Effect of various sterilization procedures on the in vitro germination of cotton seeds

In vitro manipulations of cotton often require high-quality sterile seedlings as a source of hypocotyl and cotyledon explants for initiation of embryogenic cultures or embryo apexes for shoot production. Unfortunately, in vitro seed germination is often hindered if cotton seeds were collected from the open field and stored under improper conditions. In our case a limited supply of field-grown cotton seeds was received, which necessitated the development of a more effective surface sterilization protocol. Seeds from two accessions, designated as I and II, with very high contamination levels and lowered germination rate were used in this study. These seeds were treated with the most commonly used sterilizing agents, which included commercial bleach, chlorine gas and hydrogen peroxide. Additional steps such as soap-water washes (SW), 70 % ethanol (ETH) and plant preservative mixture rinses were also included in the sterilization procedures to improve the efficiency of tested protocols. Surface sterilized seeds were germinated on Linsmaier and Skoog medium and the percentage of contamination free and well-germinated seeds were recorded for each treatment. Seeds treated with hydrogen peroxide showed significant improvement in germination rate and level of contamination when compared to identical seeds treated with chlorine gas and commercial bleach. The most effective sterilization protocol for all genotypes tested consisted of SW wash followed by ETH rinse and H₂O₂ sterilization for 7 h. This protocol was successfully used to sterilize seeds of 55 cotton lines.     

THE SURFACE DECONTAMINATION OF SEEDS TO PRODUCE AXENIC SEEDLINGS

A variety of sterilizing agents were tested to develop a standard procedure for surface decontaminating seeds to produce axenic seedlings. The use of calcium hypochlorite (0.5% phosphate buffer, pH 6) for 10 min followed by three sterile water rinses was among the most effective agents, and it did not injure some species as did sodium hypochlorite, formaldehyde, ethylene oxide and mercuric chloride. Some species contained internal microbes requiring severe treatments which killed or injured the seedling, while other species were “decontaminated” with a sterile water rinse. The percentage of seeds with internal microbes varied considerably among plant species, seed lot, and the length of seed storage. Thus, with seeds not easily decontaminated, screening of additional seed lots would be more profitable than testing additional decontamination agents. Release of microbes from the seed's interior is associated with germination, and microbial testing must last at least 11 days. Nutrient agar permitted growth, although the seedlings outgrew petri plates too quickly for adequate certification. These seedlings were transferred to nutrient agar in quart jars in which an internal pool of broth was periodically agitated to permit microbial sampling of the leaves while the plant grew.     

Contamination control of microbe Ziziphus spina [christti] seed in vitro culture

Ziziphus spina [christti] is a naturally distributed tree in subtropical, arid and semi-arid parts of Iran. It is ecologically and economically important due to its tolerance to drought and salinity. Most tree seeds are infected with parasitic and saprophytic microorganisms which decrease the seed germination and seedling establishment. The goal of this paper was to evaluate the ability of selected chemical solutions to inhibit the growth of variety of microbial contaminants in Z. spina [christti] seeds and to enhance the seed germination. Different chemical treatments were used in surface sterilization of seeds: (Treatment 1) sodium hypochlorite (NaOCl) in concentrations of 1, 2 and 4% for 20 min. (Treatment 2) hydrogen peroxide (H₂O₂) in concentration of 4, 8, 12% treated for 10 min. (Treatment 3) 1% mercuric chloride (HgCl₂) at duration 10, 15, 20, 25 min. Seeds were scarified and aseptically, planted on agar Murashige and Skoog (MS) medium. Contaminants were identified according to their morphological and cultural characteristics. Bacterial contaminants included Xanthomonas sp. While Fungal isolates were Fusarium, Penicillium, Alternaria, Rhizopus, and Aspergillus. Our experiment reveals that 4% NaOCl followed by benomyl is the best sterilization treatment for Z. spina [christti] seeds, since the highest number of germination and highest number of sterilized seeds was observed after this treatment.     

