维吾尔药茶昆仑雪菊ISSR分子标记体系的建立

研究适用于昆仑雪菊的ISSR-PCR反应体系,建立ISSR分子标记技术评价昆仑雪菊生态多样性体系。采用改良CTAB法、CTAB区室法、改良CTAB区室法、试剂盒法提取昆仑雪菊的新鲜叶片、干花以及种子的DNA;以827为引物,研究浓度、循环数、退火温度对ISSR-PCR反应体系的影响,以不同实验材料检测其适宜性。结果表明,昆仑雪菊新鲜叶片、干花以及种子的DNA的最佳提取方法为改良CTAB区室法,ISSR-PCR反应体系的最适引物浓度为1.0μmol·L-1,退火温度为54.6℃,循环数为30。ISSR-PCR反应体系适用于评价昆仑雪菊的生物多样性。     

A general,fast, and sensitive micromethod for DNA determination: Application to rat and mouse liver,rat hepatoma,human leukocytes,chicken fibroblasts,and yeast cells

A fluorometric method using 3,5-diaminobenzoic acid for DNA determination in tissues, cultured cells, nucleated blood cells, and yeast cells is described. The method is general, simple, and rapid, and does not require prior DNA extraction, since tissue is directly solubilized in Triton X-100 and ammonia. The procedure is highly sensitive, and is able to measure rather accurately as little as 10 ng of DNA. It is applicable to all types of DNA structure. The DNA content determined in various tissues and cells was: 2.50 mg/g fresh rat liver, 3.32 mg/g rat diethylnitrosamine-induced hepatoma, 2.49 mg/g fresh mouse liver, 8.76 μg/10⁶ human leukocytes, 3.37 μg/10⁶ chicken fibroblasts, 2.97 μg/10⁸ haploid yeast cells, and 2.84 μg/10⁸ haploid yeast protoplasts.     

DNA Extraction from small blood volumes and the processing of cellulose blood cards for use in polymerase chain reaction

This article describes two procedures for the purification of genomic DNA from small blood volumes of whole blood using DNAzol^®BD. In the first procedure, DNA is isolated from 1–20 μL of whole blood using a fast and simple protocol that is appropriate for the simultaneous extraction of a large number of samples. The isolated DNA is suitable for gel electrophoresis and polymerase chain reaction (PCR). In the second procedure, cellulose blood cards containing approx 5 μL of dried blood are treated with DNAzol BD in order to retain DNA on the cellulose matrix while removing other cellular components. The blood card with DNA subsequently serves as template in PCR. The blood card processing and amplification procedures are performed in the same PCR tube without any centrifugation steps, making the combined procedures amenable for automated DNA preparation and amplification in a single tube.     

Practical methodology for gametophyte proliferation and sporophyte production in green penny fern (Lemmaphyllum microphyllum C. Presl) using mechanical fragmentation

Propagation of gametophytes and sporophytes using mechanical fragmentation has been considered a suitable method for mass production of ferns. This study aimed to develop a practical propagation method for Lemmaphyllum microphyllum C. Presl, which is a fern of significant ornamental and medicinal value. Gametophytes were obtained through in vitro spore germination and used for propagation experiments. The gametophyte was mechanically fragmented using a scalpel into small fragments, which were then used to investigate gametophyte proliferation. In addition, the gametophyte was fragmented using a blender and then used to study sporophyte formation. Optimal proliferation conditions of the gametophyte were determined using Murashige and Skoog (MS) basal medium (double-, full-, half-, quarter-strength), Knop medium, and medium components (sucrose, nitrogen sources, activated charcoal), at various concentrations. The fresh weight of the gametophyte was 14-fold higher than that of gametophytes (300 mg) used as culture material, when cultured on double-strength MS. Moreover, 1 g of the gametophyte fragmented in 25 mL of distilled water formed more than 430 sporophytes in a soil mixture in an area of 7.5 cm². The sporophytes were successfully cultivated in the greenhouse after acclimation. A large-scale production method for L. microphyllum that can be easily implemented in a fern production farm is outlined.

