12.

Ginkgoales

Ginkgoales is an ancient group which appeared in the Permian, and was

well-represented and nearly worldwide in distribution in the Mesozoic.
Their maximal diversity occurred during the Middle Jurassic, and remains
have been collected from many countries: Alaska, Greenland, Scandinavia,
Siberia, Mongolia, England, Europe, China, Japan, Australia, New Zealand,
India, tip of South Africa and South and North America. These plants
flourished for over 150 million years. By the beginning of the Oligocene
(Tertiary), only 2 out of ca. 19 genera survived; Ginkgo adiantoides is one
of these. This species became extinct by the Pliestocene (Tertiary), and
Ginkgoales has been represented by the extant Ginkgo biloba-the "living
fossil". It appeared in the Jurassic, and became extensively distributed in
the Tertiary. In later periods, it was abundant in the more northern latitudes.
At present, its natural occurrence is restricted to a small inaccessible region
in southeastern China (see Chap. 1).

Fossil Taxa
Abundant compression-impression leaf remains have been collected, the
reproductive organs are meagre. There are about seven or eight Mesozoic
leaf genera included in Ginkgoales (Harris 1935, 1974). Ginkgoites and Baiera
have the maximal number of species. These two taxa are indistinguishable
in their cuticular and stomatal characteristics. However, Baiera lacks a petiole
and its lamina is comparatively more wedge-shaped, than that of Ginkgoites.
The latter has a distinct petiole with two traces which diverge into the basal
edge of a bilobed lamina. The fossil leaves, which cannot be distinguished
from those of the extant Ginkgo, are included in this taxon, but the species
are different. Those leaves which can be distinguished by their morphological
and anatomical characteristices (size and shape of epidermal cells, distribution
of stomata, structure of subsidiary cells, mesophyll and distribution of resin
bodies) are placed in the genus Ginkgoites.
Generally, the primitive leaves were linear and deeply dissected. The
leaves of Arctobaiera show a deeply dissected to entire margins
(Fig. 12.1 A, B). Sphenobaiera (Lower Permian to Lower Cretaceous) has
a characteristic dissected lamina (Fig. 12.1 C) with several dichotomised
 Ginkgoales 99

T'I

··:
..'·
~

i.•
1,'
' t• t
ii .,

B
A c

Fig. 12.1 A-K. Leaves and stomata. A, B Arctobaeira. C Sphenobaiera. D, E Baiera

spectabilis. F Ginkgoites minuta. G G. taeniata. H Ginkgoidium . I Ginkgo digitata.
J G. lamariensis. K Ginkgoites lunzensis, stomatal apparatus . (A·C after Florin 1951,
D, F-H after Harris 1935, E after Schenk 1867, I, J after Brown 1943, K after Andrews
1961)

veins. Baiera (Middle Jurassic to Lower Cretaceous) had wedge-shaped

leaf with an indistinct petiole (Fig. 12.1 D, E). The extent of lobing of
lamina and variations in venation pattern in Ginkgoites .spp. (Late Triassic)
can be arranged in a series (Harris 1935). The leaf in G. minuta is highly
dissected by equal dichotomies (Fig. 12.1 F), in G. taeniata the unequal
dichotomies make each half of the leaf appear three-lobed (Fig. 12.1 G),
and in Ginkgoidium the lamina is entire except for a median notch, and
prominent marginal and unbranched secondary veins (Fig. 12.1 H). Thus,
100 The Gymnosperms

there is a general trend from deeply dissected Jurassic leaves to those of the
Tertiary, which tend to be entire except for a median sinus (see Stewart
1983). This series may have an evolutionary significance, as shown by
Mesozoic species of Ginkgo: G. digitata (Early Mesozoic) has a deeply
dissected wedge-shaped petiolate leaf (Fig. 12.1 1), G. lamariensis
(Cretaceous) has undissected wedge-shaped leaves (Fig. 12.1 J), and in
G. adiantoides (Tertiary) the leaves are indistinguishable from those of
extant G.' biloba.
The stomata are haplocheilic. There are four to six subsidiary cells each
with a blunt papilla which tends to overarch the guard cells as in Ginkgoites
lunzensis (Fig. 12.1 K).
There are very few verified reports of reproductive structures. Numerous
dispersed ovules of Allicospermum (Harris 1935) -similar to those of cycads
and G. biloba-are associated with Jurassic and Cretaceous Ginkgo-like
remains. Kp,rkenia is an ovulate fructification from the Cretaceous of Argentina
(Archangelsky 1965). It is a short stalk crowded with 100 or more ovules
associated with Ginkgoites tigrensis. Paired ovules joined by a pad of tissue
with Ginkgo-like stomata have been reported from Yorkshire Jurassic beds
(Harris 1976), where abundant Ginkgoites huttoni occurs. In the same beds,
small pollen-bearing catkins are present. Each consists of a stalk (ca. 5 mm
long) to which are attached rather lax stalks with pairs of microsporangia
at the tips. The pollen grains are monocolpate like those of G. biloba.
The Ginkgo line can be trac~d to the Lower Permian Trichopitys (often
placed in its own order/family). Florin (1949) investigated T. heteromorpha,
known since 1875 from the Lower Permian of southern France, and interpreted
it to be the earliest member of the group (see Phylogenetic Considerations).
It has spirally arranged dichotomously branched leaves without lamina
(Fig. 12.2 A). There are small branched ovuliferous trusses (branch system,
Fig. 12.2 B) in the axil or on the upper surface of the leaf base. Each
ultimate branch bears a terminal, recurved ovule (Fig. 12.2 C), unlike Ginkgo.
The male organs of Trichopitys are not known. However, Sphenobaiera
furcata (Triassic) bears clusters of microsporangia at the branch tips of a
bifurcating axis, which are borne in tum on short shoots, along with leaves
similar to Trichopitys.

Ginkgoaceae
Ginkgoaceae is a monotypic family. The deciduous leaves are fan-shaped
with parallel veins. The tree is dioecious; male flowers are catkin-like
while the female is long-stalked with (usually) two ovules. The male gametes
are motile, and the fruit is drupaceous.

