A Cytochemical Study of DNA, RNA, and Protein in the Developing Maize Anther: II. Observations

Changes in acetic-alcohol fixable DNA, RNA, and protein werefollowed in the tapetum, sporogenous tissue, and spores of thedeveloping maize anther using standard cytochemical methodsand microdensitometry. In the tapetum, early nuclear divisionsoccur without prior DNA synthesis, giving a population of IC nuclei. Subsequent synthesis produces the equivalent of 34,000C amounts per pollen sac, 20 times more than is present in thespores before pollen mitosis. The main tapetal RNA synthesisis during the meiotic prophase, with a further period of accumulationin the interval, tetrad to young spores. In the meiocytes, theprincipal accumulation is in the early prophase, with no synthesisduring the meiotic divisions or through the tetrad period. Proteinaccumulation occurs in the tapetum up to mid-meiotic prophase;after this there is a pause, followed by further synthesis frommeiotic metaphase I to the final dissolution of the tissue.In the meiocytes, protein is accumulated through the early prophase;there is no synthesis during the meiotic mitoses or in the tetradperiod, but active accumula-tion occurs in the developing spores. The implications of these observations are discussed in relationto the function of the tapetum.     

Identification of the Genomic Constitution ofMusaL. Lines (Bananas, Plantains and Hybrids) Using Molecular Cytogenetics

The genomic constitutions of someMusaL. lines (bananas, plantainsand artificial hybrids) were identified using molecular cytogenetictechniques. Double targetin situDNA:DNA hybridization to chromosomespreads using as probes, total genomic DNA isolated from diploidMusalinesof known AA (labelled with biotin-11-dUTP) and BB (labelledwith digoxigenin-11-dUTP) genome constitution was carried out.The use of 60% acetic acid combined with heating over a flamegave high quality chromosome spreads free of cytoplasm forinsituhybridization. Total genomic A DNA labelled broad centromericregions of all 22 chromosomes of the diploid line, Calcutta4 (M. acuminataColla. ssp.burmanniccoides; A genome) with somechromosomes showing stronger hybridization. Labelled DNA fromthe B genome hybridized strongly to the centromeric regionsof all 22 chromosomes of Butohan 2 (M. balbisianaColla; B genome).The two satellited chromosomes of genome B labelled stronglywith genomic A DNA.In situhybridization of labelled A and Bgenomic DNA to metaphase chromosomes of triploid AAB and ABBcultivars discriminated between A and B genome chromosomes.The plantains Agbagba, Obino l'Ewai and Mbi Egome showed 22genome A and 11 genome B chromosomes while the cooking bananasBluggoe and Fougamou showed 11 genome A and 22 genome B chromosomes.Hybridization of labelled A and B genomic DNA to chromosomesof the hybrids showed that TMP2x 2829-62 has all 22 genome Achromosomes while TMPx 4698-1 has 33 genome A and 11 genomeB chromosomes.In situhybridization of labelled total genomicDNA to chromosomes has immense potential for identificationof chromosome origin and can be used to characterize cultivarsand hybrids produced inMusabreeding.Copyright 1997 Annals ofBotany Company Genomicin situhybridization; banana; plantain; hybrids; plant breeding; genome organization; biodiversity     

Tubulin and Male-gamete Interconnections in the Pollen Tubes of the Grass Alopecurus pratensis

Tubulin was localized in pollen tubes of the grass Alopecuruspratensis by immunofluorescence using a rat monoclonal yeastantitubulin as a primary antibody. In tubes fixed in activegrowth with paraformaldehyde fixative and permeabilized withdetergent, the two male gametes were found to be linked by tubulin-containingcytoplasmic bridges. The connections appeared to persist throughoutthe growth of the tube, but they were highly mutable, the distancebetween the gamete pair varying greatly, the gametes sometimesactually overlapping in the tube. Systems of longitudinally disposed, discrete microtubule strandscomparable with those seen in generative cells of species withbicellular pollen grains were not observed in the gametes. However,an envelope of antitubulin-binding material was apparent atthe cell boundary in many instances, possibly to be interpretedas a sheath of closely packed microtubules seen in profile.Irregular masses, probably representing pools of unpolymerizedor partly polymerized tubulin, were also present in most ofthe gametes. Alopecurus pratensis, tubulin, pollen tubes, grass male gametes     

Sites of Origin of the Peripheral Microtubule System of the Vegetative Cell of the Angiosperm Pollen Tube

