Highest Transformation Frequency Research Articles

High-frequency transformation of oat via microprojectile bombardment of seed-derived highly regenerative cultures

A highly efficient and reproducible transformation system for oat ( Avena sativa L. cv. GAF/Park-1) was developed using microprojectile bombardment of highly regenerative tissues derived from mature seeds. Callus was induced under dim light conditions on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and high cupric sulfate. Highly regenerative tissues, generated from embryogenic callus, were used as a transformation target. From 327 individual explants bombarded with the β-glucuronidase gene ( uidA; gus) and a hygromycin phosphotransferase gene ( hpt), 84 independent transgenic events were obtained after an 8–12-week selection period on hygromycin. All events were regenerable, giving an effective transformation frequency of 26%; co-expression of GUS activity occurred in 70% of the independent events. Presence of the foreign genes in DNA from leaf samples of T 0 and T 1 plants was confirmed by polymerase chain reaction (PCR) amplification and/or DNA blot hybridization. Fertility of the plants from the transgenic lines was 63% (24/38) and the transgene(s) was stably transmitted to T 1 and T 2 progeny.

Agrobacterium-mediated high frequency transformation of an elite indica rice variety Pusa Basmati 1 and transmission of the transgenes to R2 progeny

Among rice, indica varieties are considered recalcitrant to tissue culture and genetic manipulation. We report here Agrobacterium-mediated transformation of an economically important, elite indica variety Pusa Basmati 1. A large number of morphologically normal and fertile transgenic plants have been obtained. Molecular and genetic analysis of transgenic plants reveals the integration, expression and inheritance of transgenes in the progeny of these plants. Twenty seven percent plants contain single copy gene insertion and copy number of transgenes has been found to vary from 1–4 in transgenic plants. Mendelian as well as non-Mendelian inheritance patterns of the introduced genes have been obtained in the R1 progeny.

Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation.

The visual marker GUS has been utilized in this study to understand the Arabidopsis thaliana vacuum infiltration transformation process by Agrobacterium tumefaciens. High transformation frequencies of up to 394 transgenic seeds per infiltrated plant were achieved. The results showed that the majority of the transgenic seeds from single infiltrated plants were from independent transformation events based on Southern analysis, progeny segregation, distribution of transgenic seeds throughout the infiltrated plants and the microscopic analysis of GUS expression in ovules of infiltrated plants. GUS expression in mature pollen and anthers was monitored daily from 0 to 12 days post-infiltration. In addition, all ovules from a single infiltrated plant were examined every other day. GUS expression frequencies of up to 1% of pollen were observed 3-5 days post-infiltration, whereas frequencies of up to 6% were detected with ovules of unopened flowers 5-11 days post-infiltration. Most importantly, transgenic seeds were obtained only from genetic crosses using infiltrated plants as the pollen recipient but not the pollen donor, demonstrating Agrobacterium transformation through the ovule pathway.

Parameters interacting with mannose selection employed for the production of transgenic sugar beet

