a result, they were able to reproduce themselves in a cyclic fashion. Subculture of BCT on medium supplemented with a combination of GA 3 ,cytokininsandauxinsresulted in the production of rooted shoots. In-vitro plants were transferred to the greenhouse foracclimatisationand further development. All tested pea cultivars ('Espace', 'Classic', 'Solara', and 'Puget') responded in the same way.Culture of seeds for a relatively long time (Chapter 4) on TDZ supplemented medium resulted in the production of very high numbers of shoots together with BCT which was identical to the BCT described in Chapter 3. This protocol resulted in the faster production of BCT as compared to the protocol described in Chapter 3.The regeneration protocol from Chapter 3 was combined with genetic modification (Chapter 5). Transgenic pea plants were obtained after co-cultivation of bud containing tissue (BCT) andshootyBCT withAgrobacteriumtumefaciens strain AGL0(pG49A). The binary vector pG49A contained an interrupted inverted repeat of aleguminA gene, flanked by the promoter of thetrypsin/chymotrypsininhibitor gene, together with theluciferasegene for selection of transgenic tissue.Luciferasepositive tissue was identified, isolated, andsubculturedon TDZ-supplemented medium. On this medium, BCT can be multiplied, and theshootyBCT will become pure BCT again. Theluciferasebased selection procedure was repeated until (almost) completeluciferasepositive BCT cultures were obtained. Plants (S 0 ) of 23 transgenic lines were grown in the greenhouse. The S 0 plants were smaller in size and produced less seeds than the control plants. All lines producedluciferasepositive seeds. The transgenic nature of 5 S 0 plants was further confirmed using Southern blot analysis. Protein analysis with SDS PAGE electrophoresis of the seeds of 8 lines indicated differences in protein composition, although our data were not conclusive on whether the amount ofleguminA or otherleguminswas affected. Further experiments should show whether the protein compositional changes resulted from silencing of theleg Agene, or other factors such as genetic or epigenetic changes in the genetically modified plants, caused by the tissue culture procedures.Seeds of 6 lines were grown to produce S 1 . The S 1 plants were comparable in height to control plants. However, the number of seeds per plant was significantly lower.The developed transformation protocol is highly repeatable. Each experiment resulted in genetic modified plants, in contrast to other systems, which have low repeatability. However, our system is more time consuming than those developed by others. Therefore, the regeneration system, which produces BCT directly from the seeds without the need for production of in-vitro plants, should be combined with genetic modification in the future. Furthermore, the selection of transgenic tissue should be optimised using selectable marker genes such asnpt IIor pat .
Factors influencing gene silencing of granule-bound starch synthase in potato
Heilersig, H.J.B. - \ 2005
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Anne-Marie Wolters. - [S.l.] : S.n. - ISBN 9789085042471 - 107
solanum tuberosum - aardappelen - genen - genetische modificatie - genexpressie - transgene planten - genetische transformatie - antisense rna - solanum tuberosum - potatoes - genes - genetic transformation - genetic engineering - gene expression - antisense rna - transgenic plants
In the past, antisense RNA technology was used to modify the composition of potato tuber starch. Potato starch comprises amylose and amylopectin, polymers of glucose. Amylose production in potato is completely dependent on the presence of granule-bound starch synthase I (GBSSI). Inhibition of GBSSI has been achieved by transformation with antisense and sense GBSSI constructs. However, the percentages of transformants showing strong silencing were relatively low which implicated that large numbers of transformants needed to be generated in order to obtain sufficient transformants showing strong silencing. This was an undesirable situation since in practical breeding many transformants would have to be made, making the effort too time consuming and costly. Therefore, an approach that would enhance the silencing efficiency was desired.This thesis describes the application of inverted repeat constructs for the modification of potato tuber starch. Transcription of inverted repeat constructs results in the formation of double stranded RNA (dsRNA). These dsRNA molecules are cleaved in small interfering RNAs (siRNAs) by a dsRNA-specific nuclease named Dicer. Subsequently, one strand of the siRNA is incorporated into the RNA Induced Silencing Complex (RISC) which is guided to the target mRNA through conventional base-pairing interactions. The target mRNA is then cleaved opposite the centre of the guide siRNA and finally, the cleaved mRNA is degraded.The antisense strand of the siRNA can also act as a primer on target mRNA after which new dsRNA is synthesized by the action of a cellular RNA-directed RNA polymerase (RdRP). The RdRP-synthesized dsRNA will be recognized by Dicer and degraded to secondary siRNAs. These secondary siRNAs can be derived from regions upstream of the targeted regions and can thus induce RNA silencing of sequences that were not initially targeted. This phenomenon is called transitive RNA silencing.First, an extensive study on the Post-Transcriptional Gene Silencing (PTGS) effects of eight different cDNA inverted repeat constructs was performed. These cDNA inverted repeat constructs were designed in such a way that the effect of size, orientation and sequence could be determined. The orientation of the inverted repeat did not affect silencing efficiency but the size and sequence did. Small inverted repeat constructs with a repeat size of 500-600 bp were more efficient silencing inducers than the large inverted repeat constructs with a repeat size of 1.1 or 1.3 kb. The two large inverted repeat constructs comprised the
Quantitative modelling in design and operation of food supply systems
Beek, P. van - \ 2004
In: Bayesian Statistics and Quality Modelling in the Agro-Food Production Chain / Boekel, van, Stein, A., Bruggen, van, Dordrecht : Kluwer (Wageningen UR Frontis series vol. 3) - ISBN 9781402019166 - p. 141 - 144.
voedselvoorziening - genetische transformatie - modellen - ketenmanagement - logistiek - food supply - genetic transformation - models - supply chain management - logistics
During the last two decades food supply systems not only got interest of food technologists but also from the field of Operations Research and Management Science. Operations Research (OR) is concerned with quantitative modelling and can be used to get insight into the optimal configuration and operation of food supply systems. In this contribution a short general introduction is given. Also three extended abstracts of case studies from the field will be presented: the milk-chilling chain, the impact of genetic-modification (GM) techniques on the structure of the cassava chain and the optimization of the production/distribution system of a dairy company in Hungary. Finally some conclusions are formulated
New technology adoption in food chains: a review with special reference to GMO applications in livestock production
Novoselova, T. ; Meuwissen, M.P.M. ; Huirne, R.B.M. - \ 2004
In: Dynamics in chains and networks, Proceedings of the sixth International Conference on chain and network management in agribusiness and the food industry, Ede, 27-28 May 2004. - Wageningen : Wageningen Academic Publishers - ISBN 9789076998404 - p. 33 - 39.
