Efficient production of <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-transformed roots and composite plants in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Recalcitrance of most large-seeded legumes, such as peanut, to regeneration and genetic transformation has hampered studies on gene function and efforts for genetic improvement. Agrobacterium rhizogenes-mediated transformation provides a system for rapid and efficient transformation of plant tissues. In this study, embryonic axes along with cotyledons of peanut were injected with a suspension culture of A. rhizogenes using microliter syringes. The influence of several factors such as plant genotype, A. rhizogenes culture stage, co-culture period of A. rhizogenes, and acetosyringone concentration in the co-cultivation medium have been evaluated. It is found that A. rhizogenes-mediated transformation of peanut is genotype-independent. Up to 61% transformation was recorded when embryonic axes were co-cultivated with 5 × 10⁷ A. rhizogenes cells from logarithmic phase for 2 days on co-culture medium containing 50 μmol l⁻¹ acetosyringone. Composite plants with transgenic roots were harvested after 45 days of treatment. Furthermore, this method was applied to assess the insecticidal activity of a synthetic cry8Ea1 gene against Holotrichia parallela in transgenic roots of peanut.     

Demonstration of IS<Emphasis Type="Italic">711</Emphasis> transposition in <Emphasis Type="Italic">Brucella ovis</Emphasis> and <Emphasis Type="Italic">Brucella pinnipedialis</Emphasis>

Background  

The Brucella genome contains an insertion sequence (IS) element called IS711 or IS6501, which is specific to the genus. The copy number of IS711 varies in the genome of the different Brucella species, ranging from 7 in B. abortus, B. melitensis and B. suis to more than 30 in B. ovis and in Brucella strains isolated from marine mammals. At present, there is no experimental evidence of transposition of IS711, but the occurrence of this element with a high copy number in some species, and the isolation of Brucella strains with "ectopic" copies of IS711 suggested that this IS could still transpose.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.     

Plant growth-promoting activity in newly isolated <Emphasis Type="Italic">Bacillus thioparus</Emphasis> (NII-0902) from Western <Emphasis Type="Italic">ghat</Emphasis> forest,India

A Gram-positive rod-shaped bacterium isolated on nutrient agar plates incubated at 28 ± 2°C. The identity of the bacterium was confirmed by sequencing of the 16S rRNA gene and it reveals that it shares highest similarity with Bacillus thioparus CECT 7196^T (99.08%). It was capable of growing at temperatures ranging from 4 to 40°C, but optimum growth was observed at 28 ± 2°C. Strain NII-0902 is endowed with multiple plant growth promotion attributes such as phosphate solubilization, Indole acetic acid (IAA), siderophore and HCN production, which were expressed differentially at sub-optimal temperatures (5–40°C). It was able to solubilize phosphate (17.7 μg ml⁻¹), and produce IAA (139.7 μg ml⁻¹) at 28 ± 2°C. Qualitative detection of siderophore production and HCN were also observed. At 5°C it was found to express all the plant growth promotion attributes except HCN production. The ability to colonize roots is a sine qua non condition for a rhizobacteria to be considered a true plant growth-promoting rhizobacteria (PGPR). Bacillus sp. NII-0902 has a potential ability to colonize roots visualized by transparency, bacterial growth (turbid, milky and narrow zone) along and around roots and truly supported by scanning electron micrograph. Hence, it is proposed that, Bacillus thioparus sp. NII-0902 could be deployed as an inoculant to attain the desired results of bacterization.     

Potential plant growth-promoting activity of <Emphasis Type="Italic">Serratia nematodiphila</Emphasis> NII-0928 on black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.)

