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.     

Direct and indirect effects of seed related characters on number of seed in guava (Psidium guajava L.) fruits

Dessert quality of guava fruit is considerably reduced by high seed content. Number of seeds in guava is associated with different seed and fruit characters. Influence of different seed characters on number of seeds per fruit (NSPF) was studied by analysing character association and direct and indirect effects on NSPF and seed related traits for 68 genotypes of guava collected from diverse sources and conserved at National Active Germplasm Site, CISH, Lucknow. Seed related characters were studied at the colour break stage of the fruits using image analysis software for counting number of seeds. The data was subjected to path analysis to find out direct and indirect effects of different characters on number of seeds in the fruits. At genotypic and phenotypic levels, NSPF was significantly and positively associated with seed weight per fruit (SWPF), number of seeds 100 g⁻¹ pulp (NSPHP) and fruit weight (FW). The genotypic (0.0029) and phenotypic (0.0563) residual values were fairly very low, which revealed that variables included in this study had significant contribution in determining NSPF. The NSPHP exhibited high positive direct effect on NSPF. The maximum direct response (P = 0.737, G = 1.004) of this component was attributed to the indirect positive effects of the SWPF and fruit:seed weight proportion (FSWP). SWPF also had a very high positive direct effect (P = 0.521, G = 0.694) on the NSPF. Correlation and path coefficient analysis revealed that SWPF, NSPHP and 100-seed weight (100SW) were deciding factors for realizing improvement for NSPF. The importance of small seeded genotypes in selection of less seeded varieties was emphasized.     

Breaking the intergeneric hybridization barrier in Carica papaya and Vasconcellea cauliflora

The present investigation was undertaken to develop PRSV (Papaya ringspot virus) resistant hybrids through intergeneric hybridization. Intergeneric hybridization was done involving nine Carica papaya cultivars as female and Vasconcellea cauliflora as male. To break the intergeneric hybridization barrier, various nutrient combinations were used. Among the combinations used, sucrose 5%, sucrose 5% + boron 0.5% and sucrose 5% + CaCl₂ 0.5% improved the fruit set and seed set percentage. A total number of 1197 flowers were pollinated and 308 fruits were obtained. On extraction, 721 seeds were obtained from CO 7, Pusa Nanha and CP 50. Out of 721 F₀ seeds (crossed seeds) sown, 419 seeds germinated and artificial screening for PRSV was carried out 27 days after sap inoculation. Out of 29 F₁ hybrid plants from CO 7 x V. cauliflora cross, only six plants namely CO 7V1 to CO 7V6 were found free from PRSV symptoms. Similarly, out of 55 F₁ hybrids from cross involving Pusa Nanha x V. cauliflora only 23 plants namely PNV1 to PNV23 were found free from the symptoms and 70 plants namely CPV1 to CPV70 out of 335 plants of CP50 x V. cauliflora cross were found free from PRSV symptoms. Among the crosses, Pusa Nanha x V. cauliflora had higher yield under PRSV infected conditions, however, total soluble solids and total sugars were found lesser than the CO 7 x V. cauliflora cross. The hybridity of the progenies were confirmed by using ISSR (Inter Simple Sequence Repeats) primers by the amplification of DNA from progenies and their parents. ISSR primers UBC 856, UBC807 and ISSR primer combinations UBC 856-817, UBC 810-817, UBC 861-817, UBC 856-810, UBC 861-810 and UBC 856-817 clearly amplified specific bands of the male parent, which were present in F₁ progenies, but it was absent in female parents.     

