Effect of nickel on regeneration in <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. and assessment of genotoxicity using RAPD markers

The aim of the present study was to determine the effect of nickel on shoot regeneration in tissue culture as well as to identify polymorphisms induced in leaf explants exposed to nickel through random amplified polymorphic DNA (RAPD). In vitro leaf explants of Jatropha curcas were grown in nickel amended Murashige and Skoog (MS) medium at four different concentrations (0, 0.01, 0.1, 1 mM) for 3 weeks. Percent regeneration, number of shoots produced and genotoxic effects were evaluated by RAPD using leaf explants obtained from the first three treatments following 5 weeks of their subsequent subculture in metal free MS medium. Percent regeneration decreased with increase in addition of nickel to the medium up to 14 days from 42.31% in control to zero in 1.0 mM. The number of shoot buds scored after 5 weeks was higher in control as compared to all other treatments except in one of the metal free subculture medium wherein the shoot number was higher in 0.01 mM treatment (mean = 7.80) than control (mean = 7.60). RAPD analysis produced only 5 polymorphic bands (3.225%) out of a total of 155 bands from 18 selected primers. Only three primers OPK-19, OPP-2, OPN-08 produced polymorphic bands. The dendrogram showed three groups A, B, and C. Group A samples showed 100% genetic similarity within them. Samples between groups B and C were more genetically distant from each other as compared to samples between groups A and B as well as groups A and C. Cluster analysis based on RAPD data correlated with treatments.     

A two-step protocol for shoot regeneration from hypocotyl explants of oilseed rape and its application for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

A two-step protocol for improving the frequency of shoot regeneration from oilseed rape (Brassica napus L.) hypocotyl explants was established. The protocol consists of a pre-culture on callus induction medium (CIM) and a subsequent shoot regeneration on shoot induction medium (SIM). The SIM was Murashige and Skoog medium supplemented with different concentrations of 6-benzylaminopurine (BA; 2–5 mg dm⁻³) and naphthaleneacetic acid (NAA; 0.05–0.15 mg dm⁻³). Maximum frequency of shoot regeneration (13 %) was on the SIM medium containing 4 mg dm⁻³ BA and 0.1 mg dm⁻³ NAA, but it increased to 24.45 % when 20 μM silver thiosulphate (STS) was added. Strikingly, an extremely high frequency of shoot regeneration up to 96.67 % was reached by a two-step protocol when hypocotyl explants had been pre-cultured for 7 d on a CIM medium containing 1.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid. In addition, the shoot emergence was also 7 d earlier than that observed by use of the one-step protocol. The two-step protocol was also applied for regeneration of transgenic plants with cZR-3, a nematode resistance candidate gene. As a result, 43 plants were generated from 270 shoots and from these 6 plants proved to be transgenic.     

Influence of different ethylene inhibitors on somatic embryogenesis and secondary embryogenesis from <Emphasis Type="Italic">Coffea canephora</Emphasis> P ex Fr.

The effect of cobalt chloride, salicylic acid, and silver nitrate for embryogenesis was studied in in vitro cultures of Coffea canephora. Murashige and Skoog (in Physiol. Plant. 15:473–497, 1962) medium containing 20 and 40 μM either of cobalt chloride, silver nitrate, or salicylic acid supplemented with 1.1 μM N ⁶ benzyladenine and 2.85 μM indole-3-acetic acid was used for the study. At 20 and 40 μM silver nitrate treatment, 35–48% explants responded for embryogenesis, and 38 ± 7 and 153 ± 27 embryos were produced from each callus mass, respectively, whereas only 5% control explants responded on medium devoid of silver nitrate, cobalt chloride, or salicylic acid. Secondary embryogenesis was observed in 70–90% of the explants, and around 100–150 embryos were produced from each explant cultured on a medium containing silver nitrate, and only a 3% response was noticed in control embryo explants. Yellow friable embryogenic calluses were obtained from the cut edges of most of the tissues grown in a medium supplemented with cobalt chloride. The results clearly demonstrated that, among the tested ethylene inhibitors, silver nitrate is very effective in reprogramming the cellular machinery toward embryogenesis.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of blueberry (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis> L.)

