DNA delivery into Eucalyptus globulus zygotic embryos through biolistics: optimization of the biological and physical parameters of bombardment for two different particle guns

Summary DNA transfer into zygotic embryos of Eucalyptus globulus by microprojectile bombardment was studied with two devices: a gunpowder apparatus and a compressed-helium system. Using, as a test, the transient expression of a reporter gene, we optimized the physical and biological conditions of bombardment. Six-day-old cultured embryos were found to be the best target material, and osmotic treatment increased the expression rate. Conditions of bombardment (particle acceleration and quality of the particle: DNA mix) were studied. In optimal conditions, we were able to obtain up to 130 GUS expression events per embryo with a good distribution over the tissue.In our transient expression experiments, the gunpowder and helium devices exhibited similar efficiencies, reliabilities and reproducibilities.Abbreviations GUS -glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid     

High-efficiency,microprojectile-mediated cotransformation of sugarcane,using visible or selectable markers

Transient expression of the maize anthocyanin regulatory elements,R andC1, was used to optimise parameters for microprojectile-mediated delivery of DNA into sugarcane embryogenic callus. Osmotic treatment of target tissues and particle acceleration in a high-pressure helium pulse increased the frequency of transient expression to 5–8×10³ cells per bombardment, with minimal tissue damage. An average of 0.34% of transiently expressing cells developed into stably transformed, anthocyanin-pigmented proembryoids which subsequently regenerated into plantlets. However, constitutive expression ofR andC1 proved deleterious, and no anthocyanin-pigmented plant survived beyond 3 cm in height. We also compared selective subculture of callus portions showing luciferase activity with antibiotic selection on medium containing G418 or phosphinothricin, upon bombardment of callus with constructs driving strong expression ofluc, aphA orbar genes. Selective subculture based on luciferase activity enabled recovery of 1.4±0.5 independent transgenic plants per bombardment, compared to 19.8±3.7 independent transgenic plants per bombardment from an optimised G418 selection regimen, and no transformed plants from phosphinothricin selection. Whenluc andaphA on separate plasmids were coprecipitated onto microprojectiles before bombardment, 67–79% of callus lines selected for G418 resistance also showed luciferase activity detectable under a low-light camera. Southern analysis confirmed a very high cotransformation frequency, with variable copy numbers of introduced genes. The high efficiencies of gene transfer, selection and cotransformation in the optimised system, coupled with the simple initiation and regeneration of embryogenic callus, provide an effective tool for practical genetic transformation of sugarcane.     

Transfection by particle bombardment: Delivery of plasmid DNA into mammalian cells using gene gun

Background

Recently, particle bombardment has become increasingly popular as a transfection method, because of a reduced dependency on target cell characteristics. In this study, we evaluated in vitro gene transfer by particle bombardment.

Methods

gWIZ luciferase and gWIZ green fluorescent protein (GFP) plasmids were used as reporter genes. Mammalian cell lines HEK 293, MCF7 and NIH/3T3 were used in the transfection experiments. Transfection was performed by bombardment of the cells with gene-coated gold particles using the Helios Gene Gun. The technology was assessed by analyzing gene expression and cell damage. Cell damage was evaluated by MTT assay.

Results

This technology resulted in efficient in vitro transfection, even in the cells which are difficult to transfect. The gene expression was dependent on the gene gun's helium pressure, the sizes of the gold particles, the amount of the particles and DNA loading, while cell viability was mostly dependent on helium pressure and amount of the gold particles.

Conclusions

This technology was useful to transfection of cells. Optimal transfection conditions were determined to be between 75 and 100 psi of helium pressure, 1.0 to 1.6 μm gold particle size and 0.5 mg of gold particle amount with a loading ratio of 4 μg DNA/mg gold particles.

General significance

These findings will be useful in the design of gene gun device, and bring further improvements to the in vitro and in vivo transfection studies including gene therapy and vaccination.     

Transient expression of a lysine-rich vicilin gene ofVicia faba in barley endosperm detected by immunological tissue printing after particle bombardment

Summary Using immunological tissue printing we detected transient expression of a faba bean vicilin gene with or without introns driven by the B1 hordein promoter in barley endosperm after particle bombardment. The described method generally allows the analysis of transient expression of genes without depending on reporter gene constructs and specifically suggests correct splicing of dicot introns by a monocot splicing machinery.Abbreviations GUS -glucuronidase - NPTII neomycin phosphotransferase II - ELISA enzyme-linked immunosorbent assay     

The effect of the parameters of biolistic transformation of spring barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) on the level of transient expression of <Emphasis Type="Italic">GFP</Emphasis> reporter gene

The effect of the parameters of biolistic transformation (rupture disk pressure of helium, vacuum pressure, stopping screen to target tissue distance, material (gold or tungsten) and size of particles, and duration of explant culturing before bombardment) on the level of transient expression of GFP reporter gene was studied in barley embryos. The highest transient expression was observed after explant preincubation for 12–14 days and bombardment with 1 μm gold particles at the helium pressure of 61.24–74.85 atm, vacuum pressure of 0.064 atm, and distance to target of 9 cm.     