Responses of non-primed or primed seeds of ‘Marketmore 76’ cucumber (Cucumis sativus L.) slurry coated with Trichoderma species to planting in growth media infested with Pythium aphanidermatum

Aqueous slurries of commercial preparations of Trichoderma harzianum Rifai strain KRL-AG2 G41 (Th), T. virens Strain G-41 (Tv), or their combination (ThTv, at half rates each of the single application rate) were applied to ‘Marktetmore 76’ cucumber seeds (Cucumis sativus L.) that were non-primed or primed for 3 days at 25 °C either osmotically (−2.5 MPa from 0.337 molal Ca(NO₃)₂) or osmomatrically (−1.0 MPa from 0.135 molal Ca(NO₃)₂ plus −1.5 MPa from 50% water in exfoliated grade 5 vermiculite). Slurries were applied to seeds (1 mg per seed) either before or after priming. Seeds were sown in soilless, peat-based media with or without inoculation with Pythium aphanidermatum (Pa). Protection against damping-off caused by high pressures of Pa (16% emergence in non-coated, non-primed seeds) was increased by slurry coating Th on non-primed (76.4% emergence) or on osmotically primed seeds, with coating either before or after priming having no effect on efficacy (average 62.6% emergence). Slurry coating Th on osmomatrically primed seeds failed to increase final emergence percentage (FEP). Colony forming units per three seeds (CFU, all 10³) was 2.8 for non-primed seeds, and 3.2 and 2.6, respectively, when osmotically and osmomatrically primed seeds were coated after priming. In a second study with lower disease pressure (58.1 FEP from non-coated, non-primed seeds), slurry coating of non-primed or osmotically primed seeds with Th, Tv or ThTv reduced percentage damping-off and increased FEP. The combination coating eliminated damping-off only in non-primed seeds, and tended to reduce percentage damping-off in osmotically or osmomatrically primed seeds compared to coating with Th or Tv alone. In a third study using only non-primed seeds, slurry coatings with mefenoxam fungicide, Th, Tv, or ThTv decreased total damping-off to 2.6%, 7.4%, 2.0%, and 0%, respectively, from the 30.1% occurring in non-coated seeds. Th, Tv or ThTv applied to growth media at the same rate as the seed coating (1 mg per seed) were generally as effective as the seed coatings, and only the ThTv growth medium application eliminated damping-off. A fourth experiment revealed that Th, Tv or ThTv remained viable on non-primed seeds for up to 4 weeks (the longest storage duration) at 21 or 4 °C, but 21 °C storage resulted in faster seed germination by week 3 and higher CFU per three seeds by week 4. In summary, coating of non-primed seeds with Th, Tv or ThTv was more effective than coating primed seeds in reducing percentage damping-off. While priming treatments generally led to faster seedling emergence and greater seedling shoot fresh weight than was achieved with non-primed seeds, only for non-primed seeds was damping-off eliminated by the ThTv seed coating or growth medium application.     

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.     

In vitro asymbiotic seed germination and seedling growth of Ansellia africana Lindl.

The effects of sodium hypochlorite treatment and growth medium on in vitro seed germination and seedling growth of the leopard orchid (Ansellia africana Lindl.) were investigated. Forty minutes treatment of seeds with 1.75% sodium hypochlorite stimulated germination (70.6%) and seedling development when measured at Stage 5 (emergence of first leaf) on P668 medium after 8 weeks of culture. The growth medium played a significant role in determining the germination response of A. africana seeds. Dark pretreatment of seed cultures significantly enhanced the germination percentage and the growth of rhizoids on the protocorms. Leaf growth in terms of length was substantially enhanced on P668 medium. It was significantly reduced in modified Knudson C medium after 16 weeks of culture. Seedlings developed on P668 medium showed a significantly better growth performance in relation to leaf length, leaf number, root length, fresh and dry weights per plant in vermiculite after 12 weeks of ex vitro growth in a mist house with 90% relative humidity. Further studies developing a suitable method for in vitro symbiotic germination could assist in reintroduction of this threatened orchid species into the wild.     

Ovules that failed to form seeds in zinnia (Zinnia violacea Cav.)

The structure of ovules that failed to form seeds after compatible pollination in double-flowered zinnias (Zinnia violacea Cav.) was investigated by observing histological sections, and comparing the normal and abnormal development of the seeds. In an embryo sac of zinnia at anthesis, a large nucleus of fused polar nuclei was clearly recognized. Success in double fertilization was determined by the disappearance of this nucleus. In normally developing seeds, the cellular endosperm became vague at 5–6 days after pollination (DAP) and the developing embryos occupied the entire portion of the seeds up to 10 DAP. A number of ovules failed in fertilization for the lack of an embryo sac. In most of the aborted seeds, the embryo sacs degenerated, whereas the aborted embryos were still alive in a small number of aborted seeds. A small percentage of the aborted seeds exhibited an aborted embryo and an unfertilized fused nucleus of two polar nuclei or ‘single fertilization’. The three major problems suggested for the failure in seed formation in pollinated florets of zinnia included failure in fertilization, ovules lacking embryo sacs, and abortion of developing seeds.     