Transient expression studies using blueberry leaf explants and monitored by -glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 M for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)₃AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 M AS. Explants were then placed on modified WPM supplemented with 1.0 mg l^–1 thidiazuron, 0.5 mg l^–1 -naphthaleneacetic, 10 mg l^–1 kanamycin (Km), and 250 mg l^–1 cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 E m^–2 s^–1 at 25°C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.     

Fertile transgenic Lotus corniculatus resistant to the non‐selective herbicide phosphinothricin

Resistance to the non‐selective herbicide dl ‐phosphinothricin (PPT) was introduced into commercial Lotus corniculatus cv. Bokor by co‐cultivation of cotyledons with Agrobacterium tumefaciens AGL1 harbouring the binary vector pDM805 which contains the bialaphos resistance gene (bar) from Streptomyces hygroscopicus encoding phosphinothricin acetyltransferase (PAT) and the uidA gene encoding β‐glucuronidase. The half‐cotyledon explants were precultured on regeneration Murashige and Skoog's (MS) medium supplemented with 6‐benzyladenine (BA) and 1‐naphthaleneacetic acid (NAA) at 0.5 mg L^?1 each, 3 days prior to infection. Upon co‐cultivation, the explants were cultured on PPT‐free regeneration medium for 10 days, and then subcultured on regeneration/selection media with increasing PPT concentrations (5–7 mg L^?1) for about 18 weeks. Out of 480 initially co‐cultivated explants, 272 regenerated shoots survived the entire PPT selection procedure. Resistant shoots were grown further, multiplied by tillering that was additionally promoted by PPT and rooted on hormone‐free MS medium containing 5 mg L^?1 PPT. Established shoot cultures, continuously maintained on the same medium, have preserved PPT resistance up to now (more than 2 years). Transformed plants assessed in vitro and in a greenhouse were tolerant to the herbicide PPT at 300 mg L^?1 equivalent to more than twofold the recommended field dosage for weed eradication. Applied PPT treatment did not affect the activities of glutamine synthetase (GS; EC 6.3.1.2) and NADH‐dependent glutamate dehydrogenase (NADH‐GDH; EC 1.4.1.2) in transformed plants. However, PPT did increase the mobility of glutamine synthetase isoforms GS1 and GS2 as well as the inhibition of an additional high mobility GS (hmGS) activity. In untransformed plants, PPT treatment reduced total GS activity by 4.4‐fold while contrary the activity of NADH‐GDH was increased by ninefold. All transformed herbicide‐resistant plants were phenotypically normal and exhibited genomic stability, as were the untransformed plants analysed by flow cytometry. Under greenhouse conditions, they grew to maturity, flowered and set seeds. Stable integration and expression of the bar gene in T0 and T1 plants were confirmed by Southern and Western blot analysis, while integration of the reporter uidA gene did not occur. The bar gene was inherited in a Mendelian fashion by the progeny, as detected by PPT resistance. The production of PPT‐resistant plants may have significant practical applications in weed control in fields of L. corniculatus.     

Cytokinins induce direc somatic embryogenesis of <Emphasis Type="Italic">Dendrobium</Emphasis> chiengmai pink and subsequent plant regeneration

