Recovery of Basta-resistant Sedum erythrostichum via Agrobacterium-mediated transformation

Sedums are used as groundcover, in rock gardens and flower borders, and for greening the top floor of buildings, cottages, and thatched roofs. In this study, Agrobacterium-mediated genetic transformation of Sedum erythrostichum was studied by introducing a herbicide-resistant gene (phosphinothricin-N-acetyl-transferase) and a reporter gene (#-glucuronidase, GUS). Following co-cultivation with Agrobacterium on MS medium supplemented with 0.5 mg/l !-naphthaleneacetic acid (NAA) and 2 mg/l 6-benzylaminopurine (BA) for 3 days, leaf segments were transferred onto medium containing 300 mg/l cefotaxime. When adventitious shoots developed directly near the margins of explants after 3 weeks, they were transferred to selection medium with 25 mg/l kanamycin. Of a total of 640 infected leaf explants, 24 (3.75%) produced kanamycin-resistant adventitious shoots; of these, 2.5% were GUS-positive. Transgenic plantlets were confirmed using polymerase chain reaction, Southern, and Northern analyses. Ninety-four percent of the transgenic plantlets were successfully transferred to soil and produced flowers. All GUS-positive transgenic plants were strongly resistant to Basta (phosphinothricin at 200 mg/l) after spraying.     

Transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] plants obtained by Agrobacterium-mediated transformation of somatic embryos

A protocol for the production of transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] was developed via Agrobacterium-mediated genetic transformation of somatic embryos. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the nptII gene and gus-intron were evaluated as vector systems. A number of parameters were tested with respect to maximizing transformation efficiency. While pre-culture, wounding and acetosyringone treatment were inhibitory, the bacterial growth phase (optical density; OD₆₀₀ = 0.6), cell density (10⁹/ml), co-cultivation period (5 days) and pH of the co-cultivation medium (5.6) had positive effects on transformation. Following co-cultivation, globular somatic embryos were placed on multiplication medium and stressed with kanamycin (50 µg/ml). Further selection occurred in the maturation and germination medium at an elevated kanamycin level (75 µg/ml). An average of 40% transient expression was evident based on the GUS histochemical assay. Kanamycin-resistant, GUS-positive embryos were germinated, and the resulting microshoots were multiplied in vitro. Integration of the transgenes into the tea nuclear genome was confirmed by PCR analysis using nptII- and gus-specific primers and by Southern hybridization using an nptII-specific probe. The transgenic shoots were micrografted onto seed-grown rootstocks of cv. Kangra Jat and eventually hardened in a walk-in polyhouse. This is the first report on the production of transgenic tea.     

Optimization of in vitro micropropagation and regeneration for Populus × interamericana and Populus × euramericana hybrids (P. deltoides, P. trichocarpa, and P. nigra)

A rapid and efficient micropropagation method has been established for six European poplar cultivars of economic interest - four Populus 2 interamericana and two Populus 2 euramericana. Using a three-step procedure, we were able to regenerate plantlets from callus and acclimate them within 4 months. In the first step, callogenesis was induced when explants were cultured for 25 days on culture medium supplemented with 10 µM !-naphthaleneacetic acid and 5 µM N⁶(2-isopentenyl)adenine. Bud regeneration followed by shoot elongation was then obtained from callus tissue by combining the cytokinin-like compound thidiazuron with the surfactant Pluronic F-68 at concentrations adjusted for each cultivar. The usefulness of this procedure in the area of genetic engineering is discussed.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated production of transgenic plants of<Emphasis Type="Italic"> Muscari armeniacum</Emphasis> Leichtl. ex Bak.

A system for the production of transgenic plants has been developed for the Liliaceous ornamental plant Muscari armeniacum Leichtl. ex Bak via Agrobacterium-mediated transformation of embryogenic cultures. Leaf-derived embryogenic cultures were co-cultivated with each of three A. tumefaciens strains, all of which harbored the binary vector carrying the neomycin phosphotransferase II (nptII), hygromycin phosphotransferase (hpt) and intron-containing #-glucuronidase (gus-intron) genes in the T-DNA region. Following co-cultivation, the embryogenic cultures were cultured on a medium containing 500 mg l^-1 cefotaxime for 1 week followed by a medium containing 75 mg l^-1 hygromycin in addition to cefotaxime. After 4-5 weeks, several hygromycin-resistant (Hyg^r) cell clusters were produced from the co-cultivated embryogenic cultures. The highest efficiency of production of Hyg^r cell clusters was obtained when embryogenic cultures were inoculated with A. tumefaciens EHA101/pIG121Hm in the presence of 100 µM acetosyringone (AS) and 0.1% (v/v) of a surfactant (Tween20) followed by co-cultivation in the presence of 100 µM AS. Hyg^r embryogenic cultures developed into complete plants via somatic embryogenesis, and most of them were verified to be transgenic by GUS histochemical assay and polymerase chain reaction analysis. Southern blot analysis revealed the integration of one to five copies of the transgene into the genome of transgenic plants, but most of them had one or two copies.     