Seed germination and in vitro propagation of Maytenus canariensis through regeneration of adventitious shoots from axillary and apical buds

Seed germination and micropropagation protocols of the medicinal species Maytenus canariensis (Loes.) G. Kunkel & Sunding were optimized. In vitro seed germination occurred (86 to 94.7 %) only after treatment of the seeds with H₂SO₄, followed by surface sterilization and culture on solid nutrient medium without any growth regulators. Micropropagation failed when explants were taken from mature trees, and browning of the nutrient medium frequently occurred despite testing many growth media. Nonetheless, adventitious shoot regeneration was achieved employing axillary or apical buds taken from 2–2.5 months old plantlets obtained after in vitro germination of seeds, following culture on nutrient media supplemented with benzylaminopurine, kinetin and naphthalenacetic acid (NAA), attaining up to 3.9 shoots per explant, after 4–6 months. Root induction was best on a medium containing 4.0 mg dm⁻³ NAA, achieving a 100 % induction. After hardening of rooted plants, survival after transfer to soil was 71.43 %.     

Antisense-transformation reveals novel roles for class I beta-1,3-glucanase in tobacco seed after-ripening and photodormancy

Little is known about the molecular basis for seed dormancy, after-ripening, and radicle emergence through the covering layers during germination. In tobacco, endosperm rupture occurs after testa rupture and is the limiting step in seed germination. Class I beta-1,3-glucanase (betaGLU I), which is induced in the micropylar endosperm just prior to its penetration by the radicle, is believed to help weaken the endosperm wall. Evidence is presented here for a second site of betaGLU I action during after-ripening. Tobacco plants were transformed with antisense betaGLU I constructs with promoters thought to direct endosperm-specific expression. Unexpectedly, these transformants were unaffected in endosperm rupture and did not exhibit reduced betaGLU I expression during germination. Nevertheless, antisense betaGLU I transformation delayed the onset of testa rupture in light-imbibed, after-ripened seeds and inhibited the after-ripening-mediated release of photodormancy. It is proposed that betaGLU I expression in the dry seed contributes to the after-ripening-mediated release of seed dormancy.     

A new method for the identification and isolation of genes essential forArabidopsis thaliana seed germination

Seed viability, dormancy and germination efficiency are very important aspects of the life cycle of plants and their potential to survive and spread in the environment. To characterize the genes controlling these processes, we have devised a technique for the selection of mutants impaired in seed germination. Selection for such a trait is complicated by physiological factors that interact with these processes and affect seed germination efficiency. The distinction between low seed germination potential due to physiological factors that interfere with seed maturation or germination and germination deficiency due to genetic factors was based on screening for tagged mutations.Arabidopsis thaliana T-DNA primary transformants obtained by an in planta transformation technique are all heterozygotes. We screened for lack of germination of 1/4 of the seeds in the progeny of independent transformants, and simultaneously for the abnormal segregation (2:1 instead of 3:1) of a kanamycin resistance marker carried by the T-DNA inserted into the genome of these primary transformants in the plants that germinate. This yielded several mutants affected in the germination processes. One of the mutants, designated ABC33, was further characterized. Once the viable embryos from non-germinating seeds were removed from their testa, they grew and displayed a dwarf phenotype which could be complemented by providing gibberellic acid. A genetic and molecular analysis, based on the characterization of the flanking genomic sequences of the T-DNA insert, showed that ABC33 is a new loss-of-function allele at theGA ₁ locus.     

Enhanced attachment of Bradyrhizobium japonicum to soybean through reduced root colonization of internally seedborne microorganisms