Summary Four auxins (2,4-dichlorophenoxyacetic acid [2,4-D], indole-3-acetic acid [IAA], indole-3-butyric acid [IBA], and naphthaleneacetic acid [NAA]), and five cytokinins (N ⁶-[2-isopentenyl]-adenine [2iP], N ⁶-benzyladenine [BA], 6-furfurylaminopurine [kinetin], 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea [TDZ], and 6-[4-hydroxy-3-methylbut-2-enylamino]purine [zeatin]) were examined for their effects on direct embryo induction from leaf explants of Dendrobium cv. Chiengmai Pink cultured on 1/2 Murashige and Skoog (MS) medium. Whether in light or darkness, explants easily became necrotic and no embryos were obtained on growth regulator-free or auxin-containing media after 60 d of culture. By contrast, five cytokinins tested induced direct embryo formation from leaf explants, and explants cultured in light had a higher embryogenic response compared with those cultured in darkness. The best condition for direct embryo induction was at 18.16 μM TDZ cultured in light for 60 d, where 33% of explants formed a mean number of 33.6 embryos per explant. During subculture on growth regulator-free 1/2 MS medium, embryos gradually developed into plantlets. Secondary embryogenesis was occasionally found on sheath leaves of embryos. Regenerated plantlets were successfully transplanted and grown in a greenhouse environment.     

Electromagnetic field effects on <Emphasis Type="Italic">Artemia</Emphasis> hatching and chromatin state

The influence of magnetic fields on hatching and chromatin state of brine shrimp, Artemia sp., was investigated. Dry Artemia cysts were exposed to a magnetic field of intensity 25 mT for 10 min. The magnetic field was applied in different variants: constant field, rotating field of different directions (right-handed and left-handed) and different magnet polarization. The effect of ultra wideband pulse radiation and microwave radiation was also investigated. The energy density on the surface of object exposed to ultra wideband pulse radiation was 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵ and 10⁻⁶ W/cm², the power of microwave radiation was 10⁻⁴ and 10⁻⁵ W/cm², exposure time - 10 s. After incubation of the cysts for 48 hours in sea water the hatching percentage of Artemia from exposed cysts was higher than in controls. The number of heterochromatin granules was significantly higher in the nauplia (newborn larvae of Artemia) developed from cysts that had been exposed to magnetic and electromagnetic fields. The data obtained demonstrate an increase in percentage hatching of Artemia cysts after treatment with magnetic and electromagnetic fields and chromatin condensation in nauplia. We have also shown different effects of right-handed and left-handed rotating magnetic fields on these processes.     

Intracellular levels of adenosine triphosphate in hamster trachea organ cultures exposed toMycoplasma pneumoniae cells or membranes

Summary The amount of adenosine triphosphate (ATP) in hamster trachea organ cultures was determined with a technique based on light emission from a luciferin/luciferase/ATP reaction. The amount of ATP, expressed as ng per mg dry weight, was consistent in tracheal explants prepared from various animals and changed negligibly when explants were cultivated in vitro for several days. The amount of ATP was related directly to cellular activity and integrity in the epithelium since inactivation by heat or freeze-thaw rapidly depleted measurable ATP, and ciliary activity and ATP content were related directly. When tracheal explants were infected with 10⁵ to 10⁷ CFU of virulentMycoplasma pneumoniae cells, both ciliary activity and ATP content in the tissue dropped dramatically after approximately 5 to 8 days (up to 85% and 60% decreases, respectively). Exposure of explants to 50 to 200 μg per ml of purifiedM. pneumoniae membranes also caused significant decreases in ciliary activity and ATP. When explants were infected with attenuated or nonvirulent mycoplasmas, ciliary activity was only slightly decreased, while ATP values often rose slightly. The technology associated with the determination of ATP levels in tracheal explants should prove useful as a new, objective, analytical approach to cell viability in organ cultures. This investigation was supported in part by the National Institutes of Health (PHS Grant AI 12559), by a Biomedical Sciences Support Grant made to the University of Illinois School of Life Sciences, and by the University Research Board.     

