Seed inoculation with Azospirillum mitigates NaCl effects on lettuce

Data on the growth-promoting effects of Azospirillum on lettuce exposed to either normal or saline conditions, is scarce. Lactuca sativa L., cv Mantecosa seeds were colonized with A. brasilense Sp245 cells during imbibition. Germination percentages were determined after 7 d treatments with 0, 30, 50 or 80 mol m⁻³ NaCl. In another experiment, seeds germinated in Hoagland were irrigated for 30 d with 0, 30, 50 or 80 mol m⁻³ NaCl supplemented media. Vegetative growth proceeded in a growth chamber with a 13–11 h day–night cycle. Buffer-imbibed seeds were considered non-inoculated controls. Plant samples were taken at 0, 14, 20, and 30 d after the onset of NaCl treatments and dissected in aerial and root portions. The weights of both tissues were measured. Azospirillum-inoculated seeds had significantly higher germination percentages than controls in all treatments. Inoculated dried seeds stored up to 30 d maintained such characteristic in most of the treatments, particularly at 80 mol m⁻³ NaCl. Plants grown from inoculated seeds and irrigated with saline media displayed higher total fresh and dry weights and biomass partition to the aerial portion, than non-inoculated controls. Azospirillum-inoculated lettuce seeds had better germination and vegetative growth than non-inoculated controls after being exposed to NaCl.     

Genetic transformation of Prunus domestica L. using the hpt gene coding for hygromycin resistance as the selectable marker

The study and development of transformation technology with new selection schemes is important for various fundamental studies and for crop trait improvement via genetic engineering. Here we have shown that hygromycin resistance is an effective system for plum genetic transformation. Embryonic axes of mature seeds were co-cultivated with Agrobacterium strain LBA4404 containing the pC1381 plasmid carrying the hygromycin phosphotranferase gene (hpt) and β-glucuronidase (GUS) gene or with strain EHA105 containing the plasmid pC1301 carrying the same marker and reporter genes. The latter strain containing a pC2301 plasmid carrying the neomycin phosphotransferase gene (nptII) gene was used as a control. Infected explants were placed on shoot induction medium containing either 5 mg L⁻¹ hygromycin or 75 mg L⁻¹ kanamycin for selection. Green shoots developed from the explants under hygromycin pressure. These shoots showed continued and vigorous growth and development upon transfer onto fresh hygromycin medium. PCR using hpt sequence primers, and Southern blot analysis using a probe from the hpt gene, confirmed the presence of the transgenes and their stable integration in regenerated plants. Full transgenic plants were obtained in a greenhouse. Hygromycin selection was very effective and no escapes were observed. The study demonstrated that hygromycin resistance can be used as an effective selectable marker for plum transformation. The new system developed here is important and useful for multiple gene transformation in plum.     

A strain of Bacillus subtilis stimulates sunflower growth (Helianthus annuus L.) temporarily

Preliminary studies showed that a Bacillus subtilis strain stimulates plant growth. We investigated how inoculating seeds of a sunflower cultivar (Helianthus annuus L.) with this strain stimulated plant growth, soil properties and emissions of greenhouse gasses, i.e. carbon dioxide (CO₂) and nitrous oxide (N₂O), when cultivated in a greenhouse. Unfertilized sunflowers or fertilized with urea served as controls. After one month, root length and fresh and dry root weight of the sunflower was significantly higher in the bacteria amended plant than in the urea and unfertilized plants. However, at harvest, no positive effect was observed. The number of seeds per plant and seed weight was not significantly different between the treatments, but total plant N was significantly higher in urea-amended plants than in unfertilized plants. The CO₂ production rate was not affected by treatment, but the N₂O emission rate was significantly higher in soil amended with urea plus bacteria soil compared to the unfertilized treatments. It was found that the B. subtilis strain used in this study had a positive, but only temporarily effect on growth of the sunflower cultivar used.     