The mannose selection system employs the phosphomannose isomerase (PMI) gene as selectable gene and mannose, converted to mannose‐6‐phosphate by endogenous hexokinase, as selective agent. The transgenic PMI‐expressing cells have acquired the ability to convert mannose‐6‐phosphate to fructose‐6‐phosphate, while the non‐transgenic cells accumulate mannose‐6‐phosphate with a concomitant consumption of the intracellular pools of phosphate and ATP. Thus, certain steps of mannose selection depend on the cells’ own metabolism which may be affected by a number of factors, some of which are studied here using Agrobacterium tumefaciens‐mediated gene transfer to sugar beet cotyledonary explants. Four frequently employed saccharides (sucrose, glucose, fructose, and maltose) were tested at various concentrations and were found to interact strongly with the phytotoxic effect of mannose, glucose being able to counteract nearly 100% of an almost complete mannose‐induced growth inhibition. Sucrose, maltose, and fructose also alleviated significantly the mannose‐induced growth inhibition, but were 4‐, 5‐, and 7‐fold less potent than glucose, respectively (calculated as hexose equivalents). The transformation frequencies were also dependent on the nature and concentration of the added carbohydrates, but in this respect sucrose resulted in the highest transformation frequencies, about 1.0%, while glucose and fructose gave significantly lower frequencies. The selection efficiencies were highest in the presence of maltose where no non‐transgenic escapes were found over a range of concentrations. The effect of the light intensity was also investigated and the transformation frequencies were positively correlated to light intensity, although the relative impact of light on growth in the presence of mannose appeared not to be dependent on the mannose concentration. Additional phosphate in the selection media had a strong positive effect on the transformation frequencies, suggesting phosphate limitation during selection. The mannose selection system was found to be relatively genotype‐independent, provided a slight optimization of the mannose concentrations during selection. Analysis of F1‐offspring showed that all studied primary transformants resulted in PMI‐expressing plantlets and that the segregational patterns were in accordance with expectations in at least 50% of the transformants, confirming the stable and active inheritance of the PMI‐gene.

Transgenic Central American, West African and Asian Elite Rice Varieties Resulting from Particle Bombardment of Foreign DNA into Mature Seed-derived Explants Utilizing Three Different Bombardment Devices

We developed a novel system based on particle bombardment for the transfer of foreign genes into eight important rice varieties grown in Central America, West Africa and Asia. Novel features of this improved protocol, which we believe will replace alternative systems currently utilizing immature embryos, embryogenic cell lines or suspension cultures include: (a) bombardment of target tissue 5 d after seed germination; (b) no requirement to select for embryogenic callus prior to bombardment; (c) high transformation frequencies (up to 14%); (d) ability to recover transgenic material at similar frequencies irrespective of bombardment devices used. Southern blot analysis and enzymatic assays in primary transformants and progeny confirmed the stable integration and expression of introduced transgenes in all engineered cultivars.

Ku70: A Candidate Tumor Suppressor Gene for Murine T Cell Lymphoma

We present evidence that inactivation of the Ku70 gene leads to a propensity for malignant transformation both in vitro and in vivo. In vitro, Ku70 −/− mouse fibroblasts displayed an increased rate of sister chromatid exchange and a high frequency of spontaneous neoplastic transformation. In vivo, Ku70 −/− mice, known to be defective in B but not T lymphocyte maturation, developed thymic and disseminated T cell lymphomas at a mean age of 6 months with CD4 +CD8 + tumor cells. These findings directly demonstrate that Ku70 deficiency facilitates neoplastic growth and suggest a novel role of the Ku70 locus in tumor suppression.

Transgenic peanut plants containing a nucleocapsid protein gene of tomato spotted wilt virus show divergent levels of gene expression

The nucleocapsid protein (N) gene of the lettuce isolate of tomato spotted wilt virus (TSWV) was inserted into peanut (Arachis hypogaea L.) via microprojectile bombardment. Constructs containing the hph gene for resistance to the antibiotic hygromycin and the TSWV N gene were used for bombardment of peanut somatic embryos. High frequencies of transformation and regeneration of plants containing the N gene were obtained. Southern blot analysis of independent transgenic lines revealed that one to several copies of the N gene were integrated into the peanut genome. Northern blot, RT-PCR and ELISA analyses indicated that a gene silencing mechanism may be operating in primary transgenic lines containing multiple copy insertions of the N transgene. One transgenic plant which contained a single copy of the transgene expressed the N protein in the primary transformant, and the progeny segregated in a 3 :1 ratio based upon ELISA determination.

The malignant capacity of skin tumours induced by expression of a mutant H-ras transgene depends on the cell type targeted.

