Stable Genetic Transformation Research Articles

An efficient method for the production of transgenic plants of peanut ( Arachis hypogaea L .) through Agrobacterium tumefaciens-mediated genetic transformation

Cotyledon explants from mature peanut seeds ( Arachis hypogaea L.) were optimized to obtain adventitious shoot buds with high frequencies (>90%). Efficient transformation of these cotyledons by using Agrobacterium tumefaciens strain C58 carrying neomycin phosphotransferase II ( nptII) and ß-glucuronidase ( GUS; uidA), or coat protein gene of the Indian peanut clump virus (IPCVcp) and nptII on binary vectors (pBI121; pROKII:IPCVcp) led to the production of a large percentage (55%) of transgenic plants. Transformed individuals were obtained through selection on medium containing 125 mg l −1 kanamycin. A large number of independently transformed plants (over 75) were successfully transplanted to the glasshouse. Integration of the transgenes and stable genetic transformants in the progeny were assessed by PCR amplification of 700-bp fragment of nptII and 585-bp of IPCVcp genes, and Southern blot hybridizations in the T1 generation of transgenic plants. Analysis of 35 transgenic plants of T1 generation from the progeny of a single transformation event suggested the segregation of a single copy insert in a 3:1 Mendelian ratio. On an average, 120–150 days were required between the initiation of explant transformation and transfer of rooted plants to the greenhouse. The cotyledon regeneration system proved to be an excellent vehicle for the production of a large number of independently transformed peanut plants. Shoot formation was rapid and prolific, and a large proportion of these shoots developed into fertile plants. The method reported here provides new opportunities for the crop improvement of peanut via genetic transformation.

Agrobacterium-mediated transformation of opium poppy, Papaver somniferum, via shoot organogenesis

Summary An efficient Agrobacterium -mediated protocol has been developed for the stable genetic transformation of intact opium poppy, Papaver somniferum L., plants via shoot organogenesis. Excised cotyledons were cocultivated with the disarmed A. tumefaciens strain GV3101 carrying the pBI121 binary vector, and incubated on an optimized shoot induction medium consisting of B5 salts and vitamins, 30 g L −1 sucrose, 2 mg L −1 6-benzylaminopurine, 5 mg L −1 AgNO 3 , and 3 g L −1 Gelrite. Except for the cocultivation medium, all formulations included 30 mg L −1 paromomycin as the selective agent, and 200 mg L −1 timentin to eliminate the Agrobacterium . Eight-week-old, paromomycin-resistant shoots were transferred to an optimized root induction medium consisting of B5 salts and vitamins, 0.5 mg L −1 indole-3-acetic acid, 0.5 mg L −1 indole-3-butyric acid, and either 5 mg L −1 AgNO 3 or 40 mg L −1 putrescine. About 15 % of the regenerated shoots developed roots within eight weeks. Regenerated plants were transferred to soil, where they grew normally and set seed. Detection of the neomycin phosphotransferase gene, the high levels of β-glucuronidase (GUS) mRNA and enzyme activity, and the cytohistochemical localization of GUS activity in all organs, confirmed the genetic transformation of the regenerated plants. The transformation process did not alter the normal alkaloid content of opium poppy; thus, the reported protocol could serve as a valuable tool to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis.

Taming abiotic stresses in plants through genetic engineering: current strategies and perspective

During the last decade, major advances have been made in plant genetic engineering. The methods for stable genetic transformation as well as for regulation of introduced trans-genes have been optimised to a great deal. The major limiting factor in the widespread application of genetic engineering is the availability of the target genes. This is particularly true for engineering tolerance against abiotic stresses (such as those caused by high levels of salts in soils, reduced/excess availability of water and sub- and supra-optimal temperature regimes). In spite of this, the past 5 years (1993–1998) have witnessed significant achievements in terms of generating transgenics for enhanced tolerance to these stresses. For future work on producing plants with still higher level of tolerance, there is a need to expand the information on the stress-induced genes so that appropriate genes can be pyramided. The current upsurge in genomic research has the potential to catalyse efforts in elucidating new stress-responsive genes. There are also possibilities of engineering the whole cascade of multiple genetic changes through manipulation of the regulatory genes.

