Expression and inheritance pattern of two foreign genes in petunia

Transgenic petunia (Petunia hybrida Vilm.) plants were obtained from Agrobacterium-mediated shoot apex transformation. Studies at the phenotypic as well as molecular level established both the presence of the NPT II (neomycin phosphotransferase II) and GUS (-glucuronidase) genes and their level of activity. Twenty-nine primary transformed plants showed varying patterns of phenotype expression of both genes. NPT II and GUS expression in 7 primary plants over a 4-month interval showed varying levels of gene expression within and among individual plants. All primary transgenic plants were self-pollinated and backcrossed to establish the inheritance patterns of both genes. Mendelian and non-Mendelian inheritance patterns for both genes were observed. Analysis of the progeny showed poor transmission of the foreign genes through the pollen especially when two or more bands were present in the Southern hybridization. Most plants whose progeny segregated in Mendelian ratios for either the NPT II or GUS gene had just one copy of the gene. In this study where both foreign genes were examined in both self and test crosses, no transgenic plant showed Mendelian patterns of inheritance for both foreign traits.Department of Plant Pathology and Microbiology     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

Generation of transgenic Lolium temulentum plants by Agrobacterium tumefaciens-mediated transformation

Lolium temulentum L. (Darnel ryegrass) has been proposed to be used as a model species for functional genomics studies in forage and turf grasses, because it is a self-fertile, diploid species with a short life cycle and is closely related to other grasses. Embryogenic calluses were induced from mature embryos of a double haploid line developed through anther culture. The calluses were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harboring pCAMBIA1301 and pCAMBIA1305.2 vectors were used to infect embryogenic callus pieces. Hygromycin was used as a selection agent in stable transformation experiments. Hygromycin resistant calluses were obtained after 4–6 weeks of selection and transgenic plants were produced in 10–13 weeks after Agrobacterium-mediated transformation. Fertile plants were readily obtained after transferring the transgenics to the greenhouse. Transgenic nature of the regenerated plants was demonstrated by Polymerase chain reaction (PCR), Southern hybridization analysis, and GUS staining. Progeny analysis showed Mendelian inheritance of the transgenes. The transformation system provides a valuable tool for functionality tests of candidate genes in forage and turf grasses.     

Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation

Camelina sativa is an alternative oilseed crop that can be used as a potential low-cost biofuel crop or a source of health promoting omega-3 fatty acids. Currently, the fatty acid composition of camelina does not uniquely fit any particular uses, thus limit its commercial value and large-scale production. In order to improve oil quality and other agronomic characters, we have developed an efficient and simple in planta method to generate transgenic camelina plants. The method included Agrobacterium-mediated inoculation of plants at early flowering stage along with a vacuum infiltration procedure. We used a fluorescent protein (DsRed) as a visual selection marker, which allowed us to conveniently screen mature transgenic seeds from a large number of untransformed seeds. Using this method, over 1% of transgenic seeds can be obtained. Genetic analysis revealed that most of transgenic plants contain a single copy of transgene. In addition, we also demonstrated that transgenic camelina seeds produced novel hydroxy fatty acids by transforming a castor fatty acid hydroxylase. In conclusion, our results provide a rapid means to genetically improve agronomic characters of camelina, including fatty acid profiles of its seed oils. Camelina may serve as a potential industrial crop to produce novel biotechnology products.     

Agrobacterium-mediated transformation of pepino and regeneration of transgenic plants

Regeneration of pepino (Solanum muricatum Ait.) shoots was achieved both by organogenesis and by embryogenesis. Shoots derived via organogenesis were easily rooted and most regenerated plants appeared phenotypically normal. Transgenic plants were obtained using the binary vector pKIWI110 in the avirulent Agrobacterium tumefaciens strain LBA4404. Optimization of transformation protocols was rapidly achieved by monitoring early expression of the GUS (-D-glucuronidase) reporter gene carried on pKIWI110. Transgenic plants expressed GUS and selectable marker genes for kanamycin resistance and chlorsulfuron resistance. PCR (polymerase chain reaction) and Southern analysis provided molecular evidence for transformation.     

