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Sex Plant Reprod (2004) 17: 117–124
DOI 10.1007/s00497-004-0219-7
ORIGINAL ARTICLE
A. A. Estrada-Luna
.
M. García-Aguilar
.
J.-Ph. Vielle-Calzada
Female reproductive development and pollen tube growth
in diploid genotypes of
Solanum cardiophyllum
Lindl.
Received: 2 February 2004 / Accepted: 17 May 2004 / Published online: 2 July 2004
# Springer-Verlag 2004
Abstract We have characterized female gametophyte
(megagametophyte) development and the kinetics of
pollen tube growth in self-pollinated diploid genotypes
(2n=2x=24) of Solanum cardiophyllum Lindl. that show
normal seed formation. In this species megasporogenesis
and megagametogenesis give rise to a female gametophyte
of the Polygonum type composed of two synergids, an egg
cell, a binucleated central cell and three antipodals;
however, asynchronous abnormalities resembling mechan-
isms that prevail during the formation of second division
restitution gametes were observed. In self-pollinated pistils
at least 1–2% of germinating pollen tubes were able to
reach the megagametophyte 60–84 hours after pollination
(hap). Although the egg cell acquired a zygote-like
morphology 60 –84 hap, division of the primary endo-
sperm nucleus was only observed 84 hap. The analysis of
genetic variability in full-sib progeny confirmed that seeds
are derived from sexual reproduction. These observations
suggest that diploid genotypes of S. cardiophyllum can
serve as an ideal system to genetically investigate true seed
formation in a tuber-bearing Solanum species.
Keywords Solanum
.
Megagametophyte
.
Pollen tube
.
Megasporogenesis
.
Megagametogenesis
Introduction
The commercial production of potato (Solanum tuberosum
L.) has generally relied on vegetative propagation and the
use of tubers for planting, with several disadvantages that
include high storage and transport costs, vulnerability of
fresh tissue to pathogen transmission and long-term
physiological degeneration (Almekinders et al. 1996;
Martin 1988). These sorts of problems have generated
renewed interest in the potential use of true potato seed
(TPS) as the initial material for field establishment,
especially in developing countries that do not have access
to good quality seed tubers (Jaworsky et al. 1988; Ortiz
1997). The eventual use of TPS technology for potato will
require a solid understanding of female reproductive
development and seed formation in the genus Solanum
since vegetative propagation will have to be replaced by
the production of sexually derived seed.
The use of diploid wild species has simplified genetic
analysis and made the study of reproductive development
more feasible in Solanum spp.; however, rapid progress
has been hindered by self-incompatibility at the diploid
level. Most diploid tuber-bearing Solanum species are
characterized by a gametophytic self-incompatibility
system controlled by an S-ribonuclease active in the
haploid pollen grain and in the diploid style (Xu et al.
1990; de Nettancourt 2001). Self-incompatibility comple-
tely prevents self-fertilization, as well as fertilization by
any pollen grain sharing an S allele with the female parent.
Although gametophytic self-incompatibility prevails as a
general rule, the identification of self-compatible geno-
types has been documented in several species and some
interspecific crosses. For example, Cipar (1964) found
self-compatible variants in S. chacoense, S. goniocalyx, S.
kurtzianum, S. neohawkesii, S. phureja, S. pinnatisectum,
S. raphanifolium, S. sancta-rosae and S. stenotomum.A
self-compatible mutation inhibiting S-allele activity was
identified in dihaploid lines of S. tuberosum (Thompson et
al. 1991), and self-compatible diploid hybrids have been
obtained by crossing S. phureja to dihaploids of S.
tuberosum ssp. andigena (Cipar et al. 1964). Additional
self-compatible genotypes have been characterized
through the identification of a modifier locus (Hosaka
and Hanneman 1998) or the molecular characterization of
modifier genes that affect self-incompatibility in the
Solanaceae (McClure et al. 1999;O’Brien et al. 2002).
Solanum cardiophyllum Lindl. is a wild Mexican potato
that grows throughout the warm and semi-arid highlands
A. A. Estrada-Luna
.
M. García-Aguilar
.