Germination of synthetic seeds of pineapple (Ananas comosus L. Merr.)

. Tufts of multiple shoots were produced from dormant, axillary buds of pineapple in vitro. Tiny shoots (2-5 mm) isolated from the tuft of multiple shoots were encapsulated in 3% sodium alginate prepared using hormone-free Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol and 0.06 M sucrose. The encapsulated shoots represented synthetic seeds that germinated and formed roots in vitro after subculture onto one of the following media solidified with 0.8% agar: (1) hormone-free Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol and 0.06 M sucrose (Pin1), (2) Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol, 0.06 M sucrose, 9.67 µM 1-naphthalene acetic acid, 9.84 µM indole-3-butyric acid and 9.29 µM kinetin (Pin2), and (3) White's basal medium, White's vitamins, 0.56 mM myo-inositol, 0.03 M sucrose, 0.54 µM 1-naphthalene acetic acid and 1.97 µM indole-3-butyric acid (Pin3). Pretreatment of shoots in either liquid Pin3 or Pin4 medium (White's basal medium, White's vitamins, 0.56 mM myo-inositol, 0.03 M sucrose, 10.8 µM 1-naphthalene acetic acid and 39.4 µM indole-3-butryic acid) was required for development into plantlets with roots after culture on either Pin1, Pin2 or Pin3 media. One hundred percent germination of synthetic seeds to plantlets occurred after pretreatment of shoots in liquid Pin4 medium for 12 h followed by culture of synthetic seeds on Pin2 medium. Synthetic seeds stored at 4°C remained viable without sprouting for up to 45 days. Plantlets produced in vitro from synthetic seeds were successfully established in soil. The protocol provides an easy and novel propagation system for pineapple, an otherwise vegetatively propagated fruit crop.     

Transformation of elite white maize using the particle inflow gun and detailed analysis of a low-copy integration event

Elite white maize lines W506 and M37W were transformed with a selectable marker gene (bar) and a reporter gene (uidA) or the polygalacturonase-inhibiting protein (pgip) gene after bombardment of cultured immature zygotic embryos using the particle inflow gun. Successful transformation with this device did not require a narrow range of parameters, since transformants were obtained from a wide range of treatments, namely pre-culture of the embryos for 4-6 days, bombardment at helium pressures of 700-900 kPa, selection-free culture for 2-4 days after bombardment and selection on medium containing bialaphos at 0.5-2 mg l^-1. However, bombardments with helium pressures below 700 kPa yielded no transformants. The culture of immature zygotic embryos of selected elite white maize lines on medium containing 2 mg l^-1 2,4-dichlorophenoxyacetic acid and 20 mM L-proline proved to be most successful for the production of regenerable embryogenic calli and for the selection of putative transgenic calli on bialaphos-containing medium after transformation. Transgenic plants were obtained from four independent transformation events as confirmed by Southern blot analysis. Transmission of the bar and uidA genes to the T₄ progeny of one of these transformation events was demonstrated by Southern blot analysis and by transgene expression. In this event, the transgenes bar and uidA were inserted in tandem.     

Identification of genetic factors controlling the efficiency of Agrobacterium rhizogenes-mediated transformation in Brassica oleracea by QTL analysis

We have identified quantitative trait loci (QTL) for transgenic and adventitious root production using an Agrobacterium rhizogenes-mediated co-transformation system in conjunction with a Brassica oleracea double haploid (DH) mapping population. Three QTL for green fluorescent protein (GFP)-fluorescent root production and four QTL for adventitious root production were identified as accounting for 26% and 32% of the genetic variation in the population, respectively. Two of the QTL regions identified were common to both transgenic and adventitious root production. Two different methods of QTL analysis were employed (marker regression and interval mapping) and with the exception of one region on linkage group O7 for transgenic root production, both techniques detected the same regions of the genome. The regions we identified to be associated with the control of transgenic root production following A. rhizogenes-mediated transformation are the first to be detected using a QTL mapping approach. In addition, this is the first study to identify genetic regions that co-regulate both transgenic and adventitious root production within the constraints of an A. rhizogenes-mediated transformation process. We have identified plant genotypes that do not produce any transgenic roots that may be deficient for T-DNA integration via illegitimate recombination and that may also be potentially important for the development of homologous recombination protocols. Conversely, we have also identified plant genotypes with high rates of transgenic root production that will be critical in the development of high throughput transformation systems.     

