Developing transgenic barley lines producing human lactoferrin using mutant alfa-tubulin gene as selective marker gene

Method of biolistic transformation was used for genetic improvement of commercial barley cultivars (Oksamitoviy, Vodogray and Getman). The plasmid pHLFTuBA was used for particle bombardment that consists of the hLF gene under the control of the barley glutelin B-1 promoter and a selectable marker gene, alpha-tubulin conferring resistance to trifluralin (dinitroanilinr herbicide). Preliminary screening of different trifluralin concentration range from 0,1 to 30 microM was tested for determination of effective selective agent concentration. Two transgenic barley line of genotype Oksamitiviy and transgenic callus line of cultivar Getman were obtained after selection on 10 microM of trifluralin. To confirm the transgenic nature of regenerated plants, the PCR analysis was carried out. The 734bp length fragment of hLFgene was amplified from both regenerated plants.     

Effects of growth regulators and incubation period on in vitro regeneration of adventitious shoots from gerbera petioles

An effective system for in vitro regeneration of adventitious shoots from callus for the transformation or mutation of gerbera was developed. Callus was produced from petioles of the youngest 3–4 leaves detached from auxillary shoots produced in vitro. Induction medium, on which leaves were incubated over 3 or 6 days, contained 2.3 μM thidiazuron and 0.53 μM α-naphthaleneacetic acid. Explants were than transferred to one of three regeneration media with lower levels of growth regulators. Regeneration was quantified over four (4-weeks each) passages at the time of explant transfer to fresh medium. Direct shoot regeneration occurred during the first 4 weeks, and after these shoots were discarded a semi-compact organogenic callus was produced. Effectiveness of shoot regeneration depended on four criteria: the cultivar (three cultivars were tested), the sequence of passage on regeneration medium, the growth regulators in regeneration medium and the duration of the induction period. Regeneration potential from calli of all cultivars increased from the first to the fourth passage. Duration of the incubation period on induction medium (3 or 6 days) influenced regeneration to varying degrees, depending on the cultivar used and the regeneration medium contents. There were no differences between two of the regeneration media – B, containing 2.2 μM 6-benzyladenine and 0.3 μM indole-3-acetic acid and C, containing 4.4 μM 6-benzyladenine, 4.6 μM zeatin and 0.6 μM indole-3-acetic acid. Cultivar Mariola was the most productive and regenerated more than seven shoots per callus in the fourth passage. Regeneration on medium B was further evaluated on four additional gerbera cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Okadaic acid and trifluoperazine enhance <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in eastern white pine

Mature zygotic embryos of recalcitrant Christmas tree species eastern white pine (Pinus strobus L.) were used as explants for Agrobacterium tumefaciens strain GV3101-mediated transformation using the uidA (β-Glucuronidase) gene as a reporter. Influence of the time of sonication and the concentrations of protein phosphatase inhibitor (okadaic acid) and kinase inhibitor (trifluoperazine) on Agrobacterium-mediated transformation have been evaluated. A high transformation frequency was obtained after embryos were sonicated for 45–50 s, or treated with 1.5–2.0 μM okadaic acid or treated with 100–200 μM trifluoperazine, respectively. Protein phosphatase and kinase inhibitors enhance Agrobacterium-mediated transformation in eastern white pine. A 2–3.5-fold higher rate of hygromycin-resistant callus was obtained with an addition of 2 μM okadaic acid or 150 μM trifluoperazine or sonicated embryos for 45 s. Stable integration of the uidA gene in the plant genome of eastern white pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable and enhanced transformation system has been established in eastern white pine and this system would provide an opportunity to transfer economically important genes into this Christmas tree species. Communicated by W. H. Wu     

Node-derived cultures with high-morphogenic competence in barley and wheat

A successful regeneration system is presented for elite cultivars in barley and wheat based on nodes. Nodal explants were excised from in vitro and ex vitro grown plants. A combination of 8.28 μM 4-amino-3,5,6-trichloropicolinic acid and 4.54 μM to 22.71 μm thidiazuron (TDZ) used in MS-based medium containing 60 g l⁻¹ maltose favoured induction of clumps of multiple shoots/buds with or without callus formation in the primary cultures. Within 8–10 weeks upon further subcultures, the proliferation into callus with rapid and continuously forming adventitious buds containing clusters of meristemoids, termed meristematic bulk tissue (MBT) was obtained. Lowering the levels of growth regulators resulted in redifferentiation of shoots, which elongated, rooted, developed into morphologically normal plants and set seeds normally. With a frequency ranging between 37 and 82% the nodes raised from in vitro grown plants were proliferated into MBT independent of TDZ concentration, cultivar and species. The average number of shoots per responding node in different cultivars was 7–15 in barley and 1–6 in wheat after 12–14 weeks. Nodes from greenhouse grown plants mainly responded for callus formation with poor development of MBT.     