. Pinpointing the cells from which tumours arise is a major challenge n tumour biology. Previous work has shown that the targeted expression of a mutant ras gene within the interfollicular cell compartment of mouse skin induces the formation of benign papillomas, but these do not spontaneously progress to malignancy. We have investigated the carcinogenic effects of expressing the same oncogene in a different population of epidermal cells. Expression of mutant ras from a truncated keratin 5 gene promoter, which directs expression to the follicular and interfollicular cells of newborn mice and the hair follicle cells of adults, stimulated the development of acanthotic areas in newborn mice. Within one week of birth, the acanthotic skin developed areas of carcinoma in situ and adult mice developed papillomas and keratoacanthomas, the latter having a high frequency of spontaneous malignant transformation to squamous and occasionally spindle carcinomas. The benign tumours that arose had several hallmarks of tumours at a high risk of malignant progression, including suprabasal cell proliferation and heterogeneous expression of keratin 13. In contrast to tumours induced by expressing mutant ras under the control of the keratin 10 or keratin 1 gene promoters, the formation of these lesions was not dependent on wounding or a tumour promoter. Benign tumours that are at a risk of malignant conversion are primarily derived from cells located within the hair follicle, and the nature of the cell in which tumour initiation occurs is a major determinant of malignant potential.

Integrative transformation of the dimorphic yeast arxula adeninivorans LS3 based on hygromycin B resistance.

A transformation system has been developed for the dimorphic yeast Arxula adeninivorans based on a stable integration of the donor DNA into ribosomal DNA. For this purpose a cassette was constructed which contains the E. coli hph gene, conferring hygromycin B resistance, fused to the 5' expression signals of the A. adeninivorans TEF1 gene, encoding the translation elongation factor EF-1alpha, and the transcription termination region of the Saccharomyces cerevisiae PHO5 gene. This cassette was fused into the 25S rDNA of A. adeninivorans. Linearization of this vector was required for high transformation frequencies. The vector was integrated in multiple copies into the 25S rDNA by homologous recombination. Copy number was not altered even after the growth of transformants for 15 generations under non-selective growth conditions. Microscopical analyses revealed that integration of the transformed plasmid did not influence the dimorphism, which is triggered at 42 degrees C for both transformed and non-transformed cells.

Extracellular enzymes ofErwinia carotovora eliminate the need for azacytidine treatment for high frequency transformation ofArabidopsis thaliana

This paper reports a part of our studies on large-scale T-DNA-mediated gene tagging inArabidopsis thaliana. To enhance the chance of tagging specific stress-responsive genes of this species by monitoring the preferential insertion of the T-DNA into the actively transcribed loci, we exposed the root explants to low temperature (LT), abscisic acid (ABA), and extracellular enzymes (EXE) of the plant pathogenErwinia carotovora prior to transformation byAgrobacterium tumefaciens. Both LT and ABA reduced the frequency of transformation; with these treatments, the average transformation frequencies were 8.1% and 2.6%, respectively. However, in explants pretreated with EXE the transformation frequency was 89.0%, similar to that obtained in control materials (92.6%). Transgenic calli developed from these explants did not require any treatment with azacytidine (azaC) for efficient shoot regeneration. Furthermore, this treatment enhanced multiple insertion of the T-DNA into the plant genome; within a population of EXE-treated transgenic plants, the number of lines harboring at least three copies of the integrated T-DNA was much higher (61%) than that observed in an untreated population (34%).

Electrotransformation of Lactobacillus plantarum using linearized plasmid DNA.

Evidence is presented that linearized plasmid DNA is capable of electrotransforming Lactobacillus plantarum at a frequency 500-fold lower than with the covalently closed circular molecule. When the linearized plasmid was religated prior to transformation the transformation efficiency was < 10-fold higher, suggesting that open circular plasmid was only slightly more efficient in the transformation of Lact. plantarum than linear DNA. This observation has implications for direct cloning into this species since the high background transformation frequency produced by the linear DNA could potentially obscure the recovery of clones. Nevertheless, using positive selection for enhanced chloramphenicol resistance, cloned fragments of Lact. helveticus DNA were obtained using the shuttle vector pGKV110.

