A comparison of methods for direct gene transfer into maize (Zea mays L.)

Summary Techniques for transforming intact tissues of cereals were evaluated for their efficacy in transforming immature embryos and Type II callus of maize (Zea mays L.). The techniques used were particle bombardment, tissue electroporation, tissue electrophoresis, and silicon carbide fibers. Each method was assessed in terms of transient β-glucuronidase (GUS) expression. High levels of GUS expression were observed in A188 Type II callus using both tissue electroporation and particle bombardment, with means of 417.8 and 954.5 blue expression units (beu) per g fresh weight (FW) callus, respectively. Only particle bombardment resulted in high transient gene expression in immature embryos, with a mean transformation frequency of 34.8 b.e.u. per embryo. Very low levels of GUS expression were achieved with silicon carbide-mediated gene transfer, even when employing tissues used in the original publication (Black Mexican Sweet suspension cells). GUS expression was not obtained following tissue electrophoretic gene delivery.     

Whisker-mediated plant transformation: An alternative technology

A number of different methods, involving direct DNA delivery are now available for plant transformation. Here we review the most recently developed technique which involves the mixing of silicon carbide whiskers with plant cells and plasmid DNA. Fertile transgenic plants have now been produced using whisker-mediated transformation, and this method can now be considered as a simple, inexpensive alternative for plant transformation. A brief review on transformation of animal cells andChlamydomonas using whiskers technology is also included.     

Plant transformation by microinjection techniques

Several techniques have been developed for introducing cloned genes into plant cells. Vectorless delivery systems such as PEG-mediated direct DNA uptake (e.g. Pasz-kowski et al. 1984), electroporation (e.g. Shillito et al. 1985), and fusion of protoplasts with liposomes (Deshayes et al. 1985) are routinely used in many experiments (see several chapters of this issue). A wide range of plant species, dicotyledonous as well as monocotyledonous, has been transformed by these vectorless DNA transfer systems. However, the availability of an efficient protoplast regeneration system is a prerequisite for the application of these techniques. For cells with intact cell walls and tissue explants the biological delivery system of virulent Agrobacterium species has been routinely used (for review see Fraley et al. 1986). However, the host range of Agrobacterium restricts the plant species, which can be transformed using this vector system. In addition, all these methods depend on selection systems for recovery of transformants. Therefore a selection system has to be established first for plant species to be transformed. The microinjection technique is a direct physical approach, and therefore host-range independent, for introducing substances under microscopical control into defined cells without damaging them. These two facts differentiate this technique from other physical approaches, such as biolistic transformation and macroinjection (see chapters in this issue). In these other techniques, damaging of cells and random manipulation of cells without optical control cannot be avoided so far. In recent years microinjection technology found its application in plant sciences, whereas this technique has earlier been well established for transformation of animal tissue culture cells (Capecchi 1980) and the production of transgenic animals (Brin-ster et al. 1981, Rusconi and Schaffner 1981). Furthermore, different parameters affecting the DNA transfer via microinjection, such as the nature of microinjected DNA, and cell cycle stage, etc, have been investigated extensively in animal cells (Folger et al. 1982, Wong and Capecchi 1985), while analogous experiments on plant cells are still lacking.     

针叶松遗传转化操作的研究进展(英文)

1986年从火炬松方面获得了首例针叶松愈伤组织以后,针叶松遗传转化研究取得了明显的进展。1991年从Larix decidua方面获得了第一个转基因再生植株。最近,通过农瘤杆菌介导的转化方式在Larix kaempferi ( L. Decidua杂交树种上获得了转基因针叶松,通过鸟枪法在Picea glauca、Picea mariana、Picea abies、Larix laricina、Pinus radiata上亦获得了转基因植株。已经有多种办法用于转化针叶松。本文就目前在针叶松上所用的遗传转化方法??诸如：T－DNA转化法、农瘤杆菌介导转法化、鸟枪法、电转化法等给以粗略的评论。     