voedseltechnologie - kwaliteit voor voedselverwerking - genetische transformatie - transgene planten - veevoederindustrie - ketenmanagement - food technology - food processing quality - genetic transformation - transgenic plants - feed industry - supply chain management
De rol van heterotrimere G-eiwitten in de ontwikkeling en virulentie van Phytophthora infestans
Latijnhouwers, M. - \ 2003
Gewasbescherming 34 (2003)5. - ISSN 0166-6495 - p. 171 - 174.
solanum tuberosum - aardappelen - phytophthora infestans - plantenziekteverwekkende schimmels - virulentie - pathogenesis-gerelateerde eiwitten - genetische transformatie - solanum tuberosum - potatoes - phytophthora infestans - plant pathogenic fungi - virulence - pathogenesis-related proteins - genetic transformation
Onderzoek naar de ziekteverwekker Phytophthora infestans die de aardappelziekte veroorzaakt. Samenvatting van het proefschrift van Maita J.M. Latijnhouwers getiteld: 'The role of heterotrimeric G-proteins in development and virulence of Phytophthora infestans'
The role of heterotrimeric G-proteins in development and virulence of Phytophthora infestans
Latijnhouwers, M. - \ 2003
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Francine Govers. - [S.I.] : S.n. - ISBN 9789058088130 - 158
solanum tuberosum - aardappelen - phytophthora infestans - plantenziekteverwekkende schimmels - virulentie - pathogenesis-gerelateerde eiwitten - genetische transformatie - solanum tuberosum - potatoes - phytophthora infestans - plant pathogenic fungi - pathogenesis-related proteins - virulence - genetic transformation
Ever since the 1840s, when the disease first appeared in Europe and the United States, the threat of new outbreaks of late blight has terrified potato growers. In years when cool and wet weather conditions are prevailing, the disease is most destructive and nearly impossible to control. Decades of breeding efforts failed to yield resistant cultivars able to satisfy potato producers and consumers. In addition, many of the present-day fungicides are either ineffective or hazardous to human health or the natural environment. Phytophthora infestans , the organism causing late blight, is an oomycete. The class oomycetes encompasses eukaryotic pathogens and saprophytes with morphological similarities to fungi. Since fungi and oomycetes are evolutionary unrelated organisms, the ability to infect plants also evolved independently in both classes of organisms.
Heterotrimeric G-proteins are ubiquitously occurring signaling components that function in the translation of extracellular cues into intracellular signals and are indispensable for cells to adapt to alterations in environmental conditions. Animals, plants, fungi and lower eukaryotes such as the slime molds, all possess heterotrimeric G-proteins. In fungal plant pathogens, heterotrimeric G-proteins have been shown to control developmental processes such as mating, sporulation and formation of infection structures, including appressoria. Moreover, the virulence of plant pathogenic fungal species, in which genes encoding G-protein subunits were disrupted, was often reduced. To gain more insight in the regulation of development and virulence in P. infestans , we aimed at elucidating the role of conserved signaling pathways in this pathogen. We set off by studying the role of heterotrimeric G-proteins and the results are described in this thesis.
Heterotrimeric G-proteins consist of three subunits (a,bandg) of which theaandbsubunits are highly conserved. This high level of conservation was utilized to isolate P. infestans genes encoding these two subunits ( chapter 2 ). Degenerate primers corresponding to conserved regions in Gawere used in a PCR resulting in the amplification of a fragment of a Phytophthora Gasubunit gene. The gene was named Pigpa1 . A Gbgene, Pigpb1 , was isolated based on sequence information from a P. infestans mycelium EST database with high homology to Gbgenes from other organisms. Pigpa1 and Pigpb1 expression was developmentally regulated. Both genes were transcribed at the highest level in sporangia, the asexual spores of P. infestans . Both genes displayed lower levels of expression in zoospores and cysts. In mycelium, Pigpb1 was only lowly expressed while now expression of Pigpa1 could be detected.
Introduction of extra copies of Pigpa1 in the P. infestans genome triggered gene-silencing in a subset of the transformants, resulting in mutants that no longer transcribed the Pigpa1 gene and failed to synthesize the PiGPA1 protein ( chapter 3 ). Sporangial cytoplasmic cleavage and zoospore release was less efficient in these Pigpa1 -silenced mutants compared with the wildtype strain. Wildtype zoospores generally swim in straight lines and form aggregates at the liquid surface. The zoospores of the Pigpa1 -silenced mutants turned much more frequently than wildtype and showed no preference to move to the liquid surface. Neither did they aggregate and chemotaxis towards amino acids was absent in mutant zoospores. This indicates that PiGPA1 is involved in the regulation of flagellar movements, perception of chemotactic compounds, or both. The Pigpa1 -silenced mutants were severely reduced in virulence. In contrast, PiGPA1 gain-of-function mutants expressing a constitutively active form of PiGPA1, showed increased virulence in three out of four of these mutants. These observations point to a role for PiGPA1 in virulence of P. infestans .
Pigpb1 -silenced mutants were generated using a similar procedure as used for obtaining Pigpa1 -silenced mutants ( chapter 4 ). Two transformation methods were used to introduce copies of the Pigpb1 gene. The traditional PEG-mediated protoplast transformation resulted in a higher frequency of gene silencing than transformation by electroporation of zoospores. Pigpb1 -silenced mutants formed only very few sporangia when cultured on rye sucrose agar. Sporulation in the completely Pigpb1 -silenced mutants was even below 1% of wildtype sporulation. Instead, the mutants produced a denser mat of aerial mycelium than wildtype on the same growth medium. Pigpb1 expression in mycelium was induced under starvation conditions. PiGPB1 seems to represent an important link between starvation and sporulation.
Activation of G-proteins triggers a signaling cascade, which eventually results in changes in expression of particular genes. In chapter 5, experiments are described that aimed at finding genes, the expression of which is under control of the G subunit PiGPA1. Gene expression in sporangia of the wildtype strain, one of the PiGPA1 gain-of-function mutants and three Pigpa1 -silenced mutants was profiled using cDNA amplified fragment length polymorphism (cDNA-AFLP) analysis. Gene expression in wildtype mycelium was used as an internal control. Seventy-seven transcript-derived fragments (TDFs) were generated that were present in the wildtype profile and absent in the profile of the Pigpa1 -silenced mutants. Vice versa , 11 TDFs were present in the Pigpa1 -silenced mutants but absent in the wildtype strain. These two groups of TDFs are highly interesting as they are potentially derived from genes the expression of which is normally up- or downregulated by PiGPA1, respectively. The expression patterns of a subset of the genes from which those TDFs are derived were confirmed by RT-PCR and northern blot analysis. Twenty-seven of the differentially expressed TDFs were cloned and sequenced. A subset of these 27 clones showed homology with sequences present in P. infestans EST databases. Further analysis of these genes will reveal their possible functions in signaling and virulence.