Internally seedborne microorganisms are those surviving common surface sterilization procedures. Such microbes often colonize the radicle surface of a germinating soybean (Glycine max) seed, introducing an undefined parameter into studies on attachment and infection by Bradyrhizobium japonicum. Bacterial isolates from surface-sterilized soybean seed, cv. Williams 82 and cv. Maverick, used in our studies, were identified as Agrobacterium radiobacter, Aeromonas sp., Bacillus spp., Chryseomonas luteola, Flavimonas oryzihabitans, and Sphingomonas paucimobilis. Growth of these microbes during seed germination was reduced by treating germinating seeds with 500 micrograms/mL penicillin G. The effects of this antibiotic on seedling development and on B. japonicum 2143 attachment, nodulation, and nitrogen fixation are reported here. Penicillin G treatment of seeds did not reduce seed germination or root tip growth, or affect seedling development. No differences in nodulation kinetics, nitrogen fixation onset or rates were observed. However, the number of B. japonicum attached to treated intact seedlings was enhanced 200-325%, demonstrating that other root-colonizing bacteria can interfere with rhizobial attachment. Penicillin G treatment of soybean seedlings can be used to reduce the root colonizing microbes, which introduce an undefined parameter into studies of attachment of B. japonicum to the soybean root, without affecting plant development.     

A new method for the identification and isolation of genes essential forArabidopsis thaliana seed germination

Seed viability, dormancy and germination efficiency are very important aspects of the life cycle of plants and their potential to survive and spread in the environment. To characterize the genes controlling these processes, we have devised a technique for the selection of mutants impaired in seed germination. Selection for such a trait is complicated by physiological factors that interact with these processes and affect seed germination efficiency. The distinction between low seed germination potential due to physiological factors that interfere with seed maturation or germination and germination deficiency due to genetic factors was based on screening for tagged mutations.Arabidopsis thaliana T-DNA primary transformants obtained by an in planta transformation technique are all heterozygotes. We screened for lack of germination of 1/4 of the seeds in the progeny of independent transformants, and simultaneously for the abnormal segregation (2:1 instead of 3:1) of a kanamycin resistance marker carried by the T-DNA inserted into the genome of these primary transformants in the plants that germinate. This yielded several mutants affected in the germination processes. One of the mutants, designated ABC33, was further characterized. Once the viable embryos from non-germinating seeds were removed from their testa, they grew and displayed a dwarf phenotype which could be complemented by providing gibberellic acid. A genetic and molecular analysis, based on the characterization of the flanking genomic sequences of the T-DNA insert, showed that ABC33 is a new loss-of-function allele at theGA ₁ locus.     

Sterilization of culture media for orchids using a microwave oven

Production of orchid seedlings often requires complex laboratory infrastructure; therefore, a simple and low-cost method would be of general benefit to many small-scale producers and orchid enthusiasts. This article describes a protocol for preparing culture media using a domestic microwave oven. Two alternative culture media were evaluated for a range of factors, including the duration of boiling, the efficacy of antiseptics applied to jars and media, the concentration of the antiseptic hydrogen peroxide required for sterilization, and the growth of Oncidium cebolleta and Phalaenopsis amabilis plantlets in media prepared using a microwave oven compared with conventionally autoclaved media. It was found that addition of 2 mL of 30% hydrogen peroxide (Peridrol® solution) per liter of culture medium, an 8-min boiling time in the microwave oven, and 8 g/L agar were sufficient to produce solidified culture media, which facilitated orchard seed germination and growth without contamination. Furthermore, the microwaved media exhibited superior plantlet growth to autoclaved media.     

Asymbiotic seed germination and in vitro seedling development of the threatened orchid Hoffmannseggella cinnabarina

Hoffmannseggella cinnabarina has not been found in the wild for the last 70?yr in the State of S?o Paulo and, therefore, wild populations of this native orchid are thought to be extinct. This investigation studied seed storage at a low temperature, in vitro germination, and seedling development of H. cinnabarina in order to establish an optimized protocol for propagation, and thus assure species conservation. Seeds of different ages were incubated on Knudson C (KC), Murashige and Skoog, and Vacin and Went media with or without 1???M of N⁶-benzyladenine (BA) and exposed to either 12 or 16?h of light (30???mol?m^?2?s^?1 at 26?±?2°C). Seed surface sterilization was deleterious to 3-mo-old seeds and severely reduced the viability of the 4-mo-old seeds. More mature seeds were not affected by the sterilization procedure. In general, the germination of 4-, 5-, 6-, and 7-mo-old seeds increased when BA was added to the culture medium especially under 16?h of light. Germination rates were highest with 8- and 9-mo-old seeds, and application of BA failed to enhance germination rates further. Developmental studies revealed that this cytokinin reduced seed and protocorm mortality rates; however, protocorm development was negatively affected in its presence. Seedling development was more pronounced when KC medium with 16?h of light was used. Long-term seed storage at 4°C did not provide promising results. The protocol described in this study proved to be efficient and relevant to in vitro seed germination and initial development of H. cinnabarina, and thus will contribute to conservation of this orchid species.     