Genotype-dependent Stable Genetic Transformation in German Spring Wheat Varieties Selected for High Regeneration Potential

Summary Five commercial German spring wheat cultivars ( Triticum aestivum L.), four of them selected for high regeneration potential, were used for particle bombardment of embryogenic scutellar calli with plasmid pAHC25 containing the bar and the uidA gene, each under the control of the maize ubiquitin-1 promoter. In transient expression of uidA , no statistically significant differences between the varieties used could be detected. From four of the cultivars used, 16 transformants were obtained. Variery ‹Combi›> showed the highest transformation frequency (0.96 %), followed by ‹Turbo› (0.58 %), ‹Hanno› (0.48 %) and ‹Naxos› (0.15 %). ‹Thasos› proved to be recalcitrant. At least one copy of both the uidA and the bar gene had been integrated into the genome of each transformant. The bar gene was expressed in all T 0 -plants, whereas the uidA gene had been silenced in 4 of them. From most transformants the transgenes were transmitted unchanged in a Mendelian manner to the T 1 - and T 2 -generations, and their expression was maintained. Only three transformants were unstable. In the T 1 , they had lost either the bar or the uidA gene. In all of the other T 2 - and - first results - T 3 -rgenerations, the transgenes proved to be stable, and no additional silencing was observed. The data indicate stable transformation in most of the commercial varieties used. In addition, two more general conclusions can be drawn: First, in comparing the present data with the results of earlier Agrobacterium -mediated gene transfer attempts, using the same wheat variery ‹Turbo› and the same coding regions, convincing evidence for the superiority of biolistic methods in wheat transformation could be obtained. Second, there was no parallelism between the frequencies of regeneration and transformation, indicating genorype dependent transformation potentials.

Stable genetic transformation of white pine (Pinus strobus L.) after cocultivation of embryogenic tissues with Agrobacterium tumefaciens

A genetic transformation procedure for white pine has been developed after cocultivation of embryogenic tissues with Agrobacterium tumefaciens. This efficient transformation procedure led to an average of four independent transformed lines per gram of cocultivated embryogenic tissue and up to 50 transformed lines can be obtained in a routine experiment. Constructs bearing the uidA gene or the green fluorescent protein (GFP) gene were introduced and β-glucuronidase (GUS) activity was followed over time. The expression of the uidA gene was lowest with a 35S-gus-intron construct and was 20-fold higher with a 35S-35S-AMVgus::nptII construct. The addition of scaffold attachment region (SAR) sequences surrounding the gus::nptII fusion did not significantly enhance the GUS activity. Transformed mature somatic embryos have been germinated and plantlets are presently being acclimatized.

Analysis of RNA Editing in Plastids

Chloroplast mRNAs can be subject to posttranscriptional pyrimidine-to-pyrimidine conversions at highly specific sites. This RNA modification mechanism shows a high degree of similarity to plant mitochondrial editing but differs markedly from, and is most likely evolutionarily unrelated to, all other RNA editing systems. The study of RNA editing processes in chloroplasts has been largely hampered by the lack ofin vitroediting systems; however, considerable insights into the recognition mechanisms of individual editing sites have come fromin vivoapproaches. Chloroplast transformation proved to be a particularly useful tool to study plastid RNA editing. In this article, specific methods for the analysis of chloroplast RNA editing are discussed. Detailed experimental procedures are provided for (i) the purification of chloroplasts and (ii) the stable genetic transformation of higher plant plastids.

Transfection of Malaria Parasites

The stable genetic transformation of three phylogenetically diverse species ofPlasmodium,the parasitic etiological agent of malaria, is now possible. The parasite is haploid throughout the vast majority of its life cycle. Therefore with the single selectable marker activity and protocols currently available, it is possible not only to express introduced transgenes but also to study the effects of site-specific homologous recombination such as gene knockout. Transgene expression will allow the detailed study of many aspects of the cellular biology of malaria parasites, for example, the mechanisms underlying drug resistance and protein trafficking. We describe here the methods for propagation of the two animal models (Plasmodium bergheiandPlasmodium knowlesi) and for transfection of these two species and the human parasite,Plasmodium falciparum.Examples of transgene expression are given.