Ginkgo

Morphology
Ginkgo biloba is a tree more than 30 m high and exceeds 1.5 m in diameter.
 Ginkgoales 101

8
c

Fig. 12.2 A-C. Trichopitys. A Portion of shoot with sterile telome trusses (leaves) and
axillary ovule-bearing shoot. B Ovule-bearing shoot. C Ovule. (After Florin 1949, 1951)

It resembles a conifer in its general habit. The crown becomes broad,

irregular with age, and shows a variable pattern of branching. The main
axis branches profusely. There are two kinds of shoots: (a) long shoots
which elongate rapidly and bear scattered leaves, and (b) dwarf shoots
which grow slowly and bear a terminal cluster of leaves; the older portion
is covered with leaf scars of previous years (Fig. 12.3 A, D, F). Bud scales
cover young spur (dwarf) shoots. Frequently, the dwarf shoots become
more active and tum into a long shoot, while (more rarely) the terminal
growth of a long shoot may be retarded and resemble a lateral spur shoot
(Gunckel and Wetmore 1946). This suggests that there is not much
fundamental difference between them. The apical meristems of the two
types of shoots are also essentially similar; the difference between them
depends on the duration of cell division and cell elongation in the stem
tissues derived from the shoot apex (Foster 1938). There is experimental
evidence to show that the dwarf shoots are formed due to the inhibitory
effects of auxins produced by the tissues of the long shoots (Gunckel et al.
1949).
The shape and venation of the deciduous foliage leaves are unique. The
102 The Gymnosperms

Fig. 12.3 A-F. Ginkgo biloba. A Dwarf shoot bearing male strobilus. B, C
Microsporophylls. D, F Dwarf shoot bearing young strobili (D) and seeds (F).
E Longisection female strobilus, (A·E After Ganguli and Kar 1982, F after Andrews
1961).

petiole is long, smooth, black and slender, traversed by two collateral vascular
bundles. The lamina is broadly wedge-or fan-shaped, variously lobed, and
the venation is conspicuously dichotomous-(Fig. 12.4 D). The leaves resemble
those of Adiantum (maidenhair fern) in form and venation, hence the popular
name maidenhair tree. The old leaves are shed in autumn, when they change
colour to a golden yellow; the new leaves appear in spring. There is
considerable difference in the lobing of the leaves on the same tree. They
may be nearly entire or two-lobed due to a conspicuous, often deep, apical
notch. They are mostly bilobed on long shoot and entire on short shoot. In
seedlings the leaves have several notches which give a palmatifid appearance
(as in the extinct taxa).
 Ginkgoales 103

Fig. 12.4 A-E. Ginkgo biloba. Stem anatomy. A, B Transection long (A) and dwarf
shoot (B). C radial longisection, secondary wood shows circular bordered pits, bars of
sanio, and ray cells with cross-field pits. D, E Leaf. D Single leaf shows venation.
E Vertical section (A, B, E After Ganguli and Kar 1982, C, D after Stewart 1983)
104 The Gymnosperms

Anatomy

Root. The young roots are usually diarch. The endodermis has conspicuous
thickenings on its radial walls and there is a broad pericycle. Older roots
may be tetrach or hexarch. A radiallongisection shows that the spiral elements
of the protoxylem are followed successively by traceheids with (a) reticulate
pitting, (b) transversely elongated simple pits and (c) bordered pits.
Secondary growth occurs, but annual rings are not pronounced. The
tracheids have thinner walls. Some xylem parenchyma cells include crystals,
and thick-walled fibres are abundant in phloem. The medullary rays are
one to several cells high and often show crystals.

Stem. The shoot apex consists of superficial apical initials, a prominent

subapical zone of central mother cells, and a zone of rib meristem around
it on its proximal side (Foster 1938).
A young stem comprises an epidermis, a parenchymatous cortex and
pith, and vascular cylinder. The stele is an endarch siphonostele. A long
shoot has a thick zone of wood and comparatively narrow pith and cortex
(Fig. 12.4 A). In the dwarf shoots, the ring of wood is narrow and there is
a broad cortex and pith (Fig. 12.4 B). The primary xylem consists of a
number of separate strands. The phloem forms a broad zone. The double
leaf traces are very conspicuous.
A radial section of the wood .shows tracheids with numerous round ai:l.d
opposite pits separated by bars of Sanio (Fig. 12.4 C). The uniseriate rays
are one to three cells deep ip. the long shoots, and much deeper in dwarf
shoots. Occasionally, a few xylem parenchyma cells contain calcium oxalate
crystals. In secondary wood the tracheidal overlap is not extensive, and
several tracheids end at the same level. This makes the wood very brittle,
and in high wind storms the trees suffer heavily. When a tree is felled. the
log frequently shatters as it strikes the ground (see Bierhorst 1971.
The secondary phloem is composed of sieve elements, parenchyma strands
and fibres in the axial system and rays in the radial system (Srivastava
1963a). The albuminous cells associated with the sieve cells lack starch
and are crushed in the old phloem. They are connected to the sieve cells
by one-sided sieve areas. Lateral connections between the sieve elements
and albuminous cells could not be traced, as callose was not present in
sufficient quantity. These cells also have plastids but do not store normal,
detectable starch. The fibers are elongated, tapering, tangentially flattened
and non-septate. They have narrow lumen, the wall is thick, lamellated and
appears to be cellulosic. They do not stain positive with phloroglucinol and
HCl and are strongly birefringent under polarized light '(see Paliwal 1992).
Outside the phloem there is a ring of sclereids, probably pericyclic, and
an inner ring of thickened cells, whose walls appear to be gelatinized.
Throughout the plant numerous mucilage canals occur in the pith and the
cortex.
 Ginkgoales 105

Leaf. The leaf has a double trace. A transection of the petiole shows two
endarch vascular bundles. The primary xylem of the stem branches
sympodially when the leaf traces are given off. The two traces to any leaf
therefore arise ind~pendently from two different primary strands. They
divide at the base of the blade and the resultant four strands fork repeatedly
to form the dichotomous system of veins which occasionally anastomose in
the lamina (see Stewart 1983). The venation of each of the two halves of
the leaf is completely independent. Mucilage canals are present even between
the veins of the leaf.
A vertical section of the lamina shows: (a) a thick cuticle, (b) stomata
mostly on the lower surface of the leaf, (c) a distinct palisade only in the
leaves on the long shoots (Fig. 12.4 E), (d) mucilage canals and (e) \!lsually
endarch vascular bundles with traces of centripetal xylem represented by
one or two tracheids. The bundles are surrounded by a sheath of thick-
walle<;l cells.
On the lower epidermis, stomata occur irregularly scattered between the
veins. They are haplocheilic, surrounded by four to six ·subsidiary cells,
each with a blunt papilla which projects over the guard cells (see Stewart
1983). The characteristic accessory cells of the stomata are also recognizable
in the extinct taxa.