     

An Efficient and Reliable Method of DNA Extraction from Meyna spinosa — A Traditional Medicinal Plant from North-East India

A simple, efficient and reliable CTAB method is standardized for genomic DNA isolation from fresh young leaves of a traditional medicinal plant Meyna spinosa. Key steps in the modified procedure include additional chloroform: isoamyl alcohol (24:1, v/v) extraction, addition of 4% PVP in the extraction buffer and an overnight isopropanol precipitation at room temperature. This procedure yields a high amount (46 μg DNA g^?1 fresh leaf tissue) of good quality DNA free from contaminants. The isolated DNA is suitable for digestion with EcoRI and HindIII restriction enzymes and can be used in other DNA manipulation techniques.     

Identification of genes expressed in the angiosperm female gametophyte

Until recently, identification of gene regulatory networks controlling the development of the angiosperm female gametophyte has presented a significant challenge to the plant biology community. The angiosperm female gametophyte is fairly inaccessible because it is a highly reduced structure relative to the sporophyte and is embedded within multiple layers of the sporophytic tissue of the ovule. Moreover, although mutations affecting the female gametophyte can be readily isolated, their analysis can be difficult because most affect genes involved in basic cellular processes that are also required in the diploid sporophyte. In recent years, expression-based approaches in multiple species have begun to uncover gene sets expressed in specific female gametophyte cells as a means of identifying regulatory networks controlling cell differentiation in the female gametophyte. Here, recent efforts to identify and analyse gene expression programmes in the Arabidopsis female gametophyte are reviewed.     

Squash Techniques in the Cytological Investigation of Mosses

Abstract

Photosynthetic activity of attached sporophytes is very low (a few per cent or less) compared with that of associated gametophyte structures (perianth, bracts and uppermost leaves) in Cephalozia bicuspidata and Lophocolea heterophylla, or with even small areas of thallus in Pellia epiphylla. Photosynthetic uptake of ¹⁴CO₂ by developing sporophytes of P. epiphylla, C. bicuspidata and L. heterophylla is at most a few per cent of the ¹⁴C translocated subsequently from the gametophyte, and could be negligible. In L. heterophylla, the perianth, bracts and uppermost leaves appear to play only a limited role in nutrition of the sporophyte, the leafy shoots making a major contribution. In C. bicuspidata the perianth and leaves of the short archegonial shoot may provide a substantial part of the nutrition of the sporophyte. There is some indication in all three species that translocation from the gametophyte is most active when the sporophyte reaches full size but is still green, declining in the final stages of maturation of the capsule.     

The development of the placenta in the anthocerote Phaeoceros laevis (L.) Prosk

The development of the placenta in the anthocerote Phaeoceros laevis (L.) Prosk. was studied by transmission electron microscopy. By the time the sporophyte emerges from the involucre, a conspicuous placental region is formed by the intrusive growth of sporophyte foot haustorial cells into the adjacent gametophyte vaginula tissue. The separation of gametophyte cells by haustorial cells and their incorporation into the placenta are preceded by the loosening and swelling of their walls and the formation of a periplasmic space. This process causes the disruption of the plasmodesmata, and may eventually result in the complete isolation and consequent degeneration of the cells. Crystals are commonly observed in the vacuoles of gametophyte placental cells. Crystals become more abundant during cytoplasmic degeneration, and are released in the placental lacunae that result from the complete dissolution of gametophyte cells. During the subsequent phase of capsule elongation, the gametophyte placental cells that retain the symplastic connection with the adjoining gametophyte parenchyma develop a wall labyrinth typical of transfer cells. Obliteration of the wall labyrinth by deposition of lightly staining wall material is observed later in sporophyte development, in concomitance with capsule dehiscence. Crystals are negative to the periodic acid/thiocarbohydrazide/silver proteinate test for carbohydrates whilst they are completely digested by pepsin or protease, denoting protein composition.Abbreviation PATAg periodic acid/thiocarbohydrazide/silver proteinate     

Meiospores produced in sori of nonsporophyllous laminae of Macrocystis pyrifera (Laminariales,Phaeophyceae) may enhance reproductive output

Different lamina of Macrocystis pyrifera sporophytes (i.e., sporophylls, pneumatocyst‐bearing blades, and apical scimitars) in a wave‐sheltered site were found to be fertile. We quantified their sorus surface area, reproductive output (number of spores released) and the viability of released spores (germination rate). Sorus area was greatest on the sporophylls, with sporangia developing on >57% of the total area and smallest on the pneumatocyst‐bearing blades with 21% of the total area bearing sporangia. The apical scimitar released the greatest number of meiospores (cells · mL^?1 · cm^?2) and the sporophylls the least. Meiospores produced from all types of fertile laminae were equally viable. This reproductive plasticity may enhance reproductive output, and contribute to short and long‐distance spore dispersal and the cryptic gametophyte propagule bank for the next generation of sporophytes.     