Chapter 6 describes the first steps in the characterization of phospholipase D (PLD) signaling in P. infestans . PLD hydrolyzes phospholipids that are abundant in membranes, such as phosphatidylcholine or phosphatidylserine. The products are phosphatidic acid (PA) and the respective headgroup. PLD and PA are involved in vesicle trafficking in animals and possibly also in plants. PLD is activated by a variety of stress conditions in plants and conversion of PA to diacylglycerolpyrophosphate (DGPP) is often observed, presumably as a means to quench the PA signal. Treatment of P. infestans sporangia, zoospores, cysts and mycelium with the G-protein-activating peptide mastoparan resulted in increases in the levels of both PA and DGPP. This can be due to activation by mastoparan of the phospholipases PLD, PLC, PLC, or both. If available, PLD favors alcohols over water as the 'acceptor' of the phosphatidyl moiety, producing phosphatidylalcohol instead of PA. This feature was exploited to monitor PLD activity in P. infestans . Mastoparan treatment in the presence of butanol resulted in an increase in the level of phosphatidylbutanol, evidencing activation of PLD by mastoparan. Similarly, treatment with primary and secondary alcohols also activated PLD. Furthermore, it was demonstrated that mastoparan did not activate PLC in sporangia of P. infestans . PLD was activated during zoospore encystment. All PLD activators tested, as well as the product of PLD, PA, induced encystment. Taken together, we consider it likely that PLD plays a role in encystment.
Chapter 7 is a treatise on differences and similarities between strategies of fungal and oomycete pathogens to colonize plants. These groups are taxonomically unrelated. The comparison of the infection strategies of these two groups based on published data led us to conclude that they are more similar than anticipated. There are clear resemblances in infection structures, virulence factors and signal transduction pathways that govern development and virulence. Of course, there are also differences and we mention in this chapter the presence of certain classes of virulence factors that are described in fungi but have not (yet) been detected in oomycetes. We concluded that convergent evolution has had a large impact on the development of infection of plants in the two groups.
Production of polyhydroxyalkanoates (PHAs) in transgenic potato
Romano, A. - \ 2002
Wageningen University. Promotor(en): L.H.W. van der Plas; R.G.F. Visser; H. Mooibroek. - S.l. : [s.n.] - ISBN 9789058087263 - 149
solanum tuberosum - polymeren - polyesters - transgene planten - genetische transformatie - genetische modificatie - gentransfer - polyhydroxyalkanoaten - solanum tuberosum - polymers - polyesters - transgenic plants - genetic transformation - genetic engineering - gene transfer - polyhydroxyalkanoates
Polyhydroxyalkanoates (PHAs) represent a large class of microbial polyesters which are widely distributed in prokaryotes. Because of the current environmental concerns related to the use of mineral-oil-based plastics, PHAs gained a considerable interest for their possible use as biodegradable polymers. In recent years the possible fields of application for PHAs have broadened and a considerable number of novel uses of PHAs as rubber material, coating material, binder in paints and several medical applications have been developed. However, high costs related to the fermentative production of PHAs in natural or recombinant microorganisms hampered the production of PHAs on large scale and the expansion of the utilisation of PHAs into society. The use of transgenic plants for the production of PHAs has been considered an excellent and elegant strategy to accumulate bulk amounts of PHAs at low costs. In this thesis, the possibility to produce PHAs in transgenic potato was investigated. A number of metabolic engineering strategies has been considered and the accumulation of low amounts of PHAs in transgenic potato lines has been achieved.
PHA production is one of those traits that require the coordinate expression of several genes. Because the minimum gene set required for PHA accumulation was not completely known at the beginning of the experiments described in this thesis, a flexible system was required for gene-transfer into the potato genome in order to avoid time-consuming or technically difficult approaches (multiple genes plant vector construction, crossing, re-transformation). It was advantageous to introduce simultaneously into the potato genome all the genes of interest delivered via separate DNA molecules. This allowed the easy combination of different sets of genes to establish the minimum gene-set required for PHA accumulation in potato. Although Agrobacterium -mediated transformation is a well established technique to deliver alien genes into potato, our preliminary analyses showed that it was not suitable for simultaneous co-transformation using separate plasmids. Thus, we developed a particle bombardment-mediated procedure to introduce simultaneously into the potato genome several genes from separate plasmids or separate linear DNA fragments. The particle bombardment protocol established during the course of this project proved to be very efficient and comparable with Agrobacterium -mediated transformation. We observed that transgenic potato plants obtained by particle bombardment showed relatively simple patterns of integration of the transgenes and integration of different transgenes at independent sites into the plant genome. The system also proved applicable for co-transformation of potato using gene cassettes, i.e. DNA fragments comprising promoter - gene - terminator.
Particle bombardment-mediated co-transformation was subsequently used to introduce the genes involved in the PHA biosynthetic pathways. Four methods were considered for the production of PHAs in potato.
Method 1. The accumulation of the homopolymer polyhydroxybutyrate (PHB) was achieved after Ralstonia eutrophaphb B and phb C genes coding for an NADPH-dependent acetoacetyl-CoA reductase and for the PHB-polymerase, respectively, were expressed in transgenic potato lines. Because the minimum gene set for PHB production in plants was well documented when this project started, the possible production of PHB seemed to be the simplest model to test the ability of potato to accumulate PHAs. Indeed, PHB formation in small amounts was observed.
Subsequently, we attempted the production of the heteropolymer medium-chain-length (mcl)-PHA in potato.
Method 2. To study the expression of the Pseudomonas oleovorans mcl-PHA-polymerase in potato, the polymerase was first expressed in the cytoplasm, where no post-transcriptional modification of the polypeptide is needed. Proper expression of the mcl-PHA polymerase was indeed observed in selected transgenic lines. However, because the cytoplasm of potato does not contain suitable substrates for the polymerase (mcl-hydroxy fatty acids), substrate feeding experiments were performed. These experiments resulted in the accumulation of low amounts of mcl-PHA.