Genetic transformation of the C3-C4 intermediate plant, Flaveria pubescens (Asteraceae).

The dicot genus Flaveria (Asteraceae), besides species with C3 or C4 photosynthesis, contains taxa with a broad range of different states of transition between the two major photosynthetic types. We have developed a reproducible and efficient Agrobacterium-mediated method for the stable genetic transformation of the C3-C4 intermediate species F. pubescens. Fusion constructs of the reporter gene β-glucuronidase (uidA, GUS) to several plant promoters, mainly derived from genes encoding subunits of the glycine cleavage system (gdcs), have been used to confirm the reproducibility and efficiency of the method. The stable integration of the foreign DNA has been examined by Southern analysis, kanamycin resistance, GUS enzyme activities and histochemical staining. Transformed shoots can be routinely obtained within 8-10 weeks after co-cultivation with A. tumefaciens.

Agrobacterium rhizogenes-mediated transformation and plant regeneration of four Gentiana species

Shoots of micropropagated Gentiana acaulis, G. cruciata, G. lutea, and G. purpurea were inoculated with suspensions of Agrobacterium rhizogenes cells, strains ATCC 15834 or A4M70GUS. Adventitious roots appeared at the sites of inoculation in all 4 species. Root tips were excised and cultured on growth regulator-free media for 2-6 years. They exhibited very high branching and plagiotropism. Spontaneous bud initiation occurred in roots of G. cruciata. Roots of G. lutea, G. acaulis and G. purpurea were cultured on media with high kinetin concentration, which induced the formation of friable callus tissues. Only in G. purpurea were these calluses organogenic. Regenerated shoots of G. cruciata and G. purpurea gave rise to plants, that displayed the typical phenotypes of A. rhizogenes-transformed plants: short internodes and rolled leaves. In the roots of G. acaulis and G. cruciata, transformed with A. rhizogenes A4M70GUS, a positive reaction with X-gluc indicated the activity of β-glucuronidase. The DNA extracted from hairy roots and from the roots of transgenic plants hybridized with the appropriate genomic probes in Southern blotting. This is taken as evidence of the stable genetic transformation in the 4 Gentiana species.

Pantropic retroviral vectors integrate and express in cells of the malaria mosquito, Anopheles gambiae.

The lack of efficient mechanisms for stable genetic transformation of medically important insects, such as anopheline mosquitoes, is the single most important impediment to progress in identifying novel control strategies. Currently available techniques for foreign gene expression in insect cells in culture lack the benefit of stable inheritance conferred by integration. To overcome this problem, a new class of pantropic retroviral vectors has been developed in which the amphotropic envelope is completely replaced by the G glycoprotein of vesicular stomatitis virus. The broadened host cell range of these particles allowed successful entry, integration, and expression of heterologous genes in cultured cells of Anopheles gambiae, the principle mosquito vector responsible for the transmission of over 100 million cases of malaria each year. Mosquito cells in culture infected with a pantropic vector expressing hygromycin phosphotransferase from the Drosophila hsp70 promoter were resistant to the antibiotic hygromycin B. Integrated provirus was detected in infected mosquito cell clones grown in selective media. Thus, pantropic retroviral vectors hold promise as a transformation system for mosquitoes in vivo.

Use of viral replicons for the expression of genes in plants.

Autonomously replicating virus-based vectors have been investigated as a means of introducing heterologous genes into plants. This approach has a number of potential advantages over stable genetic transformation, particularly in terms of speed and levels of expression that can be obtained. Several groups of plant viruses, with genomes consisting of both DNA and RNA, have been investigated as possible gene vectors. In the case of DNA viruses, it has generally been possible to identify nonessential regions of the genome that can be replaced by foreign sequences. However, there appear to be limitations on the size of insert which can be tolerated. In the case of RNA viruses, replacement of viral sequences usually has a drastic effect on the viability. However, in several cases it has proved possible to substantially increase the size of the viral genome by the direct insertion of additional sequences while still retaining the ability of the viruses to multiply and spread in plants. These RNA virus-based systems appear to have the greatest potential as gene vectors.