Efficient regeneration of <Emphasis Type="Italic">Brassica napus</Emphasis> L. hypocotyls and genetic transformation by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

An efficient system for shoot regeneration and Agrobacterium-mediated gene transfer into Brassica napus was developed through the modification of the culture conditions. Different concentrations of benzyladenine (1.5, 3.0 and 4.5 mg dm^–3) and thidiazuron (0.0, 0.15 and 0.30 mg dm^–3) were evaluated for shoot regeneration of 7, 14 and 21-d-old hypocotyl explants. Maximum shoot regeneration frequency was obtained in 21-d-old explants using 4.5 mg dm^–3 benzyladenine and 0.3 mg dm^–3 thidiazuron. Under above culture condition, the highest percentage of shoot regeneration frequency was 200 %. Agrobacterium-infected explants grown on the selection medium gave rise to transgenic shoots at a frequency of 11.8 %. Transformed shoots rooted when cultured on a medium supplemented with 2 mg dm^–3 of indolebutyric acid and 10 mg dm^–3 kanamycin. The rooted plantlets were successfully established in the soil and developed fertile flowers and viable seeds. Evidences for transformation were confirmed by GUS assay and PCR analysis.     

Genetic transformation and regeneration of <Emphasis Type="Italic">Sesbania drummondii</Emphasis> using cotyledonary nodes

Sesbania drummondii (Rydb.) Cory is a source for phytopharmaceuticals. It also hyperaccumulates several toxic heavy metals. Development of an efficient gene transfer method is an absolute requirement for the genetic improvement of this plant with more desirable traits due to limitations in conventional breeding methods. A simple protocol was developed for Agrobacterium-mediated stable genetic transformation of Sesbania. Agrobacterium tumefaciens strain EHA 101 containing the vector pCAMBIA 1305.1 having hptII and GUS plus genes was used for the gene transfer experiments. Evaluation of various parameters was carried out to assess the transformation frequency by GUS expression analysis. High transformation frequency was achieved by using 7-day-old precultured cotyledonary node (CN) explants. Further, the presence of acetosyringone (150 μM), infection of explants for 30–45 min and 3 days of cocultivation proved to be critical factors for greatly improving the transformation efficiency. Stable transformation of S. drummondii was achieved, and putative transgenic shoots were obtained on medium supplemented with hygromycin (25 mg l⁻¹). GUS histochemical analysis of the putative transgenic tissues further confirmed the transformation event. Genomic Southern blot analysis was performed to verify the presence of transgenes and their stable integration. A transformation frequency of 4% was achieved for CN explants using this protocol.     

In vitro propagation of the nitrogen-fixing tree-legume Acacia mangium Willd.

In vitro propagation was initiated from 2-week-old and 7-month-old explants of Acacia mangium. Juvenile explants (2 week-old) of 5- to 10-mm lengths composed of two leaves were cultured on Murashige and Skoog (MS) medium containing 1.0 or 2.0 mg L^-1 6-benzyladenine (BAP). After 6 weeks, most explants had formed a large cluster of 14–18 axillary shoots produced by prolific branching of the primary axillary shoot after elongation. The maximum multiplication rate (40) was obtained in the first subculture; the rate decreased to 10–20 in the second one. The mean length of shoots was not significantly affected by BAP concentrations during the subsequent cultures. Rooting ability of juvenile explants was greatly affected by BAP concentrations used in the multiplication medium. When both types of explants were multiplied on a MS medium containing 1.0 mg L^-1 BAP and transferred to a half-strength MS medium containing 0.05 mg L^-1 IBA, only 10% of the juvenile explants were rooted versus 70% of the 7-month-old explants. Rooted plants transferred onto artificial substrate were all nodulated, when inoculated with a specific Bradyrhizobium sp. strain.     