A potential bacterial strain designated as NII-0928 isolated from Western ghat forest soil with multiple plant growth promoting attributes, and it has been identified and characterized. Plant growth promoting traits were analyzed by determining the P-solubilization efficiency, Indole acetic acid production, HCN, siderophore production and growth in nitrogen free medium. It was able to solubilize phosphate (76.6 μg ml⁻¹), and produce indole acetic acid (58.9 μg ml⁻¹) at 28 ± 2°C. Qualitative detection of siderophore production and HCN were also observed. At 5°C it was found to express all the plant growth promotion attributes except HCN production. The ability to colonize roots is a sine qua non condition for a rhizobacteria to be considered a true plant growth-promoting rhizobacteria (PGPR). 16S rRNA gene sequencing reveals the identity of the isolate as Serratia nematodiphila with which it shares highest sequence similarity (99.4%). Seed bacterization with black pepper cuttings in greenhouse trials using Sand: Soil: FYM with three individual experimental sets with their respective control showed clearly the growth promoting activity. Hence, Serratia nematodiphila NII-0928 is a promising plant growth promoting isolate showing multiple PGPR attributes that can significantly influence black pepper cuttings. The result of this study provides a strong basis for further development of this strain as a bioinoculants to attain the desired plant growth promoting activity in black pepper growing fields.     

Large-scale somatic embryogenesis and regeneration of <Emphasis Type="Italic">Panax notoginseng</Emphasis>

An efficient system for inducing somatic embryogenesis in Panax notoginseng was established using shaker flasks and bioreactor cultures; furthermore, regenerated plantlets were successfully transferred to ex vitro soil conditions. Embryogenic callus was induced from segments of adventitious roots incubated on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 5 weeks of culturing. The highest frequency (100%) of somatic embryogenesis, with a mean of 32.7 somatic embryos per callus, was obtained on embryogenic callus incubated on a medium containing 0.5 mg/L 2,4-D. To scale-up somatic embryo formation, 10 g (~1.65 × 10⁴) of early globular-stage somatic embryos were incubated in a 3 L airlift bioreactor containing 1.5 L 1/2 MS medium without plant growth regulators (PGRs) for a period of 4 weeks; these globular-stage somatic embryos then developed into cotyledonary embryos. When maintained on PGR-free medium, the cotyledonary embryos developed roots but did not develop shoots. However, when they were treated with gibberellic acid (GA₃), they continued to germinate and transformed into plantlets after 2 weeks of culture. Plantlets with well-developed shoots and roots were transferred to an autoclaved vermiculite and perlite mixture, acclimatized for a period of 3 months and successfully transferred to forest mountain soil. Following overwintering, these plants produced new growth.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

High efficiency <Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from cotyledon explants of <Emphasis Type="Italic">Incarvillea sinensis</Emphasis>

Summary A simple and effective procedure has been developed for plantlet regeneration from cotyledon-derived callus of the medicinally important herb and ornamental species, Incarvillea sinensis. An average of 18.4 adventitious shoots per explant were obtained from 100% cotyledon explants cultured on half-strength Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ 6-benzylaminopurine for 3 wk, followed by another 4 wk on hormone-free 1/2×MS medium. The cotyledon explants continued to expand and regenerate new shoots upon repeated subculturing onto fresh medium. Most regenerated shoots (66.9%) were rooted on 1/4×MS mediumcontaining 1.0 mg l⁻¹ indole-3-acetic acid, with an average of about 3.8 roots per shoot. Regenerated plants with well developed shoots and roots were successfully acclimatized in soil and were normal phenotypically.     

Associations between common variants in <Emphasis Type="Italic">GC</Emphasis> and <Emphasis Type="Italic">DHCR7</Emphasis>/<Emphasis Type="Italic">NADSYN1</Emphasis> and vitamin D concentration in Chinese Hans

