Planta
Planta <1994)193:115-122
9 Springer-Verlag 1994
Populations of plastids and mitochondria during male reproductive
cell maturation in Nicotiana tabacum L.: A cytological basis for
occasional biparental inheritance
Hong-Shi Yu, Scott D. Russell
Department of Botany and Microbiology,Universityof Oklahoma, Norman, OK 73019, USA
Received: 16 August 1993 / Accepted: 21 September 1993
Abstract. The dynamics of plastid and mitochondrial
populations in male reproductive cells of tobacco (Nicotiana tabacum L.) were examined during development using serial ultrathin sections and transmission electron microscopy to reconstruct 58 generative cells and 31 sperm
cells at selected stages of maturation from generative cell
formation through gametic fusion. The first haploid mitosis resulted in incomplete exclusion of plastids providing an average of 2.81 plastids and 82.7 mitochondria for
each newly formed generative cell. During generative-cell
maturation, plastid content decreased to an average of
0.48 plastids/generative cell at anthesis owing to autophagy of organelles. Plastids were present in low frequency within generative and sperm cells in the pollen
tube and appeared to be transmitted, according to observations immediately prior to fertilization. This forms a
cytological basis for genetic reports of occasional biparental plastid inheritance. In contrast, mitochondria
were transmitted in larger numbers, and approximately
80 mitochondria per generative cell or sperm cell pair
were retained throughout development. This provides a
potentially stable source for the transmission of male mitochondrial DNA, if present at fertilization.
Key words: Autophagy- Cytoplasmic inheritance Generative cell - Nicotiana (reproductive cells) - Plastid
diminution Sperm cell
Introduction
The inheritance of plastids in flowering plants has been
categorized into three types: maternal, paternal and biparental. Uniparental, maternal inheritance is the prevalent pattern, found in the majority of angiosperms. Biparental inheritance occurs in a minority of higher
Abbreviations: GC - generativecell; SC = sperm cell
Correspondence to: S.D. Russell; FAX: 1 (405) 325 7619
plants, and exclusively paternal inheritance has just been
reported recently in a few taxa (for reviews, see Kirk and
Tilney-Bassett 1978; Sears 1980; Smith 1988; Russell
1992).
A number of cytological mechanisms have been proposed for uniparental maternal inheritance of plastids,
including: (i) exclusion of plastids during the first and/or
second post-meiotic mitosis (Hagemann 1981), (ii) degeneration or alteration of plastids during the maturation of
the generative cell (GC) and sperm cell (SC), and (iii)
elimination of plastids during the process of fertilization
(Hagemann and Schr6der 1989). For species to display
biparental plastid inheritance, both male and female
gametes must contain plastids that are transmitted into
the next generation. In the case of exclusively paternal
plastid inheritance (Medicago, Zhu et al. 1993; Daucus,
Hause 1991), maternal plastids are distributed toward the
micropylar end of the egg cell, apparently excluding maternal plastids from the lineage of the embryo proper at
the first division of the zygote.
Small proportions of hybrid offspring have been reported recently in plants previously thought to exhibit
exclusively maternal plastid inheritance (Cornu and
Dulieu 1988; Dulieu et al. 1990; see also Table 9.3 in
Smith 1988). Smith (1988) therefore distinguishes species
with biparental plastid inheritance into those species
with "regular" occurrence of paternal plastids (>95%
with paternal plastid expression) from those with "occasional" transmission (< 5 % with paternal plastid expression).
Medgyesy et al. (1986) revealed a very low frequency
of paternal plastid expression in the progeny of Nicotiana
tabacum (0.07% for interspecific crosses, and 2.5% for
intraspecific crosses) thereby indicating that tobacco conforms to the latter pattern. In the current study, the organelle content of generative and sperm cells was examined during development to determine the dynamics of
plastid and mitochondrial populations and to evaluate
whether there exist in the male gamete transmissible plastids and mitochondria which may be inherited in the embryo.