FACTORS AFFECTING DORMANCY,GERMINATION, AND SEEDLING DEVELOPMENT OF AEGINETIA INDICA L. (OROBANCHACEAE)

Dormancy in seeds of the parasitic phanerogam Aeginetia indica L. can be broken by chemical treatment with sodium hypochlorite, which also helps to control contaminating microflora. A germination medium was developed that suppressed microbial contamination and permitted long-term observation of these slow-germinating seeds. The medium consisted of 10 ppm streptomycin, 10 ppm penicillin, and 10⁻⁵ m indole-3-acetic acid (IAA) (or other growth regulator) in 1 % water agar. Optimum germination range was 25–30 C. Dormancy could also be broken by exposure on agar for several days at 3-5 C (stratification), or by brief exposures (15 min) to 50 C. Continuous light as low as 0.1 ft-c completely inhibited germination on this growth medium. Brief, intermittent light exposures depressed germination. Germination and growth in vitro of nondormant seed of Aeginetina indica L. can be described in five stages: (1) Germination: expansion of spheroidal cells or nodule at micropylar end of the seed, stimulated by growth regulators. Unique stimulation of germination by IAA was noted; (2) Tendril formation: production of a root-hairlike protrusion in response to seedling roots or extract of pea seedlings; (3 and 4) Tendril septation and branching: induced by pea extract; (5) Seedling extension growth: cell growth and division adjacent to nodule by action of various carbohydrate-containing growth factors. This type of growth may bypass tendril formation.     

无菌猕猴桃种子采集方法研究

无菌的猕猴桃种子是猕猴桃胚乳培养、实生苗微嫁接等技术的基础材料,利用消毒剂灭菌是常用的无菌种子采集手段,应用最广泛的消毒剂为升汞(mercuric chloride)和次氯酸钠(NaClO)。为了避免使用消毒剂,该研究提出了一种新的无菌猕猴桃种子采集方法——无菌搅拌法,同时为探索其准确性和应用性,比较了0.2%升汞灭菌20 min、10%次氯酸钠灭菌20 min、无菌搅拌法三种方式采集无菌猕猴桃种子的效果,并对种子萌发和幼苗形成的影响进行了研究。结果表明:无菌搅拌法、0.2%升汞灭菌20 min是稳定有效的无菌猕猴桃采集方式,10%次氯酸钠灭菌20 min的采集效果不稳定;在相同的时间内,无菌搅拌法的种子发芽率最高,为89.90%,但发芽势较低,均可正常成苗; 10%次氯酸钠灭菌20 min的发芽率次之,与无菌搅拌法的种子无显著差异,发芽势和成苗率最高,分别为47.47%和67.86%,且有打破猕猴桃种子休眠的作用,整体作用类似于赤霉素(GA_3)浸种的效果; 0.2%升汞灭菌20 min对猕猴桃种子的萌发有抑制作用,各项指标均显著低于无菌搅拌法种子,且生长缓慢。此外,无菌搅拌法是物理处理,对种子、操作人员、环境均无害。这证实了无菌搅拌法的实用性和优势,发现了次氯酸钠在打破猕猴桃种子休眠方面的作用,为其它同类型果实的无菌种子采集提供了参考。     

The significance of the methods of stigmatal and placental pollinationin vitro inAntirrhinum majus L.; seed and callus formation on placentae