Comparative development of the sporophyte–gametophyte junction in six moss species

Developmental anatomy of the sporophyte–gametophyte junction in six moss species is described with special reference to sporophyte penetration into the gametophytic tissue. The sporophyte–gametophyte junction in mosses is classified into two types based on vaginula morphology: in the “true vaginula” type, the junction involves only an epigonium derived from the archegonium, and in the other “shoot vaginula” type, it involves a shoot and an epigonium. In both of the types, the sporophyte penetrates into an epigonial tissue accompanied by degeneration of epigonium cells under the developing sporophyte. In the “shoot vaginula” type, the sporophyte further penetrates into the conducting strand or similar cells that seem to be induced by stimulation of fertilization. It is likely that the difference in growth rate between the epigonium and the capped sporophyte is a mechanical force for sporophyte penetration.     

African Hepatics: VI. Euosmolejeunea

Abstract

(1) The ratio of ¹⁴CO₂ uptake of fully expanded capsules to that of corresponding gametophytes in the species studied was about 1 : 15 in Mnium hornum, 1 : 35 in Pleurldium acuminatum and around 1 : 1 in Funaria hygrometriea.

(2) Translocation of photosynthate from gametophyte to sporophyte took place within 1 to 4 days of exposure to ¹⁴CO₂; in expanded capsules the final activity was at least equal to that remaining in the gametophyte.

(3) In M hornum there was no evidence of reverse translocation from sporophyte to gametophyte.

(4) Translocation from gametophyte continued at a high level throughout the expansion and differentiation of the capsule in M. hornum. The activity reaching the sporophyte was approximately proportional to its fresh weight.

(5) Both in situ photosynthesis and translocation to the capsule of F. hygrometrica rose to a maximum during the latter part of capsule expansion and ensuing differentiation, subsequently declining as the green tissues of the capsule senesced.

(6) Fully expanded (but premeiotic) capsules of M. hornum contributed approximately 20% of the assiinilate necessary for their growth. The corresponding figure for P. acuminatum was about 10%, and for F. hygrometrica about 50%.     

ROLE OF SETTLEMENT DENSITY ON GAMETOPHYTE GROWTH AND REPRODUCTION IN THE KELPS PTERYGOPHORA CALIFORNICA AND MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

Laboratory studies were used to examine how variation in the density of spore settlement influences gametophyte growth, reproduction, and subsequent sporophyte production in the kelps Pterygophora californica Ruprecht and Macrocystis pyrifera (L.) C. Ag. In still (non-aerated) cultures, egg maturation in both species was delayed when spores were seeded at densities 300 · mm^?2. Although the density at which this inhibition was first observed was similar for both species, the age at which their eggs matured was not. P. californica females reached sexual maturity an average of 4 days (or ～ 30%) sooner than did M, pyrifera. As observed previously in field experiments, per capita sporophyte production was negatively density dependent for both species when seeded at spore densities of 10 · mm^?2. Total sporophyte production (i.e. number · cm^?2) for both species, however, was greatest at intermediate densities of spore settlement (～ 50 spores · mm^?2). In contrast, total sporophyte production by P. californica steadily increased with increasing spore density in aerated cultures; highest sporophyte density was observed on slides seeded at a density of 1000 spores · mm^?2. Preliminary experiments with P. californica involving manipulation of aeration and nutrients indicate that inhibition of gametophyte growth and reproduction at higher densities of spore settlement in non-aerated cultures was probably caused by nutrient limitation.     

Distribution of Potassium, Calcium and Magnesium in the Gametophyte and Sporophyte Generations of Funaria hygrometrica Hedw

Analyses have been made of the K, Ca and Mg in separated sporophytes,spores and gametophytes of the moss Funaria hygrometrica duringsporophyte development. Degeneration of the gametophyte wasaccompanied by loss of K and a gain in Ca while the K contentof the developing sporophyte increased more rapidly than thatof Ca. The presence of an air–gap in the expanding capsulesignificantly influenced the observed cellular location of ions.Relative to the sporophyte the spores were shown to have a higherK and a lower Ca concentration. The behaviour of Mg was intermediatebetween K and Ca throughout. Funaria hygrometrica, potassium, calcium, magnesium, cation distribution, gametophyte, sporophyte     

