Cryopreservation of Fern Spores and Pollen.

Fern spores and pollen are haploid plant germplasm of microscopic nature that can be used to regenerate full plants through germination (fern spores) or to fertilize seed-bearing plants through breeding programs (pollen). Due to their short life span in conventional storage (i.e., dry at -20 °C), the use of cryopreservation has been indicated for long-term ex situ conservation. While fern spores of most species and pollen from many seeded plants tolerate desiccation and can be stored dry at liquid nitrogen temperatures, some pollen is desiccation sensitive, and cryopreservation protocols require controlled drying and cooling and some level of cryoprotection. In this chapter we describe the cryopreservation process for fern spores used in the Millennium Seed Bank of Royal Botanic Gardens, Kew, including some details of the fern spores harvest and cleaning methods. In addition, two protocols for pollen cryopreservation are described, one generic for desiccation-tolerant pollen that can be used for multiple species and one specific for a desiccation sensitive pollen (Zea mays).

Macrocyclic Trichothecenes from Myrothecium roridum Strain M10 with Motility Inhibitory and Zoosporicidal Activities against Phytophthora nicotianae.

The cytotoxicity of the extract obtained from Myrothecium roridum M10 and a characteristic (1)H signal at δH ∼8 led to the assumption that verrucarin/roridin-type compounds were present. Upscaling on rice medium led to the isolation of four new metabolites: verrucarins Y (1) and Z (6) (macrocyclic trichothecenes), bilain D (12) (a diketopiperazine derivative), and hamavellone C (14) (an unusual cyclopropyl diketone). In addition, nine known trichothecenes [verrucarin A (3), 16-hydroxyverrucarin A (5), verrucarin B (7), 16-hydroxyverrucarin B (8), verrucarin J (2), verrucarin X (4), roridin A (9), roridin L-2 (10), and trichoverritone (11)] and a bicyclic lactone [myrotheciumone A (15)] were identified. Their structures and configurations were determined by spectroscopic methods, published data, Mosher's method, and considering biosyntheses. Some trichothecenes showed motility inhibition followed by lysis of the zoospores against devastating Phytophthora nicotianae within 5 min. Compounds 2, 3, 7, and 9 also exhibited potent activities against Candida albicans and Mucor miehei.

Posttranscriptional control over rapid development and ciliogenesis in Marsilea.

Marsilea vestita is a semiaquatic fern that produces its spores (meiotic products) as it undergoes a process of natural desiccation. During the period of desiccation, the spores mature, and produce large quantities of pre-mRNA, which is partially processed and stored in nuclear speckles and can remain stable during a period of extended quiescence in the dry spore. Rehydration of the spores initiates a highly coordinated developmental program, featuring nine successive mitotic division cycles that occur at precise times and in precise planes within the spore wall to produce 39 cells, 32 of which are spermatids. The spermatids then undergo de novo basal body formation, the assembly of a massive cytoskeleton, nuclear and cell elongation, and finally ciliogenesis, before being released from the spore wall. The entire developmental program requires only 11 h to reach completion, and is synchronous in a population of spores rehydrated at the same time. Rapid development in this endosporic gametophyte is controlled posttranscriptionally, where stored pre-mRNAs, many of which are intron-retaining transcripts, are unmasked, processed, and translated under tight spatial and temporal control. Here, we describe posttranscriptional mechanisms that exert temporal and spatial control over this developmental program, which culminates in the production of ∼140 ciliary axonemes in each spermatozoid.

[Cytogenetic peculiarities of cell genesis in apical meristems under gametophytic apomixis (using autonomous apomicts of the Asteraceae as an example)].

Cytogenetic peculiarities of cell genesis in apical meristems of apomicts has been analyzed using a series of the Asteraceae species as an example. The extent to which aneu- and mixoploids are spread among plants in the investigated populations of the Asteraceae species is so high (up to 30-60% of the studied plants and their offspring), that it seems reasonable to suppose that their rise is a natural phenomenon. It has been shown that in the aposporous facultative apomict Pilosella officinarum microgametophyte is a relatively stable element of the seed reproduction system from the point of view of caryotypical variation.

[Observation on gametophyte development of Cibotium barometz].

OBJECTIVE: To observe the spores germinating process of Cibotium barometz, and understand the growth principle provided for experience for indoor culturing and further research. METHOD: The spores of C. barometz were cultured both in inorganic medium and in the soil from original habitat, and the whole process of spores germination and the development of gametophytic were observed under microscope. RESULT: The spores germinated about 1-2 weeks after being sowed, and the type of germination belonged to Vittaria-type. The prothallial plates formed in 25 days after being sowed, while hairs developed after the formation of the prothallial plate. The gametophyte formed about 40 days after being sowed. But the type of mature prothalli was cordate. The antheridia formed in 60 days after inoculation, while the archegonia developed in 10 days after the formation of antheridia. CONCLUSION: Soil based indoor culturing of C. barometz spores is practical and can be used for cultivation of C. barometz.