In vitro regeneration of cereals based on multiple shoot induction from mature embryos in response to thidiazuron

The in vitro competency of mature cereal embryos (winter, spring and durum wheats, oat, barley and triticale) was assessed for direct multiple shoot production on culture media containing the plant growth regulators, thidiazuron (TDZ) and/or 6–benzylaminopurine (BAP). Mature embryos of CDC Dancer oat showed the best response, with 69 shoots per explant on culture medium containing a combination of 4.5 μM TDZ and 4.4 μM BAP. TDZ alone induced about 16 shoots per explant from the oat. Among the wheat genotypes, durum wheat showed the most number of shoots (35) per explant on culture medium containing 4.5 μM of TDZ and 4.4 μM of BAP. With TDZ alone, shoot regeneration for durum wheat ranged from 27–32 shoots per explant. The regeneration frequency from the three winter wheat genotypes ranged from 11–25 shoots per explant and was highest on culture medium containing 9.1 μM TDZ and 4.4 μM BAP. The latter culture medium was also effective for a triticale genotype, inducing 34 shoots per explant. The regeneration from mature embryos of barley genotypes ranged from 5–9 shoots per explant. The mature embryos of all the cereals tested could be used for in vitro regeneration with TDZ and TDZ+BAP combinations.     

An improved procedure for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of trifoliate orange (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.) via indirect organogenesis

Highly efficient Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata (L.) Raf.) was achieved via indirect shoot organogenesis. Stable transformants were obtained from epicotyl segments infected with Agrobacterium strain EHA 105 harboring the binary vector pBI121, which contained the neomycin phosphotransferase gene (NPTII) as a selectable marker and the β-glucuronidase (GUS) gene as a reporter. The effects of regeneration and selection conditions on the transformation efficiency of P. trifoliata (L.) Raf. have been investigated. A 7-d cocultivation on a medium with 8.86 μM 6-benzylaminopurine (BA)+1.43 μM indole-3-acetic acid (IAA) was used to improve callus formation from epicotyl segments after transformation. A two-step selection strategy was developed to select kanamycin-resistant calluses and to improve rooting of transgenic shoots. Transgenic shoots were multiplied on shoot induction medium with 1.11 μM BA + 5.71 μM IAA. Using the optimized transformation procedure, transformation efficiency and rooting frequency reached 417% and 96%, respectively. Furthermore, the number of regenerated escape shoots was dramatically reduced. Stable integration of the transgenes into the genome of transgenic citrus plants was confirmed by GUS histochemical assay, PCR, and Southern blot analysis.     

Somatic embryogenesis and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Bixa orellana</Emphasis> L.

Establishment, maintenance, regeneration, and transformation of somatic embryos by both direct and indirect means (callus-mediated) was achieved for Bixa orellana, a tropical plant whose seeds produce commercially edible ‘annatto pigment,’ which mainly constitutes an apocarotenoid called bixin. Callus-mediated methodology was found to be efficient in producing a greater number of embryos in a short time. The maximum of 28 somatic embryos were produced in 16–18 weeks when immature zygotic embryonic stalks were inoculated onto Murashige and Skoog (MS) medium containing B5 vitamins supplemented with 0.44 μM benzyladenine (BA), 0.054 μM α-naphthaleneacetic acid (NAA), 2.89 μM gibberellic acid (GA₃), 0.02 μM triiodobenzoic acid (TIBA), and 0.011 μM triacontanol (TRIA). Callus initiation from hypocotyl explants was obtained on MS medium supplemented with 1.07–2.14 μM NAA and 10.2 μM BA. In 3 months, somatic embryos were produced when callus was inoculated onto MS medium supplemented with 4.44 μM BA, 40 μM AgNO₃, and 0.011 μM TRIA. Somatic embryos were efficiently regenerated on MS basal solid and liquid media supplemented with 0.44–4.4 μM BA, 0.54–2.69 μM NAA, 4.92 μM 2iP, 2.1 μM calcium d-pantothenate, 0.21 μM biotin, 227.7 μM cysteine HCl monohydrate, and 108.6 μM adenine sulfate. Agrobacterium tumefaciens GV 3101 harboring pCAMBIA 1305.2 binary vector-mediated stable transformation of somatic embryos exhibited a transformation frequency of 2.56%. As somatic embryogenesis in any perennial system is useful in terms of both commercial and scientific nature, this somatic embryo-based transformation protocol for the commercially important dye-yielding tropical plant B. orellana is useful for its improvement through genetic engineering.     