116
H.-S. Yu and S.D. Russell: Plastids and mitochondria in tobacco pollen
Materials and methods
Plants of Nicotiana tabacum L. cv. Da Qing Ye (Solanaceae) used for
this study were kept in cultivation under controlled growth conditions (19-28~ 80-90% humidity, 15 h daylength) in growth chambers.
Selected stages of male gametophyte development were examined including: (i) early, mid and anthesis GCs in the pollen grain
prior to germination, (ii) pre-mitotic GCs in the pollen tube, (iii)
newly formed SCs (8.5 h after pollination), (iv) later SCs when the
pollen tube has elongated to 2/3 of the stylar length (24.5 h after
pollination), and (v) discharged SCs in the degenerated synergid
after pollen-tube arrival (45 h after pollination).
Anthers were fixed at three stages of maturity estimated by the
length of the flower bud, including the early GC, maturing GC and
mature pollen at anthesis. For GCs in pollen tubes, emasculated
flowers were hand-pollinated with mixed pollen from several individuals. For semi-vivo studies, styles and attached stigmas were
excised just ahead of the growing pollen tubes and floated on a 10%
sucrose-0.01% boric acid medium at 28 30~ for approximately
1.5 h. When pollen tubes began emerging from the cut end of the
styles, 1-mm-long stylar segments were collected, chemically fixed
and prepared for electron microscopy (Yu et al. 1989, 1992). For SCs
within the ovule, whole ovules were collected and fixed from handpollinated emasculated flowers. All materials were fixed, dehydrated and embedded as previously described (Yu and Russell 1992a).
Serial sections of anthers, pollen grains, styles and embryo sacs
were cut at 100 nm using a diamond knife, collected, stained, and
Fig. la, b. Electron micrographs showing part of a pollen grain of
tobacco containing a lenticular generative cell (GC). a Overview.
GN, generative nucleus; M, mitochondria; P, plastids; V, vacuole;
observed as previously described. Each micrograph was printed at
the same magnification and numbered according to its order in the
series (Yu and Russell 1992b).
The number of organelles was determined by a direct comparison of organelle profiles throughout the series, counting each organelle once. For materials in pollen grains, serial electron micrographs of nine newly formed GCs were taken and the organelles
counted using the micrographs. The counting of plastids in 12 additional newly formed GCs and 21 GCs at anthesis was performed by
analyzing serial sections in the electron microscope. Statistical comparisons among groups of samples from different developmental
stages were made using an analysis of variance (ANOVA).
Results
Ultrastructural aspects of the development of male reproductive cells. O n e week before a n t h e s i s , the m i c r o s p o r e
undergoes an unequal division, forming a lens-shaped
G C w h i c h is a t t a c h e d to the i n t i n e wall (Fig. la). T h e
n e w l y f o r m e d G C c o n t a i n s n u m e r o u s m i t o c h o n d r i a , dict y o s o m e s , e n d o p l a s m i c r e t i c u l u m , a n d s o m e t i m e s a few
p l a s t i d s (Fig. la). T h e u l t r a s t r u c t u r e a n d size of the m i t o c h o n d r i a a n d p l a s t i d s are i n i t i a l l y s i m i l a r to t h o s e in the
v e g e t a t i v e cell. S o m e G C p l a s t i d s c o n t a i n s m a l l s t a r c h
grains and plastoglobuli. Mitochondrial constrictions
VC, vegetative cell; VN, vegetative nucleus, x 11 400; Bar = 2 lam.
b Dividing mitochondrion, x 30 000; Bar = 0.5 ~tm
H.-S. Yu and S.D. Russell: Plastids and mitochondria in tobacco pollen
117
frequently occur in the GC providing the appearance
that they are dividing (Fig. lb).