Recent studies have identified common variants in or near GC, CYP2R1 and NADSYN1/DHCR7 to be associated with 25-hydroxyvitamin D [25(OH)D] levels in European populations. We aimed to examine whether these variants also influence 25(OH)D levels in Chinese. Seven common variants were successfully genotyped and tested for associations with plasma 25(OH)D levels in a population-based cohort of 3,210 Chinese Hans from Beijing and Shanghai. Six common variants at GC (rs4588, rs7041, rs2282679 and rs1155563) and NADSYN1/DHCR7 (rs3829251 and rs1790349) loci were all significantly associated with lower plasma 25(OH)D levels (−0.036 ≤ β ≤ −0.076 per risk-allele, P ≤ 5.7 × 10⁻⁵), while CYP2R1-rs2060793 showed a trend toward association with 25(OH)D levels in the Shanghai subpopulation (P = 0.08), but not in the Beijing subpopulation (P = 0.82). Haplotype-based association analyses of the four GC variants showed that only the haplotype that contained all risk-alleles (TACC) was significantly associated with lower plasma 25(OH)D levels (β = −0.085, P = 2.3 × 10⁻⁹), while the haplotype containing the risk-alleles of rs4588 and rs2282679 (TATC) was marginally associated with lower 25(OH)D levels (β = −0.054, P = 0.0562) when compared with GCTA haplotype carrying the four protective alleles. Most notably, conditional analyses showed that only GC-rs4588 and GC-rs2282679 (r ² = 0.97) remained significantly associated with 25(OH)D concentrations (P ≤ 1.9 × 10⁻⁵) after adjusting for the other two SNPs in GC. In conclusion, GC and NADSYN1/DHCR7 loci individually and collectively contribute to variation in plasma vitamin D levels in Chinese Hans.     

Root-promoting rhizobacteria in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> cuttings

Cloned Eucalyptus spp. plantations are based in greenhouse production of plants generated by vegetative propagation. Diverse studies have demonstrated that rhizospheric bacteria can stimulate plant growth, and more recently that they can increase rooting in vegetative material. Considering this potential, the objective of this study was to verify the effect of bacterial strains on rooting Eucalyptus globulus. A total of 132 bacterial strains isolated from the rhizosphere of E. globulus and Eucalyptus nitens were studied. The bacterial inoculums in a concentration of 4 × 10⁸ cfu/ml were applied to the rooting substrate at the cutting installation and 45 days after by irrigation. Rooting was evaluated on days 60 and 75 after cutting installation, considering the number of roots as well as their fibrosity and roots biomass. Of the 132 strains evaluated, 26 significantly increased cutting rooting in a range of 191.4–69.4% with respect to the control. Additionally, some strains stimulated the development of fine roots and incremented the roots biomass. The strains identificated that produced a rooting effect were: Bacillus firmus, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, B. subtilis/amyloliquefaciens, Bacillus circulans, Brevibacillus brevis, Paenibacillus lautus and Stenotrophomona maltophilia. These first trials suggest the potential of these bacteria to be used in clonal production programs for E. globulus.     

Combined field efficacy of <Emphasis Type="Italic">Paranosema locustae</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var. <Emphasis Type="Italic">acridum</Emphasis> for the control of sahelian grasshoppers

Field trials were conducted to evaluate the efficacy of wheat bran bait formulations of Paranosema locustae and Metarhizium anisopliae for controlling grasshoppers in southeast Niger. Treatments consisted of wheat bran baits mixed with M. anisopliae, P. locustae + M. anisopliae or with P. locustae spores and P. locustae + sugar. Oedaleus senegalensis, Pyrgomorpha cognata and Acrotylus blondeli were the predominant species at the time of application representing ca. 94% of the total population. Bran application was done when O. senegalensis (ca. 75% of the population) was at its early developmental stages, with first, second and third instars accounting for 64–85%. Grasshopper population reduction, P. locustae prevalence and level of infections in the predominant species were monitored. Manual application of P. locustae and M. anisopliae formulated in wheat bran has proven to induce consistent pathogen infection in grasshopper populations. Population density over the three weeks monitoring, typically decreased by 44.7 ± 6.9%, 52.8 ± 8.4%, 73.7 ± 5.5% and 89.1 ± 1.8% in P. locustae, P. locustae + sugar, M. anisopliae and P. locustae + M. anisopliae treated plots respectively. Paranosema locustae prevalence in surviving adult grasshoppers at 28 after application was 48.1 ± 2.3%, 28.9 ± 4.8% and 27.4 ± 3.7%, with infection level of 6.2 ± 0.8 × 10⁶, 2.3 ± 0.3 × 10⁴ and 2.1 ± 0.3 × 10³ spores mg⁻¹ host weight in O. senegalensis, A blondeli and P. cognate respectively. Other species that each accounted for <2% of the community, namely Aiolopus thalassinus, A. simulatrix, Acorypha glaucopsis, Acrotylus patruelis, Anacridium melanorhodon, Diabolocatantops axillaris, Kraussaria angulifera and Schistocerca gregaria were found to show sign of infection. The results from this study suggest that wheat bran application of M. anisopliae and P. locustae alone or in combination, targeting early instars grasshopper could be a valuable option in grasshopper control programs.     