Methods 3 and 4. Afterwards, mcl-PHA accumulation was attempted in plastids, by re-directing hydroxy fatty acid precursors from FAB to mcl-PHA biosynthesis. A truncated form of E. coli thioesterase-I ( tes A gene. Method 3) and P. putida ACP-CoA-transacylase ( pha G gene. Method 4) were expressed in combination with the Pha-C1 polymerase. All heterologous proteins were targeted to the plastid by N terminal fusion with the Rubisco transit peptide. The two combinations of genes (Pha-C1 plus thioesterase, and Pha-C1 plus transacylase) represent theoretically the minimum gene set for mcl-PHA accumulation deriving precursors from FAB. The double transformants which expressed the Pha-C1 polymerase and the thioesterase-I did not accumulate mcl-PHA because a fewb-oxidation steps may be required to provide the proper precursor to the polymerase. However, simultaneous expression of the Pha-C1 polymerase and the ACP-CoA-transacylase did result in the synthesis of low amounts of mcl-PHA in leaves of intact plants.
We established several possible routes to achieve the accumulation of PHAs in transgenic potato, based on the use of the endogenous pool of acetyl-CoA, on feeding approaches of cell suspension cultures or on using precursors derived from de novo FAB. Although improvements in these systems are still required in order to increase the yields of PHAs, the results open new prospects for the accumulation of PHAs in potato. Final conclusions on the future development of production of PHAs in plants are overviewed. Advantages and disadvantages of fermentation-based-PHAs, plant-based-PHAs, other biodegradable polymers and oil-based-plastics are compared. Novel and future possible interesting applications of PHAs not based on bulk productions, are also described.
Cell cycle and storage related gene expression in potato tubers
Verhees, J. - \ 2002
Wageningen University. Promotor(en): L.H.W. van der Plas; A.R. van der Krol; D. Vreugdenhil. - S.l. : S.n. - ISBN 9789058086587 - 133
solanum tuberosum - aardappelen - knollen - genexpressie - celcyclus - methodologie - genetische transformatie - luciferasen - plantenfysiologie - in vivo experimenten - solanum tuberosum - potatoes - tubers - gene expression - cell cycle - methodology - genetic transformation - luciferases - plant physiology - in vivo experimentation
Potato tubers are vegetative structures aimed at survival of the period when conditions are not optimal for the growth of the potato plant. Understanding the process of tuber formation in potatoes ( Solanum tuberosum ) is important, both from a scientific view, and for practical applications. During the initial stages of tuber formation an enhanced synthesis of starch and storage proteins is observed at the site of the stolon that develops into a tuber. Besides an enhanced synthesis of storage products, longitudinal cell division and cell enlargement is observed resulting in a radial expansion of the stolon. Because cell division and the synthesis of storage products are two important events during tuber development we analyzed the expression of two sets of genes: two cell cycle genes (cycB1;1 and CDC2a) and two storage related genes (AGPaseS andlPat21). To analyze cell cycle and storage related gene expression we fused the promoter sequences of these genes to the firefly luciferase reporter gene from North American firefly ( Photinus pyralis ). Luciferase (LUC) activity can be observed non- destructively in individual tubers and correlates with the luciferase mRNA level in both in vitro grown as well as in soil grown tubers, indicating that in planta LUC activity reflects the ongoing expression level of the reporter gene. More important, LUC activity correlates with the mRNA level of the endogenous genes.
In chapter 2 we describe the expression of cell cycle and storage related gene expression in in vitro grown explants in relation to the onset of swelling marking the morphological onset of tuber formation. The average activity quantified in multiple tubers indicated that the storage related genes were upregulated well before visible swelling, viz., four days before swelling for AGPaseS, and three days before swelling forlPat21, while the average activities of both cell cycle genes showed an upregulation after the onset of swelling. However with the ability to observe changes in gene expression in individual tubers a variable upregulation for both storage related genes could be shown in relation to the moment of visible swelling, varying between four days before till ten days after the onset of swelling. The results indicate that tuber development does not occur via a fixed sequential order of events, but consists of independent individual programs that occur in the same region of the stolon resulting in the formation of a potato tuber.
When after tuber initiation, environmental conditions become adverse for tuber induction, second growth may occur. Adverse tuber-inducing conditions may result in the outgrowth of the apical bud (second growth) and a reduced growth of the tuber (primary growth). We mimicked second growth inducing conditions by transferring developing in vitro tubers to medium with gibberellin (chapter 3) or to medium with a low level of ethanol (chapter 4). GA-induced second growth resulted in the outgrowth of the apical bud followed by the formation of a secondary stolon. Transfer of tubers to medium supplemented with ethanol resulted in the formation of a secondary tuber when combined with 8% sucrose, whereas ethanol combined with 1% sucrose resulted in the formation of a secondary stolon. Preceding bud growth cycB1;1, AGPaseS andlPat21 expression declined in the complete tuber both in young (1-5 days after visible swelling) and in mature tubers (25-30 days after visible swelling) after transfer to medium with GA 4+7 or medium with ethanol, whereas CDC2a activity only declined when tubers were transferred to medium with ethanol. Second growth inducing conditions did not affect the activity of the CaMV35S promoter, indicating that the decline in the cell cycle and storage related promoter activity in the tubers during second growth is not related to a general effect on gene expression. Although a decline in cell cycle and storage related gene expression is observed before second growth, there is no causal relation between this decline and the outgrowth of the bud, as concluded from analysis of tubers transferred to medium with GA or ethanol combined with ABA which prevents outgrowth of the bud. Under these conditions cell cycle and storage related gene expression also declined. Transferring tubers to medium with different alcohols and combining ethanol with an inhibitor of alcohol dehydrogenase (4-methyl pyrazole) showed that ADH activity is a key factor during ethanol-induced second growth and the changes observed in gene expression in bud and tuber region.
Preceding second growth, a higher cycB1;1 and AGPaseS expression is observed in the bud region as compared to the activity in the tuber region. CycB1;1 expression in the bud region indicates a specific induction of the cell cycle genes during second growth. However, a relatively higher cycB1;1 expression in the bud is not sufficient for bud growth both during GA or ethanol-induced second growth. These results indicate that bud growth is not controlled by cycB1;1. AGPaseS expression in the bud region during second growth possibly indicates the enhanced import of sugars into the bud region during second growth. Preventing bud outgrowth by combining GA with ABA counteracts the difference in AGPaseS expression between bud and tuber in mature tubers, indicating that tuber and bud region respond differentially upon GA and ABA. During ethanol-induced second growth, relatively higher AGPaseS expression was observed in the bud region in young tubers when transferred to medium with ethanol and 8% sucrose whereas tubers transferred to medium with ethanol and 1% sucrose showed a decline in AGPaseS expression both in bud and tuber region during second growth. These results indicate that the type of second growth and the enhanced AGPaseS expression in the bud region during ethanol-induced second growth are related to the amount of sugars available for the bud. Mature tubers transferred to medium with ethanol showed higher AGPaseS expression in the bud region, irrespectively of the sucrose concentration in the medium. Combining ethanol with ABA could not prevent AGPaseS expression in the bud region during ethanol-induced second growth. These results indicate that ABA affects sugar import in the bud region during GA-induced second growth whereas ABA does not affect sugar import during ethanol-induced second growth.