J.-P. Vielle-Calzada (*)
Laboratory of Reproductive Development and Apomixis,
CINVESTAV—Plant Biotechnology Unit,
Apartado Postal 629, CP 36500 Irapuato Gto, Mexico
e-mail: vielle@ira.cinvestav.mx
Tel.: +52-462-6239634
Fax: +52-462-6245849
of central Mexico (Zacatecas, San Luis Potosí, Guanajua-
to, Jalisco and Aguascalientes; Luna-Cavazos 1997). It is a
herbaceous annual plant characterized by a short flowering
time (4–6 weeks) and the formation of small edible tubers
that usually start differentiating 4 weeks after planting.
The species shows great phenotypic variation associated
with the wide range of environmental conditions in which
it grows (Galindo-Alonso 1982). Four groups of taxono-
mically related populations with variable ploidy levels
have been morphologically and cytogenetically character-
ized (Luna-Cavazos 1997). Whereas diploid genotypes
present the highest fertility among these populations,
tetraploid individuals usually show reduced male fertility
and poor seed set (Luna-Cavazos et al. 1988). Although
pollen morphology and viability has received some
attention, a detailed characterization of ovule and female
gametophyte development has not been documented in
this species.
To explore the potential use of S. cardiophyllum as a
model system for studying the developmental biology of
female gametophyte development and seed formation, we
collected a wide range of its naturally grown variants and
identified several diploid genotypes (2n=2x=24) that form
viable seeds after self-pollination (Estrada-Luna et al.
2002). In this paper we report the reproductive character-
ization of sexual diploid self-compatible genotypes of S.
cardiophyllum suitable for genetic studies. We describe the
mechanism of megasporogenesis and megagametogenesis
at the cellular level and determine the timing for pollen
tube arrival into the female gametophyte and the initiation
of embryo and endosperm development. The identification
of these genotypes and their reproductive characteristics
indicate that S. cardiophyllum is an ideal system for the
investigation of the genetic basis and molecular mechan-
isms regulating female reproductive development and TPS
formation in a tuber-bearing Solanum species.
Materials and methods
Plant collection and micropropagation
Individual plants were selected from a mixed population of
Solanum cardiophyllum collected in different regions of
Central Mexico and micropropagated through culture of
young shoots (0.5 cm length) originally obtained from
germinating tubers. Initially, the young shoots were
dissected from tubers and washed in a solution of 10%
v/v Clorox (6% active Cl) and Tween-20 (0.02%) for
15 min. Propagules were transferred to culture containers
(baby jars) in groups of five shoots per jar after having
been rinsed several times with distilled-sterile water. Basal
salt formulation of Murashige and Skoog (1962),
supplemented with sucrose (3%), agar (0.7%), and
pH 5.7, were used in all steps of micropropagation. The
cultures were maintained in a culture room under a 16-h
light photoperiod with 200 μmol m
2
s
−1
photosynthetic
photon flux density (PPFD), and 23±2°C temperature.
After micropropagation, plantlets with eight pairs of leaves
were selected to be transplanted and transferred to the
glasshouse. The plantlets were taken out from culture
containers, washed with tap water and transplanted to
plastic pots (2 l capacity previously filled with a mix of
peat-moss:perlite (1:1)). To avoid excessive dehydration
and plantlet death, each pot was covered with a plastic bag
for 8 days and daily punctured to allow plant acclimati-
zation. Once a week 100 ml fertilizer solution was applied
to each pot (5 g Peter’s 9-4.5-15 in 1,000 ml water).
Growth temperature and light conditions were maintained
at 25±5° C and 1,000 μmol m
2
s
−1
PPFD, respectively.
Histological analysis of ovule and female
gametophyte development
Selected diploid plants were transplanted from tissue
culture containers and cultured under glasshouse condi-
tions until blooming. Flower buds and mature flowers
were dissected and classified according to their size (0.4–
0.8 mm, 0.8–1 mm, 1–1.8 mm, 1.8–2 mm, 2–2.8 mm, 2.8–
3.5 mm, 3.5–4 mm, 4–4.5 mm, 4.5–5.5 mm, 5.5–6.5 mm,
6.5–7.5 mm, 7.5–8.5 mm, 8.5–9.5 mm, flower at anthesis,
flower 1 day after anthesis, flower 3 days after anthesis).
For analysis of cleared ovules, individual ovaries from
each class were dissected and fixed in FAA (1% formal-
dehyde, 0.5% acetic acid, 50% ethanol) for 24 h at room
temperature and subsequently stored in 70% ethanol.