Efficient separation and purification of allophycocyanin from <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

Biodegradation of malathion by <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain KB2 and <Emphasis Type="Italic">Bacillus cereus</Emphasis> strain PU

We report here the degradation of a pesticide, malathion, by Brevibacillus sp. strain KB2 and Bacillus cereus strain PU, isolated from soil samples collected from malathion contaminated field and an army firing range respectively. Both the strains were cultured in the presence of malathion under aerobic and energy-limiting conditions. Both strains grew well in the medium having malathion concentration up to 0.15%. Reverse phase HPLC–UV analysis indicated that Strain KB2 was able to degrade 72.20% of malaoxon (an analogue of malathion) and 36.22% of malathion, while strain PU degraded 87.40% of malaoxon and 49.31% of malathion, after 7 days of incubation. The metabolites mal-monocarboxylic acid and mal-dicarboxylic acid were identified by Gas chromatography/mass spectrometry. The factors affecting biodegradation efficiency were investigated and effect of malathion concentration on degradation rate was also determined. The strain was analyzed for carboxylesterase activity and maximum activity 210 ± 2.5 U ml⁻¹ and 270 U ± 2.7 ml⁻¹ was observed for strains KB2 and PU, respectively. Cloning and sequencing of putative malathion degrading carboxylesterase gene was done using primers based PCR approach.     

Benzyladenine Promotes Flowering in <Emphasis Type="Italic">Doritaenopsis</Emphasis> and <Emphasis Type="Italic">Phalaenopsis</Emphasis> Orchids

Two experiments were performed to determine how application of the cytokinin benzyladenine (BA) influenced flowering in Doritaenopsis and Phalaenopsis orchid clones. In the first experiment, two vegetative orchid clones growing in 15-cm pots were transferred from a 28°C greenhouse that inhibited flowering to a 23°C greenhouse for flower induction (day 0). A foliar spray (0.2 L m⁻²) containing BA at 100, 200, or 400 mg L⁻¹ or 25, 50, or 100 mg L⁻¹ each of BA and gibberellins A₄ + A₇ (BA+GA) was applied on days 0, 7, and 14. Plants treated with BA alone at 200 or 400 mg L⁻¹ had a visible inflorescence 3–9 days earlier and had a mean of 0.7–3.5 more inflorescences and 3–8 more flowers per plant than nontreated plants. The application of BA+GA had no effect on inflorescence number and total flower number at the rates tested. In the second experiment, three orchid clones received a single foliar spray of BA at 200 mg L⁻¹ at six time points relative to time of transfer from 29°C to 23°C (−1, 0, +1, +2, +4, or +6 weeks). A separate group of plants received a BA application at week 0 but was maintained at 29°C. Inflorescence number was greatest in all three orchid clones when plants were treated with BA 1 week after the temperature transfer. Plants that were sprayed with BA and maintained at 29°C did not initiate inflorescences. The promotion of flowering by the application of BA suggests that cytokinins at least partially regulate inflorescence initiation of Doritaenopsis and Phalaenopsis, but its promotion is conditional and BA application cannot completely substitute for an inductive low temperature.     