Reproduction
Ginkgo is dioecious. 1 The male cones are pendant and catkin-like, borne on
short shoots in the axil of normal leaves or scale leaves (Fig. 12.3 A).
The ovulate cones are borne in groups at the apex of the dwarf shoot
(Fig. 12.3 D, F). They are reduced, and each shoot bears two ovules on a
long peduncle in the axil of a scale leaf (Fig. 12.3 D, E).

Male Cone
A male cone comprises 40-50 microsporophylls (Fig. 12.3 A). Each
microsporophyll has a terminal knob, which contains a mucilage sac
(Fig. 12.3 B, C), and there are two (occasionally three to seven) pendulous
microsporangia which dehisce by longitudinal slits (Fig. 12.3 C).

Microsporangium. The strobili are initiated in summer, and appear as

small papilae in the axils of bracts. Wolniak (1976) examined more than
300 cones, and observed: (a) There is no acropetal or basipetal progression
in sporangia! development; (b) There is no correlation between the size of
the sporangium and its development. There is, however, variation in

1ln the Botanical garden at lnsbuck (Austria), on a female tree of Ginkgo, a branch from
a male tree was grafted. The male cones developed and produced fertile pollen grains,
pollination and fertilization occurred normally, and numerous (apricot-coloured) ripe
seeds developed on the female tree (BMJ, pers. observ. 1957).
106 The Gymnosperms

development, and in a sporangia! pair the rnicrosporocytes are ontogenetically

similar. Earlier stages have not been observed but there is evidence of a
single hypodermal cell which divides by anticlinal and periclinal divisions.
The outer cells form a wall of four to seven layers, and the inner cells give
rise to a large group of sporogenous cells. A tapetum surrounds the
sporogenous tissue, and a peritapetal membrane has been reported (Pettitt
1966).
An· endothecium differentiates, which is an exception, since in
gymnosperms only an exothecium is reported. The endothecium develops
from one to three layers of subepidermal cells, becomes thick-walled and
develops fibrous thickenings (Fig. 12.5).

Fig. 12.5 Ginkgo biloba. Longisection microsporangium and a part of microsporophyll,

several layers of wall show endothecial thickenings . (After Jeffrey and Torrey 1916)
 Ginkgoales 107

Microsporogenesis. Meiosis in microspore mother cells coincides with

the opening of the bud scales of the spur shoot. The distribution of starch
in the microsporangium is specific, and first appears at the archesporia!
stage. Prior to meiosis, starch reappears in the sporogenous cells and then
in tapetal cells. During meiosis I starch grains accumulate at the equatorial
plate at metaphase, move to the opposite poles during anaphase and telophase,
and become equally distributed after the completion of meiosis. After
telophase I, besides starch, entire plastids and mitochondria shift to the
equatorial region of the microspore until nuclear divisions cease. Microtubules
and ER proliferations appear to stabilize.the organelle distribution through
meiosis II. Tetrads are formed by centripetal wall formation, and the organelles
become distributed equally (Wolniak 1976). A callose wall has been observed
during microspore mitosis (Gorska-Brylass 1968).

Male Gametophyte. The rnicrospore nucleus cuts off two prothallial cells
(Fig. 12.6 A, B); the first cell towards the wall is ephemeral while the
second persists. The antheridial initial divides and forms a smaller antheridial
cell, which remains attached to the intine, and a larger tube cell, which
becomes vacuolate and has a conspicuous nucleus (Fig. 12.6 C, D). The
antheridial cell divides periclinally to form the stalk cell (toward the pollen
wall) with a distinct wall, and the body cell (Fig. 12.6 E). The stalk and
body cells persist in situ. The persistent prothallial cell remains active and
grows into the stalk cell which lies next to it. The stalk cell thus appears
to form a jacket around the protruding prothallial cell (Fig. 12.6 D-F).
The microsporangium dehisces by a longitudinal slit along the inner
face. The pollen is shed at the four-celled stage: two prothallials, one
antheridial and a tube cell.

Ovule
The peduncle bifurcates and bears on each branch a single sessile ovule
with a fleshy collar around its base (Fig. 12.3 E). The morphology of the
collar has been variously interpreted (see Chamberlain 1935); it does not
grow after pollination.
Usually there are only two ovules on each peduncle, occasionally three,
four or more. Whatever the number of ovules, the peduncle always has
twice the number of vascular bundles.
The morphology of the meristem which gives rise to the ovule needs a
critical reinvestigation. The ovule is orthotropous with a beaked nucellus
which has a heavily cutinized epidermis. The nucellus has a well-differentiated
strand of elongated cells and extends almost to its entire length (Fig. 12.7 A).
Its degeneration forms a narrow, deep pollen chamber (De Sloover-Colinet
1963). The inner cells degenerate first followed by the epidermis
(Fig. 12.7 B, C). There is a single integument, which is free from the nucellus
at the apex. Two unbranched vascular strands supply the base of the
integument.
108 The Gymnosperms

A 8

Fig. 12.6 A·F. Ginkgo biloba. Male gametophyte. A·F Development of male
gametophyte. al antheridial cell, be body cell, pr prothallial cell, sc stalk cell, tn tube
nucleus. (A·D After Chamberlain 1935, E. F after Favre-Duchartre 1956)

Megasporogenesis. One or more megaspore mother celVs become distinct

by their prominent nuclei and dense cytoplasm. Due to a considerable
thickening of the middle lamella (Fig. 12.8 A), and the development of a
double-layered wall, the wall of the mother cell becomes thick and two-
layered (Stewart and Gifford 1967). The latter has densely staining outer
layer which resembles the circumjacent nutritive tissue, and an inner layer
which is similar to the middle lamella except for a tighter arrangement of
the fibrillar structure. The young megaspore mother cell is spherical, and
has a large nucleus in the centre. Its cytoplasm occasionally shows a small
vacuole, relatively scanty endoplasmic reticulum (Stewart and Gifford 1967),
 Ginkgoales 109

8
c
Fig. 12.7 A·C. Ginkgo biloba. A·C Longisection nucelli show successive stages of
development of pollen chamber. (After De Sloover-Colinet 1963)

and randomly placed starch-bearing plastids, mitochondria and dictyosomes.