The G C then detaches from the intine and becomes a
free cell within the vegetative cell cytoplasm. At anthesis,
the GC is spindle-shaped, associated with the vegetative
nucleus and contains a complete complement of organelles, except that plastids are less frequently observed
at this stage (Fig. 2a,b). Plastids in both the G C and vegetative cell stain lightly at this stage with rare internal
lamellae and some paracrystalline inclusions (Fig. 2a,b).
By 9 h after pollination, the highly elongated GC undergoes mitosis, producing two SCs (Yu and Russell
1993). The GC before and during this division has the
same organellar composition as at maturity (Fig. 3a). The
plastids in the G C and in the SCs contain darkly staining
contents and small starch grains during pollen-tube
growth (Figs. 3a,b and 4). Mitochondria in the SCs are
well formed and contain numerous cristae (Fig. 4).
Autophagic activity in the newly formed GC. A conspicuous feature of the lenticular GC is the presence of variably sized vacuoles which occupy a considerable volume
of the cell. These vacuoles contain abundant electrondense bodies of presumed cytoplasmic origin, suggesting
an autophagic function (Fig. 5a). In addition to abundant
amorphous, darkly stained inclusions in these vacuoles,
double-membrane-bound structures are also observed,
which are presumably mitochondria according to their
size and internal membranes (Fig. 5b). Up to 40 recognizable mitochondria were observed in the autophagic vacuoles of one GC. Individual mitochondria or plastids in
the GC are apparently engulfed by ER, fully enclosing
them within small vacuoles, along with the surrounding
cytoplasmic components of the G C (Fig. 5c-e). Small
vacuoles are also observed attached to and fusing with
larger vacuoles (Fig. 5f), apparently releasing their inclusions into the vacuole (Fig. 5 g). Endoplasmic-reticulum
lamellae also remain associated with these vacuoles.
Plastid abundance during development. Plastids were observed throughout GC and SC development, but are
most abundant in the newly formed GC, with an overall
frequency of 2.81 _+ 1.33 plastids/cell. Of 21 GCs examined, five contained a single plastid, three contained two
plastids, and one each contained 4, 7, 10 and 27 plastids.
By anthesis, the average is 0.48 _+ 0.19 plastids/GC
(Fig. 6a), which is significantly less than at G C inception
(P < 0.05) and decreases to 0.19 __+ 0.19 before GC mitosis (one G C had three plastids, whereas the other 15 did
not have any). In newly formed SC pairs, no plastids were
observed (n = 7), although plastids were seen at 18 h after pollination using winter flowers with a frequency of
1.00 _+ 0.53 plastids/SC pair (Yu et al. 1992) and 0.29 _+
0.29 plastids/SC pair at 26 h after pollination. No statistically significant differences were noted in the plastid population during the last three developmental stages. Inside
the embryo sac, no plastids were observed in three serially reconstructed SCs, but this is probably a result of the
small sample size available.
Mitochondrial abundance during development. The newly
formed GC contains an average of 82.7 _+ 8.64 mitochon-
Fig. 2a, b. Electron micrographs of pollen grain of tobacco at anthesis. a Spindle-shaped GC containing a nucleus (GN), microtubules
(MT), and a plastid (P). Note the association between the GC and
the vegetative nucleus (VN). • 8900; Bar = 2 p.m. b Another section of the same GC as in a, showing the edge of the nucleus (GN),
mitochondrion (M), and plastids (P). x 33 700; Bar = 0.5 Bin. ER,
endoplasmic reticulum; VC, vegetative cell
118
H.-S. Yu and S.D. Russell: Plastids and mitochondria in tobacco pollen
Fig. 3a, b. Electron micrographs showing GC and sperm pair (S,,,
and Su, ) in pollen tubes of tobacco, a Dividing GC at 9 h after
pollination containing two groups of chromatids (Ch), plastids (P),
and mitochondria (M). b Sperm pair at 26 h after pollination, with
a plastid (P) in Sua. SN, sperm nuclei; VN, vegetative nucleus.