Life Cycle and Morphology of Bryopsidella ostreobiformis spec. nov. (Bryopsidaceae,Chlorophyta) from the Mediterranean,under Culture Conditions,with Comments on the Phylogeny of the Bryopsis/Derbesia Complex

Bryopsidella ostreobiformis sp. nov. is presented as the second species of the genus. The latter is amended to accomodate this new species. The derbesioid sporophyte is capable of producing three- to eight-rayed stellate propagules. The dioecious gametophyte is a heterotrichous thallus with branched, prostrate filaments and unbranched, erect ones, and does not resemble Bryopsis. The life cycle is distinctly diplohaplontic, with a diploid sporophyte and a haploid gametophyte. B. ostreobiformis is postulated as the starting point of a hypothetical evolutionary sequence of life histories in the Derbesia/Bryopsis complex.     

<Emphasis Type="Italic">Macrocystis</Emphasis> mariculture in Chile: growth performance of heterosis genotype constructs under field conditions

Recent progress in Macrocystis mariculture is based on clonal stock cultures of gametophyte parents. Batches of up to 10⁵ genetically identical sporophyte seedlings can be produced at any time in the laboratory and explanted in the field for production of biomass. Sexual crosses of selected Macrocystis pyrifera gametophyte parents of different geographic origin along the coast of Chile showed heterosis and produced sporophyte batches with superior growth performance. Starting from zygotes, after 10 weeks in the laboratory and 5 months in the sea, our best hybrid genotypes grew up to 11 kg fresh weight per frond, which corresponds to 66 kg m⁻¹ of line in a commercial mariculture installation. In contrast, average yields of 14.4 and 22 kg m⁻¹ are reported in the literature for traditional methods. Additional experiments, including inter-specific crosses M. pyrifera × M. integrifolia and their performance in different climate zones of Chile, confirm that heterosis is a powerful tool for crop improvement in Macrocystis. It opens the possibility to construct tailor-made heterosis genotypes with maximum productivity and/or other desired properties for any given locality.     

Generating Autotetraploid Sporophytes and Their Use in Analyzing Mutations Affecting Gametophyte Development in the Fern Ceratopteris

The haploid gametophytes of the fern Ceratopteris richardii are autotrophic and develop independently of the diploid sporophyte plant. While haploid genetics is useful for screening and characterizing mutations affecting gametophyte development in Ceratopteris, it is difficult to assess whether a gametophytic mutation is dominant or recessive or to determine allelism by complementation analysis in a haploid organism. This report describes how apospory can be used to produce genetically marked polyploid sporophytes whose gametophyte progeny are heterozygous for mutations affecting sex determination in the gametophyte and a known recessive mutation affecting the phenotype of both the gametophyte and sporophyte. The segregation ratios of wild-type to mutant phenotypes in the gametophyte progeny of polyploid sporophyte plants indicate that all of the mutations examined are recessive. The presence of many multivalents and few univalents in meiotic chromosome preparations of spore mother cells confirm that the sporophyte plants assayed are polyploid. The DNA content of the sperm of their progeny gametophytes was also found to be approximately twice that of sperm from wild-type haploid gametophytes.     

Protein composition of different stages in the life cycle of Ulva mutabilis,Føyn

Summary Soluble protein, about 15% of the total cellular protein, was extracted from different stages in the haplodiplontic life cycle of Ulva mutabilis. The electrophoretic band pattern of the protein extracts from the haploid gametophyte and the diploid sporophyte were found to be the same, except for one slow moving band present in the gametophyte, but lacking in the diploid sporophyte. This band was also missing in the extract from the haploid parthenosporophyte, but is seen in the extract from the zoospores. It was found that the synthesis of the protein in this band occurred during most of the preparation period preceding meiosis. The band is not seen in extracts from gametes, and it is inferred that this protein is broken down during the period preceding the mitotic gametangial division in the gametophyte. So far the protein making up this band behaves as should be required for a factor determining the shift in generation during the life cycle.     