A simple procedure for the isolation of streptomycin resistant plants in Solanaceae

A system has been developed for rapid selection of streptomycin resistant mutants, as adventitious shoots arising from explants of several Solanaceous species. Efficient mutagenesis was achieved by incubating shoot culture-derived leaf strips with 1 or 5 mM nitroso-methylurea, for 90 or 120 min. In Nicotiana tabacum and Lycopersicon peruvianum these treatments resulted in white or variegated adventitious shoots from up to 3.5% of explants placed on medium promoting shoot regeneration. Chlorophyll deficiencies were only observed very rarely in Solanum nigrum. Streptomycin resistant shoots were obtained from leaf explants placed on medium containing 500 mg l^-1 streptomycin sulphate, under which conditions explants are bleached and adventitious shoot development suppressed. Green adventitious s shoots appeared at a frequency dependent both on the mutagenic treatment and on the species. The best response was with S. nigrum where >70% of the explants produced streptomycin resistant shoots, most of which retained their resistance on subsequent testing. Maternal inheritance of streptomycin resistance has been confirmed for several N. tabacum and S. nigrum mutants, and there is also evidence for paternal transmission in the latter species. The procedure has been successfully extended to other species, including N. sylvestris and N. plumbaginifolia, and also to obtain spectinomycin resistant mutants.Communicated by R. Hagemann     

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.     

Selection of atrazine-resistant plants by <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

The effects of atrazine on cotyledon cultures of Capsicum annuum (L.) cv. G₄ were investigated with a view of establishing a system for in vitro selection of resistant mutants. At low levels of herbicide produced little growth inhibition, some chlorophyll loss occurred associated with the production of albino shoots. At 20 mg l⁻¹ atrazine bleaching was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated with severe suppression of growth. Mutagenized cotyledon explants resulted in production of herbicide-resistant plants on medium containing selective levels of sucrose (0.5%) and atrazine (20 mg l⁻¹). Differential morphogenetic responses were observed when the levels of sucrose (0.5–5%) were altered. Shoot regeneration was maximum in 2 sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3%–5%). However, lowering of sucrose concentration from 2 to 0.5% caused complete bleaching of explants and permitted the selection of herbicide-resistant plants. Complete atrazine-resistant plantlets were obtained after rooting of regenerated green shoots on rooting medium containing 10 mg l⁻¹atrazine, 1.0 mg l⁻¹IAA and 0.5% sucrose. Leaf-segment assay of differentiated plants revealed that all regenerants were resistant to the atrazine. Reciprocal crosses between atrazine-resistant and -sensitive plants showed a non-Mendelian transmission of resistance trait.     

Differential Reactivity in Epidermal Cells of Begonia rex Excised and Grown in vitro

Thin explants composed of the epidermis and underlying collenchyma excised from leaf veins of Begonia rex and cultured in vitro are capable of neoformation of unicellular hairs, roots and buds. Unicellular hairs were formed over the entire surface of the explant when 10⁻⁶M indole acetic acid or 10⁻⁷M naphthaleneacetic acid (NAA) was added to the basal medium; each epidermal cell was potentially involved. The epidermis was most sensitive to a NAA treatment during the first few days of culture but 30% of the explants could still react after 4 days of culture without NAA. When NAA (5 × 10⁻⁷M) and a cytokinin, zeatin (10⁻⁷M), were added together, roots were formed from epidermal tissue after numerous divisions in the original cells. Their initiation was not related to particular cells. Buds were formed when a cytokinin (10⁻⁶M) was added to the basal medium; bud meristems were formed from small groups of cells surrounding basal cells of glandular hairs. Hair formation was inhibited by either high (32–27°C) or low (12°C) temperatures applied continuously. 32–27°C seemed to inhibit elongation of the hairs specifically, whereas 12°C inhibited earlier phases in hair formation. This hypothesis was supported by short temperature treatments applied at different times during hair formation.     