Changes in enzymes involved in photosynthesis and other metabolic processes in the fruit of Opuntia ficus-indica during growth and ripening

The aim of this study was to investigate changes in the abundance of a number of enzymes in the peel, core and seeds of fruits of Opuntia ficus-indica (L.) Miller during development. The enzymes studied were phosphoenolpyruvate carboxylase (PEPC; EC: 4.1.1.31), ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO; EC: 4.1.1.39), aldolase (EC: 4.1.2.13), pyruvate, orthophosphate dikinase (PPDK; EC: 2.7.9.1), phosphoenolpyruvate carboxykinase (PEPCK; EC: 4.1.1.49) and aspartate aminotransferase (AspAT; EC: 2.6.1.1). To detect these enzymes, antibodies specific for each enzyme were used to probe Western blots of sodium dodecyl sulphate polyacrylamide gels. Fruit weight increased throughout development, and during ripening there was both an accumulation of total soluble solids and a decrease in both total titratable acidity and chlorophyll content. In the early stages of its growth the polypeptide pattern and enzyme composition of the fruit chlorenchyma was similar to that of the cladode. In the peel of the fruit PEPC, RUBISCO, plastidic aldolase and PPDK decreased to undetectable amounts as ripening progressed. PEPCK was not detected in either the chlorenchyma of the fruit or cladode. This showed that O. ficus-indica is not a PEPCK type CAM plant and that in this fruit PEPCK is not involved in the metabolism of organic acids. Cytosolic aldolase and AspAT were present in both the peel and core throughout development. The seeds accumulated storage proteins before the fruit ripened, and the abundance of all the enzymes studied declined once the accumulation of storage proteins was complete.     

Interspecific variations in seed germination of Corylopsis

This study was initiated to investigate the differences in germination percentages and rates between Corylopsis coreana Uyeki and Corylopsis sinensis var. calvescens Rehder & E.H. Wilson following a warm stratification (WS) and cold stratification (CS), and to study the effect of different WS temperatures interacting with different durations of CS. Warm stratification at 10 °C, 15 °C, 20 °C, and 25 °C was given for 1 month (1 M 10 °C, 15 °C, 20 °C, and 25 °C WS) followed by 0 M, 1 M, 2 M, and 3 M of CS at 5 °C (0 M, 1 M, 2 M, 3 M CS) and seeds were germinated in an air conditioned greenhouse maintained at 18.5 °C/18 °C. On average, less than 1% of C. coreana seeds germinated when sown without any WS and CS or with 1 M 15 °C, 20 °C, and 25 °C WS without CS treatment. However, 26% C. coreana seeds germinated after 1 M 10 °C WS without any CS treatment. Germination was not affected by WS temperatures when followed by 2 M 5 °C CS. It is concluded that C. coreana exhibited low seed germination at 10 °C and that this temperature could be considered the upper limit of CS for C. coreana. Only 2 M CS was required for more than 90% seeds to germinate. However, C. sinensis var. calvescens required longer than 3 M CS for more than 29% seeds to germinate. This clearly shows that there is an interspecific variation in optimum dormancy-breaking requirements.     