Transfection by Electroporation

AbstractElectroporation‐‐the use of high‐voltage electric shocks to introduce DNA into cells‐‐can be used with most cell types,yields a high frequency of both stable transformation and transient gene expression,and,because it requires fewer steps,can be easier than alternate techniques. In this unit,the basic protocol describes the electroporation of mammalian cells and an alternate protocol outlines modifications for preparation and transfection of plant protoplasts.

Identification of Autonomously Replicating Sequence (ARS) Elements in Eukaryotic Cells

Autonomously replicating sequence (ARS) elements were first identified in the budding yeastSaccharomyces cerevisiaeas chromosomal DNA fragments that promoted high frequency of transformation and extrachromosomal maintenance of plasmid DNA. These specific sequence elements were subsequently shown to function as origins of DNA replication. Detailed analysis of the structure and function of ARS elements has been limited largely toS. cerevisiaeand more recently the fission yeastSchizosaccharomyces pombe.Characterization of ARS activity in other eukaryotes is far less complete. Here we describe the ARS assay developed in yeast and its application to the study of origin function in other eukaryotes. Other available methods for detecting autonomous replication in these systems are also presented.

Tagging Genes and Trapping Promoters inToxoplasma gondiiby Insertional Mutagenesis

Plasmid vectors that incorporate sequence elements from the dehydrofolate reductase–thymidylate synthase (DHFR-TS) locus ofToxoplasma gondiiintegrate into the parasite genome with remarkably high frequency (>1% of transfected parasites). These vectors may—but need not—include mutantDHFR–TSalleles that confer pyrimethamine resistance to transgenic parsites. Large genomic constructs integrate at the endogenous locus by homologous recombination, but cDNA-derived sequences lacking long stretches of contiguous genomic DNA (due to intron excision) typically integrate into chromosomal DNA by nonhomologous recombination. Nonhomologous integration occurs effectively at random; and coupled with the high frequency of transformation, this allows a large fraction of the parasite genome to be tagged in a single electroporation cuvette. Genomic tagging permits insertional mutagenesis studies conceptually analogous to transposon mutagenesis in bacteria, yeast,Drosophila,etc. In theory (and, thus far, in practice), this allows identification of any gene whose inactivation is not lethal to the haploid tachyzoite form ofT. gondiiand for which a suitable selection or screen is available. Transformation vectors can be engineered to facilitate rescue of the tagged locus and to include a variety of reporters or selectable markers. Genetic strategies are also possible, using reporters whose function can be assayed by metabolic, visual, or immunological screens to “trap” genes that are activated (or inactivated) under various conditions of interest.

Uptake and processing of DNA by Acinetobacter calcoaceticus--a review.

In natural transformation, DNA in the form of macromolecular fragments can be translocated across the cell envelope of prokaryotic microorganisms. During the past two decades, several, largely mutually contradictory, hypotheses have been forwarded to explain the molecular mechanism and bioenergetics of this translocation process. Other biomacromolecules are translocated across the bacterial cell envelope as well, such as polysaccharides and proteins, the latter for instance in the process of the assembly of type-IV pili. This brings up the question whether or not common components are involved. Here, we review analyses of DNA translocation in Acinetobacter calcoaceticus, a Gram-negative eubacterium that is able to migrate through twitching motility, and also shows a high frequency of natural transformation. DNA uptake in this organism is an energy-dependent process. Upon entry into the cells, the DNA fragments are integrated into the resident chromosome when a sufficiently large region of mutual homology is available (200 to 400 bp). However, this process is rather inefficient, and on the average 500 bp of each incoming fragment is degraded through exonuclease activity. Upon covalent attachment of a bulky protein molecule to the transforming DNA, the DNA-translocation machinery becomes blocked in further translocation activity. Since A. calcoaceticus is not well suited for transposon mutagenesis, a random mutagenesis procedure has been developed, based on the ligation of an antibiotic-resistance marker to random fragments of chromosomal DNA. This method was used to generate several mutants impaired in the natural transformation process. Three of these have been characterized in detail. No components, common to the translocation of macromolecules through the cell envelope of Acinetobacter, have been detected in this screen.