Fixed samples were cleared according the protocol
established by Jongedijk (1987) with slight modifications.
The ovaries were dehydrated for 1 h in 100% ethanol and
immersed in a series of ethanol:methyl salycilate solutions
(3:1, 1:1, 1:3) for 30 min each. They were transferred to
methyl salycilate (100%) until microscopic observation.
Cleared ovaries were carefully peeled off using 1 ml
disposable syringes. The ovules were separated from the
placenta and mounted on a few drops of methyl salicylate.
Microscopical analysis under Normarsky illumination was
conducted using a Leica DMR microscope (Leica
Microsystems, Wetzlar, Germany). Micrographs were
acquired with a Leica DC 300 digital camera system
using a Leica IM50 Software, version 1.20, release 19.
For analysis of semi-thin sections, fixation was in 3%
glutaraldehyde in 50 mM cacodylate buffer (pH 7.2) for
2 h at room temperature (T=25°C). Following three rinses
in the same buffer (10 min each), samples were postfixed
in 2% OsO
4
in cacodylate buffer for 2 h at room
temperature, dehydrated in an acetone series (10% steps)
and flat embedded in Spurr’s low viscosity resin (Spurr
1969). Semi-thin sections (1–2 μm) were cut in a Leica
UltraCutR microtome (Leica Microsystems, 9435 Heer-
brugg, Switzerland) using glass knives and mounted on
poly-
L-lysine-coated slides. Semi-thin sections were
stained with 1% toluidine blue in sodium metaborate
buffer and observed under conventional brightfield illu-
mination.
118
Observation of pollen tube growth
Mature flowers 1 day prior to anthesis were labeled and
either self-pollinated or reciprocally crossed to the same or
different diploid genotypes. Individual flowers were
collected and their whole gynoecium was dissected and
fixed in FAA at 4, 8, 18, 42, 60, or 84 h after pollination.
For pollen tube staining we followed the protocol
described by Martin (1959). Samples were washed in
distilled water, softened with NaOH (8 N) for 24 h at room
temperature and rinsed with distilled water for 1 h. Pollen
tubes were stained for 4 h with 0.1% aniline blue in 0.1 N
K
3
PO
4
. Stained samples were mounted in slides with
several drops of the same staining solution and observed in
an Olympus BX60 (Model BX60F5) (Olympus Optical,
Japan) microscope equipped with an epifluorescence UV-
filters set (excitation filter at 365 nm). Images were
acquired using Image Pro-Plus Software, version 4.0
(Media Cybernetics, Carlsbad, Calif.).
AFLP analysis
Genomic DNA was extracted from young leaf tissues of 3-
week-old seedlings according to the procedure described
by Doyle and Doyle (1987) and purified in a series of
phenol-chloroform solutions. AFLP analysis was per-
formed using the method described by Vos et al. (1995)
and the commercial IRDye Fluorescent AFLP kit for large
plant genome analysis and protocol (Li-Cor Biosciences,
Lincoln, Neb.), which employs EcoRI and MseIas
restriction enzymes. For selective amplification, the primer
combination included EcoRI+AAG/MseI+CAA (700) and
EcoRI+AGG/MseI+CAA (800), and the fragments ob-
tained were resolved on a 6.5% polyacrylamide gel, which
was run on an IR
2
DNA analyzer device (Li-Cor). For
each fragment only clear and unambiguous bands were
scored and numbered according to their molecular weight.
Results
Early ovule formation
The flowers of S. cardiophyllum are pentamerous with an
ovary formed by two carpels and two semi-spheric
placentas separated by a centrally located septum
(Fig. 1A, B). The septum is the origin of the bicarpelloid
ovary. Sub-epidermal divisions of the placental tissue give
rise to the ovule primordia that initially adopt a
campylotropous form; however, a limited development
of the highly vascularized funiculus results in a mature
ovule with classical anatropous morphology. Ovules are
tenuinucellar and unitegmic, as a single integument starts
to develop and increases its number of cell layers during
ovule formation. Before meiosis, the young nucellus is
composed of a small number of cells that do not proliferate
during subsequent ovule development. A single arche-
osporial cell differentiates in the subepidermal layer of the
nucellus. The archeosporial cell quickly increases in
volume, acquires a dense cytoplasm and a large nucleus
to differentiate into a megaspore mother cell (MMC:
Fig. 2A, F).