x 7 1 5 0 a ; x 5 7 0 0 b ; B a r s = 2~tm
Fig. 4. Electron micrograph showing part of the sperm cell (SC) in
the tobacco pollen tube (P?). Plastids (P) contain darkly stained
matrix and small starch grains, distinguishing them from mitochondria (M). SN, sperm nucleus; V, vacuole, x 26 000; Bar - 0.5 gm
Fig. 5a-g. Electron micrographs showing the activity of autophagic
vacuoles (AV) in newly formed generative cells (GC) of tobacco, a
Overview of a GC containing a nucleus (GN), mitochondria (M),
plastids (P) and several autophagic vacuoles containing a b u n d a n t
degenerating cytoplasmic structures, x 9900; Bar = 2pro. b
Higher magnification showing three degenerating mitochondria in
autophagic vacuole. • 36 000; Bar = 0.5 lam. c Mitochondrion (M)
apparently being enclosed by ER membrane. Note numerous dictyosomes (D) in the cytoplasm of the GC. x 19 000; Bar = I pm. d
Plastid enclosed within newly formed autophagic vacuole.
x 38 000; Bar = 0.5 ~tm. e Mitochondrion (M) enclosed within
newly formed autophagic vacuole. • 38 000; Bar = 0.5 pro. f Two
small autophagic vacuoles (arrowheads) attached to a large autophagic vacuole, x 15 600; Bar = 1 ~tm. g Small autophagic vacuole fusing (arrows) with a larger one, engulfing a cytoplasmic structure (arrowhead). x 15 000; Bar = 1 pm. VC, vegetative cell
H.-S. Yu and S.D. Russell: Plastids and mitochondria in tobacco pollen
119
120
H.-S. Yu and S.D. Russell: Plastids and mitochondria in tobacco pollen
E
"53
,///'
z 2
/ ////
//, /
/ / /
//
I(n=21) II (n=21) Ill (n=16) IV(n=7) V(n=7)
Developmental Stages
Vl(n=l)
100
q-
-q-
80
o
o
60
/////
4o
r,////
/ ,/
/
/
(',,,
' ///'
Z
9
>~ 20
<
/////
...., ,
K//;'%
I (n=9)
, .,..j.;
,,_
III (n=16) IV (n=7)
V (n=7)
Developmental Stages
Vl (n=l)
Fig. 6a, b. Comparison of organelle content in generative cells and
sperm pairs at different stages of development in Nicotiana tabacum.
All of the values (mean _+ SE) are from analyses of complete series
of ultrathin sections, a Plastid abundance, b Mitochondrial abundance. Developmental stages: 1, pollen grain containing newly
formed GCs; II, pollen grain at anthesis; III, pollen tube containing GC at 9 h after pollination (just before mitosis); IV, pollen tube
containing newly formed SCs; V, pollen tubes containing SCs at
26 h after pollination; V/, SCs inside the embryo sac
dria. This frequency remains essentially unchanged
throughout pollen maturation and pollen-tube growth
(Fig. 6b), with about 80 mitochondria/SC pair observed
up to 26 h after pollination. After discharge from the pollen tube, mitochondria are still evident in the SC (Huang
et al. 1993); one SC pair in the degenerated synergid contained 56 mitochondria. During fusion with the central
cell, sperm mitochondria can still be discriminated. One
reconstructed SC contained 25 mitochondria, suggesting
that SCs retain their mitochondria through fertilization.
Discussion
Plastid exclusion and diminution during GC formation and
maturation. Although plastids are common throughout
the cytoplasm of the microspore, only 57% of the newly
formed GCs contain any plastids. That plastids are absent in 43 % of the GCs supports the concept of physical
exclusion of plastids during GC formation (for reviews,
see Hagemann 1981, Hagemann and Schr6der 1989). In
tobacco, plastid leakage occurs at low frequency (1-2
plastids/GC). Penetration of paternal plastids has also
been proposed in other species with maternal plastid inheritance (Clauhs and Grun 1977; Schr6der and Hagemann 1986; Schmitz and Kowallik 1987; Bednarska
1988; Schr6der and Oldenburg 1990). The present study
suggests that paternal plastid leakage, although not assured in a given individual, may be relatively common on
a populational basis. Factors controlling plastid distribution during GC formation are reported to include microtubule involvement (Van Went 1984; Tanaka 1991), microfilaments (Schr6der et al. 1988) and biochemical gradients (Van Went 1984; Schr6der 1985); further study is
needed confirm the validity and importance of these different models in plastid exclusion.
The most intense period of plastid diminution occurs
before anthesis, with smaller decreases in paternal plastid
frequency thereafter (Fig. 6a). Similar declines during
pollen maturation have also been reported based on
qualitative and stereological analyses of other species
with maternal plastid inheritance (Pisum, Clauhs and
Grun 1977; Gasteria, Schr6der 1986; Convallaria,
Schr6der and Hagemann 1986; Hyacinthus, Bednarska
1988), although in Epilobium (Schmitz and Kowallik
1987), plastid diminution did not occur until after GC
division. Although some periods of plastid diminution
seem to be more intense than others, plastid frequency
seems to diminish throughout GC and SC development.
Whatever mechanisms act to diminish the content of
plastids in the male cells presumably function at differing
intensities during development.
Mechanisms of plastid diminution during GC maturation.
Several structural mechanisms have been proposed by
which plastids could be eliminated from the male cytoplasm; however, the most attractive involve the intracellular digestion of GC plastids, as in the pollen grains of
Convallaria (Schr6der 1986) and Hyacinthus (Bednarska
1988) and as in the pollen tubes of Epilobium (Schmitz
and Kowallik 1987) and Lolium (Pacini et al. 1992). Most
of the plastid elimination observed in the GC of Nicotiana appears to be accomplished by a process of autophagy in the pollen grain9
In a classical study on root meristematic cells of Euphorbia, Marty (1978) proposed a cooperative role for
Golgi bodies, endoplasmic reticulum and lysosomes in
the formation of autophagic vacuoles9 Using this model
and those established for autophagic activities in animal
cells (Holtzman 1989), we presume that the autophagic
mechanism for plastid diminution in the GC includes: (i)
the enclosure of mitochondria or plastids by membranes
derived from the endoplasmic reticulum, creating an autophagosome; (ii) the fusion of lysosomal vesicles with
the autophagosome forming an autophagolysosome in
which the enclosed organelles are digested through the
activity of hydrolases; (iii) successive fusions between autophagosomes and/or autophagolysosomes resulting in
the formation of a large autophagic vacuole; and (iv)
ultimately, the fusion of all of the lysosomal compartments, feeding into one large vacuole. In this way, both
H.-S. Yu and S.D. Russell: Plastids and mitochondria in tobacco pollen
GC mitochondria and plastids are digested. Apparently,
higher rates of mitochondrial replication offset the loss of
mitochondria, and therefore the net outcome of autophagy is a selective elimination of plastids.
Plastids are presumably also diminished through the
production of plastid-containing enucleated cytoplasmic
bodies (ECBs), which are pinched-off from the surface of
the cell. The production of ECBs has been implicated in
the diminution of cytoplasmic organelles during the maturation of GCs in the pollen grains of Plumbago (Russell
and Yu 1991) and Cymbidium (Yu and Russell 1992b),
and during SC maturation before anthesis in barley
(Mogensen and Rusche 1985). This is also observed in the
GCs and SCs in the pollen tubes of Nicotiana (Yu et al.
1992; Yu, unpublished data), but not in the pollen grains.