Stable genetic transformation ofPicea mariana (black spruce) via particle bombardment

Stable genetic transformation ofPicea mariana (black spruce) was obtained via particle bombardment into two target tissues, mature cotyledonary somatic embryos and suspensions from embryonal masses, with the Biolistic PDS-1000/He device. Seven transgenic embryogenic cell line were obtained from the mature cotyledonary somatic embryos after secondary somatic embryogenesis from two different cell lines (R4F14 and 119794-014). The suspension culture from embryonal masses produced five transgenic cell lines from one cell line (R4F14). Expression of the introduced β-glucuronidase (GUS) and neomycin phosphotransferase II (NPT II) genes was detected by histochemistry and fluorometry, and by ELISA in 10 of the lines. Two lines showed only NPT II gene expression. Four of the five lines obtained after bombardment of suspensions of embryonal masses showed lower levels of expression of GUS and NPT II. The integration of the foreign genes was confirmed by polymerase chain reaction analyses and Southern hybridization for GUS and NPT II, and complex hybridization patterns were observed. The 12 transgenic lines obtained had a typical embryogenic morphology and were capable of maturation and germination. Over 40 transgenic trees were regenerated from one of the transgenic lines, and they have a normal phenotype.

Agrobacterium-mediated Transformation of Hibiscus syriacus and Regeneration of Transgenic Plants.

Hibiscus syriacus leaf tissues were transformed and transgenic plants were regenerated. Leaf pieces from in vitro grown plantlets were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary plasmid pBI121 containing β-glucuronidase (GUS) reporter and neomycin phosphotransferase II (NPT II) selectable marker genes. Twenty eight percent of inoculated leaf pieces produced kanamycin-resistant calli when cultured on MS medium supplemented with 0.2mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 2.0mg/l benzyladenine (BA) and 50mg/l kanamycin (Km). Shoot regeneration was achieved by transferring the calli to MS medium containing 2.0mg/l 2-isopentenyladenine (2ip), 0.1mg/l naphthylacetic acid (NAA) and 50mg/l Km. Regenerated shoots were rooted in medium with 0.5mg/l indolebutyric acid (IBA) and 25mg/l Km. GUS activity was detected in leaves, stems and roots of transgenic plants by both histochemical and fluorometric assays. Northern hybridization analysis provided molecular evidence for stable genetic transformation.

GENETIC TRANSFORMATION OF THE DIATOMS CYCLOTELLA CRYPTICA AND NAVICULA SAPROPHILA

ABSTRACT Two species of diatoms were genetically transformed by introducing plasmid vectors containing the Escherichia coli neomycin phosphotransferase II (nptII)gene. Expression of the bacterial nptII gene in the diatoms was achieved using the putative promoter and terminator sequences from the acetyl‐CoA carboxylase gene from the centric diatom Cyclotella cryptica T13L Reimann, Lewin, and Guillard. The vectors were introduced into C. cryptica and the pennate diatom Navicula saprophila NAVIC1 Lange‐Bertalot and Bonik by microprojectile bombardment. Putative transformants were selected based on their ability to grow in the presence of the antibiotic G418, and production of the neomycin phosphotransferase protein by the transformed cells was confirmed by western blotting. The foreign DNA integrated into one or more random sites within the genome of the transformed algal cells, often in the form of tandem repeats. This is the first report of reproducible, stable genetic transformation of a chlorophyll c‐containing alga.

Chapter 26 Electroporation of Plant Protoplasts and Tissues

Publisher Summary This chapter discusses the electroporation of plant protoplasts and tissues. Electroporation utilizes short, high-voltage electric shocks to make cells permeable to exogenous molecules. Although direct physical evidence is still lacking, it is widely believed that pores form transiently in the cell membrane in response to these shocks and that the uptake of exogenous molecules occurs through these pores. Electroporation is used to induce the cellular uptake of reporter dyes, enzyme substrates, nucleic acids, and even antibodies and viruses. In plants, electroporation is used primarily to stimulate the uptake of plasmids for stable and transient genetic transformation. Protoplasts are normally utilized in plant electroporation because the cell wall is a major barrier to the diffusion of macromolecules. However, recent work indicates that plasmid DNAs can be introduced into walled plant cells by electroporation. Most of the instruments currently used for electroporation are capacitive-discharge instruments; a capacitor bank is charged by a high-voltage power supply and then discharged into a chamber containing the experimental material.