The effect of sucrose on the expression of the VvTFL1 and VFL genes during flower development in the “Xiangfei” grapevine

VFL and VvTFL1 genes expression patterns and the effects of sucrose on the expression of VFL and VvTFL1 genes in different organs of the “Xiangfei” grapevine (Vitis vinifera L.) were investigated. VvTFL1 gene expression was detected in the meristem of the apical bud and lateral bud, but was not detected during inflorescence differentiation and flower organ development. After sucrose treatment, VvTFL1 gene expression increased in the apical bud, but decreased in the lateral bud. These results suggested that the VvTFL1 gene might be mainly involved in the apical growth process of shoots, and exogenous sucrose had an effect on the VvTFL1 gene by increasing shoot apical meristem initiation of apical buds. The VFL gene was expressed primarily during inflorescence differentiation and early flower organ development, but it gradually reduced in later flower development. After sucrose treatment, VFL gene expression increased in the inflorescence and small or middle flower, but a little change was seen in the large flower. These results suggested that the VFL gene plays important roles in the initiation of inflorescence meristems and the morphological formation of flower organs. Exogenous sucrose had an effect on VFL gene expression at the early stage of flower development.     

Ethylene influences development and flowering of Ptilotus spp. in vitro and ex vitro

The genus Ptilotus has immense potential for ornamental horticulture but its commercial development has been hindered by propagation limitations. Poor seed quality and germination are reported. Cutting propagation is limited by cutting supply as the juvenile phase of Ptilotus is short. Micropropagation has been used in an attempt to overcome these difficulties but explants become floral in vitro and this causes plantlets to elongate. Ethephon has been used to control flowering of stock plants of many ornamental species. This study investigated the effect of ethephon applied to young (3-week-old, deflasked from tissue culture) and mature (1-year-old) Ptilotus plants in a greenhouse. A system of applying gaseous ethylene at 0, 100, 200 and 300 mg l⁻¹ to the headspace of in vitro plantlets in glass jars was developed and the response of in vitro plantlets to ethylene studied. One-year-old Ptilotus plants were treated with 500 mg l⁻¹ ethephon 2 days before pruning or 1 or 2 weeks after pruning. Ethephon application 2 days before pruning decreased the number of inflorescences and increased the number of shoots (compared to the control) but was phytotoxic. Ethephon applications of 150 or 300 mg l⁻¹ applied weekly or fortnightly to 3-week-old plants deflasked from tissue culture reduced plant height and number of inflorescences and at low concentrations increased the number of new shoots. A fortnightly application at 150 mg l⁻¹ is recommended. Previous reports on the effects of ethylene on inflorescence production on plantlets in vitro are limited. Our study showed that exposure of in vitro plantlets of P. nobilis to ethylene gas at 100 mg l⁻¹ for 1 h significantly increased the number of shoots and plant height but this did not occur for plantlets of P. spicatus. Plantlets of P. spicatus exposed to transient ethylene at 200 and 300 mg l⁻¹ showed significantly greater rooting (52.4%) than the control (13.6%).     

A SCAR marker linked to the N gene for resistance to root knot nematodes (Meloidogyne spp.) in pepper (Capsicum annuum L.)

The root-knot nematodes (Meloidogyne spp.) are important nematode pests and cause serious diseases in pepper in the world. No molecular markers linked to the nematodes resistance N gene have been reported. In this paper, ‘Carolina Wonder’ (Capsicum annuum L.), a sweet pepper line resistant to root-knot nematode with N gene, ‘20080-5-29’ (C. annuum L.), an inbred line susceptible to root-knot nematode with good horticultural characteristics, and their F₂ progeny with 320 individuals were used as materials. Evaluation of resistance and susceptibility of parental lines, F₁ and F₂ progeny inoculated with root-knot nematodes (Meloidogyne incognita) were carried out. ‘Bulked segregant analysis’ method was used to search for polymorphic markers from 512 pairs of AFLP primers. Based on the assessment of resistance and susceptibility and polymorphism of the AFLP marker in F₂ population, the genetic linkage distance between the AFLP marker and the N gene was estimated. One AFLP marker E39/M41-339 was obtained and transferred to a SCAR marker amplifying a 315 bp DNA fragment linked to the N resistant allele and a 331 bp fragment linked to the N⁺ susceptible allele. The distance between the molecular marker and the nematodes resistance N gene is 6.3 cM. This research delivered a valuable tool for the marker assisted selection of nematodes resistance in pepper.     