From earlier published work it is known that microtubules inthe vegetative cell of angiosperm pollen tubes mainly occurin the form of longitudinally disposed strands closely associatedwith the plasmalemma. This peripheral cytoskeleton is extendedapically at a speed matching the growth rate of the tube. Immunofluorescencelocalization shows that in the actively elongating tube it originatesin the sub-apical zone in bands or ribbons up to 2 µmwide, interpreted here as comprising aggregates of apposed,axially oriented microtubules. These appear first in the corticalcytoplasm in close association with the wall in the part ofthe tube where the callose inner lining can first be detected.The bands do not extend apically into the region of the pecticsheath of the extreme tip. In the course of normal growth, theperipheral microtubule investment remains in the older partsof the tube from which the bulk of the cytoplasm has been withdrawn,indicating that tubulin is probably not recycled. If the growth of the tube is retarded, the inner callosic layerextends apically. The acropetal movement of callose is accompaniedby a migration of the limit of detectable tubulin towards theextreme tip, and the axially oriented bands are replaced bya confused mass of granules and short spicules. It is suggested that the bands represent nucleation zones associatedwith the stabilizing plasmalemma in the sub-apical stretch ofthe tube where the insertion of wall-precursor material is diminishing,and that it is from these zones that the microtubule cytoskeletonof the pollen tube originates. Since during growth the nucleationzones progress rapidly forward into association with new membrane,it is considered unlikely that their sites are determined bylocal differentiations of the plasmalemma. An alternative possibilityis that the distribution of the zones is related to the calciumion gradient known to be present in the apical stretch of theextending pollen tube. Microtubules, pollen tube growth, Lilium auratum     

Germination of Monocolpate Angiosperm Pollen: Effects of Inhibitory Factors and the Ca2+-Channel Blocker, Nifedipine

Hydration of pollen of Narcissus pseudonarcissus was retardedand germination blocked in media with supra-optimal concentrationsof osmoticum. Activation of the grains, expressed in circulatorymovement in the vegetative cell, was not blocked. Wall developmentwas disrupted, and pectic material and callose were depositedthroughout. In the absence of calcium many grains burst on hydration.The survivors showed evidence of activation, but few tubes wereformed. In medium with supra-optimal Ca²⁺, activation proceeded,but where tube tips were produced they became occluded withcallose, which eventually formed a general lining to the intine.Nifedipine, a Ca²⁺-blocker, did not prevent activation at 10^–4M, but reduced callose deposition and inhibited polarized movementin the vegetative cell. Prominences formed at the germinationsites were mostly low and rounded. During recovery in normalmedium, tube tips with normal callose linings were formed. Colchicine,a microtubule inhibitor, had no effect on activation or germination.Cytochalasin D, an actin inhibitor, prevented activation ofthe vegetative cell, but did not arrest all wall deposition.Movement began soon after transfer to normal medium, and somegrains produced adventitious tube tips. While Ca²⁺ appears notto be essential for activation, these results may be interpretedas indicating links in the normal course of germination betweenthe initial Ca²⁺ influx at the potential germination sites and:(a) polarization of movement in the vegetative cell, probablyrelated to re-orientation of the actin cytoskeleton; and (b)patterned deposition of callose, which appears to have an importantmorphogenetic role. Narcissus pseudonarcissus, pollen activation, pollen germination, osmotic effects, actin cytoskeleton, nifedipine, cytochalasin D, colchicine, role of Ca²⁺ flux     

Pollen--stigma Interaction in the Leguminosae: the Secretory System of the Style in Trifolium pratense L.

The canal that traverses the upper part of the style of Trifoliumpratense is derived lysigenously. The core tissue of the veryyoung style consists of elongated cells similar to those ofthe transmitting tissue of solid-style families such as theSolanaceae; as the style matures, these cells separate to formthe canal, which receives secretions both from the core tissueand the inner wall cells. The early secretion of proteins intothe intercellular spaces is associated with the presence ofparamural bodies (lomasomes) in the adjacent cells. In the cellsin the immediate vicinity of the canal, vesicles, probably derivedfrom the Golgi system enlarge during later development and accumulatea protein-carbohydrate content, which is later passed into thecytoplasm where it forms densely packed fibrillar nodules. Withthe dissolution of the cell membranes, this material is passedinto the canal, where it is progressively diluted by continuedingress of water until the cavity reaches its final volume. Leguminosae, Trifolium pratense L., pollen—stigma interaction, self-incompatibility, stylar secretion, protein secretion