Megasporogenesis
Soon after differentiation of the MMC, the first meiotic
division results in two haploid megaspores (Fig. 2B, G).
The chalazally located megaspore is invariably smaller
than the megaspore located at the micropylar pole. We also
noticed frequent asynchrony in the initiation of the second
meiotic division, as the larger micropylar megaspore
usually divides before the smaller chalazal megaspore. As
a result, it is often possible to find young ovules
containing three haploid cells rather than four. The
integument grows substantially during megasporogenesis,
covering at least half of the nucellus by the end of
meiosis I. During meiosis II, the integument completes its
Fig. 1A,B Ovule formation in
Solanum cardiophyllum Lindl.
A Longitudinal section of an
immature ovary showing the
semi-spheric placentas (P) and
the initial ovule primordia
(arrow). B Longitudinal section
of an immature ovary showing
young ovule primordia at the
onset of megasporogenesis
(arrow). O Ovary wall; P pla-
centa; Bar 20 μm
119
Fig. 2A–T Megasporogenesis and megagametogenesis in S.
cardiophyllum Lindl. A–E, K–O Cleared ovules; F–J, P–T
sectioned ovules. A, F Young ovules showing the megaspore
mother cell (arrow, MMC) prior to meiosis. B, G First meiotic
division; young ovules showing a dyad (Dy) of haploid megaspores.
The chalazal megaspore (CM) shows a reduced size. C, H
Differentiation of the functional megaspore (FM); arrows remnants
of three degenerated megaspores. At this stage the integument
completely covers the nucellus and gives rise to a long micropyle
(Mi). The endothelium (En) is fully differentiated and the megaspore
shows a centrally located nucleus (N). D, I Differentiation of a
highly vacuolated (V) mono-nucleated (N) megagametophyte. E, J
Two-nucleated megagametophyte. The first mitotic division gives
rise to two haploid nuclei: the chalazal nucleus (CN) and the
micropylar nucleus (MN). K, P Four-nucleated megagametophyte.
The second mitotic division gives rise to four haploid nuclei: two
chalazally located nuclei (CN ) and two nuclei at the micropylar pole
(MN). L, Q Cellularizing eight-nucleated megagametophyte show-
ing two unfused polar nuclei ( PN ) closely associated with the central
cell wall. M, R
Cellularized megagametophyte showing the unusual
location of the polar nuclei (PN), the egg cell (EC) and two
synergids (arrows) at the micropylar pole. N, S Fully differentiated
megagametophyte showing the two synergids (Sy) prior to degen-
eration in close association to the micropyle (Mi). O, T Megaga-
metophyte showing precocious degeneration of the antipodals (A)
and partial fusion of polar nuclei (PN). Bars 20μm
120
growth by completely covering the nucellus. At the end of
megasporogenesis, most of the nucellar tissue has been re-
absorbed by growth of the four haploid cells, but a small
portion of the chalazal nucellus prevails throughout ovule
development. A highly vacuolized layer of L1 cells is in
direct contact with the megaspores. At this stage the ovule
has almost completed its anatropous rotation and cells of
the internal layer of the integument acquire a dense
cytoplasm and large nuclei as they differentiate in a
classical endothelium that prevails throughout megagame-
togenesis.