[Reactive oxygen forms and luminescence of intact microspore cells]. / Aktivnye formy kisloroda i liuminestsentsiia intaktnykh kletok mikrospor.

The participation of reactive oxygen species (ROS) in luminescence (chemiluminescence and autofluorescence induced by ultraviolet light of 360-380 nm) was analyzed. Microspores, the pollen (male gametophyte) of Hippeastrum hybridum, Philadelphus grandiflorus, and Betula verrucosa and vegetative microspores of the spore-breeding plant Equisetum arvense served as models. It was found that the addition of the chemiluminescent probe lucigenin, which luminesces in the presence of superoxide anionradicals, leads to intensive chemiluminescence of microspores. No emission was observed in the absence of lucigenin and in the presence of the dye luminol as a chemiluminescent probe. The emission decreased significantly if superoxide dismutase, an enzyme of the superoxide anionradical dismutation during which this radical disappeared, was added before the dye addition. The autofluorescence intensity of microspores decreased in the presence of both superoxide dismutase and peroxidase, an enzyme destroying hydrogen peroxide and organic peroxides. The most significant effect was noted after the addition of peroxidase, which indicates a greater contribution of peroxides to this type of emission. The fumigation with ozone, which increases the amount of ROS on the cell surface, enhanced the intensity of the chemiluminescence of microspores with lucigenin, but decreased the intensity of the autofluorescence of microspores. Exogenous peroxides (hydrogen peroxide and tert-butylhydroperoxide) stimulated the autofluorescence of pollen and vegetative spores in a concentration-dependent manner. It was shown that the formation of ROS contributes to the luminescence of plant microspores, which reflects their functional state.

[Peculiarities of embryonal development in vitro of Brassica napus pollen grains]. / Osobennosti embriogennogo razvitiia in vitro pyl'tsevykh zeren Brassica napus.

Literature data concerning the problem of the embryogenesis in microspore cultures of Brassica were reviewed. The influence of stress factors on the microspore cultures was examined. The some cytological and morphological peculiarity microspore entering in the embryogenesis developmental pathway were described.

The Arabidopsis thaliana gametophytic mutation gemini pollen1 disrupts microspore polarity, division asymmetry and pollen cell fate.

Pollen development and male gametogenesis are critically dependent upon cell polarization leading to a highly asymmetric cell division termed pollen mitosis I. A mutational approach was adopted in Arabidopsis thaliana to identify genes involved these processes. Four independent gemini pollen mutants were isolated which produce divided or twin-celled pollen. The gemini pollen1 mutant was characterized in detail and shown to act gametophytically resulting in reduced transmission through both sexes. gemini pollen1 showed an incompletely penetrant phenotype resulting in equal, unequal and partial divisions at pollen mitosis I. The division planes in gemini pollen1 were shown to be aligned with the polar axis (as in wild type) and evidence was obtained for incomplete nuclear migration, which could account for altered division symmetry. gemini pollen1 also showed division phenotypes consistent with spatial uncoupling of karyokinesis and cytokinesis suggesting that GEMINI POLLEN1 may be required for the localization of phragmoplast activity. Cell fate studies showed that in both equal and unequal divisions a vegetative cell marker gene was activated in both daughter cells. Daughter cells with a range of intermediate or hybrid vegetative/generative cell fates suggests that cell fate is quantitatively related to cell size. The potential mode of action of GEMINI POLLEN1 and its effects on cell fate are discussed in relation to proposed models of microspore polarity and cell fate determination.

[The results of the cytogenetic monitoring of the species of angiosperm plants growing in the area of the radionuclide contamination after the accident at the Chernobyl Atomic Electric Power Station]. / Rezul'taty tsitogeneticheskogo monitoringa vidov pokrytosemennykh rastenii, proizrastaiushchikh v zone radionuklidnogo zagriazneniia posle avarii na Chernobyl'skoi AES.

Results of investigations of meiosis in microsporogenesis, sporads and pollen grains as well as mitosis in root meristematic cells in 94 species of angiosperms (from 28 families) growing in different ecological conditions in the zone with higher radionuclide pollution after the Ch.NPS accident are present. The low percentage (0.5-2.0%) of anomalies on investigating parameters has been revealed in the majority of species, only in certain species (8-10%) the correlation between an increase of the number of anomalies and an increase of the gamma-radiation level was shown. The data obtained make evident the resistance of the seed reproduction system in higher plants to chronic irradiation by the low doses of ionising radiation.

Spore appendages and taxonomy of Clostridium sordellii.

Twenty-five strains of Clostridium sordellii were divided into two groups on the basis of spore fine structure. Sixteen strains formed spores with smooth tubular appendages, and nine strains formed spores which lacked appendages. The other properties of the 25 strains were relatively constant. Since the minor strain variability which was encountered did not correlate with spore appendage status, fragmentation of this species on the basis of spore appendage status is not advocated.

Association of the crystalline inclusion of Bacillus thuringiensis with the exosporium.

Thin sections of sporulating cells of Bacillus thuringiensis show a marked association between the crystalline inclusion and the exosporium.