High-frequency transformation of cottonwoods (genus Populus) by Agrobacterium rhizogenes

Many species of Populus, particularly cottonwoods of sections Aigeiros and Tacamahaca, remain recalcitrant to genetic transformation. We demonstrate that transgenic trees can be readily produced in several poplar genotypes using wild-type Agrobacterium rhizogenes. Hairy roots were produced in a variety of clones that included Populus trichocarpa Torr. & A. Gray, Populus deltoides Bartr. ex Marsh., P. trichocarpa × P. deltoides hybrids, and Populus alba L. × Populus grandidentata Michx., some of which were otherwise recalcitrant to regeneration or transformation. The frequency of hairy roots ranged from 17 to 92%; nearly all hairy roots could be regenerated into transgenic plants. Among 18 transgenic lines that were confirmed by Southern analysis and grown into small trees, 5 showed severe dwarfism and other phenotypic abnormalities, while the majority grew normally. Agrobacterium rhizogenes appears to be an effective means for transformation of diverse poplar genotypes and may be useful in genotypes where disarmed strains of Agrobacterium or regeneration protocols are inadequate to produce transgenic plants. Resume : Plusieurs espces du genre Populus, et plus particulirement celles des sections Aigeiros et Tacamahaca, demeurent rOfractaires ‡ la transformation gOnOtique. Les auteurs dOmontrent que des arbres transgOniques peuvent Œtre obtenus facilement pour plusieurs gOnotypes de peuplier ‡ liaide diune souche sauvage de Agrobacterium rhizogenes. Des racines avec poils absorbants ont OtO obtenues pour un nombre important de clones reprOsentatifs des espces Populus trichocarpa Torr. & A. Gray et Populus deltoides Bartr. ex Marsh., ainsi que des hybrides de P. trichocarpa × P. deltoides et de Populus alba L. × Populus grandidentata Michx., dont certains Otaient rOfractaires ‡ la rOgOnOration ou ‡ la transformation. La frOquence ‡ laquelle des racines avec poils absorbants furent obtenues variait de 17 ‡ 92% et presque toutes les lignOes de racines avec poils absorbants pouvaient Œtre rOgOnOrOes en plantes transgOniques. Parmi les 18 lignOes transgOniques qui furent confirmOes par analyse de type Southern et qui se sont dOveloppOes jusquiau stade de petit arbre, 5 ont affichO un nanisme sOvre ainsi que diautres anormalitOs phOnotypiques, alors que la majoritO des lignOes se sont dOveloppOes normalement. Liutilisation de A. rhizogenes apparaOt donc Œtre un moyen efficace pour transformer divers gOnotypes de peuplier. Cette stratOgie pourrait Œtre utile pour la production de plantes transgOniques dans le cas des gOnotypes pour lesquels la transformation par des souches dOsarmOes de Agrobacterium ou les protocoles de rOgOnOration siavrent inadOquats. (Traduit par la ROdaction)

Role of the host cell cycle in theAgrobacterium-mediated genetic transformation ofPetunia: Evidence of an S-phase control mechanism for T-DNA transfer