A study was made on certain problems connected with the application of methods of stigmatal and placental pollinationin vitro in the snapdragon. Germinable pollen without microbial contamination could be obtained only from the flowers which were left, after surface sterilization, on the stem till the dehiscence of anthers. The germination of pollen, and especially the growth of pollen tubes was better in a 0.3 M lactose solution with 10^?3 per cent H₃BO₃ than in usual sucrose media. The seedsin vitro could be obtained only after normal pollination and the successive artificial cultivation of the entire pistil (the method of stigmatal pollinationin vitro). The seeds did not differ either in their morphology or in their size from those developed under natural conditions, they were viable and without dormancy. Though the pollinated pistils were cultivated in the medium without growth substances, callus formation from uncovered ovules and placenta was observed in some cases. A dependence was revealed of the proliferation on pollination and on the cultivar employed. When using the method of placental pollinationin vitro we failed to obtain seeds. The main difficulty in the use of this method seems to consist in the fact that pollen tubes are not capable of growing from the surface of ovules and placenta into the micropyle.     

The Role of White Mold-Infected White Bean (Phaseolus vulgaris L.) Seeds in the Dissemination of Sclerotinia sclerotiorum (Lib.) de Bary

Sclerotinia sclerotiorum survived in infected seeds of white beans as dormant mycelium in testa and cotyledons. The rate of survival averaged 85 to 89% and did not change appreciably over a 3-year period. When the infected bean seeds were sown in soil or sand, 88 to 100% failed to germinate. The seeds that failed to germinate, depending on the severity of seed infection, were rotted by S. sclerotiorum. In place of each seed, 3 to 6 sclerotia were formed. A low percentage of these sclerotia germinated carpogenically with or without preconditioning, (2.5 and 11.5% respectively). Myceliogenic germination of sclerotia with and without preconditioning was 35.5% and 70.5% on water agar and 81.0% and 93.0% on glucose agar, respectively. Both, preconditioning and nonpreconditioned sclerotia which were scattered on soil surface could germinate myceliogenically and infect bean leaves by contact. It is therefore, concluded that dormant mycelia in the infected seeds play an important role notonly in dissemination of the fungus but also in epidemiology of the disease.     

Growth conditions influence regeneration of encapsulated carrot somatic embryos into plantlets

Summary Carrot somatic embryos were encapsulated in calcium alginate beads, with or without a maturation step, to produce synthetic seeds. Germination and plantlet regeneration frequencies were compared for liquid or solid nutritive media, and with sucrose supply and sterilization or not. Germination rates were greater than 80% in all treatments except in non-sterilized sand. No conversion occured on a liquid MS medium. Normal plantlets were 7–22% on agar and 4–40% on sand. Abnormal plantlets were only observed on agar due to secondary embryogenesis. A sterilized sand growth support supplied with sucrose appears to be most appropriate to assess morphologically normal plantlet regeneration.Corresponding author     

A simple and effective plating method to screen polycyclic aromatic hydrocarbon-degrading bacteria under various redox conditions

Agar plates with a polycyclic aromatic hydrocarbon (PAH) layer have been used to screen for microorganisms that degrade PAHs, leaving clear zones around colonies; however, there are several problems with previous methods such as undesired contamination in the fume hood and difficulty in controlling the amount of PAH on the plates. In this study, we developed a modified screening method to address the drawbacks encountered with previous screening methods. A uniform white layer of PAHs was generated by spreading PAHs dissolved in volatile solvents over a surface of solidified agar medium, followed by the evaporation of the solvents. An inoculation was then performed by spreading a molten agar medium containing microbial samples over the solidified agar medium with a PAH layer. Subsequently, the white PAH layer migrated to the surface of the molten agar medium. This essential modification enabled us not only to solve problems of the previous screening methods but also to prepare an agar plate with a PAH layer without a complicated experimental scheme in the anaerobic chamber. After solidification of the molten agar medium and incubation of the plates, clear zones were successfully detected around colonies with aerobic and anaerobic PAH-degrading microbial cultures.     