In chapter 5 we described the relation between the expression of the cell cycle and storage related genes in the tubers and the differences in tuber growth rate of tubers growing on complete plants, to determine if the expression of these genes can be used as a measure for differences in sink strength and sink activity between tubers. Tuber growth rate was determined during the complete period of tuber development as an indication for sink strength and the correlation was determined with the level of gene expression quantified per tuber and per unit tuber area. By using hydroponics pot assemblies we were able to observe tuber growth and to quantify gene expression non-destructively during the complete period of tuber development. Three different stages of tuber growth were distinguished viz., initiation, linear tuber growth and maturation. During initiation, a low correlation was observed between tuber growth rate and the expression level of the cell cycle and storage related genes. During the period of linear tuber growth a good correlation was observed forlPat21 while during maturation the expression of cycB1;1, AGPaseS andlPat21 correlated well with the sink strength, as reflected by the growth rate. The results presented in chapter 5 indicate that patatin gene expression is a good predictive marker for differences in sink strength between tubers initiated on the same potato plant.
It has become clear that non-invasive methods to study gene expression are useful tools for developmenta l studies as described in this thesis on potato tuber development. Changes in gene expression can be observed in individual tuber samples, showing the exact relation between changes in gene expression and developmental changes.
Analysis of post-harvest deterioration in tuberous roots of cassava (Manihot esculenta Grantz)
Huang, J. - \ 2001
Wageningen University. Promotor(en): R.G.F. Visser; E. Jacobsen. - S.l. : S.n. - ISBN 9789058085481 - 103
manihot esculenta - cassave - bederf na de oogst - bewaarfysiologie - biochemie - enzymen - genetische merkers - genetische transformatie - moleculaire genetica - transgene planten - manihot esculenta - cassava - postharvest decay - postharvest physiology - biochemistry - enzymes - genetic markers - genetic transformation - molecular genetics - transgenic plants
This thesis deals with the analysis of postharvest physiological deterioration (PPD) in cassava tuberous roots at the physiological, biochemical and molecular level. By setting up a uniform visual system to monitor the onset and maintenance of PPD under the experimental conditions, amongst others physico-chemical properties of starch were characterized. Furthermore using this system, around 6,000 TDFs (transcript derived fragments) were screened via 100 primer combinations using cDNA-AFLP. 70 TDFs showing an up-regulated, a down regulated or a transiently expression pattern were isolated following the first 72 hours of PPD. Based on the sequence information, a functional catalogue of these TDFs was established. By concentrating on enzymes possibly involved in oxidative stress, biochemical results indicated that PPD may be a peroxidase-mediated process. Using a reverse genetics approach, the putative cassava dad1 (defender against cell death gene 1) homologue was transformed into cassava FEC (friable embryogenic callus) lines. Transgenic plants were produced and characterized.
Towards onions and shallots (Allium cepa L.) resistant to beet armyworm (Spodoptera exigua Hübner) by transgenesis and conventional breeding
Zheng, S.J. - \ 2000
Agricultural University. Promotor(en): E. Jacobsen; C. Kik; F.A. Krens. - S.l. : S.n. - ISBN 9789058082961 - 146
uien - sjalotten - allium cepa - plaagresistentie - insectenplagen - spodoptera exigua - plantenveredeling - genetische transformatie - transgene planten - onions - shallots - allium cepa - pest resistance - insect pests - spodoptera exigua - plant breeding - genetic transformation - transgenic plants
Onion ( Allium cepa L. group Common Onion) and shallot ( A. cepa L. group Aggregatum) are two subspecies of A. cepa . Both onion and shallot together with other Allium species like garlic ( A. sativum ), leek ( A. porrum ) and bunching onion ( A. fistulosum ) are very important vegetable crops on a worldwide scale. A. cepa is cultivated mainly as a biennial but some types are treated as perennials. It is propagated by seeds, bulbs, or sets (small bulbs). TThe bulbs of common onion are large, normally single, and plants are reproduced from seeds or from seed-grown sets. By contrast the bulbs of shallot are smaller than common onions, they form aggregated clusters as a result of the rapid formation of lateral bulbs or shoots. Reproduction of shallot is almost only vegetative via daughter bulbs although seed production is possible.
The cultivation of onion and shallot is sometimes severely limited due to the occurrence of diseases and pests. The most important pest in A. cepa cultivation for (sub) tropical zones is the beet armyworm ( Spodoptera exigua Hübner). The beet armyworm is an extremely polyphagous insect. In this research programme, two different strategies were followed to develop plant material, which is resistant to S. exigua , namely via marker-assisted breeding (MAB) and via genetic transformation. For marker-assisted breeding, first of all a reliable bio-assay needs to be developed. Secondly, suitable sources of resistance must be identified. Thirdly, the genetic basis (and the mechanism of resistance) needs to be uncovered and fourthly, molecular markers must be linked to the resistance gene(s), and finallyourthly an efficient means of screening large populations for the molecular markers should be available and the screening technique should have high reproducibility.
In marker-assisted breeding, we succeeded to develop an in vivo and in vitro bio-assay for the identification of resistance to beet armyworm in A. cepa and its wild relatives (Chapter 2). In the in vivo bio-assay the lowest larval survival (36 %) and the lowest fresh weight (10.3 mg per larva) were found on A. roylei, a wild relative of Allium cepa L.. This was not, however, significantly different from the resistance in the tropical shallot cultivar Bawang Bali. Furthermore, in the in vitro bio-assay no toxic insecticidal compound was present in A. roylei because no significant differences were found in mean fresh weight per larva and mean survival of larvae among different accessions. There were also no significant differences in pupal weight and developmental time. All larvae became pupae 10 days after inoculation. Therefore, a marker-assisted breeding approach to introduce beet armyworm resistance in A. cepa was abandoned.
For genetic transformation, first of all a reliable plant regeneration system from callus cultures and suspension cultures must be established. Secondly, an efficient and stable transformation system should be developed. Thirdly, molecular characterization techniques of transgenic plants should be available and fourthly, a construct carrying specific B. thuringiensis cCry or other gene sequences effective against S. exigua should be available. We started with a systematic study on the effects of subspecies, cultivar, basal medium, sucrose concentration and 2,4-dichlorophenoxyacetic acid concentration on callus induction, propagation and subsequent plant regeneration in Allium cepa. A reliable regeneration system was developed based on mature zygotic embryo-derived callus. It was shown that regeneration in 45.4% of the callus lines using an optimal combination of factors influencing tissue culture response in Allium could be obtained (Chapter 3).