The mother cell elongates as it matures. The cytoplasm in the micropylar
half shows a large and complex system of ER (Fig. 12.8 B), while it is
relatively meagre and there is no definite pattern in other parts of the cell.
However, all over the cell, ER is only sparsely associated with ribosomes.
In the mature mother cell the micropylar ER becomes reticulated with
several "loops" and "circles" (Fig. 12.8 C). A vacuole appears below the
nucleus of a mature mother cell, and several small vacuoles in the micropylar
part. From the micropylar half, the plastids and mitochondria shift laterally
to the chalazal end of the maturing mother cell (Fig. 12.8 D); the micropylar
ER may have a role in the migration of these organelles. In a mature cell
the plastids with a few mitochondria are restricted to the region below the
nucleus, the mitochondria lie just below the plastids (Figs. 12.8 D; 12.9).
Other organelles, like dictyosomes, lipid droplets, dense bodies bounded by
unit membrane, and an occasional multivesicular body, do not show polarity
in their distribution.
The nuclear envelope of the mother cell shows a large interruption in the
lateral wall of the cell (Fig. 12.9), and remains identifiable until the onset
of meiosis I. Starch accumulates only in the chalazal region of the megaspore
mother cell. Following meiosis I, plastids and mitochondria become restricted
to the chalaza} dyad. Generally, a linear tetrad of megaspores is formed. In
a triad-upper undivided dyad and two megaspores-it is the functional
chalaza} megaspore which has most of the plastids. The starch grains are
consumed during the enlargement of the megaspore. It appears that starch
begins to accumulate, much in advance, at the site of the functional megaspore.

Female Gametophyte. The female gametophyte develops from the haploid

110 The Gymnosperms

Fig. 12.8 A-D. Ginkgo biloba. Electron micrographs, megaspore mother cell. A Portion
of megaspore mother cell (mgmc), outer (cw) and inner (i/) layer of mgmc wall, middle
lamella (mdl), and spongy cells (sg). B, C Part of micropylar half of mother cell.
B Extensive ER. C Reticulate ER. D Part of chalaza! region of mother cell; note starch
(sgr)- bearing plastids (p), near the nucleus (nu), and mitochondria (m) lower down. (After
Stewart and Gifford 1967)
 Ginkgoales 111

Fig. 12.9. Ginkgo biloba. Megaspore mother cell (diagrammatic) . er endoplasmic

reticulum.go golgi apparatus. m mitochondria. nu nucleus. p plastid. v vacuole. (After
Stewart and Gifford 1967)

chalaza) megaspore. The gametophytic tissue, however, is not uniformly

haploid, as shown by cytological studies and cytophotometric measurements
of the DNA content (A vanzi and Cionini 1971 ). At the beginning of the
cellular stage, 5% nuclei of the gametophyte show 1C DNA content, 50%
2C, more than 40% 4C, and the remaining nuclei 8C or higher DNA content.
This variation in the DNA content is attributed to endopolyploidy. The
cells with 4C content are mostly located in the outer region of the
gametophyte. The nuclei with higher DNA content (8C or more) degenerate
in the young gametophyte. In older stages, most cells contain 2C DNA.
Free-nuclear divisions (Fig. 12.10 A-C) occur in the megaspore for about
4 weeks. According to Favre-Duchartre (1958), there are 13 successive
112 The Gymnosperms

ljij !
I

@! . • I

•
OQ··i
A ·:
\CF.; ~
I

·/;;) I
._, :
·"""·'""'• I

®··:
\.;

.r-r'.. - ~
A B 0

Fig. 12.10 A-H. Ginkgo biloba. Longisection of peripheral region of female gametophyte.
A, B Cytoplasm with free nuclei. C Nuclear divisions D-G Formation of alveoli and
initiation of walls. H Cellular gametophyte, note the thick wall. (After Favre-Duchartre
1956, 1958)

mitotic cycles, so that more than 8000 nuclei are formed. The divisions are
initiated at the chalaza! end, and proceed towards the micropyle. The
prothallial cytoplasm, throughout cenocytic phase, adheres to the megaspore
membrane. Walls are laid down at the end of the 14th mitotic wave. The
gametophyte remains colourless throughout the free-nuclear stage. Typical
alveoli are formed (Fig. 12.10 D-G), followed by cellularization (Fig. 12.10 H).
The gametophyte becomes green (due to the presence of chlorophyll) and
starch is synthesized. The female gametophyte of Ginkgo biloba is the only
seed plant with a chlorophyllous gametophyte. The relative transluscence
of the integumentary tissues of the ovule permits sufficient light to induce
the synthesis of chlorophyll (Friedman and Goliber 1986). The plastids do
not contain an organized thylakoid membrane system (Pettitt 1977). When
cell formation begins, the female gametophyte has a light green colour,
attributed to chlorophyll. EM of chloroplasts demonstrate stacking of thylakoid
 Ginkgoales 113