Mapping of a thermo-sensitive earliness <Emphasis Type="Italic">per se</Emphasis> gene on <Emphasis Type="Italic">Triticum monococcum</Emphasis> chromosome 1A<Superscript>m</Superscript>

An earliness per se gene, designated Eps-A^m1, was mapped in diploid wheat in F₂ and single-seed descent mapping populations from the cross between cultivated (DV92) and wild (G3116) Triticum monococcum accessions. A QTL with a peak on RFLP loci Xcdo393 and Xwg241, the most distal markers on the long arm of chromosome 1A^m, explained 47% of the variation in heading date (LOD score 8.3). Progeny tests for the two F_2:3 families with critical recombination events between Xcdo393 and Xwg241 showed that the gene was distal to Xcdo393 and linked to Xwg241. Progeny tests and replicated experiments with line #3 suggested that Eps-A^m1 was distal to Xwg241. This gene showed a large effect on heading date in the controlled environment experiments, and a smaller, but significant, effect under natural conditions. Eps-A^m1 showed significant epistatic interactions with photoperiod and vernalization treatments, suggesting that the different classes of genes affecting heading date interact as part of a complex network that controls the timing of flowering induction. Besides its interactions with other genes affecting heading date, Eps-A^m1 showed a significant interaction with temperature. The effect of temperature was larger in plants carrying the DV92 allele for late flowering than in those carrying the G3116 allele for early flowering. Average differences in heading date between the experiments performed at 16 °C and 23 °C were approximately 11 days (P < 0.001) for the lines carrying the Eps-A^m1 allele for early flowering but approximately 50 days (P < 0.0001) for the lines carrying the allele for late flowering. The large differences in heading time (average 80 days) observed between plants carrying the G3116 and DV92 alleles when grown at 16 °C, suggest that it would be possible to produce very detailed maps for this gene to facilitate its future positional cloning.     

Agrobacterium-mediated transformation of Campanula glomerata

A transformation system for Campanula glomerata 'Acaulis' based on the co-cultivation of leaf explants with Agrobacterium tumefaciens LBA4404 or EHA105 was developed. A. tumefaciens was eliminated when the explants were cultured on medium containing 400 mg/l vancomycin and 100 mg/l cefotaxime. Transgenic plants containing the uidA gene that codes for #-glucuronidase (gus) were obtained following co-cultivation with either strain of A. tumefaciens, LBA4404 or EHA105, both of which harbored the binary vector pGUSINT, coding for the uidA and neomycin phosphotransferase II (nptII) genes. While the transformation frequency (2-3%) was similar for both strains, A. tumefaciens LBA4404 was effectively eliminated from Campanula at a lower concentration of antibiotic as compared to EHA105. The concentration of individual antibiotics required to eliminate EHA105 resulted in a decreased rate (55-67%) of regeneration. The highest percentage of explants that regenerated plants (79%) and the highest regeneration rate was achieved with 100 mg/l cefotaxime combined with 400 mg/l vancomycin. Plants were also transformed with the isopentenyl transferase (ipt) gene using LBA4404 containing the 35S-ipt vector construct (pBC34).     

Chromosome number stability and mitotic activity of cryopreserved Hypericum perforatum L. meristems

Meristems of in vitro-grown Hypericum perforatum L. plants were precultured for 3, 10, or 14 days in the presence of 0.5 M mannitol, or 0.076 µM or 0.76 µM abscisic acid, in RM basal liquid culture medium supplemented with 0.5 mg/l 6-benzylaminopurine and subsequently subjected to cryopreservation by the slow freezing method. The survival rate - determined as the percentage of meristems capable of differentiating plantlets - varied between 10% and 48%. Chromosome number stability of the cryopreserved meristems was determined by chromosome counting. The mitotic index of the control did not significantly differ from that of the treated samples.     

