In vitro pollen germination and transient transformation of<Emphasis Type="Italic">Zea mays</Emphasis> and other plant species

To study pollen-specific gene expression, fast and convenient methods involving in vitro pollen germination and bombardment with promoter deletion constructs are needed. Unfortunately, because of variation of pollen germability and tube growth, conducting these experiments is often unsatisfying for many plant species, including maize, especially when pollen is collected at different times of the day or season. We have overcome these problems by defining a novel medium (PGM) that guarantees germination efficiencies of more than 90% for maize pollen from at least 7 genotypes (A188, AC 3572 C, B73, H99, Hi-II, Q2, Tx232). This medium is also suitable to germinate pollen of other monocot species, such asPennisetum americanum andTradescantia species, and dicot species, such asArabidopsis thaliana, Arachis hypogaea, Columnea oesterdiana, Nicotiana tabacum, Phaseolus vulgaris, Pisum sativum, Solanum lycopersicum, Solanum tuberosum, andVicia faba. On average, reproducible germination rates ranging from 50–100% were observed with all plant species tested. In addition, we report a transient transformation assay using the luciferase (Luc) reporter gene. Biolistic parameters were defined to obtain reproducibleLuc activity measurements after bombarding thick-walled pollen, such as maize pollen. For comparison, samples of germinated maize and tobacco pollen were bombarded with the reporter gene under control of the constitutive ubiquitin-and pollen-specificZmMADS2 maize promoters. The important parameters necessary to apply both in vitro pollen germination and transient transformation for a large range of plant species are discussed. An erratum to this article is available at .     

Phytotoxic effects of fumonisin B1 on maize seedling growth

Fumonisin B₁ toxin is produced by the fungusFusarium moniliforme Sheldon, which is systemic to maize (Zea mays L.) and maize seeds. The effects of zero to 100 parts per million fumonisin B₁ on the germination process of maize seeds was determined. The presence of fumonisin had no effect on percent seed germination, but fumonisin inhibited radicle elongation by up to 75% after 48 hours of imbibition. An analysis of amylase secretion in the maize endosperm indicated that fumonisins inhibited amylase production in the germinating seed. Isoelectric focusing of endosperm extracts indicated that secretion of the low pI class of amylases was affected more that other amylase isozymes. The results suggested that the presence of high levels of fumonisin in maize seed may have deleterious effects on seedling emergence.     

Characterization of diazotrophic bacteria associated with maize: effect of plant genotype, ontogeny and nitrogen-supply

Summary The population size of diazotrophic bacteria naturally associated with the maize rhizosphere can be affected by biotic and environmental factors. In this work we have evaluated the effect of two genotypes of maize, with and without nitrogen fertilization, on the population dynamics and distribution of diazotrophic bacteria associated with maize plants over different plant ontogenic stages. The study was carried out in a field experiment with and without nitrogen fertilization, using two maize cultivars (Santa Helena 8447 and Santa Rosa 3063) previously selected from 32 maize cultivars for the lowest and highest response to nitrogen fertilization, respectively. Microbiological and molecular approaches were used to characterize the diazotrophic bacterial population structure. Bacterial population was assessed by the most probable number using semi-solid N-free media, and by DNA isolation from soil and plant tissue followed by amplification of nifH gene fragments using nested PCR, and by RFLP analysis using the restriction endonucleases TaqI and HaeIII. The dynamics of the diazotrophic bacterial population were affected by the ontogenic stage of the maize plants, but not by the cultivar type. Roots were the preferred site of colonization, independent of cultivar type and growth stage. During the first stage of maize growth, addition of nitrogen fertilizer negatively affected the diazotrophic bacterial population.     

A maize defective-kernel mutant (longcell) characterized by tubular cells, severe morphological alterations and induction of cell death

Seeds of the longcell mutant in maize (Zea mays L) have a defective-kernel phenotype: the embryo aborts at the early coleoptilar stage and the endosperm is reduced in size. Mutant embryos have severe alterations in morphogenesis. They have a suspensor-, an embryo axis- and a scutellum-like structure, but the shoot apical meristem (SAM) is not formed. Scanning electron microscopy showed that most of the cells in longcell embryos are tubular and abnormally enlarged. The level of expression of several genes involved in basic metabolism is not severely affected during early and mid embryogenesis, but storage molecule accumulation is reduced. Genes which in normal conditions are only expressed after germination, are expressed during kernel development in the longcell seeds. Mutant embryos undergo cell death in late embryogenesis. Nuclei in dying embryos are TUNEL positive, and different genes coding for hydrolytic enzymes are up-regulated. The expression of genes related to oxidative stress is also altered in longcell embryos. These results lead us to suggest that the longcell mutant may be cytokinesis-defective.     