Chemical compositions and insecticidal effects of essential oils isolated from Achillea gypsicola, Satureja hortensis, Origanum acutidens and Hypericum scabrum against broadbean weevil (Bruchus dentipes)

The essential oils of aerial parts of Achillea gypsicola Hub.-Mor., Hypericum scabrum L., Satureja hortensis L., and Origanum acutidens (Hand.-Mazz.) Letswaart were analyzed in this study by GC and GC–MS and their oils were tested for toxicity against broadbean weevil (Bruchus dentipes). A. gypsicola oil contained camphor (40.17%), 1,8-cineole (22.01%), piperitone (11.29%), borneol (9.50%) and α-terpineol (1.56%) as major components. A total of 74 components were identified by GC–MS in H. scabrum oil, including α-pinene (9.26%), terpinen-4-ol (5.12%), camphor (5.94%), δ-cadinene (4.52%), pulegone (4.45%), γ-muurolene (4.12%), pinocarvone (3.97%) and β-caryophyllene (3.42%) as predominant components. The essential oils of O. acutidens and S. hortensis were characterized by high contents of carvacrol (86.99% and 55.74%), γ-terpinene (0.71% and 20.94%), p-cymene (1.95% and 12.30%), α-terpinene (0.13% and 2.04%) and β-caryophyllene (1.30% and 1.08%). All of the essential oils were toxic to adults of B. dentipes and insect mortality increased with increasing concentration of each oil. The oils (20 μl dose) brought about 100% mortality in 36 h. Although desirable insecticidal activities against the pest were achieved with the oils from all four plant species, S. hortensis and O. acutidens oils were more effective, particularly after 6 h of treatment. The current results concluded that the essential oils, in particular O. acutidens and S. hortensis oils, may be used as potential botanical insecticides against B. dentipes.     

Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker

Transgenic Citrus sinensis (L.) Osb. cv. Hamlin plants expressing the hrpN gene were obtained by Agrobacterium tumefaciens (Smith and Towns) Conn-mediated transformation. hrpN encodes a harpin protein, which elicits the hypersensitive response and systemic acquired resistance in plants. The gene construct consisted of gst1, a pathogen-inducible promoter, a signal peptide for protein secretion to the apoplast, the selection genes nptII or aacC1 and the Nos terminator. The function of gst1 in citrus was evaluated in transgenic C. sinensis cv. Valencia harboring the reporter gene uidA (gus) driven by this promoter. Histochemical analysis for gus revealed that gst1 is activated in citrus leaves by both wounding and inoculation with Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al. Genetic transformation was confirmed by Southern blot hybridization in eight cv. Hamlin acclimatized plants. RT-PCR confirmed hrpN gene expression in seven cv. Hamlin transgenic lines before pathogen inoculation. Some hrpN transgenic lines showed severe leaf curling and abnormal growth. Six hrpN transgenic lines were propagated and evaluated for susceptibility to X. axonopodis pv. citri. RT-PCR confirmed gene expression in all six hrpN transgenic lines after pathogen inoculation. Several of the hrpN transgenic lines showed reduction in susceptibility to citrus canker as compared with non-transgenic plants. One hrpN transgenic line exhibited normal vegetative development and displayed very high resistance to the pathogen, estimated as up to 79% reduction in disease severity. This is the first report of genetic transformation of citrus using a pathogen-inducible promoter and the hrpN gene. Further evaluations of the transgenic plants under field conditions are planned. Nevertheless, the evidence to date suggests that the hrpN gene reduces the susceptibility of citrus plants to the canker disease.     