Cryopreservation of alfalfa (Medicago sativa L.) cells by encapsulation-dehydration

Our objective was to establish a cryopreservation protocol for alfalfa (Medicago sativa L.) cells and study the physiological changes occurring in cells during cryopreservation treatment. Cell cultures of Pioneer cvs. 5262 (fall-dormant, winter-hardy) and 5929 (non-dormant, non-hardy) plants initiated regrowth after cryopreservation by encapsulation-dehydration (ED). Pre-treatment of the encapsulated cells for 4 days in B5 medium containing 0.75 M sucrose and dehydration for 4 h in a laminar flow hood were necessary to achieve maximum cell viability after ED and cryopreservation in liquid N₂ (EDN). Viability (measured as triphenyl tetrazolium chloride reduction) of the cv. 5262 cells after cryopreservation was two- to three-fold greater than that of the cv. 5929 cells. Cold acclimation of the cells (10 days at 2°C) improved viability after cryopreservation. The addition of 7.6 µM ABA to the B5 medium enhanced viability in ED but did not improve cell cryopreservability. Cold-acclimated cells had higher protein concentrations, but neither ABA nor cold acclimation influenced protein composition of cold-acclimated cells determined using SDS-PAGE. Encapsulated cells pre-treated for 4 days in B5 medium containing 0.75 M sucrose showed an increased concentration of cell protein and an altered protein composition. Suspension cultures were re-initiated from both ED and EDN treatments by transferring beads sequentially to B5 media containing 0.75, 0.5, 0.25 M sucrose and then to fresh B5 medium. The ED cells resumed rapid growth after two subcultures, whereas EDN cells needed four or five subcultures to resume rapid growth.     

Two rice <Emphasis Type="Italic">DMC1</Emphasis> genes are differentially expressed during meiosis and during haploid and diploid mitosis

We have cloned two rice homologues of yeast DMC1, a meiosis-specific gene required for recombination between homologous chromosomes. We show that rice DMC1A and DMC1B were produced by a gene duplication event that occurred after rice separated from the common ancestor of the cereals. The predicted proteins contain 344 amino acids, of which all but 7 are conserved between the two homologues. Between bases -1 and -245, the two promoters share six invariant blocks of sequence of 10-28 bp, interspersed in variable sequences. Both DMC1A and DMC1B are expressed in pollen mother cells coincident with meiosis, and in diploid non-meiotic tissues such as calli and root tips. DMC1B is also expressed in haploid male gametophytes during pollen maturation and in diploid zygotic embryos and endosperm after pollination. These data suggest that DMC1B, either alone or in combination with DMC1A, contributes to recombination during meiosis and during haploid and diploid mitosis.     

Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene

Ponkan (Citrus reticulata Blanco), one of the most important commercial cultivars of mandarin, is very seedy. In this study, the chimeric ribonuclease gene (barnase) driven by an anther tapetum-specific promoter (pTA29) was introduced into embryogenic callus of Ponkan by Agrobacterium-mediated transformation using the bar gene as a selectable marker. In contrast to previous reports, embryogenic calluses were used as the explant for Agrobacterium infection and transgenic plant regeneration. Selection of transformed callus was accomplished using basta. After 3 days of co-culture, calluses were transferred to MT medium with 50 mg/l basta and 400 mg/l cefotaxime. Resistant calluses were recovered and proliferated after three to four subcultures and then regenerated plantlets. A total of 52 resistant plants were recovered, of which 43 were verified to be transformants by polymerase chain reaction amplification of a fragment of the transgene. Southern hybridization of seven randomly selected transformed plants further confirmed their transgenic nature. The potential of this strategy for breeding citrus seedless types is discussed.     

Improvement of artemisinin accumulation in hairy root cultures of Artemisia annua L by fungal elicitor

The effect of fungal elicitor, derived from mycelial extracts of Penicillium chysogenum 3446, on artemisinin production in hairy root cultures of Artemisia annua L was studied. Various concentrations of elicitor were added to the culture medium after 18 days. Time course experiments were carried out using a defined concentration of elicitor after 18 days. Various ages of hairy root cultures were elicited using a defined concentration of elicitor for 3 days. Artemisinin production in 21-day hairy root cultures treated with 0.3 mg total sugar/ml medium elicitor for 3 days reached to 549.1 mg/l.     

Influence of putrescine, silver nitrate and polyamine inhibitors on the morphogenetic response in untransformed and transformed tissues of Cichorium intybus and their regenerants