Megagametogenesis
The most chalazally located megaspore enlarges as the
three others degenerate (Fig. 2C, H). The first signs of
megaspore degeneration usually occur in the micropylar
megaspore but in a few cases we observed premature signs
of degeneration in the haploid cell neighboring the
functional megaspore. The degeneration of the three
dying megaspores and of the L1 nucellar layer occurs
while the functional megaspore enlarges and becomes
vacuolated, acquiring a characteristic oval form, progres-
sively differentiating into a mono-nucleated female game-
tophyte that is highly vacuolated and contains a large
nucleus with a conspicuous nucleolus. At this stage the
integument is composed of 7–10 cell layers, giving rise to
a long micropyle. The enlarging mono-nucleated female
gametophyte rapidly invades most of the nucellar region,
establishing a direct contact with the endothelium before
its first mitotic division (Fig. 2D, I). At the two-nucleated
stage, the haploid nuclei are located at opposite ends of the
highly vacuolized cell, and a second small vacuole is
invariably formed at the chalazal end, between the chalazal
nucleus and the nucellus (Fig. 2E, J). During the second
and third mitotic divisions, most of the endothelium is
progressively reabsorbed (Fig. 2K, P). We only rarely
observed the eight-nucleated non-cellularized stage in
hundreds of cleared ovules observed, suggesting that
cellularization and differentiation of the female gameto-
phyte occurs rapidly (Fig. 2L, Q). By the end of
megagametogenesis, the female gametophyte contains
two synergids and the egg cell at the micropylar pole, a
binucleated central cell, and three antipodals at the
chalazal end (Fig. 2M, R). Both synergids are highly
vacuolized and contain a centrally located nucleus pressed
against the plasma membrane; the differentiation of the
filiform apparatus occurs well after the cellularization of
both cells (Fig. 2N, S). The egg cell has a characteristic
pear-shaped form with a nucleus located at the chalazal
pole, in close proximity to the central cell; it is extremely
vacuolized and larger than the synergids (Fig. 2M, R). A
small portion of volume around the nucleus is occupied by
its cytoplasm. The central cell accumulates large numbers
of amyloplasts in restricted cytoplasmic regions closely
associated with the lateral walls. The polar nuclei are
usually in close association but remain unfused before
fertilization (Fig. 2O, T). Interestingly, they are invariably
in direct contact with the lateral portion of the plasma
membrane of the central cell and not always in the vicinity
of the egg cell nucleus, as they can often be found far from
the egg apparatus and closer to the chalazal end (Fig. 2N–
O, S–T). The antipodals are quite ephemeral, and are only
rarely observed in the unpollinated cellularized female
gametophyte (Fig. 2O, T). Whereas their size is compar-
able to that of nucellar cells located in the chalaza, they are
highly vacuolated and contain a centrally located nucleus.
Kinetics of pollen tube growth
While diploid self-compatible variants have been reported
in several sexual Solanum species, the spatial and temporal
characterization of pollen tube growth in those genotypes
is poorly documented. To establish the kinetics of pollen
tube growth, we either selfed or crossed-pollinated
selected flowers of diploid genotypes and scored the
progression of an average of 100 pollen tubes in each pistil
(Table 1). Our results show that the rate of pollen tube
growth is nearly identical in selfed and cross-pollinated
pistils. In S. cardiophyllum the length of the style from the
base of the stigmatic papillae to the base of the ovarian
cavity is on average 8 mm. We followed pollen tube
growth by measuring their length with respect to the base
of the stigma. At 4 hap, up to 98% of pollen grains present
in the stigma had germinated and extended over a distance
of 1 mm within the style (Fig. 3A). At 18 hap, the same
percentage of pollen tubes had reached a distance of 1.5–
2.5 mm (Fig. 3B). Interestingly, at 42 hap only 40% of all
pollen tubes had reached 6.5–7.2 mm in length. This
decrease in the number of pollen tubes that were able to
Table 1 Kinetics of pollen tube growth in selfed and cross-
pollinated genotypes of Solanum cardiophyllum Lindl. The table
shows the percentage of pollen tubes having reached a specific
distance with respect to the base of the stigma after self-pollination
(cross pollination between brackets) at arbitrary time intervals
Distance with respect to the base of the stigma (mm)
Hours after pollination (hap) 0–1 1.5–2.5 4.2–5.2 6.5–7.2 End of style Ovarian cavity
4 98 (98)
8 98 (98)
18 98 (98)
42 40 (40)
60 85 (50) 2 (1) 1 (0.5)
121
pursue their growth was confirmed in pistils observed
60 hap. In these samples pollen tube growth was slightly
slower as up to 85% of pollen tubes in self-pollinated
pistils (up to 50% in cross-pollinated pistils) arrested their
growth after reaching between 4.2 and 5.2 mm (Fig. 3C).
Interestingly, at 60 hap 1–2% of pollen tubes had reached
at least the base of the style and were often found within
the ovarian cavity showing oriented growth towards the
ovules (Fig. 3D). Since several thousand pollen grains
germinated in the stigma, this proportion of pollen tubes
reaching the ovules was sufficient to ensure normal seed
set. The presence of pollen tubes within the micropyle was
confirmed by clearing ovules from samples collected at 60
and 84 hap. Although a zygote was present in the female
gametophyte at 60–84 hap (Fig. 3E), the division of the
primary endosperm nucleus was only observed 84 hap
(Fig. 3F).