Production of selectable marker-free transgenic tobacco plants using a non-selection approach: chimerism or escape,transgene inheritance,and efficiency

Public concern and metabolic drain were the main driving forces for the development of a selectable marker-free transformation system. We demonstrated here the production of transgenic tobacco plants using a non-selection approach by Agrobacterium tumefaciens-mediated transformation. A. tumefaciens-infected leaf explants were allowed to produce shoots on a shoot induction medium (SIM) containing no selective compounds. Up to 35.1% of the A. tumefaciens-infected leaf explants produced histochemically GUS⁺ shoots, 3.1% of regenerated shoots were GUS⁺, and 72% of the GUS⁺ shoots were stably transformed by producing GUS⁺ T1 seedlings. When polymerase chain reaction (PCR) was used to screen the regenerated shoots, 4% of the shoots were found to be PCR⁺ for the transgene and 65% of the PCR⁺ shoots were stable transformants. Also, generation of PCR⁺ escapes decreased linearly as the number of subculture increased from one to three on SIM containing the antibiotic that kills the Agrobacterium. Twenty-five to 75% of the transformants were able to transmit transgene activity to the T1 generation in a Mendelian 3:1 ratio, and a transformation efficiency of 2.2–2.8% was achieved for the most effective binary vector. These results indicated that majority of the GUS⁺ or PCR⁺ shoots recovered under no selection were stable transformants, and only one-third of them were chimeric or escapes. Transgenes in these transgenic plants were able to transmit the transgene into progeny in a similar fashion as those recovered under selection.     

Agrobacterium-mediated transformation of tomatillo (Physalis ixocarpa) and tissue specific and developmental expression of the CaMV 35S promoter in transgenic tomatillo plants

A protocol for the Agrobacterium-mediated transformation of tomatillo was developed. Up to 40 transgenic plants could be obtained in experiments using 60 cotyledon expiants. The transformed nature of the regenerated plants was confirmed by NPT II and Southern blot hybridization analysis. Using the b-glucuronidase system the tissue specific and developmental patterns of expression of the Cauliflower Mosaic Virus 35S promoter were determined in transgenic tomatillo plants. It was found that this promoter is developmentally regulated during fruit and seed formation.     

Efficient regeneration of transgenic plants from rice protoplasts and correctly regulated expression of the foreign gene in the plants

Summary Rice is one of the most important crops in the world with 35% of the total population (over two billion people) depending on it as their source of food. It is therefore essential to develop efficient methods for the transformation and regeneration of rice plants in order to delineate the exact regulatory sequences responsible for gene expression and to transfer beneficial genes into this plant. Here, for the first time, we present definitive evidence for the regeneration of a large number of transgenic rice plants after introduction of the bacterial -glucuronidase gene into rice protoplasts. The presence of integrated copies of this gene was detected in the genome of transgenic plants by DNA hybridization analysis. Furthermore, under the control of regulatory regions from a maize alcohol dehydrogenase sequence, -glucuronidase gene expression was detected in the roots of transgenic plants. This expression was stimulated up to six fold under anaerobic conditions.     

Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration

Various aspects of transformation and regeneration processes were examined in efforts to improve the efficiency of production of transgenic cotton (Gossypium hirsutum L.). Green fluorescent protein (GFP) proved to be a valuable tool in elucidating the timing and localization of transient gene expression and in visualizing conversion of transient events to stable transformation events. By day 4 after infection, there was maximal transient activity in the cells at the cut edge of Agrobacterium-infected cotyledon disks. We were able to visualize conversion of some of these events to stable transformation by day 8. The effects of Agrobacterium strains, acetosyringone, and temperature on stable transformation were also evaluated. Strain LBA4404 proved to be significantly better than EHA105. Acetosyringone increased significantly the stable transformation efficiency in cotton. Cocultivation at 21°C, compared to 25°C, consistently resulted in higher transformation frequencies. GFP expression in stably transformed callus was useful in studying the efficiency of selection during early stages of culture. We found that the survival of individual callus lines on selection medium was influenced by their original size and initial transgene expression status. Larger-size calluses and calluses expressing the transgene (GFP) had a higher rate of survival. Survival could be improved by an additional two-week culture on medium high in cytokinin and low in auxin before transfer to a medium to induce embryogenesis. However, this treatment delayed embryogenesis. Various other important aspects of the regeneration process are described and an overall scheme for producing transgenic cotton is presented.     