Adventitious bud regeneration from leaf explants of <Emphasis Type="Italic">Platanus occidentalis</Emphasis> L. and genetic stability assessment

The aim of this work is to develop a method of plant regeneration from leaf explants of Platanus occidentalis L. successfully. Woody plant medium (HortScience 16:453–459, 1981) and Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium were used as induced and rooted basal medium, respectively. The effects of combinations of 6-BA, IBA, NAA and KT with different concentrations on adventitious bud regeneration from P. occidentalis leaf explants were compared. The results showed that the highest shoot regeneration frequency (90%) and maximum number (13.72 ± 0.44) of shoots per explant was recorded on WPM medium supplemented with 22.20 mmol l⁻¹ 6-BA and 0.49 mmol l⁻¹ IBA. A 40-day-old explants were much more productive for shoot formation than others in this study. The regenerated shoots were cultured on MS medium supplemented with 1.33 mmol l⁻¹ 6-BA, 0.16 mmol l⁻¹ NAA and 2% (w/v) adenine, after 2-week shoots were transferred to 1/2 MS medium supplemented with 0.49 mmol l⁻¹ IBA for rooting. Hardened plantlets via acclimatization were transferred to pots and transplanted to the soil finally. To ascertain whether tissue culture had effects on the genetic stability of plantlets regenerated, the genetic diversity was assessed using RAPD marker. A total of 96 bands ranging from 0.5 to 2.2 kb with an average of 6.4 bands per primer, were obtained using 15 primers. Amplified products exhibited few of polymorphic patterns across all the plants of P. occidentalis and the overall frequency of detection of somaclonal polymorphisms was lower than 0.0104%. Yuehua Sun, Yanling Zhao, and Xiaojuan Wang contributed equally to this work.     

Direct somatic embryogenesis and synthetic seed production from<Emphasis Type="Italic"> Paulownia elongata</Emphasis>

We have developed a reproducible system for efficient direct somatic embryogenesis from leaf and internodal explants of Paulownia elongata. The somatic embryos obtained were subsequently encapsulated as single embryos to produce synthetic seeds. Several plant growth regulators [6-benzylaminopurine, indole-3-acetic acid, -naphthaleneacetic acid, kinetin and thidiazuron (TDZ)] alone or in combination were tested for their capacity to induce somatic embryogenesis. The highest induction frequencies of somatic embryos were obtained on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% Phytagel, 500 mg l^-1 casein hydrolysate and 10 mg l^-1 TDZ (medium MS10). Somatic embryos were induced from leaf (69.8%) and internode (58.5%) explants on MS10 medium after 7 days. Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal media. The maturation frequency of somatic embryos from leaf and internodal explants was 50.8% and 45.8%, respectively. Subculturing of mature embryos led to their germination on the same medium with a germination frequency of 50.1% and 29.8% from leaf and internode explants, respectively. Somatic embryos obtained directly on leaf explants were used for encapsulation in liquid MS medium containing different concentrations of sodium alginate with a 30-min exposure to 50 mM CaCl₂. A 3% sodium alginate concentration provided a uniform encapsulation of the embryos with survival and germination frequencies of 73.7% and 53.3%, respectively. Storage at 4°C for 30 days or 60 days significantly reduced the survival and complete germination frequencies of both encapsulated and non-encapsulated embryos relative to those of non-stored somatic embryos. However, the survival and germination rates of encapsulated embryos increased following storage at 4°C. After 30 days or 60 days of storage, the survival rates of encapsulated embryos were 67.8% and 53.5% and the germination frequencies were 43.2% and 32.4%, respectively. These systems could be useful for the rapid clonal propagation and dissemination of synthetic seed material of Paulownia elongata.Abbreviations BAP 6-Benzylaminopurine - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid - TDZ ThidiazuronCommunicated by H. Lörz     

Efficient<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>-mediated transformation of soybeans using an embryonic tip regeneration system