An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus

A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.     

Multiple Shoot Regeneration from Immature Embryo Explants of Papaya

A simple and rapid method for multiple shoot formation in vitro from immature embryo axis explants of Carica papaya L. cvs. Honey Dew, Washington and Co2 is described. Multiple shoot regeneration was achieved by culture of the explants on modified Murashige and Skoog (MS) medium supplemented either with thidiazuron (TDZ; 0.45–22.7 μM) or a combination of benzylaminopurine (BAP; 0.2 – 8.84 μM) and naphthalene acetic acid (NAA; 0.5 – 2.64 μM). Highest frequency of shoot regeneration occurred on medium supplemented either with 2.25 μM TDZ or a combination of BAP (4.4 μM) and NAA (0.5 μM). Composition of the basal media influenced the frequency of multiple shoot initiation. Stunted shoots regenerated at 4.5 μM and higher concentrations of TDZ. Such shoots could, however, be elongated by transfer to medium containing 5.7 μM GA₃. Rooting of the regenerated shoots was achieved in presence of indolebutyric acid (IBA; 4.92 – 19.68 μM), however, least response was in presence of 14.7 μM IBA. Rooted plants were hardened and transferred to pots. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characteristics of amino acid uptake in barley

Plants have the ability to take up organic nitrogen (N) but this has not been thoroughly studied in agricultural plants. A critical question is whether agricultural plants can acquire amino acids in a soil ecosystem. The aim of this study was to characterize amino acid uptake capacity in barley (Hordeum vulgare L.) from a mixture of amino acids at concentrations relevant to field conditions. Amino acids in soil solution under barley were collected in microlysimeters. The recorded amino acid composition, 0–8.2 μM of l-Serine, l-Glutamic acid, Glycine, l-Arginine and l-Alanine, was then used as a template for uptake studies in hydroponically grown barley plants. Amino acid uptake during 2 h was studied at initial concentrations of 2–25 μM amino acids and recorded as amino acid disappearance from the incubation solution, analysed with HPLC. The uptake was verified in control experiments using several other techniques. Uptake of all five amino acids occurred at 2 μM and below. The concentration dependency of the uptake rate could be described by Michaelis–Menten kinetics. The affinity constant (K _m) was in the range 19.6–33.2 μM. These K _m values are comparable to reported values for soil micro-organisms.     

Ultrasonic treatment stimulates multiple shoot regeneration and explant enlargement in recalcitrant squash cotyledon explants in vitro

Ultrasonic treatment (0.5–2 min) stimulated multiple shoot regeneration to high levels in vitro from recalcitrant cotyledon explants of commercial squash (Cucurbita pepo L.) cultivars Ma’yan and Bareqet, on Murashige and Skoog [Physiol Plant 15:473–497, 1962] (regeneration) medium augmented with 4.4 μM benzyladenine. At this stage, unsonicated control explants regenerated only a few very small shoots or bud-like structures. Ultrasound also stimulated massive explant growth. Ultrasound treatment resulted in further multiple shoot production (five times greater than control) after explant transfer to elongation medium (Murashige and Skoog [Physiol Plant 15:473–497, 1962] medium with 0.44 μM benzyladenine and 2.9 μM gibberellic acid). Longer ultrasonic treatments (5 or 10 min) promoted multiple shoot regeneration and explant growth accompanied by hyperhydration. Scanning electron microscope observations showed that 2 min ultrasound changed the joint area between epidermal cells and removed some of the surface from the cotyledon epidermal cells, without gross surface injury to the explants. Longer periods of ultrasound (5–10 min) caused further surface erosion. Rubbing the explant contact surface with chloroform or sandpaper emulated the effect of sonication on shoot regeneration and explant growth, demonstrating that ultrasound exerts its morphogenic influence by surface removal. Sonication of explants from other batches of squash seeds (of cultivars Ma'yan and True French), that regenerated without such treatment, reduced regeneration and caused hyperhydration. This is the first report of stimulation of in vitro regeneration by ultrasound treatment. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Transgenic Acacia sinuata from Agrobacterium tumefaciens-mediated transformation of hypocotyls