Isolation of a maize beta-glucosidase gene promoter and characterization of its activity in transgenic tobacco

The β-glucosidase gene of maize (ZmGLU1) was suggested to hydrolyze cytokinin-conjugate and release free cytokinin during plant growth and development. A clone containing the upstream region of ZmGLU1 was isolated and sequenced from a maize genomic library. The full-length ZmGLU1 promoter and a series of its 5′ deletions were fused to the beta-glucuronidase (GUS) reporter gene and transferred into tobacco. The GUS activity of transgenic plants was assayed at various developmental stages. The results showed that ZmGLU1 promoter-driven GUS gene had the highest expression level in the roots and that the expression of GUS gene declined during seed maturation and down to the lowest level in mature seeds. The ZmGLU1 promoter-driven GUS expression increased during seed germination, reaching a peak on day 11. The results also showed that this promoter could be inhibited by 6-BA, trans-zeatin, and NAA, but was not affected by GA₃, ABA, SA, cold, salt, drought, and submergence treatments. The histochemical staining revealed that GUS activity was located in vigorous cell division zones with dominant staining associated with vascular tissues. Deletion analysis showed that the promoter contained a putative leaf-specific and stem-specific negative regulative element and two putative enhancers.     

Effect of post-irradiation ageing on onion seeds

Seeds of onion (Allium cepa) cv. Ailsa Craig were exposed to various doses of gamma radiation (0, 10, 20, 40, 80 and 100 krad) and subjected to accelerated ageing (RH 100%, 42°C) for 12 h. Radiation doses significantly affected the percentage of normal seedlings, abnormality types (%) and seedling growth. Seed viability, conductivities of seed leachates, final germination (%), germination speed and time to reach 50% germination (T ₅₀) were not affected by the seed irradiation. Accelerated ageing after irradiation had significant influence on seed viability, conductivities of seed leachates, final germination (%) and percentage of normal seedlings. Germination speed, T ₅₀ and seedling growth were not affected by the accelerated ageing. However, all the parameters studied were significantly influenced by the interaction of radiation doses and accelerated ageing. Accelerated ageing generally enhanced the damaging effects of irradiation on seeds. Therefore, it was concluded that onion seeds should not be exposed to adverse environmental conditions after irradiation.     

Germination and seed water status of four grasses on moss-dominated biological soil crusts from arid lands

Biological soil crusts dominated by drought-tolerant mosses are commonly found through arid and semiarid steppe communities of the northern Great Basin of North America. We conducted growth chamber experiments to investigate the effects of these crusts on the germination of four grasses: Festuca idahoensis, Festuca ovina, Elymus wawawaiensis and Bromus tectorum. For each of these species, we recorded germination time courses on bare soil and two types of biological soil crusts; one composed predominantly of the tall moss Tortula ruralis and the other dominated by the short moss Bryum argenteum. On the short-moss crust, the final germination percentage was about half of that on bare soil. Also, the mean germination time was 4 days longer on short-mosses than on bare soil. In contrast to the short-moss crust, the tall-moss crust did not reduce the final germination percentage but increased the mean germination time. Similar results were observed in the four grasses studied. To investigate the mechanism by which moss crusts affected germination, we analyzed the water status of seeds on bare soil and moss crusts. Six days after seeding, the water content of seeds on bare soil was approximately twice that of seeds on tall- or short-moss crust. Analysis of the time course of changes in seed weight and water potential in Bromus tectorum revealed that overtime seeds on tall mosses reached higher water content than those on short mosses. The increase in the water content of seeds on tall mosses occurred as the seeds gradually fell through the moss canopy. Taken together, our results indicate that biological soil crusts with distinct structural characteristics can have different effects on seed germination. Furthermore, this study revealed that a biological soil crust dominated by short mosses had a negative effect on seed water status and significantly reduced seed germination.     

Allelochemical stress produced by aqueous leachate of <Emphasis Type="Italic">Nicotiana plumbaginifolia</Emphasis> Viv.

The effects of aqueous leachate of Nicotiana plumbaginifolia Viv. on germination, seedling growth, amylase activity, sugar and starch contents of germinated seeds of maize (Zea mays L. cv. Uttam) were examined. Effects of leachate on photosynthetic pigments, protein content, activities of nitrate reductase and some antioxidants were also studied. Higher concentration of aqueous leachate of N. plumbaginifolia reduced the germination rate (GR). However, final germination percentage remained almost unaffected. Lower concentration of leachate stimulated the amylase activity and resulted in higher sugar content and GR. The increasing concentrations of leachate inhibited the conversion of starch into sugars. Allelochemicals decreased the amount of chlorophyll a, chlorophyll b, carotenoids, protein and nitrate reductase activity (NRA). The leachate of lower concentrations stimulated the activity of peroxidase but slight decrease was recorded in higher concentration. Superoxide dismutase and catalase exhibited concentration dependent increase except in seedlings treated with 100% concentration of leachate. Impairment of various metabolic activities due to leachate resulted in decreased root and shoot length.     

