Plant Systematics Laboratory #11
Equipment & supplies needed:
Compound microscope (preferably phase contrast or D.I.C.)
Scalpel, w/ #11 blade
Cover slips (squares or circles)
Felt-tip pen, rapidograph, or diamond scribe (for marking slides)
Paper and pencil (2H)
Herr's Clearing Solution
F.A.A. [5% formalin (commercial strength), 5% acetic acid
(glacial), 63% ethanol]
Ethanol - 70%
Ethanol - 95%
Ovules must be individually removed from the ovary or a flower in order to observe histological and positional details. Flowering material may be dissected live or fixed beforehand in F.A.A. for 24-48 hours. (Replace F.A.A. with 70% ethanol after fixation.) Dissect in Syracuse dish while viewing under a dissecting scope, using needles, forceps, and scalpel. Place flower in a dish filled with 95% ethanol and detach the ovary. Carefully tear open the side of the ovary (from top or bottom) with forceps to expose the ovule(s); avoid damaging the ovule(s). Observe placentation, ovule type (if apparent), and ovule position; record information. Place the opened ovary on a small drop of ethanol on a microscope slide (Don't let dry out!). Detach the ovule(s) from the base of the funiculus with forceps or needle. (With patience and skill in dissecting, even the tiniest ovules can be successfully removed.)
Place two cover slips on either side of the ovule(s). Drain most of the ethanol (or allow to evaporate). Immediately, place 1-2 drops of Herr's Clearing fluid (Herr, 1971, 1982) directly on the ovule(s). Cover ovule with a third cover slip, which rests on the edges of the other two. This arrangement prevents the ovule from being squashed. Add more Herr's solution from the edge, as needed.
After ca. 5 minutes, place slide under brightfield, phase contrast, or D.I.C. microscope and observe. Note:
1) Placentation (axile, parietal, basal, apical, free-central, apical-axile, parietal-septate, laminar)
2) Ovule position (hypotropous, epitropous, pleurotropous; dorsal or ventral)
3) Ovule type (anatropous, orthotropous, or campylotropous & subtypes).
4) Number of integuments (unitegmic or bitegmic).
5) Number of cell layers per integument (1, 2, or more).
6) Micropyle type (amphistomal, exostomal, endostomal, or unistomal).
7) Vasculature traversing funiculus into nucellus.
8) Details of embryo sac (this usually requires careful and considerable observation).
Possible taxa to investigate:
Armeria maritima [Caryophyllales, Plumbaginaceae]:
Aptenia cordifolia [Caryophyllales, Aizoaceae]:
Centranthus ruber [Dipsacales, Valerianaceae]: apical, epitropous, anatropous, unitegmic, unistomal
Eriogonum fasciculatum [Caryophyllales, Polygonaceae]:basal, epitropous, orthotropous, bitegmic, exostomal
Erythrina sp. [Fabales, Fabaceae]: marginal, pleurotropous, anatropous, bitegmic, exostomal
Euryops pectinata [Asterales, Asteraceae]: basal, hypotropous, anatropous, unitegmic, unistomal
Hedera helix [Apiales, Araliaceae]: apical-axile, epitropous-dorsal, anatropous, unitegmic, unistomal
Malacothamnus fasciculatus [Malvales, Malvaceae]: axile, pleurotropous, anatropous, bitegmic, exostomal
Pereskia grandiflora [Caryophyllales, Cactaceae]:
Phormium tenax [Asparagales, Phormiaceae]: axile, pleurotropous, anatropous, bitegmic, exostomal
Salvia mellifera [Lamiales, Lamiaceae]: axile, pleurotropous, anatropous, unitegmic, unistomal
Pollen Tube Growth
Carefully remove the style of a pollinated flower (e. g., a composite). Place the style in a couple of drops of aniline blue stain. Cover with a cover slip and slightly squash. Observe under the fluorescent microscope. Aniline blue is preferential for callose, which is deposited in the pollen tubes. Pollen tubes appear bright yellow under fluorescence microscopy. Observe.
Female Gametophyte (Embryo Sac) Development
Observe the mature female gametophyte of Phormium tenax and of the prepared slides of Lilium, noting the 3 antipodal cells, 2 polar nuclei, and 3 cells of the egg apparatus. If time permits, observe earlier developmental stages. Draw.
Dissect out embryos or clear small seeds of a given species and observe under brightfield, phase contrast, or D.I.C. microscopy. Draw.
Taxa: Capsella bursa-pastoris Shephard's Purse or Lepidium sp. Peppergrass (both Brassicaceae)
Bean (Phaseolus vulgaris): Observe seed coat and embryo (noting that endosperm is absent). The embryo consists of two large cotyledons, the radicle (embryonic root), the hypocotyl (between the cotyledons and radicle, and the epicotyl (embryonic shoot). If available, note the transformation of these structures from seed to seedling stage. The cotyledons are lifted above the ground on the hypocotyl (called "epigeal" germination) and eventually wither away. Draw the seed:
Corn (Zea mays) grain: Observe outermost pericarp (fruit wall), seed coat, endosperm, and embryo. The embryo consists of one cotyledon, a radicle (contained within the coleorhiza), and the epicotyl (contained within the coleoptile). If available, note the transformation of these structures from seed to seedling stage. The cotyledon and seed coat remain in the ground (called "hypogeal" germination). Draw the seed: