Localization of the Ca(2+)-binding protein, Bra r 1, in anthers and pollen tubes.

Calcium plays an essential role during pollen development and pollen tube growth, and several Ca(2+)-binding proteins are expressed in anthers. We have previously reported that Brassica pollen allergens encoded by Bra r 1 and Bra r 2 show sequence similarities to Ca(2+)-binding proteins [Toriyama et al. (1995) Plant Mol. Biol. 29: 1157]. Herein, we report that both genes are expressed in the diploid tapetum and haploid microspores, as detected by in situ RNA hybridization. Immunoblot analysis revealed that Bra r 1 and Bra r 2 were accumulated in anthers during pollen development. When pollen grains were suspended in an aqueous solution, both proteins were mainly detected in the pollen extracellular fraction, indicating that Bra r 1 and Bra r 2 are released from the pollen upon hydration. Localization of Bra r 1 was further investigated in sections of anthers and pollen tubes. Bra r 1 was detected in the tapetum, microspores and pollen grains. In longitudinal sections of cross-pollinated pistils. Bra r 1 was detected throughout pollen tubes elongating in transmitting-tissue. These findings suggest that Bra r 1 may be involved in pollen-pistil interaction and pollen tube growth.

Isolation and collection of two populations of viable sperm cells from the pollen of Plumbago zeylanica.

A protocol is described for individually collecting two populations of sperm cells, Svn and Sua, from pollen of Plumbago zeylanica. Pollen grains were burst in 10 mM MOPS buffer containing 0.8 M mannitol (pH 4.6). Paired sperm cells released from pollen were separated using a microinjector. Svn and Sua were then collected individually with a microinjector, based upon known size differences. Collected sperm cells were washed with isolation medium and transferred to liquid nitrogen until use. Fluorochromatic reaction (FCR) test of isolated sperm cells showed a positive reaction, indicating that the isolated sperm cells are viable; most of the sperm cells retain viability for at least 2 h.

Isolation of viable sperm cells from tobacco (Nicotiana tabacum).

Viable sperm cells of Nicotiana tabacum were isolated by the semi-vivo technique. After pollination, excised styles were floated, cut end immersed, in a solution of 15% sucrose with 0.01% boric acid and 0.03% Ca(NO3)2 at 27 degrees C in a growth chamber until pollen tubes emerged. After sperm cells were formed (at least 8 h after pollination) tubes were immersed in a 9% mannitol solution. In this solution, sperm cells are nearly ellipsoidal and retain viability for over 6 h.

Three-dimensional ultrastructure of generative cell mitosis in the pollen tube of Nicotiana tabacum.

Generative cell mitosis was examined in stylar-grown pollen tubes of Nicotiana tabacum using serial sectioning, transmission electron microscopy and computer-assisted reconstruction. Before mitosis, the generative cell has a cage-like organization of cytoplasmic microtubules. The mitotic spindle forms when the cytoplasmic microtubules reduce in frequency and kinetochore microtubules form in an area delimited by sheets of endoplasmic reticulum; no preprophase band of microtubules is observed. At metaphase, 21 pairs of kinetochores are distributed unevenly along the length and depth of the cell without the formation of a strictly planar metaphase plate. The metaphase spindle is highly oblique, with diffuse subpoles distributed along the sides of the cell, colocalized with endoplasmic reticulum lamellae. From these dispersed subpoles the kinetochore bundles emanate, closely associated with tubular endoplasmic reticulum. Anaphase consists of three principal processes: convergence of diffused mitotic poles, shortening of the kinetochore bundles, and the elongation of the spindle by an average of nearly 50%. At mid-anaphase, a phragmoplast begins to form, mainly by the assembly of new microtubules at the equatorial area, which form as a cluster of numerous short microtubules. Cytokinesis is essentially conventional, with centrifugal cell plate formation. Cytoplasmic microtubules are restored in the newly formed "brother" sperm cells in a distribution similar to that in the generative cell but fewer in number.

Cytoskeletal organisation and modification during pollen tube arrival, gamete delivery and fertilisation in Plumbago zeylanica.

The cytoskeletal organisation of the isolated embryo sac and egg cells of Plumbago zeylanica was examined before, during and after pollen tube penetration into the embryo sac to determine the potential involvement of microtubules and actin filaments in fertilisation. Material was singly and triply stained using Hoechst 33258 to localise DNA, fluorescein isothiocyanate (FITC)-labelled anti-alpha-tubulin to detect microtubules and rhodamine-phalloidin to visualise F-actin. Microtubules in the unfertilised egg cell are longitudinally aligned in the micropylar and mid-lateral areas, aggregating into bundles near the filiform apparatus. In the perinuclear cytoplasm of the egg cell, microtubules become more or less randomly aligned. F-actin bundles form a longitudinally aligned mesh in the chalazal cytoplasm of the egg cell. In the central cell, microtubules and F-actin are distributed along transvacuolar strands and are also evident in the perinuclear region and at the periphery of the cell. During pollen tube penetration, sparse microtubule bundles near the pathway of the pollen tube may form an apparent microtubular 'conduit' surrounding the male gametes at the delivery site. Actin aggregates become organised near the pathway of the pollen tube and at the delivery site of the sperm cells. Subsequently, actin aggregates form a 'corona' structure in the intercellular region between the egg and central cell where gametic fusion occurs. The corona may have a role in maintaining the close proximity of the egg and central cell and helping the two sperm cells move and bind to their target cells. The cytoskeleton may also be involved in causing the two nuclei of the egg and central cell to approach one another at the site of gametic fusion and transporting the two sperm nuclei into alignment with their respective female nucleus. The cytoskeleton is reorganised during early embryogenesis.

A method for improved resolution for fluorescence microscopy using plastic-embedded material subjected to resin extraction.

A protocol is given that uses NaOH, benzene, acetone and methanol to extract epoxy resins from semithin sections. Such sections appear superior to paraffin or unsectioned materials for fluorescence microscopic observations. Use of ultrarapid films (e.g., Kodak T-Max P3200) at ISO 3200 minimizes fading without use of antifading agents and without introducing unacceptable photographic grain size.

Curing and commerce: changes in an indigenous medical practice in a highland Philippine town.

The intrusion of the capitalist mode of production in Third World countries is usually accompanied by the expansion of cosmopolitan medicine. In most multi-ethnic societies, indigenous medical practices continue to persist, if not thrive, under these conditions for a variety of reasons. Urbanizing areas in particular may become focal points for the emergence of innovative role adaptations among indigenous curers. This paper describes the career development of an indigenous medical specialist in a highland Philippine town. The purpose is to suggest the importance of examining the lifestyle and socio-economic strategies pursued by curers in similar contexts. Such analysis can aid in the process of understanding the way in which particular individuals can perform significant buffer roles that promote the continued viability of medically plural practices and beliefs.