Advances in Pollen Mediated Genetic Transformation

植物遗传转化技术是植物科学基础理论与应用研究的有力武器,已成为植物遗传改良的重要途径之一。但是、目前遗传转化所采用的受体系统,大都需要体外培养和植株再生过程,才能获得转基因植株。其中、基因型限制和遗传变异是该技术不可逾越的两大障碍。花粉管通道法可省去转化体的离体培养,不过、多数植物受花器结构的限制而难以经花柱注射ＤＮＡ,只能向子房注射,并不是真正的“花粉管通道”。又由于此法外源基因的导入发生在授粉之后,因此该方法亦不属于花粉遗传转化。利用小孢子胚胎发生体系进行遗传转化与利用花粉作为外源ＤＮＡ的媒介,继而、通过授粉受精获得转基因种子,是目前花粉遗传转化的两个重要方面和活跃的研究方向。前者仍需要离体再生系统,后者则可以利用植物自身的再生机制,本文称之为花粉介导法（ｐｏｌｅｎ－ｍｅｄｉａｔｅｄｔｒａｎｓｆｏｒｍａｔｉｏｎ）。该方法通过自然的胚胎发育过程获得转基因子代,避免了组织培养过程中的遗传变异和转基因植株的嵌合现象。可望成为简便快速的植物遗传转化体系。目前对花粉介导的遗传转化进行专门评述的文献较少,本文对该领域的研究分三个层次进行了综述。一、外源基因转化方法小孢子或由小孢子形成的胚状体是很有潜力的遗传转化受体     

Electroporation-mediated DNA delivery to seedling tissues ofPhaseolus vulgaris L. (common bean)

Summary DNA was delivered to intact embryonic axes of the legumePhaseolus vulgaris L. through electroporation. Expression of the ß-glucuronidase reporter gene was observed in hypocotyl and epicotyl tissue in a spot-like manner. Transgene expression was high when a single pulse of 260 ms at a field strength of 225 V·cm^–1 was applied but could be achieved within a wide range of electrical conditions. Linearization of plasmid DNA greatly enhanced transient expression levels. The procedure was successful for embryonic axes of all testedP. vulgaris cultivars, for similar explants of several large-seeded leguminous species, as well as for some other tissues ofP. vulgaris.     

Transmission of injected DNA sequences to multiple eggs of Metaseiulus occidentalis and Amblyseius finlandicus (Acari: Phytoseiidae) following maternal microinjection

The persistence of DNA injected into two species of adult female phytoseiids and its transmission to serial eggs deposited by them was assessed by the polymerase chain reaction (PCR). The effect of DNA concentration on persistence and transmission was examined in Metaseiulus occidentalis. M. occidentalis females were microinjected with plasmid DNA at three different concentrations (250, 500, 750 ng L^–1) and allowed to deposit one to five eggs before the females and their last eggs were analyzed. Plasmid DNA was found in 82% of the females assayed and in 70% of all the eggs analyzed (including the fifth eggs produced after microinjection). Transmission of DNA to multiple eggs was also examined in Amblyseius finlandicus. Females of this species are less traumatized by microinjection allowing analysis of transmission over a more extended number of eggs. Females were microinjected and allowed to deposit eggs until their death. DNA from every fifth egg was analyzed by the PCR. PCR products were amplified from 51% of the eggs and from all egg classes except the 30th egg. The persistence and presence of plasmid DNA in both eggs and females suggests that (1) maternal microinjection is a more efficient method for DNA delivery than traditional egg microinjection, (2) it may be possible to isolate transformants from fewer maternally-microinjected females than originally expected, and (3) maternal microinjection could be useful as a DNA delivery system in other phytoseiids.     

Advances in plant proteomics-key techniques of proteome

Following the completion of genome sequen-cing of model plants,such as rice (Oryza sativa L.) and Arabidopsis thaliana,the era of functional plant genomics has arrived which provides a solid basis for the develop-ment of plant proteomics.We review the background and concepts of proteomics,as well as the key techniques which include:(1) separation techniques such as 2-DE (two-dimensional electrophoresis),RP-HPLC (reverse phase high performance liquid chromatography) and SELDI (surface enhanced laser desorption/ionization) protein chip; (2) mass spectrometry such as MALDI-TOF-MS (matrix assisted laser desorption/ionization-time of flight- mass spectrometry) and ESI-MS/MS (elec-trospray ionization mass spectrometry/mass spectro-metry); (3) Peptide sequence tags; (4) databases related to proteomics; (5) quantitative proteome; (6) TAP (tandem affinity purification) and (7) yeast two-hybrid system.In addition,the challenges and prospects of pro-teomics are also discussed.     