掌叶木种皮障碍及种子各部位内源抑制活性的研究

掌叶木(Handeliodendron bodinieri)是残遗于中国的稀有单种属植物,因人为破坏、生境特殊及自身特性的影响,资源稀少,被列为国家一级重点保护野生珍稀濒危植物。该研究以掌叶木种子为材料,研究了4种不同发芽条件下(带种皮、浓硫酸处理种皮、完全去除种皮、仅露出胚根)种子萌发性、种皮透水性、掌叶木果皮、假种皮、种皮和种仁四个部位不同浓度甲醇浸提物(0、3.125、6.25、12.5、25 mg/m L)对白菜种子萌发及幼苗生长的影响以及掌叶木各部位浸提物对种子萌发的影响。结果表明:(1)掌叶木种皮具有一定的透水性,为掌叶木种子的萌发提供必要的透水透气条件,不影响种子萌发前的水分吸收,但掌叶木种皮的机械阻碍、易霉变对种子的萌发影响较大。(2)掌叶木的果皮、假种皮、种皮和种仁甲醇浸提物对白菜种子的萌发和生长都有影响,尤其对白菜幼根的生长有较强的抑制作用,抑制强度依次是种仁果皮假种皮种皮,且随着浓度的升高,抑制作用增强。该研究结果揭示了掌叶木种子难发芽、发芽率低的原因,为掌叶木的人工扩繁和保护与利用奠定了基础。     

Plant oils as grain protectants against infestations ofCryptolestes pusillus andRhyzopertha dominica in stored grain

Plant oils (cottonseed, soybean, corn, groundnut and palm) at different dosages were evaluated in the laboratory for their ability to suppress the populations ofCryptolestes pusillus andRhyzopertha dominica in maize and sorghum. Exposure of adults of both beetle species to grains treated with 10 ml/kg of the different oils induced 100% mortality within 24 h. A dose of 5 ml/kg of each oil significantly decreased the progeny produced byR. dominica. Complete protection was achieved on grains treated with 10 ml/kg. These oils also repelled the adults of both species. Percentage weight loss caused byR. dominica in grains treated with 5 ml/kg and 10 ml/kg levels were significantly lower than in untreated grains. Oil treatment did not affect the germination of, or water absorption by, maize and sorghum grains compared with untreated grains. The potential use of plant oils in the management of insect pests in traditional grain storage is discussed.     

Effect of IAA on symbiotic germination of an Australian orchid and its production by orchid-associated bacteria

Seven isolates of orchid-associated bacteria (OAB) belonging to five species were tested for their effect on mycorrhiza-assisted germination of the terrestrial orchid Pterostylis vittata. Hormone standards were also tested to evaluate their potential roles in the germination and development of the orchid. Strains of Pseudomonas putida, Xanthomonas maltophilia and Bacillus cereus promoted symbiotic germination, whereas certain strains of P. putida and an Arthrobacter species reduced it. Symbiotic germination was enhanced by IAA, inhibited by gibberellic acid and suppressed by kinetin. Each species of OAB produced IAA, although the conditions of growth affected the production of the auxin. IAA was not produced by the mycorrhizal fungus from P. vittata under the test conditions. Enhancement of symbiotic germination development may have resulted either from the production of IAA by the OAB and/or by the induction of endogenous hormones in the orchid by the metabolites of the bacteria and/or mycorrhizal fungus.     

Preferential transposition ofAc to linked sites in Arabidopsis

We have investigated the pattern of transposition of an intact, 4.6-kbAc element inArabidopsis thaliana. Because the trans-acting transposition function (transposase) ofAc is not fully penetrant in Arabidopsis, it is not possible to use it as a diagnostic feature to scoreAc genetically, as has been done in maize and tobacco. Instead, the presence or absence of a transposedAc (trAc) was monitored by Southern blots. Germinal transpositions from the marker SPT::Ac were selected using a streptomycin germination assay and scored for the presence of atrAc. Segregation of thetrAc element and the SPT donor locus was scored in the F₂ progeny of the germinal revertants, and the recombination fraction between thetrAc element and SPT was estimated by the method of maximum likelihood. We have found that, as in maize and tobacco, receptor sites fortrAcs in Arabidopsis tend to be linked to theAc donor locus.     