Identification and distribution of S haplotypes in Brassica vegetables from China

The amplification efficiency and identification diversity with gene-specific primers derived from S locus glycoprotein gene (SLG) and S locus receptor kinase gene (SRK) were compared, and the geographical distribution for S haplotypes was investigated by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) in 72 genotypes of 5 Brassica vegetables from China. The amplification efficiency and identification diversity with class I SRK primers were generally higher than that with class I SLG in most crops tested. Class I alleles were found in total 66 genotypes and they were classified into 16, 10, 7 and 10 groups for Chinese cabbage, purple flowering stalk, cauliflower and cabbage respectively. The number of amplification accessions and identification diversity using the primers of class II SLG and SRK were quite similar. Class II alleles were detected in 55 genotypes and further grouped into one type in mustard and three in other crops. The nucleotide sequences showed high similarity between identical S haplotypes determined by reciprocal pollination and PCR-RFLP tests. It demonstrated that the PCR-RFLP analysis was feasible for identification of S alleles, and SRK should be considered as a better marker for the identification of S haplotypes than SLG. The types of S haplotypes are highly diverse in Brassica vegetables from China. Nevertheless, they were geographically limited in some Brassica vegetables, so the exchange of germplasm resources should be enhanced for breeding.     

Symbiotic seed germination of Grammatophyllum speciosum Blume and Dendrobium draconis Rchb. f., native orchids of Thailand

In vitro symbiotic seed germination is an important tool not only for the study of orchid-fungus specificity but also for the production of mycobiont-infected healthy seedlings that could be valuable for both horticultural and conservation purposes. The current study compared effectiveness of eight putative orchid mycorrhizal fungi obtained from mature orchids in the genera Paphiopedilum, Cymbidium and Dendrobium, in promoting in vitro seed germination and protocorm development of Grammatophyllum speciosum Blume and Dendrobium draconis Rchb. f., native Thai orchids. The developmental stages of seeds and protocorms cultured on Murashige and Skoog (MS) medium, oat meal agar (OMA), or OMA inoculated with one of the eight fungal isolates were evaluated weekly. Two isolates of Epulorhiza repens (Bernard) Moore (=anamorphic species of Tulasnella calospora (Boud.) Juel), Da-KP-0-1 and Pv-PC-1-1, were found to be the most effective fungi in promoting protocorm development of G. speciosum. At week 13, protocorms co-cultured with either one of these two fungal isolates, on the average, were significantly more advanced than those sown on OMA. Protocorms co-cultured with isolate Pv-PC-1-1 were also significantly more advanced than those cultured on MS medium. For D. draconis seed germination, three fungal isolates of different anamorphic species of Tulasnella, C1-DT-TC-1, Pv-PC-1-1, and C3-DT-TC-2, were found to be the most effective fungi in promoting protocorm development. However, none of these fungal isolates outperformed MS medium. Additionally, the compatibility between the fungal isolates tested and the two orchid species was discussed.     

Fruit yield and quality of strawberry plants transformed with a fruit specific strawberry pectate lyase gene

Two transgenic strawberry lines (Pel 1 and Pel 3) containing the open reading frame of a fruit specific strawberry pectate lyase gene (FaplC) under the control of the CaMV35S promoter have been obtained to evaluate the role of this gene on fruit softening. Ripen fruits from both lines showed a significant down-regulation of FaplC, being the percentage of silencing of 84 and 71% on Pel 1 and Pel 3, respectively. The agronomic behaviour of transgenic plants was evaluated during two consecutive years. Fruit set increased in the two transgenic lines when compared with control plants, although Pel 1 showed a significant reduction on fruit weight. Firmness of full ripen fruits from Pel lines was significantly higher than control fruits, while color and soluble solids were not affected. The increase of firmness in Pel lines was maintained when ripe fruits were stored for 3 days at 25 °C. Histological analysis of ripe fruits showed lower intercellular spaces and a higher degree of cell to cell contact area in transgenic fruits when compared with controls. Altogether, these results suggest a direct relationship between pectate lyase gene expression and strawberry fruit softening.     

Application of chlorophyll fluorescence for the diagnosis of salt stress in tomato “Solanum lycopersicum (variety Rio Grande)”

To study the response of tomato (Solanum lycopersicum cv. Rio Grande) to salinity, the effect on plant growth, water relations, stomatal conductance and Chlorophyll fluorescence was investigated. Tomato plants were grown in peat culture under controlled conditions and submitted during 28 days to saline stress ranging from 0 to 25, 50, 100, 150 and 200 mM of NaCl. At the end of the experiment period, plant growth was significantly decreased with increasing salinity.     