Phenotype changes inherited by crossing pyrethroid susceptible and resistant genotypes from the cattle tick <Emphasis Type="Italic">Riphicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis>

Dialelic crosses and backcrosses of pyrethroid resistant (RR) and susceptible (SS) Rhipicephalus (Boophilus) microplus tick strains were carried out and the substitution (Phe-Ile) within the sodium channel gene was monitored in order to analyze the effects of the genotype on the pyrethroid resistance phenotype as measured by the larval packet test (LPT). Parental strains: susceptible (SS) and resistant (RR); dialelic crosses: RS (♂RR × ♀SS), and SR (♂SS × ♀RR); and backcrosses: RS × SS, RS × RR, SR × SS and SR × RR were infested on 280 kg calves. Resistance type (monogenic or polygenic) and effective dominance were determined based on the discriminant concentration (DC) for cipermethrine (0.5%), deltamethrine (0.09%) and flumethrine (0.01%). Allele specific PCR (AS-PCR) was used for genotyping, looking at a sodium channel mutation (Phe-Ile substitution). The mortality rates and allele frequency of susceptible and pyrethroid resistant reference strains were 0% mortality and 90% RR alleles for resistant strain, and 100% mortality and 0% RR alleles as measured by the larval packet test (LPT) and allele specific PCR (AS-PCR) respectively. Backcrossed strain SR × RR showed an effective dominance (D_ML) of 0.605 for cypermethrin, 0.639 for deltamethrin and 0.498 for flumethrin, while survival of backcrosses RS × SS, RS × RR and SR × SS showed a significant tendency to recesivity. Backcrossed strain SR × RR (69.4%) also showed a higher RR genotype frequency with regards to RS × SS (25.5%), RS × RR (36.7%) and SR × SS (32.0%), however, susceptible allele was inherited in general as an incomplete dominant trait. Monogenic inheritance hypothesis was tested and the results showed monogenic inheritance for cypermethrin and flumethrin (P < 0.05) but not for deltamethrin (P > 0.05). However, significant correlation was found between RR genotype and the survival rate for all three pyrethroids used (P < 0.05), suggesting that a single substitution on the sodium channel gene can be responsible for resistance to pyrethroids as a class, due to the high frequency for RR genotypes. Combination with different mutations or metabolic resistance mechanisms cannot be excluded.     

Horizontal Transfer of the Tetracycline Resistance Gene <Emphasis Type="Italic">tetM</Emphasis> Mediated by pCF10 Among <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> in the House Fly (<Emphasis Type="Italic">Musca domestica</Emphasis> L.) Alimentary Canal

The house fly (Musca domestica L.) alimentary canal was evaluated for the potential of horizontal transfer of tetM on plasmid pCF10 among Enterococcus faecalis. Two sets of experiments were conducted: (1) house flies without surface sterilization and (2) surface-sterilized flies. Both sets of flies were exposed to E. faecalis OG1RF:pCF10 as donor for 12 h and then E. faecalis OG1SSp as recipient for 1 h. Another group of flies received the recipient first for 12 h followed by exposure to the donor strain for 1 h. House flies were screened daily to determine the donor, recipient, and transconjugant bacterial load for up to 5 days. In addition, the sponge-like mouth parts used for food uptake (labellum) of surface-sterilized house flies were removed and analyzed for donors, recipients, and transconjugants, separately. In both groups of flies (n = 90 flies/group), transfer occurred within 24 h after exposure with a transconjugant/donor rate from 8.6 × 10⁻⁵ to 4.5 × 10¹. Transconjugants were also isolated from the house fly labellum. Our data suggest that the house fly digestive tract provides a suitable environment for horizontal transfer of conjugative plasmids and antibiotic resistance genes among enterococci. Our results emphasize the importance of this insect as a potential vector of antibiotic-resistant bacterial strains.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of embryogenic tissues of hybrid firs (<Emphasis Type="Italic">Abies</Emphasis> spp.) and regeneration of transgenic emblings

A genetic transformation system has been developed for selected embryogenic cell lines of hybrids Abies alba × A. cephalonica (cell lines AC2, AC78) and Abies alba × A. numidica (cell line AN72) using Agrobacterium tumefaciens. The cell lines were derived from immature or mature zygotic embryos on DCR medium containing BA (1 mg l⁻¹). The T-DNA of plant transformation vector contained the β-glucuronidase reporter gene under the control of double dCaMV 35S promoter and the neomycin phosphotransferase selection marker gene driven by the nos promoter. The regeneration of putative transformed tissues started approximately 1 week after transfer to the selection medium containing 10 mg geneticin l⁻¹. GUS activity was detected in most of the geneticin-resistant sub-lines AN72, AC2 and AC78, and the transgenic nature of embryogenic cell lines was confirmed by PCR approach. Plantlet regeneration from PCR-positive embryogenic tissues has been obtained as well. The presence of both gus and nptII genes was confirmed in 11 out of 36 analysed emblings.     

<Emphasis Type="Italic">In vitro</Emphasis> rooting of two <Emphasis Type="Italic">Eucalyptus urophylla X Eucalyptus grandis</Emphasis> mature clones

Abstract   The rooting capacity of microshoots derived from two mature Eucalyptus urophylla X Eucalyptus grandis half-sib clones kept for 3 y under intensive micropropagation was assessed in different in vitro conditions. A first set of experiments established that clone 147 microshoots rooted earlier and in greater proportions, while producing more adventitious roots overall than their homologs from clone 149. Modifying the composition of the basal 1/2-MS-derived rooting medium by 1/4-MS or Knop macronutrients, or reducing sucrose concentration to 10 g l⁻¹ did not enhance the rooting rates. However, together with the growth regulators added, they had a significant effect on the number of adventitious roots formed. With rooting rates reaching 81%, the higher rootability of clone 147 over clone 149 was further confirmed by the second set of experiments with significant effects of the various auxins tested and strong clone × auxin interactions on the proportions of rooted microshoots and on the number of adventitious roots. The best rooting scores were given by 5 μM indole-3-butyric acid (IBA) and 12.5 μM 1-naphthaleneacetic acid (NAA), whereas the microshoots exposed to 5 or 12.5 μM indole-3-acetic acid (IAA) were less responsive. Lower light intensities did not improve significantly root capacities, although differences might exist according to the genotype. Overall, root and shoot elongation was stimulated by light. At the end of the experiment, the rooted microshoots were markedly taller than the non-rooted ones, with significant influences of auxins and light intensity, and to a lesser extent, of the genotypes.     

Virulence of a South African isolate of the <Emphasis Type="Italic">Cryptophlebia leucotreta</Emphasis> granulovirus to <Emphasis Type="Italic">Thaumatotibia leucotreta</Emphasis> neonate larvae

Surface inoculation dose–response and time–response bioassays and detached fruit bioassays were conducted with a novel South African isolate of the Cryptophlebia leucotreta granulovirus (CrleGV-SA) against Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Noctuidae) neonate larvae. LC₅₀ and LC₉₀ values were estimated to be 4.095 × 10³ and 1.185 × 10⁵ OBs ml⁻¹, respectively. LT₅₀ and LT₉₀ values were estimated to be 4 days 22 h and 7 days 8 h, respectively, categorising the virus as a fast or type 2 granulovirus. There was a conspicuous difference in behaviour between larvae on inoculated diet and untreated diet, resulting in a significant reduction in penetration of diet. Bioassays on detached Navel oranges revealed LC₅₀ and LC₉₀ values of 9.310 × 10⁷ and 1.515 × 10⁹ OBs ml⁻¹, when using data on numbers of larvae per fruit rather than on numbers of infested fruit. Field trials will be conducted.