The development of a reliable regeneration system is thought to be of vital importance for the next step in our research, which is the development of a reliable transformation system. The aim was to identify callus lines with a high regeneration potential and to set up a suspension culture system for later transformation studies by means of particle bombardment. Suspension cultures were initiated from callus cultures of Allium cepa , which had been precultured on a solidified medium for seven months. For another three months the 83 callus lines were kept in suspension culture. Of these, 20 suspension lines showed adequate growth and were used to test the effect of cytokinins on plant regeneration. However, it was found that the plant regeneration capacity of selected lines significantly decreased in a relatively short time-period. Plant regeneration also proved to be highly dependent upon the line used. Contrary to expectations, the type and concentration of cytokinins could not increase the shoot regeneration capacity (Chapter 4). On the basis of these results it was the concludedsion that only relatively young callus (less 3 months) could be used for Allium transformation.
In Chapter 5 the development of a reliable transformation system is described using Agrobacterium tumefaciens as a vector. A number of parameters, such as callus age, callus induction medium, co-cultivation period, osmotic treatment, cultivars, selection method and Agrobacterium strains were used to evaluate transient expression of the uidA reporter gene in a series of experiments followed by evaluating stable expression conditions. Eventually, an Agrobacterium tumefaciens- mediated transformation system both for onion and shallot was developed using three-week old callus derived from mature embryos using two different strains EHA105 (pCAMBIA 1301) and LBA4404 (pTOK233) carrying a binary vector coding for uidA and hpt . This transformation protocol for onion and shallot can be used year-round because mature zygotic embryos are used as starting material. A total of 11 independent callus lines regenerating transgenic plants have been produced in several independent experiments. In one of these experiments, starting with 154 embryos, transgenic plants were recovered from three independent callus lines, giving a transformation frequency of 1.9 %. The best line produced 90 transgenic plants. Subspecies (onion and shallot) and cultivar were important factors for successful transformation: shallot was better than onion and for shallot with cv. Kuning the best results were obtained. Also, it was found that constantly reducing the size of the calli during subculturing and selection by chopping, thus enhancing exposure to the selective agent hygromycin, improved the selection efficiency significantly. The transformants were genetically characterized by means of standard PCR, genomic DNA blot hybridization and FISH (fluorescence in situ hybridization).
Genomic DNA blot hybridization is traditionally used to analyse integration of foreign genes into host genomes. . Although genomic DNA blot hybridization can be used to determine the presence of T-DNA and copy number, it becomes labour-intensive when many samples have to be assayed, and it consumes relatively large amounts of genomic DNA. Especially for large genome species crops, e.g. onion (15290 Mbp/1C), with a genome size which is 105 times larger than that of Arabidopsis. Adapator ligation PCR (AL-PCR) followed by the sequencing of the genomic DNA flanking the T-DNA borders was developed (Chapter 6). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed how T-DNA integration took place and it also provided insight into the number of T-DNA copies present. Comparison of AL-PCR and previously obtained Southern hybridization results pointed into the direction of rather complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing of the AL-PCR products, the junctions between plant genomic DNA and the T-DNA inserts were analysed into great detail. For example, it was shown in one of the transgenic lines that upon T-DNA integration a 66 bp sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover this particular target site for T-DNA integration. The target site sequence proved to be repetitive and Allium specific because a similarly-sized PCR fragment was obtained in all Allium species tested while it was not detected in closely related genera such as Lilium and Tulipa .
In Chapter 7, the general discussion, the possibilities for the development of onion and shallot resistant to Spodoptera exigua are discussed. It is argued that both MAB and genetic transformation methods are potentially very powerful and that in case of the introduction of resistance to beet armyworm into onion and shallot genetic transformation is the most promising.
Regeneration and transformation by particle bombardment in leek (Allium ampeloprasum L.)
Schavemaker, C.M. - \ 2000
Agricultural University. Promotor(en): E. Jacobsen; R.G.F. Visser. - S.l. : S.n. - ISBN 9789058082343 - 100
preien - allium ampeloprasum - verjonging - genetische transformatie - transformatie - genetische modificatie - somatische embryogenese - recombinant dna - transgene planten - allium porrum - biolistiek - leeks - allium ampeloprasum - regeneration - biolistics - genetic transformation - transformation - genetic engineering - somatic embryogenesis - recombinant dna - transgenic plants - allium porrum
In this thesis the results are presented of experiments aiming at the genetic modification of leek ( Allium ampeloprasum L.). Leek is a vegetable grown for its edible (false) stem and belongs to the Alliaceae, together with onion ( Allium cepa ) and garlic ( Allium sativum ). The production of leek is mainly confined to Europe. In the last few years production has increased along with consumer demands. It is propagated through seeds and gives rise to heterogeneous progeny. Problems in cultivation of leek are rust ( Puccinea allii , P.mixtu ), yellow stripe virus and the lack of uniformity. The most suitable system able to cope with these problems seems to be hybrid breeding. However, hybrid breeding is hampered by the lack of a suitable emasculation system or male sterility system and the severe inbreeding depression. Therefore, emphasis has been put on the application of genetic modification in order to solve some of these problems. This relatively new technique opens the possibility to add or alter traits which cannot easily be achieved with conventional breeding methods. The most important prerequisites for genetic modification were investigated during this thesis research. The first prerequisite is an efficient regeneration system. Starting from seed, a cyclic somatic embryogenesis regeneration system was developed with long term regeneration potential, providing a regeneration system where it should be possible to obtain true transformants from chimeric structures (Chapter 2).
A comparison was made between the first cycle, starting from zygotic embryos, and latter cycli, starting with somatic embryos. All genotypes tested were able to produce somatic embryos although genotypic differences in somatic embryo production occurred. The first cycle, using zygotic embryos, was the most effective in somatic embryo production compared to the later cycli using somatic embryos. On average the first cycle produced 23.8 somatic embryos per zygotic embryo and the later cycli ranged from 11.1 to 16.0 somatic embryos per initial somatic embryo. Shoot regeneration from somatic embryos was satisfactory, obtaining normal looking greenhouse plantlets.