Chimeric β-glucuronidase (GUS) gene expression in an efficientAgrobacterium-mediated transformation system utilising mesophyll cells ofPetunia hybrida synchronized with cell cycle phase-specific inhibitors (mimosine and colchicine) was used to show the absolute requirement of S-phase for transfer and/or integration of the transferred DNA (T-DNA). Flow-cytometric analysis of nuclear DNA content and immunohistological detection of bromodeoxyuridine (BrdUrd) incorporation showed that, prior to phytohormone treatment, most (98%) mesophyll cells were at GO-Gl-phase (quiescent phase) and no cell division was occurring. After 48 h and 72 h of phytohormone treatment, there was a rapid increase in S-G2-M-phase populations (> 75%) and a concomitant decrease (down to 24%) in G0–-G1-phase cells. Assays of GUS showed that maximum transformation (> 95% of explants) also occurred after this period. Our data showed that mimosine and colchicine blocked the mesophyll cells at late Gl-phase and M-phase, respectively. No transformation (= GUS expression) was observed in phytohormone-treated cells inhibited in late G1 by mimosine. However, after removal of mimosine, 82% of the explants were transformed, indicating the non-toxic and reversible effect of the inhibitor. On the other hand, a relatively high transformation frequency (65% of explants) was observed after blocking the cell cycle at M-phase with colchicine. However, only transient, but no stable, gene expression (= kanamycin-resistant callus formation) was observed in colchicine-treated M-phase-arrested cells. Similarly, endoreduplication of nuclear DNA, which occurred during the 48 h of phytohormone treatment in some mesophyll cells and cells located along the minor veins in the leaf explants, resulted in transient GUS expression only. These observations indicate a direct correlation between endoreduplication and transient GUS gene expression. Obviously, for stable GUS gene expression, cell division and proliferation are required, indicating that both DNA duplication (S-phase) and cell division (M-phase) are strongly related to stable transformation. We propose that the present system should facilitate further dissection of the process of T-DNA integration in the host genome and therefore should aid in developing new strategies for transformation of recalcitrant plants.

Transfer of the yeast salt tolerance gene HAL1 to Cucumis melo L. cultivars and in vitro evaluation of salt tolerance.

An Agrobacterium-mediated gene transfer method for production of transgenic melon plants has been optimized. The HAL1 gene, an halotolerance gene isolated from yeast, was inserted in a chimaeric construct and joined to two marker genes: a selectable-neomycin phosphotransferase-II (nptII)-, and a reporter-beta-glucuronidase (gus)-. The entire construct was introduced into commercial cultivars of melon. Transformants were selected for their ability to grow on media containing kanamycin. Transformation was confirmed by GUS assays, PCR analysis and Southern hybridization. Transformation efficiency depended on the cultivar, selection scheme used and the induction of vir-genes by the addition of acetosyringone during the cocultivation period. The highest transformation frequency, 3% of the total number of explants cocultivated, was obtained with cotyledonary explants of cv. 'Pharo'. Although at a lower frequency (1.3%), we have also succeeded in the transformation of leaf explants. A loss of genetic material was detected in some plants, and results are in accordance with the directional model of T-DNA transfer. In vitro cultured shoots from transgenic populations carrying the HAL1 gene were evaluated for salt tolerance on shoot growth medium containing 10 gl-1 NaCl. Although root and vegetative growth were reduced, transgenic HAL1-positive plants consistently showed a higher level of tolerance than control HAL1-negative plants.

High-frequency transformation of cottonwoods (genus &lt;I&gt;Populus&lt;/I&gt;) by &lt;I&gt;Agrobacterium rhizogenes&lt;/I&gt;

High-frequency transformation of cottonwoods (genus &lt;I&gt;Populus&lt;/I&gt;) by &lt;I&gt;Agrobacterium rhizogenes&lt;/I&gt;

A new protoplast culture system in Daucus carota L. and its applications for mutant selection and transformation

Petiole protoplasts from in vitro-grown carrot plants are a very good alternative to traditionally obtained protoplasts from suspension cultures. High plating and regeneration efficiencies were obtained in most of the breeding lines that were tested. The embedding of the protoplasts in alginate was crucial for initiating cell division and further development. Several streptomycin resistant and chlorophyll-deficient plant lines were selected for using the petiole protoplast system. Maternally inherited streptomycin resistance was demonstrated by sexual crosses. Protoplast fusion of several chlorophyll-deficient lines did not result in complementation, indicating the cytoplasmic nature of the mutations. Petiole protoplasts were used for direct transformation with plasmid DNA pNUNV containing NPTII as a selectable marker. High transformation frequencies (up to 1%) were obtained after PEG treatment of the protoplasts. Kanamycin resistance was shown to be inherited as a single dominant nuclear trait.