The addition of 40 mM putrescine (Put) to Murashige and Skoog's (MS) medium resulted in increased shoot multiplication and shoot growth in untransformed plants relative to transformed plants of Cichorium intybus L. Put at a concentration of 40 mM also resulted in flowering in both systems on the 28th day, with elevated titers of endogenous conjugated Put and spermine (Spm) in both untransformed and transformed plants. The addition of 40 µM AgNO₃ to untransformed axillary buds of C. intybus L. cultured on MS media resulted in increased shoot multiplication (36.9DŽ.63 shoots per culture) and increased shoot growth (7.82ǂ.76 cm) as compared to transformed ones (11.6ǂ.89 shoots per culture; 3.20ǂ.24 cm). Moreover, cultures treated with 40 µM AgNO₃ showed in vitro flowering on the 28th day in both systems, with the endogenous levels of conjugated spermine being higher in untransformed plants than in transformed ones. The morphogenetic response and the endogenous conjugated pool of polyamines were lower following !-DL-difluromethylarginine and !-DL-difluromethylornithine treatments; the addition of put (40 mM) and AgNO₃ (40 µM) restored these to normal levels. Under exogenous put feeding, ethylene production was lower in both the untransformed and transformed cultures. We believe that an interplay between polyamine and ethylene biosynthesis is involved in regulating the morphogenetic response in both transformed and untransformed shoots of C. intybus. The response to AgNO₃ and Put treatment was not altered by the transformation process.     

Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker

A selectable marker system for plant transformation that does not require the use of antibiotics or herbicides was developed. The selectable marker consists of the manA gene from Escherichia coli under the control of a plant promoter that encodes for phosphomannose isomerase, pmi. Only transgenic plants were able to metabolize the selection agent, mannose, into a usable source of carbon, fructose. Transgenic plants were produced efficiently after delivery by Biolistics™ of the pmi gene into maize and wheat tissues, with mean transformation frequencies of 45% for maize and 20% for wheat. Adjustment of the sucrose and mannose levels in the selection medium essentially eliminated escapes. Transgenic events can be identified as early as 2 months for wheat and 4 months for maize. A simple test, a modified chlorophenol red assay, was used for early identification of transgenic events expressing the pmi gene. Transformation frequencies for both crops exceeded those obtained with the bar and pat genes with selection on either Basta^® or bialaphos.     

Optimization of microbiological parameters for enhanced griseofulvin production using response surface methodology

Central composite design was used to determine the optimal levels of microbiological parameters, viz., slant age, seed age and inoculum level, for enhanced griseofulvin production by Penicillium griseofulvum MTCC 1898 and Penicillium griseofulvum MTCC 2004 in shake flask fermentation. The optimal levels of slant age, seed age and inoculum level for Penicillium griseofulvum MTCC 1898 were found to be 8.8772 days, 4.2093 days, 12% (v/v) (᷁.56 kg dry cell mass/m³) and for Penicillium griseofulvum MTCC 2004, 8.221 days, 3.4875 days and 9% (v/v) (̀.09 kg dry cell mass/m³) respectively. The yield of griseofulvin under optimal conditions was found to be 1.65 times for Penicillium griseofulvum MTCC 1898 and 1.07 times for Penicillium griseofulvum MTCC 2004 higher than that obtained using unoptimized conditions. The fermentation time for maximum production of griseofulvin by Penicillium griseofulvum MTCC 1898 and Penicillium griseofulvum MTCC 2004 decreased by 4 days and 2 days respectively.     

Transgenic herbicide- and disease-tolerant carrot (Daucus carota L.) plants obtained through Agrobacterium-mediated transformation

. Transgenic carrot (Daucus carota L.) plants expressing a rice thaumatin-like protein (tlp), phosphinothricin acetyltransferase (bar) and the hygromycin phosphotransferase (hpt) genes were obtained by Agrobacterium-mediated transformation. Petiole and hypocotyl segments of three carrot cultivars were used as the explant sources. Following infection, selection was achieved on Murashige and Skoog medium with 1 mg/l phosphinothricin or 25 mg/l hygromycin B, which was increased after 2 weeks to 10 mg/l phosphinothricin and 100 mg/l hygromycin B. The presence of the tlp and bar transgenes was confirmed by polymerase chain reaction and Southern blot analyses, and the expression of the thaumatin-like protein was demonstrated by Western blot analysis. Among 45 primary transformants, 13 were selected for assessment of herbicide and/or disease tolerance. The transgenic plants showed varying levels of tolerance to the herbicide phosphinothricin, depending on the transformation events in different lines. Four transgenic lines also showed significantly enhanced tolerance to the foliar and root pathogen Botrytis cinerea or Sclerotinia sclerotiorum when inoculated under controlled environment conditions. Two lines had significantly enhanced tolerance to the herbicide phosphinothricin as well as to both pathogens. These results demonstrate the feasibility of introducing two potentially useful agronomic traits into carrot through genetic engineering.     

High-efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation in black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper)

In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA^® (4 mg l^?1) containing MS medium followed by BASTA^® foliar spray on 15-day-old black gram plants (35 mg l^?1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.