Stable genetic transformation of<Emphasis Type="Italic"> Vigna mungo</Emphasis> L. Hepper via<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>

Vigna mungo is one of the large-seeded grain legumes that has not yet been transformed. We report here for the first time the production of morphologically normal and fertile transgenic plants from cotyledonary-node explants inoculated with Agrobacterium tumefaciens carrying binary vector pCAMBIA2301, the latter of which contains a neomycin phosphotransferase ( nptII) gene and a beta-glucuronidase (GUS) gene ( uidA) interrupted with an intron. The transformed green shoots, selected and rooted on medium containing kanamycin, tested positive for nptII and uidA genes by polymerase chain reaction (PCR) analysis. These shoots were established in soil and grown to maturity to collect the seeds. Mechanical wounding of the explants prior to inoculation with Agrobacterium, time lag in regeneration due to removal of the cotyledons from explants and a second round of selection at the rooting stage were found to be critical for transformation. Analysis of T(0) plants showed the expression and integration of uidA into the plant genome. GUS activity in leaves, roots, flowers, anthers and pollen grains was detected by histochemical assay. PCR analysis of T(1) progeny revealed a Mendelian transgene inheritance pattern. The transformation frequency was 1%, and 6-8 weeks were required for the generation of transgenics.     

FLP recombinase-mediated site-specific recombination in rice

The feasibility of using the FLP/ FRT site-specific recombination system in rice for genome engineering was evaluated. Transgenic rice plants expressing the FLP recombinase were crossed with plants harbouring the kanamycin resistance gene ( neomycin phosphotransferase II , nptII ) flanked by FRT sites, which also served to separate the corn ubiquitin promoter from a promoterless gusA . Hybrid progeny were tested for excision of the nptII gene and the positioning of the ubiquitin promoter proximal to gusA . While the hybrid progeny from various crosses exhibited β-glucuronidase (GUS) expression, the progeny of selfed parental rice plants did not show detectable GUS activity. Despite the variable GUS expression and incomplete recombination displayed in hybrids from some crosses, uniform GUS staining and complete recombination were observed in hybrids from other crosses. The recombined locus was shown to be stably inherited by the progeny. These data demonstrate the operation of FLP recombinase in catalysing excisional DNA recombination in rice, and confirm that the FLP/ FRT recombination system functions effectively in the cereal crop rice. Transgenic rice lines expressing active FLP recombinase generated in this study provide foundational stock material, thus facilitating the future application and development of the FLP/ FRT system in rice genetic improvement.     

Agrobacterium tumefaciens–mediatated transformation of shoot-buds of chicory

Vegetative propagation of chicory via axillary shoot proliferation is one of the best ways to obtain an offspring with complete genetic stability. These shoot buds were used in transformation experiments using Agrobacterium tumefaciens strains containing binary plasmids carrying the neomycin phosphotransferase gene (nptII) and the β-glucuronidase gene (uidA). Selection was carried out on basal medium containing 100 mg l⁻¹ kanamycin. Transformed plantlets were recovered at a frequency of about 10% within four weeks after co-cultivation. The presence of the uidA gene was demonstrated by transient gene expression experiments using the histochemical GUS staining procedure. Evidence for stable transformation was shown by subculturing leaf discs on kanamycin selection medium, and Southern blot analysis confirmed the integration of the nptII and the uidA genes in the plant genome. Analysis of the progenies showed that kanamycin resistance was inherited as a single dominant trait. This method for obtaining transgenic chicory plants represents an alternative to leaf disc transformation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Alteration of soil rhizosphere communities following genetic transformation of white spruce

The application of plant genetic manipulations to agriculture and forestry with the aim of alleviating insect damage through Bacillus thuringiensis transformation could lead to a significant reduction in the release of pesticides into the environment. However, many groups have come forward with very valid and important questions related to potentially adverse effects, and it is crucial to assess and better understand the impact that this technology might have on ecosystems. In this study, we analyzed rhizosphere soil samples collected from the first B. thuringiensis-transformed trees [with insertion of the CryIA(b) toxin-encoding gene] grown in Canada (Val-Cartier, QC, Canada) as part of an ecological impact assessment project. Using a robust amplified rRNA gene restriction analysis approach coupled with 16S rRNA gene sequencing, the rhizosphere-inhabiting microbial communities of white spruce (Picea glauca) genetically modified by biolistic insertion of the cryIA(b), uidA (beta-glucuronidase), and nptII genes were compared with the microbial communities associated with non-genetically modified counterparts and with trees in which only the genetic marker genes uidA and nptII have been inserted. Analysis of 1,728 rhizosphere bacterial clones (576 clones per treatment) using a Cramér-von Mises statistic analysis combined with a Monte Carlo comparison clearly indicated that there was a statistically significant difference (P < 0.05) between the microbial communities inhabiting the rhizospheres of trees carrying the cryIA(b), uidA, and nptII transgenes, trees carrying only the uidA and nptII transgenes, and control trees. Clear rhizosphere microbial community alterations due to B. thuringiensis tree genetic modification have to our knowledge never been described previously and open the door to interesting questions related to B. thuringiensis genetic transformation and also to the impact of commonly used uidA and nptII genetic marker genes.     