Single-stranded DNA used as an efficient new vehicle for transformation of plant protoplasts

In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3–10 fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts form recalcitrant plant species.Abbreviations ss single-stranded - ds double-stranded - CAT chloramphenicol acetyl transferase - NPTII neomycin phosphotransferase II - RT room temperature     

猕猴桃的组织培养和遗传转化研究进展

综述了国内外猕猴桃组织培养和遗传转化研究进展,内容包括花药培养、胚培养、胚乳培养、子叶、叶、茎段等器官培养、原生质体培养以及遗传转化等,并对生物技术在猕猴桃研究中存在的问题以及今后在猕猴桃中的应用前景进行了讨论。     

Transgenic rapeseed plants obtained by the microinjection of DNA into microspore-derived embryoids

Summary A novel method in the field of genetic engineering of higher plants is presented: microinjection into multicellular structures which have a high competence for plant regeneration through embryogenesis. Microspore-derived embryoids of Brassica napus L. were individually selected and microinjected with NPT II gene constructions. High frequency regeneration of haploid plants through embryogenesis was achieved within 8 weeks. Transformation efficiencies between 27% and 51% were determined by DNA dot blot analysis of primary regenerants. Stable integration of fulllength microinjected genes into high molecular weight DNA was proven by Southern analysis of genomic DNA isolated from regenerated plants. Transformed plants were tested for expression of the NPT II gene by enzyme assay. The chimeric nature of the primary regenerants was demonstrated after their in vitro segregation through secondary embryogenesis into pure transformants.     

Regeneration of Triticum aestivum apical explants after microinjection of germ line progenitor cells with DNA

A highly efficient method of regenerating fertile, phenotypically normal plants from shoot apex cultures of T. aestivum was developed. The hypodermal layer (L2) of the vegetative apex containing germ line precursor cells could be located with bright field microscopy and targeted for microinjection. Fluorescently labelled dextrans were used as markers to develop a microinjection procedure which did not disrupt nuclear or cytoplasmic structure. This procedure was used to inject plasmid DNA into L2 cells. Capillary microinjection did not shear the plasmid DNA and delivery of DNA was confirmed by polymerase chain reaction analysis of DNA isolated from injected apices. The significance of these findings in relation to the development of cereal transformation systems will be discussed.     

Genetic Transformation in Helicobacter pylori

Genetic transformation in Helicobacter pylori was investigated by using its chromosomal and plasmid DNAs. Six out of the eight strains exhibited the natural competence for incorporation of H. pylori chromosomal DNA, and all the strains incorporated the donor DNA efficiently by washing and concentrating the cells, with a glycerol solution. The much higher frequency of transformation was obtained in each strain by means of electroporation. Electroporation experiments were also conducted by use of the recombinant DNAs consisting of the H. pylori and Escherichia coli plasmids as the donors, and the occurrence of the homologous recombination was demonstrated between the incoming H. pylori plasmid-derived region and the corresponding region of the originally residing plasmid in H. pylori.     

Analysis and optimisation of DNA delivery into wheat scutellum and Tritordeum inflorescence explants by tissue electroporation

Wheat scutella and tritordeum inflorescences were transformed by tissue electroporation with plasmid DNA containing a β-glucuronidase (GUS) gene (gus A) under the control of the rice actin1 promoter. Factors affecting electroporation efficiency were analysed. Important factors were electroporation voltage and pulse length, the volume of electroporation buffer, the osmoticum of electroporation buffer and medium, the osmoticum of pre-electroporation culture medium, and pre-electroporation incubation time and temperature. Maximum transient gene expression was obtained with a single pulse of 550 V/cm from a 960-μF capacitor, using 200 μl of electroporation buffer, after 2–3 h culture on media with 357 mOsm for wheat scutella or 1 day on media with 222 mOsm for tritordeum inflorescences, and 0.5–1 h pre-electroporation incubation with DNA at 24 °C. Under these conditions, up to 90% of the explants showed GUS expression, and up to 149 expression signals were recorded per replicate. Electroporated explants showed high rates of survival and retained the ability to regenerate plants via somatic embryogenesis. Received: 9 August 1996 / Revision received: 11 September 1997 / Accepted: 2 July 1998     

Effects of DNA Topology on Transformation Efficiency of Bacillus subtilis ISW1214 by Electroporation

