BOT 4283 Plant Anatomy 69
EXERCISE 23 ANGIOSPERM EMBRYOGENY: DOUBLE FERTILIZATION AND
EMBRYOGENESIS
Pollen tube growth. Growing pollen tubes is simple if you are using bicellular pollen. Physiologically,
it is possible to grow many pollen grains on a fairly simple Brewbaker-Kwack medium (0.01% H3BO3,
0.03% Ca(NO3)24H2O, 0.02% MgSO47H2O and 0.01% KNO3), with from 0% to 20% sucrose
added. Obtain a slide and place a small drop of pollen growth solution on it. Then sprinkle some fresh
pollen on the drop. Wait for a minute or two for the pollen to settle into the solution and hydrate. Then,
VERY CAREFULLY add a cover slip. (Hydrated pollen grains break easily!) Now, to prevent the slide
from drying, either put it in a humid chamber or seal the edges of the cover slip by applying liquid
Vaseline to the outside edges of the cover slip. (A warm paper clip is an ideal applicator, if used
carefully.) Pollen tubes should form within 10-30 minutes. These will be visible using a compound
microscope or high magnification with a dissecting microscope. For showing rapid growth, pollen of
Tradescantia and Impatiens is excellent. To show the movement of the generative cell down the pollen
tube, Amaryllis is ideal. As these elongate, can you see the vegetative nucleus? the generative cell? a
linkage between these structures? When are the sperm cells formed?
Male germ unit. The male germ cells (either the sperm or generative cells, depending on the pollen
grain and stage of development) are typically associated with the vegetative nucleus prior to germination
and during pollen tube growth. This association has been termed the male germ unit. Examples are in
the lab. Although the exact form of the MGU varies from one species to the next, this association may
be important in delivering the two gametes into the embryo sac at the same time, preventing
heterofertilization (a condition in which sperm cells from different pollen tubes fertilize the egg and
central cell.) The two sperm cells are linked by retaining a common cell wall, as a result of generative
cell cytokinesis and both are contained within an internal vegetative cell plasma membrane. A second
physical association occurs between at least one of the sperm cells and the vegetative nucleus. This
forms a more or less intricate arrangement of complementary surfaces that is maintained up to the time
of fertilization. The grasses are exceptionally interesting in this regard, because their association is
severed early in development, but re-established just prior to fertilization. This argues strongly for the
importance of the MGU. This association appears to occur in the vast majority of angiosperms.
Fertilization. The function of the mature embryo sac is a complex process that can be divided into four
stages:
1. Progamic phase - A preparative stage in which the male and female
gametophytes are slowly modified prior to arrival of the pollen tube. This
includes, for example, the degeneration of the receptive synergid
2. Gamete discharge - Arrival and discharge of the pollen tube, constituting sperm
deposition
3. Gametic fusion - fusion of plasma membranes of the gametes, transmitting the
nuclei of the male gametes into the egg cell and central cell
4. Nuclear fusion - fusion of the nuclear envelopes of the male and female gametic
nuclei
BOT 4283 Plant Anatomy 70
Demonstrations are available on each facet of the process. The exhibit centers on results using
Plumbago zeylanica and Populus deltoides, two plants in which these processes have been clearly
elucidated. Several alternative models are also shown in another figure on display.
Embryogenesis. After the formation of the zygote and primary endosperm nucleus, considerable
development is usually required to organize the mature seed. Endosperm development, which creates
the nutritive material for the embryo, usually precedes the division of the zygote and further
embryogenesis. The processes of embryogenesis and endosperm development are illustrated in
Capsella bursa-pastoris, sheparďs purse, which is the classical plant for this research (and is similar to
Arabidopsis in its pattern of developement). Slides are available illustrating the organization of the
young embryo, including:
1. globular embryo stage - the first stage in the formation of a three-dimensional cell
division system producing tissue thickness in the embryo. Note the long
suspensor and unusual basal cell, which is found in many members of the
Brassicaceae. What is the function of the suspensor?
2. heart-shaped stage - differentiation of the early cotyledons. This stage also shows
the first conspicuous evidence of a protoderm. What is the developmental
potential of that layer?
3. torpedo stage - elongation of the embryo. The embryo becomes more
complicated at this stage and the apices are becoming organized.
4. maturational stage - growth of the embryo is completed within the sclerified
ovular integuments, forming the seed.
One caveat--Capsella has a curved megagametophyte (ana-amphitropous), which is a highly restricted
characteristic in angiosperms. The events are given in detail for Capsella in your text.
A remarkable reduction of the embryo is shown in the embryo of Monotropa uniflora, the parasitic
Indian pipe. Embryos of this plant are only two cells at maturity. In angiosperms, there is wide
variation in the organization of the embryo in different groups.
As a last exercise on the organization of embryos, examine the mature embryo of Zea mays, as a
representative of the monocots. The embryo of corn is particularly precocious compared to most
flowering plants. Observe a prepared slide and identify the scutella, shoot apex, root apex, epiblast,
coleoptile, coleorhiza and young foliage leaves. What constitutes the cotyledon? Is there any evidence
that a second cotyledon was ever present in monocots?