The impact of temperature regimes on development,dormancy breaking and germination of dwarf shrub seeds from arctic,alpine and boreal sites

It has been suggested that the infrequent sexual reproduction of arctic dwarf shrubs might be related to the harsh environmental conditions in which they live. If this is the case, then increases in temperature resulting from global climate change might drastically affect regeneration of arctic species. We examined whether recruitment of Empetrum nigrum ssp. hermaphroditum and Vaccinium uliginosum (hereafter E. nigrum and V. uliginosum) was affected by temperature during three reproductive stages: seed development, dormancy breakage and germination. Seeds were collected from an arctic, an alpine (only E. nigrum) and a boreal site with different climates; stored at different winter temperatures and incubated for germination at different temperatures. Seeds of V. uliginosum developed in the boreal region had a higher percentage germination than did seeds developed in the Arctic. In contrast, seeds of E. nigrum from the arctic site had a higher or similar percentage germination than did seeds from the alpine and boreal sites. Increased winter temperatures had no significant effect on resulting germination percentage of E. nigrum. However, V. uliginosum seeds from the arctic site suffered increased fungal attack (and thus decreased germination) when they were stratified under high winter temperatures. Seeds of both species increased germination with increased incubation temperatures. Our results suggest that both species would increase their germination in response to warmer summers. Longer summers might also favour the slow-germinating E. nigrum. However, increased winter temperatures might increase mortality due to fungal attack in V. uliginosum ecotypes that are not adapted to mild winters.     

In vitro zygotic embryo culture of wild Musa acuminata ssp.malaccensis and factors affecting germination and seedling growth

In vitro zygotic embryo culture of wild banana significantly increased the germination compared to greenhouse grown seeds. Embryo orientation and BAP concentration significantly affected germination rate. These factors together with gelling agent, dark and light conditions and coconut water, also showed variable effects on the number of roots per plant, root length, shoot length, number of days to root emergence and number of days to shoot emergence.     

Role of differential expression of <Emphasis Type="Italic">sdh1-1</Emphasis> and <Emphasis Type="Italic">sdh1-2</Emphasis> genes in alteration of isoenzyme composition of succinate dehydrogenase in germinating maize seeds

A probable mechanism of alteration of the isoenzyme composition of succinate dehydrogenase (SDH) due to differential expression of genes encoding subunit A was considered. The alteration of SDH activity during maize seed germination was investigated, and its maximal activity on day 4-5 of germination was found. The alteration of the sdh1-1 and sdh1-2 gene expression level during maize seed germination was evaluated using the quantitative polymerase chain reaction method. The presence of four forms of the studied enzymes, providing multiple SDH functions was found in maize inflorescence using electrophoresis in polyacrylamide gel.     

Infection of Barley by <Emphasis Type="Italic">Ramularia</Emphasis> <Emphasis Type="Italic">collo-cygni</Emphasis>: Scanning Electron Microscopic Investigations

Ramularia collo-cygni causes leaf spots on barley (Hordeum vulgare), a disease of growing economical importance. Scanning electron microscopy was used to study the life cycle of the fungus on barley during the vegetation period and in winter. The infectious stage started with conidium germination on the surface and the penetration into the leaf via the stomatal pore where the hyphae grew within the cells that became necrotic. The conidiophores emerged through the stomatal pore. On older leaves, however, they frequently emerged apart from it and the results suggested a pushing apart of adjacent cell walls of the epidermal cells. An assessment of the amount of conidium formation of one heavily infested barley plant resulted in 4.05 × 10⁶ conidia per plant. For the first time, conidiophores, conidium production and germination of conidia were also observed in winter on barley and on maize leaves.     

Involvement of carbohydrates and cytokinins in pathogenicity ofHelminthosporium carbonum

Significant stimulation of the number of appressoria, penetration and colonization by conidia ofHelminthosporium carbonum occurred on decolorized maize leaves when exogenous carbohydrates and leaf leachates were added. Germination and germ tube length, however, did not exhibit appreciable differences on decolorized or non-decolorized maize leaves. Lower germination was recorded by leached conidia on decolorized leaves; while appressoria, penetration and colonization were absent. Addition of exogenous nutrients (sucrose>leaf leachates>yeast extract>glucose) enabled conidia to accomplish appressoria, penetration and colonization. Optimum levels for various nutrients observed were 2% (w/v) sucrose/glucose or 0.1% (w/v) yeast extract. Higher concentrations inhibited the infection stages of the pathogen. Depletion of host carbohydrates from green islands/infection sites adversely affect appressoria formation, penetration and colonization; and the loss of carbohydrates from the spore affects germination. Cytokinin-like activity at the infection site/green islands increased with the period of incubation of the host as compared to the surrounding tissue or tissue under water drops. The culture filtrate extracts ofH. carbonum recorded cytokinin-like activity which increased with growth of the fungus. TLC (thin layer chromatography) of cytokinin-like substances (tissue extract and culture filtrate) revealed major activity was confined to Rf zones 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. These substances increase at infection sites by virtue of which carbohydrates accumulate at these sites ensuring a continuous supply to the growing pathogen.     