We report an investigation of electrotransformation by three different topological isomers, circular supercoiled (sc DNA), circular relaxed (cr DNA), and linearized (In DNA) forms of the plasmids pUB110 (4.5 kbp) and pBDR331T (12.6 kbp), of a Gram-positive bacterium, Bacillus subtilis ISW1214. Treatment of the sc DNA with calf thymus topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of sc DNA with restriction endonuclease linearized the DNA. The transformation with the sc DNA of pUB110 resulted in the maximum efficiency of (2.6±0.6) × 10⁵ transformants per μg DNA higher than that ((2.0±0.3) × 10⁴ transformants per μg DNA) for the cr DNA, using the DNA concentration of 20 μg/ml at an electric field strength of 7kV/cm and a capacitance of 10 μF with a single decayed pulse. The transformation efficiency (TE) for the In DNA was zero. The variations of TE for different topological forms of DNA reflected their relative stability in the host cells. The molecular efficiency (ME, transformants per molecule) for sc DNA was nearly one order of magnitude greater for the lower molecular size of pUB110 DNA than that for the higher molecular size of pBDR331T DNA.     

Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures

Pronuclear injection is currently the most often used method to make transgenic animals, but in some animal species it is temporally restrictive due to difficulty in visualizing pronuclei. However, the injection of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection technique of polylysine complexed DNA into pronuclear stage zygotes is described. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-l-lysine (degree of polymerization=51). Effects on transgenic frequency of both the lysine to phosphate ratio of polylysine to DNA and DNA concentration were studied. About 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysine/DNA mixture having a lysine to phosphate ratio (L:P) of 11 microinjection positive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysine/DNA were detected using agarose gel electrophoresis at the conditions which produced transgenic mice. The presence of polylysine with construct DNA altered thein vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic animals using DNA and polylysine in the absence of any other signal protein suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.     

Pressure built by DNA packing inside virions: enough to drive DNA ejection in vitro, largely insufficient for delivery into the bacterial cytoplasm

Tailed bacteriophage particles carry DNA highly pressurized inside the capsid. Challenge with their receptor promotes release of viral DNA. We show that addition of the osmolyte polyethylene glycol (PEG) has two distinct effects in bacteriophage SPP1 DNA ejection. One effect is to inhibit the trigger for DNA ejection. The other effect is to exert an osmotic pressure that controls the extent of DNA released in phages that initiate ejection. We carried out independent measurements of each effect, which is an essential requirement for their quantitative study. The fraction of phages that do not eject increased linearly with the external osmotic pressure. In the remaining phage particles ejection stopped after a defined amount of DNA was reached inside the capsid. Direct measurement of the size of non-ejected DNA by gel electrophoresis at different PEG concentrations in the latter sub-population allowed determination of the external osmotic pressure that balances the force powering DNA exit (47 atm for SPP1 wild-type). DNA exit stops when the ejection force mainly due to repulsion between DNA strands inside the SPP1 capsid equalizes the force resisting DNA insertion into the PEG solution. Considering the turgor pressure in the Bacillus subtilis cytoplasm the energy stored in the tight phage DNA packing is only sufficient to power entry of the first 17% of the SPP1 chromosome into the cell, the remaining 83% requiring application of additional force for internalization.     

Electroporation of intact embryonic leaflets of peanut: Gene transfer and stimulation of regeneration capacity

Summary Direct gene transfer into peanut intact embryonic leaflets was performed through electroporation. In transient β-glucuronidase expression assays, maximal expression was obtained by using pulses of 625 V cm⁻¹ in EPRm (modified electroporation) buffer supplemented with 75 μM NaCl. Kanamycin-resistant plants were obtained, and the presence of the nptII gene was demonstrated by PCR analysis. The positive effect of electroporation on the efficiency of in vitro regeneration was demonstrated. Explants submitted to field strengths between 500 and 625 V cm⁻¹ displayed a significantly increased number of shoots and originated faster growing calluses relative to control explants. Whereas in control explants callus formation occurred only at the petiolule, electroporated leaflets developed additional organogenic calluses on the foliar lamina. These authors have contributed equally to this work.     

Advances in vibrating probe techniques

Summary This paper summarizes the fundamentals and describes advances in the vibrating probe, a tool that was developed and introduced into biological research byJaffe andNuccitelli in 1974. The improvements reported are concerned on the one hand with the construction, micropositioning and maintenance of the probe itself and, on the other hand, with the origin of artifacts and possible ways to avoid or, at least, to detect and monitor them. The advances in construction and the elucidation of possible artifacts may help extend the usefulness of this unique tool in investigations of ionic currents which cells and tissues drive through themselves during development and movement.