Method of determining the antimicrobial activity of alcohol extracts of propolis]

It is proposed to determine the antimicrobial activity of propolys alcohol extracts by the method of subsequent dilutions in solid nutrient media. Dilution of the extracts immediately in hot agar eliminated the inhibiting effect of the extragent on the microbial growth. Opalesence appearing in the agar did not prevent estimation of the results in contrast to the method of subsequent dilutions in liquid nutrient media.     

Minituber production from immature seed suspension culture ofOrchis papilionacea

Summary Ripe and immature seeds ofOrchis papilionacea (method I and II, respectively) cultured on modified double strength Curtis medium were assayed for minituber production. Ripe seed germination both on solid and in liquid medium was low and the protocorms obtained developed into white calluses. Germination increased from, 9 to 33% when immature seed suspension culture was used. Protocorms obtained in suspension culture under light developed into minitubers, whereas those obtained on solid media developed into callus. A 30 s ultrasonication of immature seeds 1 wk after suspension, culture initiation further enhanced germination and minituber production. Minitubers had to be transferred and embedded in solid regeneration medium for normal growth.     

Influence of fungal isolates on germination and growth of perennial ryegrass

Summary The influence of fungi isolated from perennial ryegrass roots on the germination and development of seedlings of perennial ryegrass was investigated. The basic procedure employed was to sterilise the seed surface and then inoculate with fungi and plant in non-sterile soil. It was realised that the fungal isolate inoculated on to the sterile seed surface would not remain dominant in the root region of the host and would have an influence on the host which would decline with time from when the seed germinated. This was because it would have to face antagonism from the normal components of the root microflora present in the non-sterile soil.Trichoderma viride delayed the emergence of the seedlings and reduced the production of herbage, an observation consistent with results of some other investigators. A sterile hyaline fungus stimulated the emergence of the seedlings, but subsequent tests showed that the presence of the microflora of the seed coat, or the soil microflora, or the sterile hyaline fungus was effective in promoting the rate of germination of seed that had been surface sterilised. Leaching seed in water brought about an increase in the rate of seed germination, and it is suggested that there might be a germination inhibitor soluble in water present in the seed coat, which might be inactivated by saprophytic micro-organisms.     

Isolation and Characterization of Actinomycete Antagonists of a Fungal Root Pathogen

By use of selective media, 267 actinomycete strains were isolated from four rhizosphere-associated and four non-rhizosphere-associated British soils. Organic media with low nutrient concentrations were found to be best for isolating diverse actinomycetes while avoiding contamination and overgrowth of isolation media by eubacteria and fungi. While all isolates grew well at pHs 6.5 to 8.0, a few were unable to grow at pH 6.0 and a significant number failed to grow at pH 5.5. Eighty-two selected isolates were screened for in vitro antagonism towards Pythium ultimum by use of a Difco cornmeal agar assay procedure. Five isolates were very strong antagonists of the fungus, four were strong antagonists, and ten others were weakly antagonistic. The remaining isolates showed no antagonism by this assay. Additional studies showed that several of the P. ultimum antagonists also strongly inhibited growth of other root-pathogenic fungi. Twelve isolates showing antifungal activity in the in vitro assay were also tested for their effects on the germination and short-term growth of lettuce plants in glasshouse pot studies in the absence of pathogens. None of the actinomycetes prevented seed germination, although half of the isolates retarded seed germination and outgrowth of the plants by 1 to 3 days. During 18-day growth experiments, biomass yields of some actinomycete-inoculated plants were reduced in comparison with untreated control plants, although all plants appeared healthy and well rooted. None of the actinomycetes significantly enhanced plant growth over these short-term experiments. For some, but not all, actinomycetes, some correlations between delayed seed germination and reduced 18-day plant biomass yields were seen. For others, plant biomass yields were not reduced despite an actinomycete-associated delay in seed germination and plant outgrowth. Preliminary glasshouse experiments indicated that some of the actinomycetes protect germinating lettuce seeds against damping-off caused by P. ultimum.