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 -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.     

Cre/<Emphasis Type="Italic">lox</Emphasis> system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect

A binary expression vector was constructed containing the insecticidal gene Allium sativum leaf agglutinin (ASAL), and a selectable nptII marker gene cassette, flanked by lox sites. Similarly, another binary vector was developed with the chimeric cre gene construct. Transformed tobacco plants were generated with these two independent vectors. Each of the T(0) lox plants was crossed with T(0) Cre plants. PCR analyses followed by the sequencing of the target T-DNA part of the hybrid T(1) plants demonstrated the excision of the nptII gene in highly precised manner in certain percentage of the T(1) hybrid lines. The frequency of such marker gene excision was calculated to be 19.2% in the hybrids. Marker free plants were able to express ASAL efficiently and reduce the survivability of Myzus persiceae, the deadly pest of tobacco significantly, compared to the control tobacco plants. Results of PCR and Southern blot analyses of some of the T(2) plants detected the absence of cre as well as nptII genes. Thus, the crossing strategy involving Cre/lox system for the excision of marker genes appears to be very effective and easy to execute. Documentation of such marker excision phenomenon in the transgenic plants expressing the important insecticidal protein for the first time has a great significance from agricultural and biotechnological points of view.     

Transformation and regeneration of transgenic aspen plants via shoot formation from stem explants

An Agrobacterium -mediated transformation procedure for aspen ( Populus tremula L.), involving the direct regeneration of shoot-buds from stem explants, is described. Disarmed Agrobacterium tumefaciens strain EHA101 harboring the binary plasmid pKIW1105 (which carries the uidA and nptII genes, coding for β-glucuronidase [GUS] and neomycin phosphotransferase II, respectively) was used for the transformation of stem explants. An incubation period of 48 to 72 h was found to be most effective in terms of transient GUS expression on the cut surface of the stem explants. Adventitious shoots regenerated after 2–3 weeks of culture in a woody plant medium (WPM) supplemented with TDZ (1-phenyl-3-[1,2,3-thiadiazol-5-yl]-urea, Thidiazuron) and carbenicillin. Three different kanamycin-based selection schemes were evaluated for optimization of transformation efficiency: (1) Kanamycin was added only to the rooting medium (5 to 6 weeks post-inoculation), or (2) to the regeneration medium 10–14 days after inoculation, or (3) after 2 days of co-cultivation. The third selection scheme was found to be optimal for adventitious shoots with regard to both the time required and the transformation efficiency, the latter being much higher than with the other schemes. Leaf samples from kanamycin-resistant shoots and plantlets were tested for GUS expression, and subjected to polymerase chain reaction (PCR) analysis of uidA and nptII genes. A Southern blot of the corresponding PCR-amplified fragments confirmed their authenticity and Southern blots of total plant DNA confirmed integration of the nptII gene into the plant genome.     

The effect of duplications in the T-DNA on the stability of manifestation of heterologous genes in transgenic tobacco plants

The duplication of uidA gene within T-DNA was shown to disturb stability of expression of another marker gene, nptII, in the second generation (T₂) of selfed initial transformants and in F₁ hybrids of the crosses with nontransgenic tobacco. Hybridological analysis of the progeny resulting from various crosses involving T₁ plants demonstrated that the expression of nptII gene was impaired in the hybrids that were hemizygous for the inactivated copy of uidA gene.     