Transient expression of foreign genes in rice,wheat and soybean cells following particle bombardment

The development of an efficient transformation system is a prerequisite for the molecular analysis of gene expression in plants. In crop plants, this development has been hindered by difficulties encountered both in whole plant regeneration from protoplasts and in the general insusceptibility of monocots to Agrobacterium-mediated transformation. We have circumvented these difficulties by transferring foreign genes directly into the intact cells (with cell walls) of three important crop plants including rice, wheat and soybean by a particle bombardment device. Oryza sativa and Triticum monococcum cells were bombarded with accelerated tungsten particles coated with plasmids containing a -glucuronidase gene as the reporter. Blue transformed cells were detected in an in situ enzyme assay. The number of blue cells was next used as a convenient criterion to study several factors affecting gene transfer efficiency. After optimal conditions were defined, gene transfer into intact cells of O. sativa, T. monococcum and Glycine max was successfully carried out with chloramphenicol acetyltransferase (CAT) gene as the reporter.     

Expression of transferred genes during hairy root development in pea

Root border cell development and expression of reporter genes were evaluated in transgenic pea hairy roots. Successful induction of hairy roots in pea is conditioned by bacterial strain and plant genotype, as well as by developmental and environmental factors. Morphological changes sometimes occur when hairy roots are transferred from infected plants to tissue culture media, but such changes are confined to specific clones. Expression of reporter genes under the control of promoters from bean (Phaseolus vulgaris L.) stress genes encoding phenylalanine ammonia lyase and chalcone synthase were evaluated. Expression patterns vary between hairy roots taken directly from infected plants, and those grown in culture; most hairy roots taken from infected plants exhibit expression throughout all tissues, whereas expression in cultured hairy roots is most often localized to specific tissues. Patterns of expression that occur during different stages of hairy root development are very similar to those observed in transgenic plants expressing the same fusion genes. Border cell separation and release in hairy roots is normal, and expression of glucuronidase in border cells of some transgenic roots resulted in development of bright blue single cells. Cultured hairy roots should provide a very useful model for studying the effect of defined changes in root border cells on microbial associations with roots of this important legume.Abbreviations YEM yeast extract-mannitol - GUS glucuronidase - PAL phenylalanine ammonium lyase - CHS chalcone syntase     

Transient expression of a reporter gene in bulbscales and immature embryos of three Lilium species is affected by 5'upstream sequences and culture conditions

In Lilium , a transformation system has not yet been developed. For efficient selection of cells expressing transferred genes following particle bombardment, the effects of 5'upstream regions on the transient expression of the β-glucuronidase gene ( gusA ) were estimated in bulbscales and immature embryos of lily. When four plasmids having the gusA gene under the control of the cauliflower mosaic virus (CaMV) 35S, maize alcohol dehydrogenase gene and rice actin gene ( Actl ) promoters, and the castor bean catalase introm were introduced by particle bombardment, the patterns of transient expression in the bulbscales showed differences among three Lilium species, L. x formolongi, L. dauricum and L. japonicum . In immature embryos of L x formolongi , transient expression was significantly influenced by age of embryos after self-pollination, duration of culture before bombardment, and culture conditions. Moreover, the transient gusA expression driven by six different 5'upstream regions, including the maize ubiquitin gene promoter and a modified CaMV 35S promoter were compared in both bulbscales and immature embryos. Use of the Actl and modified CaMV 35S promoters resulted in the greatest number of cells that transiently expressed gusA in both types of tissue of L. x formolongi . These two promoters are efficient for use in lily transformation.     

Optimization of biolistic transformation of embryogenic grape cell suspensions

Embryogenic suspensions of Chancellor (Vitis L. complex interspecific hybrid) were bombarded with tungsten particles coated with plasmid pBI426 encoding ß-glucuronidase (GUS) and neomycin phosphotransferase (NPTII) which results in kanamycin resistance. Two d after bombardment, cultures were placed on semi-solid medium containing either 8.6 or 17.2 M kanamycin. Factors that affect biolistic transformation rates were studied. Tungsten microprojectiles with a mean diameter of 1.07 m (M10) resulted in more transient gene expression than 0.771 m diameter particles. Using M10 particles, helium pressures of 1000 and 1200 psi yielded more GUS-expressing colonies per plate than did 800 psi 2 d following bombardment. The number of transformants present after 34 d was not affected by the helium pressure. The distance between the particle launch site and the target cells, and the number of days between the last cell subculture and bombardment, did not affect the numbers of transient and long term GUS expressing colonies. The addition of 3 g/l of activated charcoal to the post-bombardment medium increased long term GUS expression four fold. Wrapping the plates after bombardment with Parafilm increased long term GUS expression three fold compared with plates wrapped with a porous venting tape. With up to 850 transformed callus colonies per plate 23 d after bombardment, the biolistic device holds much promise as a method to achieve stable transformation of grapevines.Abbreviations AC activated charcoal - GUS ß-glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - IAA-L-alanine indole-3 acetic acid L-alanine - MS Murashige and Skoog - CH casein hydrolysate - Km kanamycin - NPTII neomycin phosphotransferase II     