Transgenic herbicide tolerant Acacia sinuata plants were produced by transformation with the bar gene conferring phosphinothricin resistance. Precultured hypocotyl explants were infected with Agrobacterium tumefaciens strain EHA105 in the presence of 100 μM acetosyringone and shoots regenerated on MS (Murashige and Skoog, 1962, Physiol Plant 15:473–497) medium with 13.3 μM benzylaminopurine, 2.6 μM indole-3-acetic acid, 1 g l⁻¹ activated charcoal, 1.5 mg l⁻¹ phosphinothricin, and 300 mg l⁻¹ cefotaxime. Phosphinothricin at 1.5 mg l⁻¹ was used for the selection. Shoots surviving selection on medium with phosphinothricin expressed GUS. Following Southern hybridization, eight independent shoots regenerated of 500 cocultivated explants were demonstrated to be transgenic, which represented transformation frequency of 1.6%. The transgenics carried one to four copies of the transgene. Transgenic shoots were propagated as microcuttings in MS medium with 6.6 μM 6-benzylaminopurine and 1.5 mg l⁻¹ phosphinothricin. Shoots elongated and rooted in MS medium with gibberellic acid and indole-3-butyric acid, respectively both supplemented with 1.5 mg l⁻¹ phosphinothricin. Micropropagation of transgenic plants by microcuttings proved to be a simple means to bulk up the material. Several transgenic plants were found to be resistant to leaf painting with the herbicide Basta.     

Zinc sulphate improved microspore embryogenesis in barley

The effect of ZnSO₄ concentration on barley (Hordeum vulgare L.) microspore embryogenesis was investigated using cultivars of different androgenetic response. Concentrations from 0 (control) to 600 μM in the stress pre-treatment medium alone or in combination with 30 (control) to 600 μM in the embryo induction medium were assayed in anther culture. Incorporation of Zn²⁺ in the pre-treatment medium itself did not affect microspore embryogenesis. The optimum concentration in the stress pre-treatment and induction media was 180 μM for cultivars (cvs.) Igri and Reinette, and 90 μM for cv. Hop. A significant increase of 30 and 300% in cv. Igri and Reinette, respectively, were produced with 180 μM ZnSO₄ in both the number of embryos and green plants. In order to confirm the effect of Zn²⁺ on microspore embryogenesis this micronutrient was incorporated in the induction medium of isolated microspore cultures of cv. Igri. Concentrations of 90–300 μM ZnSO₄ resulted in an increase of 40–53% in the number of embryos and green plants. All these results indicate that the beneficial effect of Zn²⁺ is exerted mainly during the culture phase, increasing the number of embryos, leading to an increased number of green plants, but it had no effect on percentage of regeneration or green plants.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the dwarf pomegranate (<Emphasis Type="Italic">Punica granatum</Emphasis> L. var. <Emphasis Type="Italic">nana</Emphasis>)

The dwarf pomegranate (Punica granatum L. var. nana) is a dwarf ornamental plant that has the potential to be the model plant of perennial fruit trees because it bears fruits within 1 year of seedling. We established an Agrobacterium-mediated transformation system for the dwarf pomegranate. Adventitious shoots regenerated from leaf segments were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBin19-sgfp, which contains neomycin phosphotransferase (npt II) and green fluorescent protein (gfp) gene as a selectable and visual marker, respectively. After co-cultivation, the inoculated adventitious shoots were cut into small pieces to induce regeneration, and then selected on MS medium supplemented with 0.5 μM α-naphthaleneacetic acid (NAA), 5 μM N⁶-benzyladenine (BA), 0.3% gellan gum, 50 mg/l kanamycin, and 10 mg/l meropenem. Putative transformed shoots were regenerated after 6–8 months of selection. PCR and PCR-Southern blot analysis revealed the integration of the transgene into the plant genome. Transformants bloomed and bore fruits within 3 months of being potted, and the inheritance of the transgene was confirmed in T₁ generations. The advantage of the transformation of dwarf pomegranate was shown to be the high transformation rate. The establishment of this transformation system is invaluable for investigating fruit-tree-specific phenomena.     