Regeneration of transgenic shape Vitis vinifera L. Sultana plants: genotypic and phenotypic analysis

Different approaches to producing transgenic grapevines based on regeneration via embryogenesis were investigated. Embryogenic callus was initiated from anther tissue of Vitis vinifera cv. Sultana and three embryogenic culture types (embryogenic callus, tissue type I; proliferating embryos, tissue type II; and a suspension) were established. The three culture types were incolucaled with Agrobacterium tumefaciens harbouring a binary vector which contained a uidA reporter gene and either a hpt or nptII selectable marker gene or the cultures were bombarded with microprojectiles carrying a uidA/nptII binary vector. Transgenic plants were produced only from Agrobacterium transformation experiments. Transformed embryos were selected with kanamycin or hygromycin antibiotics and recovered with the highest efficiency from inoculated type I cultures. Southern analysis of genomic DNA extracted from ten transgenic plants showed that the number of T-DNA insertions in the genome ranged from 1 to at least 4. Evidence for methylation of the T-DNA at cytosine and adenine residues in transgenic plants was found by Southern analysis of DNA digested with two isoschizomer pairs of restriction endonucleases. No evidence for genotype alterations or somatic meiosis was found when DNA from 80 somatic embryos and seven plants regenerated from embryogenic culture were analysed at six sequence-tagged sites which are heterozygous in cv. Sultana. Expression of the uidA gene in in vitro grown leaves of transgenic plants was most often high and uniform but GUS staining was occasionally observed to be low and/or patchy. Transgenic plants and all plants regenerated from embryogenic culture produced red veined, lobed leaves which are uncharacteristic of the accepted ampelographic phenotype of Sultana. It is suggested that this phenotype may represent a juvenile growth stage.     

Stability of expressing the nptII gene in transgenic tobacco plants (nicotiana tabacum L.) with multiple T-DNA insertions

The stability of nptII gene expression was assessed in transgenic tobacco plants with multiple T-DNA insertions. The plants were obtained by self-pollination in the first (T1) and second (T2) generations and also in F1 from crossing T1 plants. The multiple copies showed stable Mendelian inheritance.     

An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens.

An efficient and reproducible procedure for the transformation of white spruce (Picea glauca [Moench] Voss) embryogenic tissues was developed using A. tumefaciens-mediated gene transfer. Rapidly dividing white spruce embryogenic tissues were co-cultivated with disarmed A. tumefaciens strains containing additional copies of the virulence regions from plasmid PToK47. The plasmid pBi121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the beta-glucuronidase (uidA) reporter gene, was used as binary vector. The highest frequency of transformation (15 transformed tissues g(-1) FW of treated embryogenic tissue) was obtained with 5-d-old tissues grown in liquid medium and co-cultivated with Agrobacterium for 2 d in the same medium but containing 50 microM acetosyringone. Recovery of kanamycin-resistant tissues was improved when tissues were first grown for 10 d on a timentin-containing medium (400 mg l(-1)), to prevent bacterial overgrowth, before application of the selection pressure. After 6 weeks on kanamycin-selection medium, resistant tissues were obtained and showed stable uidA expression. The presence of the transgenes was demonstrated by PCR analysis and their integration into the genome was confirmed by Southern hybridization. Transgenic plants were regenerated from transformed tissues within 4 months after co-culture.     

Agrobacterium-mediated transformation of yellow lupin to generate callus tissue producing HBV surface antigen in a long-term culture

The idea of an oral vaccine administered as a portion of plant tissue requires a high level of antigen production. An improved protocol for the induction of transgenic yellow lupin calli or tumours, reaching 44% of transformation rate, is presented here. It has been developed by using the nptII marker gene and the uidA reporter gene as well as various Agrobacterium strains and plant explants. This method of seedling and hypocotyl transformation was applied to raise calli or tumours producing a small surface antigen of Hepatitis B Virus (S-HBsAg). Lupin tissue lines were long-term cultured on selection media maintaining the growth rate and high expression level of the native form of S-HBs, up to 6 microg per g of fresh tissue.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Transformation of Papaver somniferum cell suspension cultures with sam1 from A. thaliana results in cell lines of different S-adenosyl-L-methionine synthetase activity

We have developed a procedure for opium poppy ( Papaver somniferum ) transformation using Agrobacterium tumefaciens -mediated gene delivery. Hypocotyl-derived cell suspension cultures of P. somniferum were cocultivated with A. tumefaciens strain GV3101(pMP90) harbouring either the binary vector pTHW136 or the binary vector pO35SSAM. The former contained the uidA reporter gene and the nptII selectable gene, encoding the enzymes β -glucuronidase and neomycin phosphotransferase II, respectively. The latter contained the sam1 gene encoding the enzyme S -adenosyl-L-methionine (SAM) synthetase and the nptII gene. Putatively transformed cell lines were selected on media supplemented with paromomycin. Integration of the foreign genes was confirmed by Southern blot analysis with and without PCR amplification prior to hybridization. SAM synthetase activity was measured in extracts of 5 transformed cell lines. One of them expressed a significant overactivity while two others had a lower activity than the control cell line, leading us to question the possible partial cosuppression of both the resident and the foreign sam genes. To our knowledge, this is the first report of Agrobacterium tumefaciens -mediated transformation of Papaver somniferum cell suspension cultures.     