Regulation of phenylalanine ammonia-lyase genes in carrot suspension cultured cells

Induction of anthocyanin synthesis occurs during metabolic differentiation in carrot suspension cultured cells grown in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), and is closely correlated with embryogenesis. Anthocyanin synthesis may also be induced by light-irradiation under different culture conditions. The phenylalanine ammonia-lyase (PAL) gene (TRN-PAL), which was transiently induced by the transfer effect, was also rapidly induced after light-irradiation. However, TRN-PAL was not involved in anthocyanin synthesis. A second PAL gene, ANT-PAL, was involved in anthocyanin synthesis. ANT-PAL was induced during metabolic differentiation in medium lacking 2,4-D parallel with the induction of chalcone synthase (CHS). PAL genes in the carrot genome are expressed differentially depending on the nature of the environmental stimulus, e.g. transfer effect and light, and other parameters which also affect anthocyanin synthesis.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Luc firefly luciferase - PAL phenylalanine ammonia-lyase - UV ultraviolet     

Optimizing somatic embryogenesis and particle bombardment of barley (Hordeum vulgare L.) immature embryos

Summary A highly regenerable target tissue and a high-frequency DNA delivery system are required for the routine production of transgenic barley. This project separately optimized tissue culture and particle bombardment parameters. Immature zygotic embryos (0.7 to 1.2 mm) were excised and culture on B5L solid medium. Klages and H930-36 cultivars regenerated significantly more green plants than Sabarlis and Bruce. The regeneration pathway shifted from organogenesis to somatic embryogenesis when maltose was used as the medium carbohydrate source instead of sucrose. More somatic embryos were induced on 5 mg/liter 2,4-dichlorophenoxyacetic acid than 2 mg/liter. Gene delivery was optimized using anthocyanin regulatory genes as a transient marker. A 3-mm rupture disc-to-macrocarrier gap distance, a 1-day prebombardment embryo culture period, and a maltose carbohydrate source were each significantly better than other treatments. Double bombardments per plate, a 6-mm macrocarrier fly distance, and 650-psi rupture discs each had the highest number of transiently expressing cells in individual experiments, although the results were not statistically significant compared to the other treatments. Using the optimized parameters, over 200 cells routinely expressed anthocyanin in a bombarded immature embryo. In tissue culture experiments, 350 to 400 green plants regenerated per 100 immature embryos. The improvement of green plant regeneration and gene delivery forms a strong basis to develop a practical barley transformation system.     

Development of a simple particle bombardment device for gene transfer into plant cells

A simple particle bombardment device was designed, constructed and shown to be efficient for the delivery of DNA into plant cells. High levels of transient -glucuronidase expression were observed in alfalfa suspension-cultured cells and embryogenic soybean suspension-cultured cells. Expression of -glucuronidase in alfalfa suspension-cultured cells was used to optimize the bombardment conditions for the device. Transient gene expression in alfalfa was found to be dependent on the state of the target tissue, the size of particles employed, the helium pressure used to accelerate the particles and the distance travel led by the tungsten particles carrying DNA.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MS Murashige & Skoog (1962) medium     

Calluses initiated from thin mature embryo fragments are suitable targets for wheat transformation as assessed by long-term GUS expression studies

Microprojectile- or Agrobacterium-mediated DNA delivery into calluses initiated from immature embryos has proven to be effective in transforming wheat. Yet, obtaining a large number of high quality immature embryos throughout the year is a laborious and delicate process. To circumvent these limitations, we propose an alternative technique applying the particle bombardment technology to calluses derived from fragmented mature embryos rather than immature tissues. The phosphinothricin acetyl transferase (bar) and -glucuronidase (gus) genes were used as selectable and screenable marker genes, respectively, to assess and optimise the performance of the proposed technique. Primary requirement for genetic transformation method development, the regeneration capacity of bombarded calluses was established. A preculture duration of 6 days was identified as optimal for DNA uptake and -glucuronidase (GUS) expression. The highest activity was recorded when calluses were selected. Long-term GUS expression studies (1–7weeks subsequent to bombardment), showed differentiated behaviours for tissues obtained from mature versus immature embryos. Notably, mature embryos exhibited the greatest number of cells stably expressing the reporter gene, thus providing an excellent source material for developing a stable transformation procedure.