In situ induction of chromosome doubling in vine cacti (Cactaceae)

Vine cacti have economic potential as exotic fruits in semi-arid and arid lands due to their high water use efficiency. The goal of this study was to attain autopolyploid plants by applying the antimitotic agents colchicine and oryzalin on axillary vegetative buds and germinating seeds. The diploid Hylocereus monacanthus and the tetraploid H. megalanthus; the interspecific triploid hybrid S-75, were studied. The effects of different concentrations and exposure times on bud survival and germination rate were recorded. A negative effect on vegetative bud survival was observed in the triploid hybrid. An inhibitory effect on germinating seeds was species specific, being greater on H. monacanthus with oryzalin and on H. megalanthus with colchicine. Putative polyploids were identified by comparing stomatal density between donor plants and plants arising from treated buds or seeds. Flow cytometric analysis and chromosome counts confirmed polyploidization in 14 autotetraploid H. monacanthus, 1 autohexaploid S-75 hybrid, and 14 autooctaploid H. megalanthus. This is the first report of artificial autopolyploidization in species of vine cactus, providing valuable novel plant material for further breeding programs.     

In vitro morphogenesis and growth of Narcissus in response to temperature

In vitro morphogenesis and growth of tissues of the Narcissus cultivar ‘Lord Nelson’ have been assessed in constant and alternating temperatures. Tissue of leaf-base and scape origin, producing leafy shoots and bulblets on a defined sterile medium, were grown at constant and alternating temperatures of 15, 20, 25 or 30°C and 15–25 or 20–25°C, respectively, in a 16-h daily photoperiod. The number of leaves, leaf length, volume of tissues, fresh weight (but not percentage dry matter), and the number of shoots that would ultimately produce bulbs, were maximal at a constant temperature of 25°C as compared with all other temperature-regimes tested.     

The insertion of the Agrobacterium rhizogenes rolC gene in tomato (Solanum lycopersicum L.) affects plant architecture and endogenous auxin and abscisic acid levels

In the present paper we report on the effects of the insertion of the Agrobacterium rhizogenes rolC gene in the tomato (Solanum lycopersicum L., formerly Lycopersicon esculentum Mill.) cultivar Tondino. Several transgenic lines were successfully obtained, between which two clones, rolC1 and rolC3, were chosen for the analysis of morpho-productive traits as well as of the endogenous levels of auxin and abscisic acid. Consistent with the known phenotypic effect of this gene, the transformed tomato plants were significantly shorter than the corresponding controls. On the other hand, even if yield was not affected by the transformation in terms of average number of fruits produced, fruit weight was significantly lower in the transgenics with respect to the controls. Therefore, insertion of the rolC gene does not lead to an improvement in plant productivity.     

Biocontrol of mildew with Bacillus subtilis in bitter gourd (Momordica charantia L.) seeds during germination

The bitter gourd seed has a thick, hard seed coat. Mildew often occurs during germination and causes uneven and low rates of seed germination. However, the problems caused by mildew can be overcome by treating seeds with hot water, by soaking in water, or by using microorganisms. Seeds of the ‘Ching Pi’ bitter gourd were treated in a water bath at 60 °C for 10 min and then soaked in tap water at 25 °C for 24 h. The resulting germination percentage was 86.7%, and the resulting percentage of mildewed seeds was 10%. The biocontrol potential of three commercially available Bacillus subtilis solutions was examined. For seeds primed with Huodijun B. subtilis solution, the germination percentage was 73.3% and the mildewed percentage 6%. In dual cultures, the antibiotic content in the Huodijun B. subtilis solution was significantly greater than in Yunghsing and Huolibao, the other B. subtilis solutions examined. B. subtilis effectively inhibited the growth of Rhizoctonia solani mycelium and caused abnormal mycelial growth.     

Both anti-oxidation and lipid-carbohydrate conversion enhancements are involved in priming-improved emergence of Echinacea purpurea seeds that differ in size

This study evaluated the effects of priming on emergence responses of purple coneflower (Echinacea purpurea L. Moench) seeds. The seeds that differ in seed size were either primed with moistened vermiculite (solid matrix priming) or primed in non-aerated −0.5 MPa polyethylene glycol 6000 solution at 25 °C for 6 days (osmopriming), followed by air-drying to their initial moisture level. The tetrazolium staining tests indicated that both large and small seeds were biochemically viable. No notable difference in germination percentage was found between large and small seeds. However, extensive cavity was visible in portions of small seeds in comparison with large seeds. Large seeds accumulated more antioxidants and had greater activities of anti-oxidative enzymes than small seeds. They also had greater isocitrate lyase and malate synthase activities than small seeds. As a result, large seeds had higher emergence percentage and faster emergence speed as compared to that of small seeds. Both solid matrix priming and osmopriming increased emergence percentage and shortened mean emergence time of purple coneflower seeds by increasing the activities of anti-oxidative enzymes and decreasing the levels of malondialdehyde and total peroxide accumulation. Moreover, priming also enhanced the anti-oxidative activities of treated seeds. The activities of isocitrate lyase and malate synthase were also increased in primed seeds. The enhanced anti-oxidation and lipid-carbohydrate conversion activities might explain in part why primed purple coneflower seeds emerged better than non-primed seeds.     