In Chapter 3 this cyclic somatic embryogenesis system was analyzed for its suitability in a genetic transformation system, like particle bombardment. The relatively new reporter gene luciferase was used in the transformation experiments. Leek, like most monocotyledons, seemed to be very persistent to selective media. Neither the selective agent kanamycin nor hygromycin could prevent leek cells from growing. The selective agent phosphinothricin had a better inhibitory effect on cell growth. A histological and morphological study showed that regeneration occurred from the deeper cell layers inside the somatic embryo, in the same way that leaves are produced on a mature leek plant. These cell layers are hardly reached by particles, explaining the poor results of the transformation experiments using somatic embryos.
In Chapter 4 a newly developed regeneration system from flower stalk strip explants was used to determine the optimal conditions for particle bombardment with the luciferase reporter gene. After a vernalisation period leek starts to develop a flower stalk. This flower stalk, still inside the plant, is harvested and stripped. Regeneration from these strips occurs just beneath the epidermis, which is easy to reach for the particles of the particle gun. The regeneration frequency was neither influenced by the stripping nor by the bombardments. Over 300 plantlets could be obtained from 1 flower stalk. Important factors for transformation experiments like pre-culture time, pressure, distance and coating of the particles were analyzed and optimized for these highly regenerative explants. Flower stalk strips of leek as explants, cut from the basal part of the flower stalk, pre-cultured for 2 days, should be bombarded at a distance of 5 cm, with a pressure of 1800 psi to obtain a high transient expression. Gold particles should be used and coated following the procedure of Christou (1991). After bombardment the explants should be transferred to selection medium.
In Chapter 5 these optimal conditions were applied to the flower stalk strip explants. A comparison of the use of the reporter genes GUS and luciferase was made. Both genes, combined in one plasmid, showed similar expression patterns. Expression of both genes was still present 7 weeks after bombardment, but local increases in gene activity were not observed. The reporter gene luciferase facilitates the investigations in the genetic modification research as the chemical reaction with this reporter gene is non-lethal to the plant tissue whereas the reaction with GUS-reporter gene is. The non-detrimental effect of the luciferin treatment made it possible to investigate gene expression in time. Still, using a novel reporter gene means also unexpected results like the long transient expression time of the luciferase gene product even after application of the substrate luciferin. Eventually, 16 chimeric plantlets were obtained. Probably, the regeneration from flower stalk strip explants is a multicellular event. Seeds harvested from potentially chimeric plants did not show any GUS or luciferase activity after germination.
In the near future leek transformation research has to focus on the development of embryogenic cell suspension or protoplast cultures, to obtain true transformants of the chimeric plants and an efficient selection system to select and favor the transgenic cells. To facilitate success in transformation research, efforts could also be directed at plastid transformation, in order to come to true breeding transgenic lines
The helper component-proteinase of cowpea aphid-borne mosaic virus
Mlotshwa, S. - \ 2000
Agricultural University. Promotor(en): A. van Kammen; J. Wellink; I. Sithole-Niang. - S.l. : S.n. - ISBN 9789058083401 - 111
vigna unguiculata - vignabonen - potyvirus - kousenbandrolmozaïekvirus - pathogeniteit - weerstand - dna-sequencing - dna - genoomanalyse - genetische modificatie - genetische transformatie - ziekteresistentie - vigna unguiculata - cowpeas - potyvirus - blackeye cowpea mosaic virus - pathogenicity - resistance - dna sequencing - dna - genome analysis - genetic engineering - genetic transformation - disease resistance
Cowpea aphid-borne mosaic potyvirus causes severe yield losses in cowpea, an important legume crop in semi-arid regions of Africa. We have elucidated the genomic sequence of the virus and subsequently focused our attention on the so-called helper component-proteinase (HC-Pro), a virus-encoded multifunctional protein with roles in different steps of the virus life cycle. Our study has shed more insight into some of the molecular properties of this protein. We have shown that HC-Pro is able to shut down host defense responses, and this puts HC-Pro at the core of the success of CABMV as a pathogen. The phenomenon also seems to benefit other viruses as they accumulate to higher levels and elicit enhanced symptoms in the presence of HC-Pro. On the other hand, we have found that the host does manifest an ability to counter the deleterious effects of HC-Pro. A full understanding of the molecular basis of this contest would enable the design of effective new strategies to protect plants from virus infections.
Development of two in vitro regeneration systems through leaf explant and callus culture and the application for genetic transformation in Alstroemeria
Lin, H.S. - \ 1998
Agricultural University. Promotor(en): E. Jacobsen; M.J. de Jeu. - S.l. : Lin - ISBN 9789054859062 - 119
weefselkweek - callus - explantaten - Alstroemeria - bladeren - genetische transformatie - plantenveredeling - tissue culture - callus - explants - Alstroemeria - leaves - genetic transformation - plant breeding
Alstroemeria is a popular ornamental crop cultivated for its flowers. Taxonomically, it belongs to a monocotyledonous family, the Alstroemeriaceae, and is commonly called by its genus name. An Alstroemeria plant consists of underground grown rhizomes, roots, and aerial shoots. The plant is grown perennially. Due to the good incorporation of plant breeding techniques combined with the modern greenhouse cultivation technologies of the last two decades, Alstroemeria has become a competitive greenhouse-grown cut flower in the Netherlands. Generally, the Alstroemeria plant is vegetatively propagated by rhizome division, but the multiplication rate is rather low. Therefore, the increasing demand for plantlets stimulated the development of in vitro propagation techniques. However, since the multiplication unit used in the in vitro method is limited to rhizome tips, the propagation rate is still rather low in comparison with other crops and the other plant organs seem to be of no use during subculture. In addition, a callus culture system has been developed in the last few years for plant propagation purpose. The multiplication efficiency of this system is expected to be higher than that of the rhizome culture system, but the commercial true-to-type requirement cannot be fulfilled, because the callus was initiated from zygotic embryos. Therefore, the development of an additional in vitro multiplication system based on other plant organs is considered to be desirable (Chapter 1).
Plant regeneration of cultured explants has in general two pathways, either via organogenesis (the development of shoots directly on an explant) or via embryogenesis (the development of differentiated somatic embryos on an explant). A large part of this thesis research deals with the development of two plant regeneration systems, one based on organogenesis and the other based on embryogenesis. In Chapter 2, a two-step protocol for the induction of shoot formation from in vitro grown Alstroemeria leaf explants is described. Leaf explants were cut from seedlings still containing a leaf blade and a stem node. After 10 days of culture on an induction medium, the leaf explants were transferred to a shooting medium for eight weeks. New shoots were formed directly from the area adjacent to the region between leaf base and node tissue within three weeks on shooting medium. It was histologically demonstrated that these shoots were initiated from the epidermal cells at leaf axils (Chapter 3). There were no pre-existing axillary buds ever found on the aerial leaf axils, so that this kind of organogenesis suggests an adventitious nature. The leaf explants together with newly formed shoots were subcultured several times and many normal plantlets with rhizomes were formed, which then were suitable for transferring to the soil (Chapter 2, Chapter 4).