Albumin extinction followed by de novo methylation of its gene in somatic hybrids of a rat hepatoma

We have previously identified an Msp I site at the 5′ end of the rat albumin gene whose undermethylation is necessary but not sufficient for stable albumin expression in rat hepatoma cells [1]. We have also shown that the extinction of albumin expression in somatic hybrids is not the result of methylation at this site, since for two different crosses, rapid extinction was found to occur in the absence of any de novo methylation of the previously active gene[2]. In the present study, we examine albumin expression and albumin gene methylation for independent hybrid clones isolated from crosses between albumin expressing rat hepatoma cells and cells of two different non-expressing lines. The cells from hybrid clones of both crosses are characterized by stable extinction of albumin expression. Moreover, we find that de novo methylation of the “extinguished” albumin gene can occur in somatic hybrids, but only some weeks after the gene has ceased to be expressed.     

Mosaic expression pattern of the nptII gene in transgenic tobacco plants Nu 21

Mosaic expression pattern of the nptII gene in transgenic tobacco Nu 21 leaf somatic cells was demonstrated. Inheritance of this phenotype (in T1-T4 and F1 backcrosses) was revealed. Three plant groups were distinguished, with low frequency of variegation manifestation (0-21.8%), with the high frequency of mosaic progeny (63.1 to 100%), and the intermediate type, where the frequency of the appearance of mosaic plants varied in a wide range, from 0 to 100%. The data obtained suggested the existence of two metastable states of a transgene in the leaf disk somatic cells (active and silenced), which could be associated with DNA modification, i.e., methylation of cytosine within the nptII gene sequence.     

Regulation of chimeric phosphoenolpyruvate carboxykinase genes by the trans-dominant locus TSE1.

Extinction of phosphoenolpyruvate carboxykinase (PCK) gene expression in hepatoma x fibroblast hybrids is mediated by a trans-acting genetic locus designated tissue-specific extinguisher 1 (TSE1). To identify PCK gene sequences required for extinction, hepatoma transfectants expressing PCK-thymidine kinase (TK) chimeric genes were fused with TK- fibroblasts and PCK-TK expression in the resulting hybrids was monitored. Expression of a PCK-TK chimera containing PCK sequences between base pairs -548 and +73 was extinguished in four of five hepatoma transfectants tested, although hybrids derived from one transfectant clone failed to extinguish PCK-TK expression. In contrast, crosses between hepatoma transfectants expressing the herpesvirus TK gene from its own promoter and TK- fibroblasts produced TK+ hybrids; extinction of the transfected TK gene was not observed. Thus, rat PCK gene sequences between base pairs -548 and +73 are sufficient for tissue-specific extinction in hybrid cells. Extinction of PCK-TK gene expression in transfectant microcell hybrids mapped specifically to human chromosome 17, the site of human TSE1.     

Ontogeny of gene expression in Pomoxis (pisces: Centrarchidae)

The regulation of gene expression during embryogenesis was investigated in white and black crappie (Pomoxis spp.) and their reciprocal interspecific F₁ hybrids. The schedule of morphological development and the timing of isozyme expression were compared among the two species and both reciprocal maternal half-sibling F₁ hybrids. Although absolute rates of morphological development differed in response to incubation temperature, relative rates of morphological development (normalized to the onset of retinal pigment deposition) were similar among all crosses. Furthermore, these relative rates were similar to those previously documented for other centrarchid species. To assess differences in ontogenetic patterns of gene expression among the crosses, we examined expression for 39 enzymeencoding loci. Expression was not detected in the embryos for 16 loci due to low or nonexistent activity. Enzymatic activity from eight other loci were continuously detected throughout embryogenesis as a result of maternal enzyme in the egg. However, 15 loci initiated expression during the early development period investigated (fertilization through yolk sac absorption). We observed temporal variability in expression of these 15 loci among the crosses, either in the form of differential expression between parental species or as disturbances in the ontogeny of expression in interspecific hybrids. Such variability in expression suggests that some of the gene regulating mechanisms have diverged since Pomoxis species shared a common ancestral genome. © 1994 Wiley-Liss, Inc.