membrane in the grana. Plastids located deeper in the gametophyte have

fewer thylakoid membranes and may also show prolamellar bodies.
The presence of chlorophyll has been confirmed by Burgerstein (1900).
He showed that an alcoholic extract of the gametophyte fluoresces red;
Carothers (1907) suggested it may be capable of photosynthesis. A
measurement of photosynthetic active radiation (PAR) indicates that a
gametophyte (growing within an ovule) can receive significant quantities
of light, i.e 70 f.1 mol photons m· 2 s· 1 (Friedman and Goliber 1986). This
unique ability to produce chlorophyll and perform photosynthesis results
from its·exposure to sufficient levels of light, and an inclination to react to
this stimulus by the development of functional photosynthetic apparatus.
The entire gametophytes were dissected free from the ovules. They were
capable of gross photosynthesis (not net photosynthesis) under experimental
conditions. On a dry weight basis, the maximum rate of carbon fixation, in
near-saturating light intensities, was 3.64 x 10· 3 ).1 mol C02 g·'s·' (Friedman
and Goliber 1986).
A mature prothallus contains water, nearly 60% of the whole mass (Favre-
Duchartre 1956). Reserve food accumulates in both the fertile and sterile
prothalli, and is therefore independent of fertilization. In addition to starch,
a mature prothallus also contains lipids, and its density decreases sharply
from the epidermis inwards (Fig. 12.11 A). The concentration of lipoptoteins
increases during the maturation of the prothallus. The starch grains vary
from 4-5 )1m in diameter in the epidermal cells to 15-20 )1m in the inner
cells. They are simple, irregular in outline, and have a central hilum of
indefinite shape (Fig. 12.11 B-F).
The apical part of the gametophyte regularly forms an unusual structure,
the "tent pole" (Fig. 12.11 A). The central portion of the micropylar region
grows by active cell divisions to form a column (the tent pole) which grows
into the nucellus, or may even reach the pollen chamber.
At the micropylar end of the cellular gametophyte, one or two cells
enlarge and function as archegonial initials. Each initial divides transversely
and forms a large central cell and small neck cell of the archegonium
(Fig. 12.12A). At maturity there are four swollen neck cells (Fig. 12.12 B),
which project upward and may have secretory role (Lee 1955). In the
beginning, the four neck cells are in one tier but later they may show a two-
tiered arrangement (Favre-Duchartre 1956). In a female gametophyte the
maturation of different archegonia is synchronous. The central cell nucleus
divides to form a small ventral canal cell and the egg cell (Fig. 12.12 A, B).
The mature egg cell is spherical, and measures ca. 500 )1m in diameter with
its nucleus ca. 100 )1m across (Camefort l965a). The chromatin is confined
to small globule so that the nucleus appears almost colourless with Feulgen
reaction (Fig. 12.12 B).
The single-layered archegonial jacket comprises uninucleate cells. Several
simple pits occur on the wall between the jacket cells and the central cell
114 The Gymnosperms

~'
' ~
~'

i .
~
Fig. 12.11 A-F. Ginkgo biloba. A Longisection mature female gametophyte, note two
archegonia and tent pole at the micropylar end, and distribution of starch (open circles),
lipids (black dots) and lipoproteins (radial lines). B Three cells from the margin, outer
layer is sudanophilic. C-F Cells from different layers of the gametophyte, at various
stages of development, show accumulation of starch , lipids and lipoproteins. (After
Favre-Duchartre 1958)

(Hirase 1895). Occasionally, lobes of the cytoplasm of the central cell may
project into these cells. Plasmodesmata are also present in these pits (Stopes
and Fujii 1906), and have been confirmed by ultrastructural studies (Maugini
and Fiordi 1970). Probably, the nutrients are absorbed through the jacket.
Avanzi and Cionini (1971) measured the DNA content of the jacket cells
cytophotometrically. The large uninucleotate nuclei had DNA content
corresponding to 2C, 2C-4C, 4C or 4C-8C, due to endoduplication. Cionini
( 1971) characterized the DNA in jacket cells by HCl hydrolysis curves and
observed two types of DNA complexes: (a) Feulgen-stainable after 5 min.
and (b) after 7 min. of hydrolysis. This is probably related to the functional
activity of jacket cells or formation of nascent DNA during endoduplication.
The passage of materials from the jacket into the egg, and to the coenocytic
(later cellular) proembryo has been studied using electron microscopy
 Ginkgoales 115

Fig. 12.12 A, B. Ginkgo biloba. development of archegonium. (After Favre-Duchartre

1956)

(Maugini and Fiordi 1970). The wall between the jacket and central cell of
the archegonium is fairly thick, and has prominent pits (Fig. 12.13 A). The
cytoplasm of the central cell forms short and blunt projections at the site
of the pits (Fig. 12.13 B, C). The cytoplasm is separated only by the thin
pit membrane, and they remain in contact through plasmodesmata. Soluble
materials move across this contact. Granular material, which lies outside
the plasma membrane of the egg cell, is deposited around the cytoplasmic
projection of the egg (Fig. 12.13 C). According to Maugini and Fiordi
(1970), the granular material represents the temporarily stored nutritive
116 The Gymnosperms

Fig. 12.13 A-D. Ginkgo biloba. Central and jacket cell. A Longisection shows thick
and pitted wall between central (cl) and jacket layer U). B Pit region shows plasma
membrane (pm) in contact with the pit region of the cell wall. C Later stage, central cell
cytoplasm (d) extends into the pit, laterally disposed granular material (gm), endoplasmic
reticulum (er) in the cytoplasmic appendage. D Degenerated jacket cell, the contents
pass into the proembryo through open pit. (After Maughini and Fiordi 1970)
 Ginkgoales 117

material on its way from the jacket cell to the egg cell. The plasma membrane
of the coenocytic proembryo shows small invaginations and short and irregular
microvilli. The increased surface provides a greater absorptive area for
nutritive materials. Starch and-protein granules, stored in the gametophyte,
are translocated to archegonium (in a soluble form) through the pits. When
the proembryo is free-nuclear, or immediately after cell formation, the pit
mambrane breaks down at places. Through these passages, the mitochondria,
plastids, dictyosomes, portions of endoplasmic reticulum and nuclei (whole
or in parts) flow (from degenerated jacket cells) into the egg cytoplasm
(Fig. 12.13 D). This is perhaps due to a sudden lowering of pressure inside
the egg. The contents of the jacket cells partially accumulate between the
cell wall and the plasma membrane of the proembryo.
The egg cytoplasm has the usual organelles like ER•. plastids, Golgi bodies,
ribosomes and a large number of mitochondria. Observations with an electron
microscope (Camefort 1965a) have revealed the following ·cytoplasmic
formations (so-called proteid vacuoles): (a) The morphology of small
inclusions is somewhat different from others. They are completely enclosed
in the double membrane of ER whose components stay together. An
enveloping vacuole is thus absent. (b) Microbodies are present in abundance,
have dense contents enclosed by a single membrane of ER. Their morphology
is similar to certain lysosomes in animal cells. (c) Vesicular bodies occur
seldom and comprise a mass of vescicles enclosed by a single membrane.
The amyloplasts in a mature egg are distributed at the periphery. They
are enclosed in a layer of endoplasmic reticulum, in addition to their own
membranes (Camefort 1965a). The amyloplasts continue to fragment until
the egg is mature.