Production protocol development for greenhouse cut Linaria, Lupinus, and Papaver flowers

Linaria maroccana Hook. f. Ann., ‘Lace Violet’, Lupinus hartwegii ssp. cruikshankii Lindl. ‘Sunrise’ and Papaver nudicaule L. ‘Meadow Pastels’ seeds were directly sown into 105 cell plug trays and received either ambient light or supplemental high intensity discharge (HID) lighting. For each species, a 2 × 3 × 3 factorial was used with two light intensities during propagation, three transplant stages, and three night temperatures. Seedlings were transplanted at the appearance of 2–3, 5–6, or 8–9 true leaves. Transplanted Linaria and Papaver seedlings were placed at 5/11, 10/16, or 15/21 ± 1 °C night/day temperatures and Lupinus seedlings were placed at 15/24, 18/25, or 20/26 ± 2 °C night/day temperatures. For this study, the optimum production temperature for Linaria was 10/16 °C as the cut stems produced at 15/21 °C were unmarketable and production time was excessively long at 5/11 °C. At 10/16 °C, Linaria seedlings should be transplanted at the 2–3 leaf stage to maximize stem number, stem length and profitability. For Lupinus the optimum temperature was 15/24 °C due to long stems and high profitability per plant. Lupinus seedlings should be transplanted at the 2–3 leaf stage when grown at 15/24 °C to obtain the longest and thickest stems; however, $/m² week was higher for plants transplanted at the 8–9 leaf stage due to less time in finishing production space. For Papaver, the 15/21 °C temperature was optimal as that temperature produced the longest stems in the shortest duration, resulting in the highest $/m² week. At 15/21 °C Papaver plants should be transplanted at the 2–3 leaf stage. Supplemental HID lighting had no effect on any of the species.     

Alteration of tomato fruit quality by root inoculation with plant growth-promoting rhizobacteria (PGPR): Bacillus subtilis BEB-13bs

In this study the effect of inoculation of tomato (Lycopersicon esculentum Mill.) roots with plant growth-promoting rhizobacteria (PGPR) on yield and fruit quality was evaluated. The control treatment was non-inoculated (CTL) and the PGPR treatment was inoculated with Bacillus subtilis BEB-lSbs (BS13). Yield per plant and marketable yield, as well as fruit weight and length were increased by the BS13 treatment when compared to the CTL treatment. Texture of red fruits was also enhanced by the BS13 treatment compared to that in the CTL treatment. These results demonstrated that PGPR have positive effects on tomato fruit quality attributes, particularly on size and texture.     

Chitosan specificity for the in vitro seed germination of two Dendrobium orchids (Asparagales: Orchidaceae)

The effects of different types of chitosan on seed germination and protocorm development were determined for two orchid species, Dendrobium bigibbum var. compactum and Dendrobium formosum. Six chitosan types derived from polymer or oligomer chitosan each with 70, 80 or 90% levels of deacetylation (P70, P80, P90, O70, O80 and O90, respectively), were evaluated as direct medium supplements at 0, 10, 20, 40 or 80 mg/L in modified VW medium by following seed germination and protocorm growth for 12 weeks. Chitosan of all six tested types and four concentrations were found to significantly enhance the proportion of D. formosum seeds that germinated, when compared to these germinated without chitosan. In contrast, chitosan caused no enhanced germination rate was noted for D. bigibbum var. compactum with all tested chitosans and doses tested. However, almost all types of chitosan at 10 mg/L, except O90, were able to significantly improve the growth of D. bigibbum var. compactum protocorms, whilst 10 or 20 mg/L of P70 chitosan was the best formula to enhance the growth of D. formosum protocorms. It is concluded that chitosan responses in seed germination and protocorm development were somewhat species and developmental stage dependent. Therefore, the appropriate chitosan application for each plant species should be evaluated first before use.