Mitochondrial behavior during GC and SC maturation. In
contrast to the population of plastids, larger numbers of
mitochondria are found in the newly formed GC and
throughout development. Evidently, the incorporation
and maintenance of plastids and mitochondria in the GC
are controlled by independent systems, because the polarized distribution of plastids and their apparently low
rates of replication do not seem to affect the behavior of
the mitochondria. Frequently, mitochondria are observed in autophagic vacuoles, but apparently the formation of mitochondria is counterbalanced by their continued division. Presumably, mitochondria are selectively
maintained to provide for the physiological needs of the
male reproductive cells, whereas no such critical needs
may be met by GC and SC plastids.
Although it appears likely that ECB production may
reduce the number of cytoplasmic organelles in tobacco,
the phase during which the greatest organelle diminution
occurs coincides with the greatest degree of autophagy.
Autophagy and ECB production apparently represent
redundant post-inception mechanisms for plastid
diminution in the male gamete lineage. The effectiveness
of organelle engulfment into ECBs for controlling the
plastid population, however, may be difficult to estimate
because of their low frequency.
Some insight into the underlying biochemical mechanisms of plastid diminution in flowering plants may be
found in the recent suggestion by Nakamura et al. (1992)
that nuclease C - an enzyme involved with the direct
elimination of paternal chloroplast nucleoids in the unicellular alga ChIamydomonas reinhardtii after gamete fusion (Ogawa and Kuriowa 1985) may have a role in
angiosperm plastid inheritance. Interestingly, zygotic and
post-zygotic plastid-elimination mechanisms have not
been found in angiosperms to date, and therefore, plastid
diminution appears to be restricted to developmental
stages prior to nuclear fusion. If the activity of nuclease C
is the major biochemical reason for plastid diminution
prior to anthesis, it may constitute the proposed "lethality factor" which Clauhs and Grun (1977) suggest acts to
cause the disappearance of GC plastids but not mitochondria in Solanum. According to Nakamura et al.
(1992), plants with biparental inheritance appear to lack
this nuclease.
Further mechanisms may also exist to diminish male
121
cytoplasmic organelles after the release of the SCs from
the pollen tube in the embryo sac. Huang et al. (1993)
observed numerous small ECBs containing sperm mitochondria immediately after the discharge of the pollen
tube in tobacco. Our observations of a discharged SC in
the degenerated synergid indicates that at least 25 mitochondria remain at the onset of gametic fusion with the
central cell. Although the number of observations is low,
in tobacco a significant number of mitochondria are
present in the SC and appear to be transmitted during
the process of gametic fusion (data not shown). Apparently, gametic fusion may not present a barrier for the
transmission of male cytoplasm in tobacco as it does in
some other angiosperms (for review, see Russell 1992).
Conclusions. Nicotiana tabacum appears to be intermediate between the Lycopersieon type of plastid inheritance,
which is currently understood as being purely maternal,
and the Solanum type which is biparental (Hagemann
and Schr6der 1989). In N. tabacum, plastid inheritance is
usually maternal with a significant but low frequency of
biparental inheritance (Medgyesy et al. 1986; see Smith
1988 for review). Plastids are partially prevented from
transmission into the male reproductive cells in this plant
by: (i) physical exclusion during the first post-meiotic mitosis; (ii) autophagic degradation during GC maturation
in the pollen grain; and (iii) ECB formation during GC
and SC descent in the pollen tube. Occasional "leakage"
of plastids through these elimination mechanisms results
in the low frequency of plastid occurrence in the SCs of
tobacco. These few plastids are likely to be transmitted
during fertilization. With the use of more sensitive genetic
analyses including molecular analyses on larger sample
sizes (Smith 1988), more species of the Lycopersicon type
may be found to display occasional biparental inheritance of plastids. Presumably, this will also occur as more
extensive use is made of serial sectioning techniques on
larger cell samples. Although the only genetic analysis
currently available indicates that a pure maternal inheritance of mitochondria is the case for tobacco (Medgyesy
et al. 1986), if SC mitochondria contain DNA they probably transmit it. Unpublished evidence strongly supports
that cytoplasmic organelles are transmitted in tobacco
during double fertilization, furnishing these cells with the
potential for male mitochondrial inheritance.