Pollination
The ovules are pollinated soon after megaspore formation. A pollination
drop is secreted at the micropyle of the ovule. The wind-borne pollen, after
landing on the pollination drop, imbibes nutrients from the fluid, becomes
heavy, sinks down the micropyle, and reaches the nucellar tip. Unpollinated
ovules drop from the tree about 4 weeks after anthesis.

Post-Pollination Development of Male Gametophyte. On the return of

favourable weather after the winter, the pollen grains germinate in the
pollen chamber (Favre-Duchartre 1956, De Sloover-Colinet 1'963), The exine
splits along the germinal furrow, the pollen tube emerges at the distal end,
and the vegetative (tube) nucleus may pass into it. The tube grows laterally
and horizontally into the massive nucellus and branches profusely. The
pollen tube grows in between the nucellar cells, in a hypha-like manner
(Fig. 12.14 A). The branched pollen tube in the nucellar tissue has been
studied from sections and dissections (Friedman 1987), and undoubtedly
has a haustoria! function (see Chap. 2).
118 The Gymnosperms

The proximal end of the pollen grain contains the prothallial, the stalk
and body cells/male gametes. In the beginning it is enclosed in the exine,
but later enlarges and bursts out of the exine. The short, broad end of the
"tube" is usually unbranched (Fig. 12.14 B-F), and a bunch of them grow
into the nucellus towards the female gametophyte. Consequently, the nucellus
becomes completely disorganized between the intermediary chamber
and the female gametophyte. At the grain end, the pollen tubes appear to

~ ~
q:.:: ·:&.?iL'7'§_.: . ~
\·/·.¥·::·:.~~!~?::..·
~ . .. . =: ·:.¥:f
. . .. B.
...·.....· ~ ·..... :·... :' :.: ·... ·...
· . · . ·.·. -: 0
-
c

E F

Fig. 12.14 A-F. Ginkgo biloba. A Haustoria! ramifications of pollen tube between
nucellar cells. be body cell. tn tube nucleus. B-F Diagrammatic representation of upper
portion of nucellus and female gametophyte, longisections. B-D Pollen tube and
degenerated inner portion of nucellus. E Pollen tube close to the archegonium. fd
fertilization fluid, tp tent pole. F Post-fertilized shrivelled nucellus. (A After De Sloover-
Colinet 1963, B-F after Favre-Duchartre 1958)

lie in a longitudinally continuous cavity in the middle of the nucellus

(Fig. 12.14 D-F). Thus, the pollen tube has: (a) a vegetative portion which
 Ginkgoales 119

grows laterally into the nucellus and is haustoria! in function, and (b) a
fertile portion which carries the motile gametes.
The occurrence of motile/ciliate sperms in Ginkgo was discovered by
Hirase (1895). 2 This was a landmark in the history of (embryological)
study of gymnoserms.
The ultrastructure of male gametes has been investigated by Gifford and
Lin (1975). The body cell remains spherical during maturation (Fig. 12.15
A, B). Two blepharoplasts, from which cytoplasmic/astral rays radiate,
appear on one side of the body cell (Fig. 12.15 A, B). These rays are the
microtubules, which extend from the matrix or the surface of the
blepharoplasts. The latter move through 90° and come to lie at a right angle
to the long axis of the pollen tube (from the very begnning). The blepharoplasts

bl

c
Fig. 12.15 A-E. Ginkgo biloba. Body cell and male gamete. A Body cell with irregular
nucleus; two blepharoplasts (bJ) at oppO>ite poles. B Body cell with a lens-shaped nucleus
and two osmiophilic globules ~og), one on either side. C Division of body cell. D Two
male gametes, one gamete shows a beak-like extension. E Gamete with three gyres of
ciliate band (top view). (A, B After De Sloover-Colinet 1963, C-E after Favre-Duchartre
1956)

2This tree, in the Botanical Garden, Tokyo, is still in a healthy state.

120 The Gymnosperms

increase in size and become prominent. The entire surface of the matrix is
covered by a single layer of probasal bodies (Fig. 12.16 A, B). The total
number of probasal bodies on one blepharoplast is 1000. Each probasal
body shows a hub-and-spoke arrangement in the centre and nine-fold
symmetry. A central tubule is present along the entire length of the probasal
body. Microtubules/astral rays/cytoplasmic rays originate from the interior
of the blepharoplast, pass between probasal bodies, and extend into the
cytoplasm. The probasal bodies separate from each other and form the
basal bodies of flagella. The matrix of the blepharoplast comprises dense
and less dense regions, the latter appearing to be infiltrated by a network
of tubules (Fig. 12.16 C, D).