Intergeneric hybridizations between Opisthopappus taihangensis and Chrysanthemum lavandulifolium

For the first time, reciprocal intergeneric hybridizations were produced between Opisthopappus taihangensis and Chrysanthemum lavandulifolium without emasculation. About 20% seed set was similarly obtained from reciprocal hybridization. Only 5 and 6 of the viable plants observed from 45 and 78 seedling survived present some conspicuous intermediate characteristics. Phenotypic evaluation among the progenies of the parents and the putative hybrids was performed carefully since an average of 5.3% seed set was produced in the type of self-pollination using pollen from the same flower and >10% seed set was similarly obtained in the types of self-pollination using pollen from different flowers in a plant and flowers in individual plants from different seeds. One individual of each hybrid shared the inflorescence habit with the pollen plant was confirmed further by the sequence of ncpGS. The two hybrids might be used as bridges of breeding of multi-generic hybrids.     

Assessing genetic variability in germplasm of Phaseolus vulgaris L. collected in Northern Portugal

The common bean (Phaseolus vulgaris L.) is the most consumed legume in the world. In Portugal, the geographic isolation of the region surrounded by the mountainous barrier of Peneda-Gerês, Barroso and Marão is thought to have the safeguard of a large number of adapted bean populations. In order to assess the value of this germplasm to breeding programs, a study of 20 Portuguese landraces collected in this Northern region were evaluated for agronomical (days to flowering, plant height, days to harvest, weight of seeds per plant and 100 seeds weight), chemical (crude protein) and genetic diversity (microsatellite DNA).     

Methods to break seed dormancy in Cyclocarya paliurus (Batal)Iljinskaja

Cyclocarya paliurus is native to China and is the sole species in its genus. However, the seeds remain deeply dormant for 2 years in their natural environment. We tested different pretreatments of chemical scarification and exogenous application of gibberellic acid (GA₃) for efficacy in breaking dormancy and speeding germination. In contrast to scarified seeds, non-scarified seed did not germinate, indicating that C. paliurus seeds have hard, impermeable seed coat dormancy. Exogenous application of GA₃ significantly enhanced germination of scarified seeds. Compared with seeds stratified in sand with water, the germination of seeds stratified in sand moistened with 400 ppm GA₃ for 60 days was significantly increased and germination rate was over 90% after 120 days. Analysis of variance indicated that both GA₃ concentration and stratification medium had significant effects on seed germination and final germination percentage. Germination was higher for longer stratification periods, but no significant difference in germination was observed after 90 days. These results suggested that C. paliurus seeds exhibit both exogenous and endogenous dormancy. A combination of chemical scarification and exogenous application of GA₃ alleviated seed dormancy in a relatively short period of time.     

Assessment of the stability and adaptability of waxbloom and waxless pea (Pisum sativum L.) mutant lines

Genotype–environment interactions, stability and adaptability for plant height, number of pods/plant, number of seeds/plant and seed weight/plant were analyzed by means of the model proposed by Eberhart and Russell (1966) in two cultivars and 22 mutant lines of field peas (Pisum sativum L.). The experimental design was the randomized block with three replicates. The genotypes were evaluated in 3 years. The stability parameters were: regression coefficient, variance due to regression and coefficient of determination. Differences in the response to environment were found among genotypes for the characters studied. The prevalent part of the investigated mutants and both cultivars reveal specific adaptation responding differently to environments, thus representing an initial plant material for developing individual targeted breeding programs.     

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.