Here, we report the establishment of an efficient, in vitro, shoot organogenesis, regeneration system for soybeans [Glycine max (L.) Merr.]. Mature soybean seeds were soaked for 24 h, the embryonic tips were collected and cultured on MSB5 medium supplemented with 3.5 mg l^–1 N⁶-benzylaminopurine (BAP) for 24 h, and explants were transferred to MSB5 medium supplemented with 0.2 mg l^–1 BAP and 0.2 mg l^–1 indolebutyric acid. Use of embryonic tips yielded a higher regeneration frequency (87.7%) than regeneration systems using cotyledonary nodes (40.3%) and hypocotyl segments (56.4%) as starting materials. Regenerated embryonic tips were inoculated with Agrobacterium tumefaciens strain EHA105, which contains the binary vector pCAMBIA2301, and cultured for 20 h. Our results showed that the T-DNA transfer efficiency reached up to 78.2% and the transformation efficiency reached up to 15.8%. These data indicate that the embryonic tip regeneration system can be used for efficient, effective Agrobacterium-mediated transformation.Abbreviations GUS -Glucuronidase - T-DNA Transferred DNA - BAP N⁶-Benzylaminopurine - IBA Indolebutyric acid     

Overexpression of farnesyl pyrophosphate synthase (<Emphasis Type="Italic">FPS</Emphasis>) gene affected artemisinin content and growth of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

Transgenic plants of Artemisia annua L., a medicinal plant that produces the compound artemisinin which has an anti-malarial activity, were developed following Agrobacterium tumefaciens-mediated transformation of leaf explants. A. tumefaciens strain EHA105 carrying either pCAMBIA1301 or pCAMBIAFPS was used. Both plasmids harbored the hygromycin phosphotransferase II (hptII) gene as a selectable gene, but the latter plasmid also harbored the gene encoding for farnesyl pyrophosphate synthase (FPS), a key enzyme for artemisinin biosynthesis. Shoot regeneration was observed either directly from leaf sections or via intervening callus when explants were incubated on solidified Murashige and Skoog (MS) (1962) medium containing 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 1 mg l⁻¹ N⁶-benzyladenine (BA), 30 mg l⁻¹ meropenem and 10 mg l⁻¹ hygromycin. Applying vacuum infiltration dramatically increased transformation efficiency up to 7.3 and 19.7% when plasmids with and without FPS gene were used, respectively. All putative transgenic regenerants showed positive bands of hptII gene following Southern blot analysis. Expression of FPS was observed in all transgenic lines, and FPS over-expressed lines exhibited higher artemisinin content and yield, of 2.5- and 3.6-fold, respectively, than that detected in wild-type plants. A relatively high correlation (R ² = 0.78) was observed between level of expression of FPS and artemisinin content. However, gene silencing was detected in some transgenic lines, especially for those lines containing two copies of the FPS transgene, and with some lines exhibiting reduced growth.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Eucalyptus phylacis</Emphasis> L. Johnson and K. Hill., A critically endangered relict from Western Australia

Summary In vitro methods were applied to the only remaining plant of the Meelup Mallee (Eucalyptus phylacis), a critically endangered species from the southwest of Western Australia. Shoot explants were initiated into culture using a 1/2 MS [Murashige and Skoog basal medium (BM) for all experiments] liquid medium supplemented with 1% (w/v) activated charcoal, which was replenished twice daily, followed by transfer of explants to agar medium supplemented with 0.5 μM zeatin. Explants were cultured under low intensity lighting (PPFD of 5–10 μmol m⁻²s⁻¹) to minimize blackening of tissues, and some explants were induced to produce nodular green calluses in response to BM supplemented with 5 μM thidiazuron. Nodular green calluses were induced to form adventitious shoots following transfer to medium supplemented with 0.5 μM zeatin and 1 μM gibberellic acid, A₄ isomer (GA₄). Development of shoots was completed on 1 μM zeatin + 0.1 μM 6-benzylaminopurine (BA) in vented culture tubes. Regenerated shoots were sequentially cultured on medium containing 0.5 μM zeatin + 0.2 μM indoleacetic acid (IAA) followed by either 0.5 μM zeatin + 1μM GA₄ for shoot elongation or 1 μM zeatin + 0.5 μM IAA to optimize shoot growth. Rooted microshoots were produced after 4 weeks on 5 μM indolebutyric acid (IBA) and survived acclimatization and transfer to potting mixture.