Fig. 12.16 A-D. Ginkgo biloba. electron micrographs. A Whole-mount (portion) of

grain-end of pollen tube; the body cell shows lens-shaped nucleus and two prominent
osmiophilic globules ( og) on either side of nucleus. B Osmiophilic globule and
blepharoplast (bl) in ( a part of) body cell. C Numerous probasal bodies; microtubules
in less dense regions (ldr-arrows) . D Peripheral portion of blepharoplast; microtubules
in groups extend from probasal body. (After Gifford and Lin 1975)
 Ginkgoales 121

During its maturation, the nucleus of the body cell becomes lens-shaped
(Figs. 12.15 B, 12.16 A). A vacuole, which is an osmiophilic globule (it has
no membrane around it), appears on either side of.the nucleus (Figs 12.15 B;
12.16 A) and is attached to the blepharoplasts (Lee 1955). A dumbell-
shaped lipoprotein granule arises in the cytoplasm close to the nuclear
membrane. In addition, the cytoplasm of the body cell shows other organelles,
like protoplastids (numerous, electron-dense, showing some lamellar
developments), mitochondria, small vacuoles, lipid bodies, abundant
ribosomes, and relatively sparse ER and dictyosomes (Fig. 12.16 B). The
body cell divides, the lipoprotein granule splits into two, and each half is
incorporated into a spermatozoid. The mitotic spindle of the body cell lies
at right angles to the long axis of the pollen tube, so that the two sperms,
enclosed in the wall of the body cell, lie side by side (Fig. 12.15 C, D) until
they are mature. The vacuole (osmiophillic globule), earlier attached to the
blepharoplasts, shifts to the opposite pole of the cell and becomes attached
to the sperm nucleus. Later, it acquires granular contents due to diffusion
of chromatin from the nucleus (Lee 1955). The blepharoplast forms a part
of the tapering distal part of the sperm. The spiral band has three turns/
gyres (Fig. 12.15 E). There is simultaneous division of the nucleus of the
body cell in the pollen tube and of the central cell in the archegonium.

Fertilization
In zooidogamous gynmosperms, autolysis of the nucellus, megaspore wall,
and the gametophytic tissue around the archegonia form an archegonial
chamber (Pettitt 1977). The turgid nucellar cells abruptly release their vacuolar
contents, a fluid is produced which forms a pool and floods the archegonial
chamber and the space above it (Fig. 12.14 E). The male gametes are
released in this fluid (Lee 1955). The spermatozoids, with the band of
flagella at their posterior end, swim about in the chamber with a forward
and circular motion. The four neck cells open out as the egg cell pushes
through the disintegrated ventral canal cell to form a beak. The egg nucleus
may elongate and extend into the beak. As soon as the spermatozoid becomes·
attached to the projection of the egg, the elongated nucleus withdraws·
toward the centre and the beak of the egg retreats. According to Lee (1955),
only the head of the sperm (made of a vacuole-like structure and cytoplasm)
flows through; most of its body is left behind, outside the archegonium,
and disorganizes immediately. However, Favre-Duchartre (1956) observed
portions of the ciliate band of the sperm inside the egg. Extra sperms are
prevented from entering 'into the egg cytoplasm by a thickening of the
plasma membrane of the egg (Lee 1955).
During karyogamy, the paternal chromosomes (12) become short. They
can be stained clearly by Feulgen reaction as soon as the male pronucleus
penetrates the female pronucleus. They mix with the maternal chromosomes
(12) during the first somatic prophase (Favre-Duchartre 1958).
122 The Gymnosperms

Several pollen grains may germinate in a pollen chamber. However, as

soon as one pollen tube has discharged its spermatozoids, the body cells in
the other tubes frequently fail to divide and degenerate. When two
spermatozoids are discharged, two eggs can be fertilized, but a second
embryo rarely develops.
Both fertilized and unfertilized ovules are shed from the tree at irregular
intervals. The ovules may be sterile or may enclose one or two embryos
ranging from the coenocytic to the dicotyledonous stage. Unfertilized ovules
shed from the tree can undergo fertilization and subsequent embryonic
development.

Embryogeny
The zygote nucleus divides in situ followed by several free-nuclear divisions
(Fig. 12.17 A). The nuclei become distributed throughout the young
proembryo. Sometimes, evanescent walls (Fig. 12.17 B) appear during the
free-nuclear period. During later stages, the nuclei become distributed almost
evenly in the cytoplasm. Wall formation takes place when there are 256
free nuclei; the newly formed cells fill the entire proembryo (Fig. 12.17 C).
Within the female prothallus, the cellular proembryo develops continuously;
it is considerably influenced by the prevailing temperature. The cells at the
base divide and function as embryonal cells, while the upper cells elongate
to form a massive suspensor (Fig. 12.170). There is, however, no well-
defined suspensor; it is a micropylar region of elongated cells.

Differentiation of Embryo. There is a cap-like structure formed by the

upper cells (towards the neck of the archegonium) which is pierced through
by the radicle at the time of germination (Favre-Duchartre 1958). The
embryo develops (slowly) after the seeds have been shed.
The mature embryo is dicotyledonous, occasionally three cotyledons are
present. The embryo may reach its maximum size in 3 months after
fertilization, if the development is accelerated in an incubator. In nature,
the embryos may remain healthy for 12 months if the ovules are preserved
in a damp place. This is because the embryo is surrounded by the prothallial
cells, which provide necessary humidity for its survival (Favre-Duchartre
1958). There is no after-ripening requirement, and the seeds germinate
whenever a suitable substrate is available.
The most conspicuous change in a female gametophyte is the deposition
of reserve food like fat, starch and protein. Unlike other gymnosperms, the
accumulation of food reserves in Ginkgo occurs before fertilization (cf. cycads;
Favre-Duchartre 1958).
Abundant starch as reserve food accumulates in the gametophyte even
when the archegonia are still immature. Lipids occur as variously-shaped
globules which decrease sharply from epidermal cells inwards. Lipoproteins
appear nearly 2 months before fertilization, at first in the inner layers and
 Ginkgoales 123

............···-·-····· ....................