The best induction was obtained on a Murashige and Skoog's (1962) medium supplemented with 10μM thidiazuron (TDZ) and 0.5μM indole butyric acid (IBA) (Chapter 2). The shooting medium contained MS medium with 2.2μM 6-benzylaminopurine (BAP). The shoot regeneration capacity of the excised leaf explants was related to the position of the leaf on the stem. The youngest explant which was located the nearest to the shoot apex, gave the highest response. A lower gradient response was found in the leaf explants derived from leaves cut off at a further distance from the apex. This was measured in percentage of shoot regeneration per leaf explant and in the number of shoots per regenerating explant (Chapter 3).
A demonstration experiment was carried out in the greenhouse in order to investigate the similarity of plant growth morphology in between plants, which were obtained from either rhizome multiplication or leaf explant culture system. The plants were grown in the greenhouse to flowering, and the results indicated that plants obtained from both systems were morphologically identical (Chapter 4). This implicates that the leaf culture system seems to be a reliable in vitro propagation technique for the genotype we have investigated.
Another advantage of the leaf explant culture system is that the leaf explants directly can be excised from in vivo full grown shoots, and that the disinfection of aerial shoots is easier than that of underground grown rhizomes (Chapter 4). Therefore, this technique is suitable for the initiation of in vitro propagation of existing cultivars.
In the conventional micropropagation system, only the rhizome tips are multiplied and therefore, the aerial shoots are always discarded during subculture. In this thesis research, it is concluded that not only the rhizome tips can be used as propagation units, but also the discarded shoots can be used for the initiation of the other propagation system. The first three leaves excised from each shoot have an average regeneration capacity of 87.7%, and the average number of newly formed shoots per explant was 5.3 (Chapter 4). On the other hand, the rhizomes can be multiplied simultaneously. Therefore, combining the rhizome multiplication system with the leaf explant culture system, the multiplication efficiency will be enhanced.
In Chapter 5, a somatic embryogenic callus regeneration system is described. A soft and sticky type of callus was induced initially from the stem segments of one month old seedlings of two tetraploid Alstroemeria genotypes. The soft calli turned into compact type after subculture on a medium (MS with 30 g/l sucrose) containing 6-benzylaminopurine. Subsequently, two other different morphotypes of callus, friable and granular, were obtained by subculturing the compact callus on different culture media. The friable callus can be maintained on a single medium (PCA) containing 10 mg/l picloram for a long period without loosing its friability. Subculturing the friable callus on plant growth regulator free media or on 6-benzylaminopurine containing media stimulated the granular callus formation, and the subsequent somatic embryogenesis. The somatic embryos were able to develop into complete plants.
The granular callus proved to be an intermediate between friable callus, somatic embryo, and compact callus. The friable callus could also be induced from granular callus, and vice versa. Therefore, a cyclic reproduction system was established in this research. This system provides two types of callus with a high embryogenic capability, which were initially derived from the stem segments. Thus, this system is considered to be applicable for the in vitro propagation of Alstroemeria .
In addition to the purpose of plant propagation, the development of a plant regeneration system is also considered to have the potential for genetic modification in Alstroemeria . Some characteristics, for example virus resistances, are very important in the continuously greenhouse-grown cultivars. However, virus resistance genes are not generally present in the Alstroemeria gene pool yet, so that the traditional breeding techniques are not sufficient for this purpose. Genetic transformation of Alstroemeria is considered to be useful for breeding in the future (Chapter 1). For genetic transformation, four important factors should be taken into account: a) an efficient DNA delivery system, b) the appropriate target cells competent for both transformation and regeneration, c) an adequate promoter, and d) a good selection system (Chapter 1).
In this research, the particle bombardment delivery system was chosen for the monocot A lstroemeria , because of its expectedly higher transformation efficiency than the Agrobacterium vector system (Chapter 1). The leaf explant regeneration system was tested for gene transformation by using the particle bombardment. Although the gene expression could be detected after particle bombardment, the gene activities were only transiently expressed on leaf tissues, and they disappeared within two months (Chapter 7). On the other hand, the somatic embryogenic callus regeneration system was successfully used for particle bombardment mediated gene transformation. Two tetraploid Alstroemeria genotypes were transformed, and many transgenic plants were obtained (Chapter 6).
Both granular and friable calli were used as bombardment targets, and the subsequent somatic embryogenesis resulted in the formation of complete transgenic plantlets. Two plasmids containing different selection and reporter genes were used. Firstly, a plasmid containing a firefly luciferase reporter gene, driven by the maize ubiquitin promoter ( Ubi1 ), was bombarded into both granular and friable calli. The luciferase activity was measured by a luminometer after spraying the bombarded plant material with a luciferin solution. Visual selection of the luciferase positive calli, assisted by the luminometer, was effective. This kind of selection has a nondestructive nature, without injuring the plant material, and the luciferase activity can be assayed periodically over the whole developmental process from callus to embryo and plantlet. It was shown that the granular callus is more suitable for particle bombardment mediated transformation using luciferase activity as selection marker than the friable callus (Chapter 6).
Secondly, another plasmid containing the selectable Basta (herbicide) resistance gene ( bar ) encoding phosphinotricin acetyltransferase (PAT) together with an uid A reporter gene encoding ß-glucuronidase (GUS) was used. Both genes were driven by the Ubi1 promoter. The granular calli were bombarded in this experiment. Selection of the phosphinotricin (PPT) resistant calli was accomplished by culturing the bombarded calli on a medium containing 5 mg/l PPT. The PPT resistant calli were the friable type of calli which were already regenerated from the granular calli, and they developed into somatic embryos, and subsequently into the plantlets. Stable expression of the GUS gene was confirmed by histochemical staining. The blue color was detectable in all tissues of the transgenic plants tested by the GUS assay. The PPT selection proved to be a more efficient and labor-saving method compared to the luciferase selection (Chapter 6).
The results described in this thesis are beneficial for both the in vitro propagation and the genetic modification of Alstroemeria . The use of leaf explants as in vitro propagation units is rather unique in Alstroemeria , which opens an alternative way for enhancing the plant propagation efficiency. The embryogenic callus regeneration system described in this thesis is not only applicable for plant propagation, but also for genetic transformation. The establishment of particle bombardment mediated transformation techniques will push the molecular breeding in Alstroemeria forward into a luminous future.