We thank Dr. Frank J. Sonleitner, for helpful suggestions on the
statistical calculationsand Dr. Bing-Quan Huang for technical assistance in the preparation of embryo sacs during fertilization.This
research was supported in part by U.S. Department of Agriculture
grant 91-37304-6471. We gratefullyacknowledgeuse of the Samuel
Roberts Noble Electron MicroscopyLaboratory of the University
of Oklahoma.
References
Bednarska, E. (1988) Ultrastructural transformations in the cytoplasm of differentiatingHyacinthus orientalis L. pollen cells. Acta Soc. Bot. Pol. 57, 235-245
Clauhs, R.P., Grun, P., (1977) Changes in plastid and mitochondrion content during maturation of generative cells of Solanum
(Solanaceae). Am. J. Bot. 64, 377-383
122
H.-S. Yu and S.D. Russell: Plastids and mitochondria in tobacco pollen
Cornu, A., Dulieu, H. (1988) Pollen transmission of plastid-DNA
under genotypic control in Petunia hybrida Hort. J. Hered. 79,
4~44
Dulieu, H., Derepas, A., Cornu, A. (1990) Le r61e du pollen dans la
transmission des chloroplastes et des mitochondries. Etude d'un
cas particulier chez Petunia. Bull. Soc. Bot. Fr. 137, 49-56
Hagemann, R. (1981) Unequal plastid distribution during the development of the male gametophyte of angiosperms. Acta Soc. Bot.
Pol. 50, 321-327
Hagemann, R., Schr6der, M.B. (1989) The cytological basis of the
plastid inheritance in angiosperms. Protoplasma 153, 57-64
Hause, G. (1991) Ultrastructural investigations of mature embryo
sacs of Daucus carota, D. aureus and D. muricatus - possible
cytological explanations of paternal plastid inheritance. Sex.
Plant Reprod. 4, 288-292
Holtzman, E. (1989) Lysosomes. Plenum Press, New York
Huang, B.Q., Strout, G.W., Russell, S.D. (1993) Fertilization in Nicotiana tabacum: Ultrastructural organization of propane jetfrozen embryo sacs in vivo. Planta 191, 256-264
Kirk, J.T.O., Tilney-Bassett, R.A.E. (1978) The plastids. Their chemistry, structure, growth and inheritance. Elsevier/North Holland,
Amsterdam
Marty, F. (1978) Cytochemical studies on GERL, provacuoles, and
vacuoles in root meristematic cells of Euphorbia. Proc. Natl.
Acad. Sci. USA 75, 852-856
Medgyesy, P., P~y, A., Mfirton, L. (1986) Transmission of paternal
chloroplasts in Nicotiana. Mol. Gen. Genet. 204, 195 198
Mogensen, H.L., Rusche, M.L. (1985) Quantitative analysis of barley sperm: occurrence and mechanism of cytoplasm and organelle reduction and the question of sperm dimorphism. Protoplasma 128, 1-13
Nakamura, S., Ikehara, T., Uchida, H., Suzuki, T., Sodmergen (1992)
Fluorescence microscopy of plastid nucleoids and a survey of
nuclease C in higher plants with respect to mode of plastid
inheritance. Protoplasma 169, 68-74
Ogawa, K., Kuriowa, T. (1985) Destruction of chloroplast nuclei of
the male gamete by calcium and nuclease C in a cell model of
Chlamydomonas reinhardtii. Plant Cell Physiol 26, 493-503
Pacini, E., Taylor, P.E., Singh, M.B., Knox, R.B. (1992) Development of plastids in pollen and tapetum of rye-grass, Lolium
perenne L. Ann. Bot. 70, 179 188
Russell, S.D. (1992) Double fertilization. Int. Rev. Cytol. 140, 357388
Russell, S.D., Yu, H.-S. (1991) Male cytoplasmic diminution in generative and sperm cells of flowering plants. Am. J. Bot. 78 (6,
suppl.), 34
Schmitz, U.K., Kowallik, K.V. (1987) Why are plastids maternally
inherited in Epilobium? Ultrastructural observations during microgametogenesis. Plant Sci. 53, 139-145
Schr6der, M.B. (1985) Ultrastructural studies on plastids of generative and vegetative cells in Liliaceae: 3. Plastid distribution dur-
ing the pollen development of Gasteria verrucosa (Mill.) Duval.