D
Fig.12.17 A-D. Ginkgo biloba. Proembryogeny. A Longisection free-nuclear proembryo
with the jacket layer and part of gametophyte. B Proembryo in non-median section,
formation of evanescent walls. C Cellular proembryo. D Proembryo, the upper cells
elongate to from the suspensor. em egg membrane. (A, C, D After Lyon 1904, B after
Favre-Duchartre 1956)

later towards the periphery. In a mature gametophyte, there are four zones
of storage cells: (a) The peripheral zone of three or four layers of isodiametric,
vacuolate cells containing numerous round and densely aggregated lipid
124 The Gymnosperms

droplets in the cytoplasm. Where the density of the lipid droplets is low,
the cytoplasm shows dictyosomes, a few mitochondria with short cristae,
ER and chloroplasts. ER comprises rounded or irregular vescicles. Small
starch grains occur embedded in the chloroplasts (Dexheimer 1973).- (b)
The middle zone contains lipoproteins and starch. The cells are large and
vacuolate. The lipoprotein inclusions are irregular (diameter 5-50 f.Lm). They
are either enclosed in the central vacuole or are limited by a fine membrane
in the periphery of the cpll. Amyloplasts are abundant in these cells while
other cytoplasmic organelles like ER, dictyosomes, mitochondria and
ribosomes are rather sparse. (c) The inner zone contains starch. these cells
are very large and show a thin parietal layer of cytoplasm. (d) The central
zone cells do not have any reserve food material (Dexheimer 1973).

Seed. The seed coat is contributed both by the chalaza and integument.
The integument differentiates into three zones: (a) outer (parenchymatous)
sarcotesta, (b) middle (sclerenchymatous) sclerotesta and (c) innermost (thin-
walled) endotesta. The sarcotesta is 5-6 mm thick in the equatorial region,
and 2-3 mm in the micropylar and chalaza! region; it is the only "live"
portion of the integument. The epidermis has a ca. 15-f.Lm-thick cuticle which
is interrupted above the stomata. The cells contain chloroplasts, some of
them also have druses of monohydrate calcium oxalate crystals. The bulk
of the integument is formed of large turgid cells, the latter further enlarging
towards the sclerotesta, while the number of chloroplasts and the size of
starch grains decrease (Favre-Duchartre 1958). The stomata comprise two
kidney-shaped cells filled with starch grains; the frequency is about eight
stomata per mm2 •
The sclerotesta is hard and lignified. It forms the shell, which is slightly
flattened laterally, and usually has two ribs (facing each other) which represent
the suture. It is unlignified in the micropylar half, and forms as many
valves as there are ribs.
a
The endotesta has withered appearance. In the micropylar half it adheres
to the sclerotesta-a~d is free from the nucellus. It is parchment-like, has a
golden-brown c.olq~r, and is thin and translucent. In the chalaza! half, the
endotesta unites :with the nucellus, adheres to the prothallus, but is always
separate from sclerotesta (Favre-Duchartre 1958).
The seed coat is vascularized. Several unbranched vascular bundles are
arranged in a ring, the traces enter the chalaza, pass through the basal plate
of thick-walled cells (see Schnarf 1937).
The mature seed is the size of a small apricot. The seed coat has an outer
orange-coloured fleshy portion rich in butyric acid, and emits an odour like
rancid butter. Inner to the fleshy layer is the stony layer, followed by the
innermost papery layer. The dicotyledonous embryo is in the centre of the
gametophyte, the so-called endosperm. The two cotyledons are normally
equal and hypogeal, and have stomata mainly on the adaxial surface.
 Ginkgoales 125

Germination. During germination, the plumule is pushed out of the testa

by elongation and arching of the cotyledonary base. A portion of the cotyledon
remains inside the seed, enlarges and persists through the first season, and
functions as a haustoria! organ. The first two or three leaves on the seedling
are small and scale-like. The young stem stops· its rapid elongation
during the first winter after it has formed a close crown of leaves and large
terminal bud.

Chromosome Number
In Ginkgo biloba, the haploid number of chromosomes is 12. There is
evidence of karyotypic changes within the species. The basikaryo type is
more or less constant; there are differences in the exact location of centromeres
and in number and position of the satellites (see Khoshoo 1962).

Temporal Considerations
The duration of the life cycle in G. biloba is 14 months (Favre-Duchartre
1958).
The ovules are pollinated (in Paris) at the megaspore stage in the second
half of April (first year). The pollen grains begin to germinate 3 weeks
after landing in the pollen chamber.
In the female prothallus, the coenocytic phase continues until the middle
of June, and archegonial initials can be observed by the end of the month.
Fertilization begins early in September. Both fertilized and unfertilized
ovules are shed from the tree at irregular intervals from October to April
(second year).
The cellular proembryo develops within the prothallus. As soon as the
embryo matures, it germinates in June (second year), in adequate water and
at suitable temperature, without a period of dormancy.

Phylogenetic Considerations
The fossil record provides only a few clues as to when the Ginkgoales
appeared first, and the ancestral group from which they have been derived
(see Stewart 1983).
Trichopitys is one of the Palaeozoic genera which may have affinities
with the Ginkgoales. However, there is no evidence of differentiation of
Trichopitys axes into short shoots (a feature of Ginkgo biloba). Florin (1949)
contemplates that as short shoots evolved, the ovulate branches present on
the long branches of Trichopitys were transferred to the short shoots, and
the number of ovules on a branch became reduced to one. or two. Some
support for this hypothesis can be derived from abnormal specimens of
G. biloba, where several ovules are formed on an axillary branch system
quite similar to that of the fertile truss of Trichopitys. If Trichopitys represents
an early stage in the evolution of Ginkgophytes, then the relationship of the
group appears- to be with the conifer type. This is because the fertile shoot
126 The Gymnosperms

of Trichopitys is in the axil of a foliar unit (cf. Cordaitales) and is not a

megasporophyll with ovules (Pteridospermales). On the other hand, evidence
from the abnormal production of ovules on leaves of G. biloba does not
support this view. Embryologically, too, they are similar to cycads (pollen
grains, male gametophyte, motile sperms, development of female
gametophyte). This implies that the origin of Ginkgoales from the conifer
type is far from settled. According to Stewart (1983), it is best to presume
that the Ginkgoales originated from a Palaeozoic ancestor, until there is
more evidence from the fossil record.
While we know a good deal about various aspect of biology of Ginkgo
biloba, priority attention should be paid to the gaps in our knowledge. A
detailed and critical investigation should be made by applying newer
techniques. A fascinating area of research would be the examination of
living material through cine films. Microcinematography will bring out the
sequenees of development (such as in the development of motile male
gametes) which are otherwise difficult to follow. The scope in this method
of study is endless.