Stability and inheritance of endosperm-specific expression of two transgenes in progeny from crossing independently transformed barley plants

To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T₈ plant, having uidA (or gus) driven by the barley endosperm-specific B₁-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T₄ plant, having sgfp(S65T) driven by the barley endosperm-specific D-hordein promoter (localized on the subtelomeric region of chromosome 2H). Both lines stably expressed the two transgenes in the generations prior to the cross. Three independently crossed F₁ progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F₂ seeds followed a 3:1 Mendelian segregation ratio and transgenes were localized by FISH to the same location as in the parental plants. FISH was used to screen F₂ plants for homozygosity of both transgenes; four homozygous plants were identified from the two crossed lines tested. FISH results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F₃ and F₄ generations. Thus, even after crossing independently transformed, homozygous parental plants containing a single, stably expressed transgene, progeny were obtained that continued to express multiple transgenes through generation advance. Such stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies.     

Expression of phosphinothricin acetyltransferase from the root specific par promoter in transgenic tobacco plants is sufficient for herbicide tolerance

Summary The pat gene, coding for phosphinothricin acetyltransferase (PAT) from Streptomyces viridochromogenes, was cloned behind the par promoter of the hemoglobin gene from Parasponia andersonii, Introduction into tobacco (Nicotiana tabacum) resulted in predominantly root specific PAT expression. Application of 5 l/ha BASTA^® (herbicidal component: phosphinothricin) did not effect growth morphology and vigor of the plants. After application of 20 l/ha BASTA^® the plants showed herbicide damage. Nevertheless, they all recovered by forming new undamaged leaves and resumed full growth despite virtually non-detectable expression of the PAT enzyme in the leaves.Abbreviations BAP 6-benzylaminopurine - CaMV Cauliflower Mosaic Virus - IAA indole-3-acetic acid - kb kilobases - LB Luria-Bertani - MS Murashige and Skoog - par Parasponia andersonii - PAT phosphinothricin acetyltransferase - ppt phosphinothricin - TCA trichloric acid     

Characterization of two class II chitinase genes from peanut and expression studies in transgenic tobacco plants

Two different genes encoding class II chitinases from peanut (Arachis hypogaea L. cv. NC4), A.h.Chi2;1 and A.h.Chi2;2, have been cloned. In peanut cell suspension cultures, mRNA levels of A.h.Chi2;2 increased after ethylene or salicylate treatment and in the presence of conidia from Botrytis cinerea. The second gene, A.h.Chi2;1, was only expressed after treatment with the fungal spores. Transgenic tobacco plants containing the complete peanut A.h.Chi2;1 gene exhibited essentially the same expression pattern in leaves as observed in peanut cell cultures. Expression characteristics of transgenic tobacco carrying a promoter-GUS fusion of A.h.Chi2;1 are described.     

Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation

Particle bombardment and Agrobacterium-mediated transformation are two popular methods currently used for producing transgenic maize. Agrobacterium-mediated transformation is expected to produce transformants carrying fewer copies of the transgene and a more predictable pattern of integration. These putative advantages, however, tradeoff with transformation efficiency in maize when a standard binary vector transformation system is used. Using Southern, northern, real-time PCR, and real-time RT-PCR techniques, we compared transgene copy numbers and RNA expression levels in R₁ and R₂ generations of transgenic maize events generated using the above two gene delivery methods. Our results demonstrated that the Agrobacterium-derived maize transformants have lower transgene copies, and higher and more stable gene expression than their bombardment-derived counterparts. In addition, we showed that more than 70% of transgenic events produced from Agrobacterium-mediated transformation contained various lengths of the bacterial plasmid backbone DNA sequence, indicating that the Agrobacterium-mediated transformation was not as precise as previously perceived, using the current binary vector system.     