Protoplasma 124, 123-129
Schr6der, M.B. (1986) Ultrastructural studies on plastids of generative and vegetative cells in Liliaceae: 4. Plastid degeneration
during generative cell maturation in Convallaria majalis L. Biol.
Zentralbl. 105, 427433
Schr6der, M.B., Hagemann, R. (1986) Ultrastructural studies on
plastids of generative and vegetative cells in Liliaceae. 6. Patterns of plastid distribution during generative cell formation in
Aloe secundiflora and A. jucunda. Acta Bot. Neerl. 35, 243-248
Schr6der, M.B., Oldenburg, H. (1990) Ultrastructural studies on
plastids of generative and vegetative cells in Liliaceae. 7. Plastid
distribution during generative cell development in Tulbaghia violacea Harv. Flora 184, 131-136
Schr6der, M.B., van Lammeren, A.A.M., Kieft, H. (1988) The intracellular arrangement of cytoskeletal components and organelles
in pollen grains of Gasteria verrucosa (Mill.) H. Duval. Ann. Sci.
Univ. Reims 23, 80
Sears, B.B. (1980) Elimination of plastids during spermatogenesis
and fertilization in the plant kingdom. Plasmid 4, 233-255
Smith, S.E. (1988) Biparental inheritance of organelles and its implications in crop improvement. Plant Breed. Rev. 6, 361-393
Tanaka, I. (1991) Microtubule-determined plastid distribution during microsporogenesis in Lilium longiflorum. J. Cell Sci. 99, 21
31
Van Went, J.L. (1984) Unequal distribution of plastids during generative cell formation in Impatiens. Theor. Appl. Genet. 68, 305
309
Yu, H.-S., Russell, S.D. (1992a) Generative cell mitosis in pollen
tubes of Nicotiana tabacum: ultrastructure and three-dimensional reconstruction. In: Proceedings of the 50th Annual Meeting of
the Electron Microscopy Society of America, pp. 848 849, Bailey, G.W., Bentley, J., Small, J.A., eds. San Francisco Press, San
Francisco
Yu, H.-S., Russell, S.D. (1992b) Male cytoplasmic diminution and
male germ unit in young and mature pollen of Cymbidium goeringii: a 3-dimensional and quantitative study. Sex. Plant Reprod. 5, 169 181
Yu, H.-S., Russell, S.D. (1993) Three-dimensional ultrastructure of
generative cell mitosis in pollen tubes of Nicotiana. Eur J Cell
Biol 61, 338-348
Yu, H.-S., Hu, S.-Y., Zhu, C. (1989) Ultrastructure of sperm cells and
the male germ unit of Nicotiana tabacum. Protoplasma 152, 2936
Yu, H.-S., Hu, S.-Y., Russell, S.D. (1992) Sperm cells in pollen tubes
of Nicotiana tabacum L.: Three-dimensional reconstruction, cytoplasmic diminution and quantitative cytology. Protoplasma
168, 172-183
Zhu, T., Mogensen, H.L., Smith, S.E. (1993) Quantitative, three-dimensional analysis of alfalfa egg cells in two genotypes: implications for biparental plastid inheritance. Planta 190, 143-150