CUTICLES FROM CHONDRUS CRISPUS (RHODOPHYTA)1

A simple, nondestructive physical process was developed for routinely isolating the outermost layers from female, male, and sporophyte fronds of Chondrus crispus Stack-house. Yields of pure cuticles from apical segments ranged from 0.74 to 2.35% on a dry weight basis after 5–7 d of culture. These undegraded cuticles were examined by electron microscopy (scanning and transmission electron microscopy), spectroscopy (infrared and X-ray), and chemical means. Cuticles isolated from female or male fronds were characterized by parallel arrays of electron-dense lamellae (typically 6–14) alternating with more electron-transparent regions. The thickness and uniformity of these lamellae provide the physical basis for the iridescence characteristic of C. crispus fronds. Sporophyte fronds are not iridescent. This phenomenon may be explained by the fewer electron-dense cuticular lamellae (usually three to seven) and the fact that these lamellae anastomose freely to form a thin cuticle with a highly irregular substructure. Elements detected by X-ray analysis, in addition to carbon and oxygen, included Mg, Br, S, and Ca in both gametophyte and sporophyte cuticles. Major features of FTIR spectra of all cuticles were absorbances due to proteins. A strong band, indicative of sulfate ester, occurred near 1250 cm^?1 in all cuticle preparations. Gametophyte, but not sporophyte, cuticles absorbed at 935, 846, and 800 cm^?1 consistent with the presence of kappa and/or iota carrageenan. Amino acid analyses showed that sporophyte and gametophyte cuticles were generally similar in gross composition. All contained proline as the principal residue together with significant amounts of cysteine, methionine, and lysine. Protein contents calculated from these analyses ranged from 37.6 to 44.4% on a dry weight basis as compared to 51.5–56.7% calculated from total nitrogen values. Up to 75% of the cuticle mass was solubilized by sodium dodecyl sulfate-β-mercaptoethanol. Three similar migrating bands were seen in female and male cuticle extracts on sodium dodecyl sulfate–polyacrylamide gel electrophoresis; however, none of the three weaker bands from sporophyte cuticles comigrated with those from gametophytes. Chloroform-methanol extraction removed < 3.3% of the cuticle mass, suggesting that lipids were minor components.     

Assessment of photosynthetic performance in the two life history stages of Alaria crassifolia (Laminariales,Phaeophyceae)

Understanding of the physiological responses of kelp to environmental parameters is crucial, especially in the context of environmental change that may have contributed to the decline of kelp forests all over the world. The current study presents the photosynthetic characteristics of the macroscopic sporophyte and microscopic gametophyte stages of the brown alga Alaria crassifolia from Hokkaido, Japan, as determined by examining their photosynthetic responses over a range of temperature and irradiance using dissolved oxygen and chlorophyll fluorescence measurements. Net photosynthetic rates of the sporophyte were consistently higher than those of gametophyte across temperature gradients and irradiance levels. Photosynthesis–irradiance curves at 8°C, 16°C, and 20°C revealed similar initial slopes (α = 0.4–0.9) on the two life history stages, but higher compensation (E _c = 4–7 μmol photons m^?2 s^?1) and saturation irradiances (E _k = 53–103 μmol photons m^?2 s^?1) for the sporophyte than for the gametophyte (E _c = 0–7 μmol photons m^?2 s^?1; E _k = 7–10 μmol photons m^?2 s^?1). Both stages exhibited chronic photoinhibition, as shown by the failure of recovery in their maximum quantum yields (F _v/F _m) following high irradiance stress, with greater possibility of photodamage at low temperature. Gametophytes were less sensitive to low temperatures than sporophytes, given their relatively stable F _v/F _m response. Nevertheless, temperature optima for photosynthesis of both stages coincide with each other at 20–23°C, which correspond to the growth and maturation periods of A. crassifolia in Japan. This species is also likely to suffer from thermal inhibition as both GP rates and F _v/F _m decreased above 24°C.     

A rapid and inexpensive method for isolation of total DNA from dehydrated plant tissue

We describe an inexpensive method for dehydration of plant tissue and extraction of high molecular weight DNA. Tissue is dried for 12 to 24 hours in a food dehydrator and subsequently powdered for DNA extraction. Dicot tissue can be powdered in centrifuge tubesen masse using a commercial paint mixer and glass beads. With the use of the paint mixer, tissue never touches common surfaces that might lead to cross contamination, a potential benefit when the DNA is to be used for PCR reactions. The DNA is of a quality equal to that obtained from either lyophilized or fresh frozen tissue (commonly used in many labs). The advantages of the described procedure are that it is fast, does not require expensive equipment (e.g., lyophilizer) and can be used in situations where large numbers of samples must be extracted.