Embryo rescue of interspecific hybrids of Brachiaria spp

Age of explant and six different media were evaluated with the objective of regenerating higher numbers of interspecific hybrids between sexual and apomictic Brachiaria. Immature embryos of 7–8, 9–10 and 11–12 days after pollination (DAP), from artificial hybridization between Brachiaria ruziziensis (R) as female parent, and B. brizantha (B) or B. decumbens (D) as male parent, were cultured in modified MS media (M4) – supplemented with different combinations of growth regulators and vitamins. Embryos cultured 9–12 DAP showed high percentage (85–100%) of germination for all the crosses examined. Germination and survival rates varied according to accessions within crosses. Six different media (all modified MS with different growth regulators and vitamins) were tested with the objective of inducing multiple shoots from 7 to 10 DAP embryos, from crosses between R × B. The media M1, supplemented with Kinetin (13.94 μM) and NAA (5.37 μM), and media M3, supplemented with BA (4.44 μM and IAA 2.85 μM), regenerated adventitious shoots and calli about 30–40 days after inoculation. The highest multiplication rate observed was 2.85 shoots per explant in media M1, 60–70 days after culturing. Two other media, M6, supplemented with 2,4-D (13.57 μM) and M2, with 2,4-D (9.05 μM) and BA (8.87 μM) exclusively induced the formation of calli. The described protocols proved to be efficient in regenerating healthy seedlings from immature embryos of interspecific hybrids in Brachiaria. This revised version was published online in June 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> with thaumatin II gene to control fungal diseases

Transgenic plants of hyacinth (Hyacinthus orientalis L.) cvs. Edisson and Chine Pink have been obtained by Agrobacterium-mediated transformation. Leaf explants of the both hyacinth cultivars regenerated shoots on MS medium containing 2.2 μM BAP and 0.3 μM NAA at a frequency of 95%. A. tumefaciens strain CBE21 carrying binary vector pBIThau35 was used for transformation. Plasmid pBIThau35 has been produced by cloning preprothaumatin II cDNA into pBI121 instead of uidA gene. Inoculated leaf explants formed calli and shoots at high frequency on selective medium with 100 mg l⁻¹ kanamycin. Four hyacinth transgenic lines of cv. Chine Pink and one line of cv. Edisson have been selected on medium containing 200 mg l⁻¹ kanamycin. The insertion of thaumatin II gene into hyacinth genome has been confirmed by PCR-analysis. All transgenic plants expressed substantial amounts of thaumatin II (between 0.06 and 0.28% of the total soluble protein). Hyacinth transgenic lines were assayed for resistance to the pathogenic fungi Fusarium culmorum and Botrytis cinerea. There were no significant differences between nontransformed control and transgenic leaves of both cultivars. At the same time the bulbs of the transgenic line Н7401 cv. Chine Pink showed the higher level of resistance to B. cinerea, the bulbs of the transgenic line Н7404 were more resistant to F. culmorum. In both cases the signs of the fungal disease were developed more slowly. The resistance of the bulbs cv. Edisson line to these fungi was not changed. All transgenic hyacinth plant were successfully transferred to soil for further evaluation.     

Regeneration of <Emphasis Type="Italic">Clivia miniata</Emphasis> and assessment of clonal fidelity of plantlets

An efficient in vitro micropropagation system for Clivia miniata Regel was developed using basal tissues of young petals and young ovaries as explants. For callus induction, explants were incubated on Murashige and Skoog (MS) medium containing either 2.22 μM 6-benzyladenine (BA) and 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 4.44 μM BA, 5.37 μM α-naphthaleneacetic acid (NAA), and 9.05 μM 2,4-D. Moreover, callus was induced from young ovaries when these were incubated on MS medium containing 8.88 μM BA, 10.74 μM NAA, and 9.05 or 18.10 μM 2,4-D. Subsequently, callus was transferred to MS medium supplemented with kinetin (KT) and NAA for shoot organogenesis. Frequency of shoot regeneration from petal-derived callus was highest when callus was transferred to medium containing 2.69 μM NAA with either 9.29 or 13.94 μM KT. Shoot regeneration frequency from ovary-derived callus was highest when this callus was transferred to medium containing 9.29 μM KT and 10.74 μM NAA. Overall, different explant types exhibited different organogenic capacities wherein, young petals had higher shoot regeneration frequencies than young ovaries. The highest rooting frequency (98.25 ± 3.04%) was obtained when shoots were transferred to half-strength MS medium without plant growth regulators. Regenerated plantlets were transplanted to soil mix and acclimatized, yielding a 96.80% survival frequency. Only 0.6% of regenerated plantlets exhibited morphological changes. The diploid status (2n = 22) of regenerated plantlets was determined using chromosome counts of root-tips. Moreover, inter-simple sequence repeats were used to assess the genetic fidelity of regenerated plantlets. Overall, regenerated plants shared 90.5–100.0% genetic similarities with mother plants and 89.0–100.0% similarities with each other.     

Highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of celery (<Emphasis Type="Italic">Apium graveolens</Emphasis> L.) through somatic embryogenesis

A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration from Agrobacterium tumefaciens- inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166 and XP85 and inoculated with A. tumefaciens strain EHA105 containing the binary vector pBISN1. PBISN1 has the neomycin phosphotransferase gene (nptII) and an intron interrupted β-glucuronidase (GUS) reporter gene (gusA). Co-cultivation was carried out for 4 d in the dark on callus induction medium (CIM): Gamborg B5 + 2.79 μM kinetin + 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented with 100 μM acetosyringone. Embryogenic calluses resistant to kanamycin (Km) were then recovered on CIM + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 12 weeks. Subsequently, a large number of Km-resistant and GUS-positive transformants, tens to hundreds per explant were regenerated via somatic embryogenesis on Gamborg B5 + 4.92 μM 6 (γ,γ-dimethylallylamino)-purine (2iP) + 1.93 μM α-naphthaleneacetic acid (NAA) + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 8 weeks. Using this protocol, the transformation frequency was 5.0% and 5.0% for leaf sections, 17.8% and 18.3% for cotyledons, and 15.9% and 16.7% for hypocotyl explants of cvs. XP85 and XP166, respectively. Stable integration of the model transgenes with 1–3 copy numbers was confirmed in all ten randomly selected transgenic events by Southern blot analysis of gusA. Progeny analysis by histochemical GUS assay showed stable Mendelian inheritance of the transgenes. Thus, A. tumefaciens-mediated transformation of cotyledons or hypocotyls provides an effective and reproducible protocol for large-scale production of transgenic celery plants.     

Thidiazuron-induced shoot organogenesis from mature leaf explants of scented <Emphasis Type="Italic">Pelargonium capitatum</Emphasis> cultivars

Shoot organogenesis from mature leaf tissues of two scented Pelargonium capitatum cultivars, ‘Attar of Roses’ and ‘Atomic Snowflake’, grown in the greenhouse, were optimized in the presence of thidiazuron (TDZ). The protocol involved preculture of leaf sections on basal Murashige and Skoog (MS) medium supplemented with 10 μM TDZ, 4.4 μM of 6-benzyladenine (BA) and 5.4 μM α-naphtaleneacetic acid (NAA) for a period of 2 weeks and followed by subculture of explants to a fresh medium containing 4.4 μM BA and 5.4 μM NAA. Frequency of regeneration reached approximately 93% for both cultivars, with the induction of more than 100 shoots per explant. Regenerated plantlets were rooted on half-strength MS medium supplemented with 4.4 mM sucrose and 8.6 μM of Indole-3-acetic acid (IAA). All regenerated shoots from both cultivars developed roots when transferred to organic soil mix, acclimatized, and successfully transferred to greenhouse conditions. When regenerated shoots were transferred to hydroponic conditions, frequency of survival was 76.2 and 61.9% for ‘Attar of Roses’ and ‘Atomic Snowflake’, respectively.     

In vitro propagation of Catalpa ovata G. Don

Plants of C. ovata were regenerated in vitro from shoot tips and nodal explants as well as from cotyledon-derived calluses. For shoot proliferation from shoot tips and nodal segments, Schenk and Hildebrandt (1972) or Lloyd and McCown (1980) basal media, supplemented with 6-benzyladenine (2.2–22.2 μM) alone or in combination with indole-3-acetic acid (0.6 μM), were used. Shoot regeneration through organogenesis was achieved by culturing cotyledons on Schenk and Hildebrandt medium containing indole-3-acetic acid (0.6 μM) and 6-benzyladenine (4.4 μM) or zeatin (22.8 μM). TLC and HPLC analysis showed that the multiple shoots and micropropagated plants exhibited similar iridoid patterns as those of the leaves of original plants of C. ovata. The highest levels of catalpol and catalposide (8.2 and 2.4 % of dry weight, respectively) were found in aerial parts of three-month-old in vitro regenerated plants. This revised version was published online in June 2006 with corrections to the Cover Date.