Green fluorescent protein as a vital marker for non-destructive detection of transformation events in transgenic plants

Transformation of plants is a popular tool for modifying various desirable traits. Marker genes, like those encoding for bacterial β-glucuronidase (GUS), firefly luciferase (LUC) or jellyfish green fluorescent protein (GFP) have been shown to be very useful for establishing of efficient transformation protocols. Due to favourable properties such as no need of exogenous substrates and easy visualization, GFP has been found to be superior in to other markers in many cases. However, the use of GFP fluorescence is associated with some obstacles, mostly related to the diminishing of green fluorescence in older tissues, variation in fluorescence levels among different tissues and organs, and occasional interference with other fluorescing compounds in plants. This paper briefly summarizes basic GFP properties and applications, and describes in more detail the contribution of GFP to the establishment, evaluation and improvement of transformation procedures for plants. Moreover, features and possible obstacles associated with monitoring GFP fluorescence are discussed.     

Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens

Defensins are small positively charged, antimicrobial peptides (~5 kDa in size) and some of them exhibit potent antifungal activity. We have cloned the complete cDNA containing an ORF of 243 bp of a defensin of mustard. The deduced amino acid sequence of the peptide showed more than 90% identity to the amino acid sequence of the well-characterized defensins, RsAFP-1 and RsAFP-2 of Raphanus sativus. We have generated and characterized transgenic tobacco and peanut plants constitutively expressing the mustard defensin. Transgenic tobacco plants were resistant to the fungal pathogens, Fusarium moniliforme and Phytophthora parasitica pv. nicotianae. Transgenic peanut plants showed enhanced resistance against the pathogens, Pheaoisariopsis personata and Cercospora arachidicola, which jointly cause serious late leaf spot disease. These observations indicate that the mustard defensin gene can be deployed for deriving fungal disease resistance in transgenic crops.     

Effective selection and regeneration of transgenic rice plants with mannose as selective agent

A new method for the selection of transgenic rice plants without the use of antibiotics or herbicides has been developed. The phosphomannose isomerase (PMI) gene from Escherichia coli has been cloned and consitutively expressed in japonica rice variety TP 309. The PMI gene was transferred to immature rice embryos by Agrobacterium-mediated transformation, which allowed the selection of transgenic plants with mannose as selective agent. The integration and expression of the transgene was confirmed by Southern and northern blot analysis and the activity of PMI indirectly proved with the chlorophenol red assay. The results of genetic analysis showed that the transgenes were segregated in a Mendelian fashion in the T₁ generation. The establishment of this selection system in rice provides an efficient way for producing transgenic plants without using antibiotics or herbicides with a transformation frequency of up to 41%.     

Expression of S-locus glycoprotein genes from Brassica oleracea and B. campestris in transgenic plants of self-compatible B. napus cv Westar

Summary Self-compatible Brassica napus var Westar was transformed with SLG, the S-locus-derived gene that encodes S-locus-specific glycoproteins (SLSG). Four allelic variants of SLG isolated from self-incompatible B. oleracea and B. campestris strains homozygous for different S alleles were used. We show that the transgenic plants synthesized SLSG with the same apparent charge, molecular weight, and antigenic properties as that produced by the corresponding self-incompatible strains from which the cloned SLG genes were isolated. In addition, transgene-encoded SLSG was detected specifically in the papillar cells of the stigma, and was correctly targeted to the papillar cell wall. However, SLSG was produced at reduced levels in transgenic plants relative to self-incompatible strains. The introduction of the SLG genes did not confer a self-incompatibility phenotype on the Westar cultivar.     

Improving genetic transformation of European chestnut and cryopreservation of transgenic lines

The aim of the present work was to study the effect of the developmental stage of the somatic embryos and of the genotype on the genetic transformation of embryogenic lines of European chestnut (Castanea sativa Mill.) and the cryopreservation of the embryogenic lines that are generated. As an initial source of explants in the transformation experiments, it was found that the use of somatic embryos isolated in the globular stage or clumps of 2–3 embryos in globular/heart-shaped stages was more effective (30%) than when embryos at the cotyledonary stage were used (6.7%). All of the seven genotypes tested were transformed, and transformation efficiency was clearly genotype dependent. Three transgenic lines were successfully cryopreserved using the vitrification procedure, and the stable integration of the uidA gene into the transgenic chestnut plants that were regenerated subsequent to cryopreservation was demonstrated.