Effects of temperature and light on cyst germination and germinated cell survival of the noxious raphidophyte Heterosigma akashiwo

The effects of temperature and light on the germination of Heterosigma akashiwo cysts were examined using bottom sediments collected from Hakata Bay, Japan. In a suspension of mixed sediment and seawater in the temperature range of 5–30 °C, motile cells emerged within 3 weeks, but at ≤12 °C the cell numbers were markedly lower and the emergence of motile cells delayed. When suspension samples incubated at various temperatures were moved to 20 °C and incubated, only a few additional motile cells emerged. The number of motile cells germinated in the dark was significantly lower than under light conditions. When suspension samples incubated in the dark were exposed to light, only a few additional motile cells emerged. These results indicate that the initiation of germination in Heterosigma cysts suspended in seawater is not dependent on temperature and light conditions, although the speed of the germination process is affected by temperature, and cell survival just after germination is strongly affected by temperature and light.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and axillary shoot propagation of <Emphasis Type="Italic">Centaurea zeybekii</Emphasis>

In vitro culture is an important aid for ex situ conservation of rare, endemic or threatened plants. In this work, we establish an efficient method for the seed germination, seedling development, and axillary shoot propagation of Centaurea zeybekii Wagenitz. The seeds, collected from a wild population, were surface sterilised and cultured on various in vitro germination media. The effects of photoperiod and temperature on seed germination were also investigated. Germinations were obtained after 6 weeks in culture and the radicle emergence was evaluated as a main indicator. A high frequency of germination was obtained on distilled water supplemented with vitamines and 1 mg/L GA₃. Although the seed germination frequencies were not affected by photoperiod, the highest germination frequency was obtained at 24 ± 2°C. A high frequency of axillary shoot proliferation was produced on MS medium supplemented with 1 mg/L BA. Then, the axillary shoots were separated and transferred to MS medium with or without plant growth regulators for rooting. Rhizogenezis was promoted after 6 weeks only in MS and 1/2 MS media containing 0.5 mg/L IBA. The rooting process was very slow and the percentage of shoot rooting was also very low (15%). The present study not only enables reinforcement of wild plant populations using ex situ growth of individuals, but it also helps to large number of aseptic seedling to use it in clonaly micropropagation studies.     

Salinity and Temperature Effects on Germination for Three Salt-resistant Euhalophytes, Halostachys caspica, Kalidium foliatum and Halocnemum strobilaceum

The effects of three salinities (0, 100 and 500 mM NaCl) and four constant temperatures (10, 20, 30 and 35 °C) on seed germination of Halostachys caspica (M. B.) C. A. Mey., Kalidium foliatum (Pall.) Mop. and Halocnemum strobilaceum (Pall.) Bieb. were investigated. After seeds were treated with different concentrations of NaCl at constant temperatures of 10–35 °C for 16 days, ungerminated seeds were transferred to distilled water for 10 days to investigate the total germination; after this time, the ungerminated seeds from the 10 and 20 °C treatments were then moved to 35 °C for another 5 days to determine the final germination. The three plant species in the present experiment are salt-resistant euhalophytes growing in high saline soils in the Zhungur Basin in Xinjiang, a northwest province of China.Compared with germination under control conditions, germination percentages of all three species were not affected by 100 mM NaCl at 10–35 °C, while severely inhibited by 500 mM NaCl; germination percentages were very low at 10 °C up to 100 mM NaCl for all species; the optimum temperature for germination of H. caspica and K. foliatum was 20–30 °C, while 35 °C for H. strobilaceum, up to 100 mM NaCl; seeds did not suffer ion toxicity for all species, as evidenced by the high total germination after ungerminated seeds pretreated with 500 mM NaCl were transferred to distilled water at constant temperatures of 10–35 °C for 10 days, and the high final germination after the ungerminated seeds from the 10 and 20 °C treatments were subsequently moved to 35 °C for another 5 days; Halostachys caspica had greater sensitivity to increasing temperatures from 10 and 20 °C to 35 °C compared with the other two species.