Stable genetic transformation of Arabidopsis thaliana by Agrobacterium inoculation in planta

Stable genetic transformation of Arabidopsis thaliana by Agrobacterium inoculation in planta

Stable genetic transformation of Arabidopsis thaliana by Agrobacterium inoculation in planta

Summary Stable genetic transformation of Arabidopsis thaliana was achieved by simple in planta inoculation of Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBl121. The transformation procedure, which we call in planta transformation, involves severing of apical shoots at their bases, inoculation with Agrobacterium at the severed sites, and in planta generation of shoots from the severed sites. On average, 5.5% of the newly formed shoots produced transformed progenies. These progenies (T2 generation) contained T-DNA in the genome as examined by assaying the T-DNA encoded β-glucuronidase and kanamycin resistance and by genomic Southern blot analysis, the copy number of the T-DNAs in the Arabidopsis genome being single (33%) or multiple. The genetic behavior of the transformants examined at the T3 and T4 generations or with the F2 progenies of the outcrosses between transformants and wild-type plants showed that most of the inserted T-DNA are inherited in a Mendelian fashion. This procedure provides a new approach for simple and efficient transformation of A. thaliana, obviating the need for plant regeneration from tissue explants in vitro.

Spontaneous membrane potential oscillations in Madin-Darby canine kidney cells transformed by alkaline stress.

High pH is known to be associated with normal cell growth and neoplastic transformation. We observed that Madin-Darby canine kidney (MDCK) cells grown under sustained alkaline stress (pH 7.7) develop "foci" composed of spindle-shaped cells lacking contact inhibition and exhibiting only poor adhesion to the culture support. Foci-developing (F) cells were cloned and grown in control medium (pH 7.4), where they maintained their neoplastic features indicating a stable pH-induced genetic transformation. After F cells had been fused to giant cells with polyethylene glycol, the cell membrane potential (Vm) was measured by means of microelectrodes. In contrast to non-transformed MDCK cells, Vm of F cells showed spontaneous biorhythmicity caused by periodic opening of Ca2(+)-activated K+ channels. Spiking activity was blunted by the Ca2+ channel blocker nifedipine, by the K+ channel blocker Ba2+, by the Na+/H+ exchange blocker amiloride and its analogue ethylisopropylamiloride, and by an extracellular pH of 7.6 and 6.8. We conclude that MDCK cells transformed by sustained alkaline stress have lost their stable plasma membrane potential but, instead, exhibit endogenous Ca2(+)- and pH-sensitive oscillations.

DEVELOPMENT OF TRANSFORMATION SYSTEM FOR COFFEA ARABICA USING PARTICLE GUN METHOD

Somatic embryogenesis of coffee has been studied for the purpose of obtaining target tissues for stable genetic transformation through use of a particle gun. Eight cultivars of C. arabica were selected for callus induction from leaves. Primary calli were induced within two weeks in over 98% of the leaf disks explanted on MS medium with 2,4-D and kinetin prior to treatment on a secondary culture medium. Somatic embryos were obtained from `Catuai' and `Blue Mountain' after six months from explanting. Somatic embryos were germinated in MS media and developed into plants. Somatic embryos and embryogenic calli are being used for gene transformation experiments with the helium-driven particle gun.

Detection of the neo r gene expression in animal cells after genetic transformation

A reliable assay is reported for the detection of the marker gene aminoglycoside phosphotransferase activity in cells that express this enzyme transiently or as a result of stable genetic transformation. This method combines the simplicity of the dot assays with the reliability of the more elaborate and time consuming electrophoretic or chromatographic methods. Inhibition of phosphatases and protein kinases during the reaction reduces labeled ATP consumption by these enzymes. As a result, this assay allows the detection of approx 10 times lower levels of the enzyme than currently used methods. To detect the expression of reporter genes in transformed cells aminoglycoside phosphotransferase can be used as well as the widely used chloramphenicol acetyltransferase enzyme.