Genetic variability in full-sib seedlings
The observation of meiotic abnormalities reminiscent of
developmental events occurring during 2n-gamete forma-
tion, and the slow kinetics of pollen tube growth, raised
questions about the reproductive origin of seeds formed by
diploid genotypes of S. cardiophyllum. To determine if
seeds were the result of sexual reproduction or could be
clonally derived, we performed amplified fragment length
polymorphism (AFLP) molecular marker analysis on a
population of seedlings randomly selected on individual
fruits resulting from self- or cross-pollination of a single
genotype of S. cardiophyllum (Fig. 4). The pattern of
AFLP fragments obtained for full-sib individuals indicates
that a significant level of polymorphism prevails among
seeds derived from the same parental combination. No
individuals genetically identical to the maternal parent
could be identified, confirming that most seeds, if not all,
are the result of sexual reproduction.
Discussion
Wild diploid Solanum species have been used as a source
of genetic diversity and desirable genes in potato breeding
programs (Ross 1986; Hanneman 1989; Hawkes 1990).
Diploid hybrids resulting from crosses between some of
these diploid species and dihaploid genotypes obtained
from cultivated tetraploids of S. tuberosum can be then
crossed to tetraploid cultivars by taking advantage of the
sporadic formation of 2n-gametes (i.e., gametes with the
sporophytic chromosome number) occurring in several
meiotic mutants and interspecific crosses (Ortiz 1998;
Peloquin et al. 1989a, 1989b, 1999). Despite their
importance in this type of breeding scheme, only a limited
number of studies have described female reproductive
development in a few of the more than 150 tuber-bearing
Solanum species.
We have successfully identified diploid (2n=2x=24)
self-compatible genotypes in a population of S. cardio-
phyllum native of central Mexico (Estrada-Luna et al.
2002). Our observations indicate that, in this species,
megasporogenesis and megagametogenesis give rise to a
female gametophyte of the Polygonum type by cellular
mechanisms resembling those that have been described for
Fig. 3A–F Pollen tube growth
and early seed formation in S.
cardiophyllum Lindl. A Pollen
germination in the stigma of a
self-pollinated pistil. B Pollen
tube growth in a self-pollinated
pistil 42 hours after pollination
(hap). C Pollen tube growth in a
self-pollinated pistil 60 hap; up
to 85% of pollen tubes arrest
their growth at a distance of
4.2–5.2 mm from the stigma
(asterisk). D Pollen tube growth
within the ovary of a self-
pollinated pistil 84 hap; pollen
tubes show oriented growth
towards the funiculus of the
ovule (asterisk). E Megagame-
tophyte 60 hap showing a zy-
gote (Z) and the remnants of a
degenerated synergid; FPN
fused polar nuclei. F Megaga-
metophyte 84 hap showing the
primary endosperm nucleus
(asterisk). Bars A, B, D 40 μm;
C, E, F 20 μm
122
S. tuberosum (Rees-Leonard 1935; Arnason 1948) and S.
demissum (Walker 1955). Despite these similarities,
several differences from what is generally believed to be
the normal course of female gametogenesis in Solanum
spp. were observed. The formation of a triad instead of a
tetrad of megaspores indicates that meiosis II does not
always occur in both cellular products of meiosis I. In
many cases the megaspore located close to the micropyle
did not appear to initiate meiosis II and eventually
degenerated. Asynchronous divisions of meiosis II were
also reported during megasporogenesis in diploid S.
phureja- S. tuberosum hybrids, and their consistent occur-
rence was associated with the formation of 2n female
gametes (Jongedijk 1985). Although the micropylar
megaspore does not participate in the formation of the
sexually derived female gametophyte in S. cardiophyllum,
asynchronous abnormalities during meiosis II are remi-
niscent of the mechanisms that prevail during the forma-
tion of second division restitution (SDR) gametes in
potato, a process that results in chromosome doubling in
one or both haploid cells derived from meiosis I, and that
generally gives rise to highly homozygous 2n-gametes
expected to be predominantly nonviable due to chromo-
some imbalance (Mendiburu et al. 1974; Peloquin 1983;
Hermsen 1984). Normal seed set in diploid S. cardio-
phyllum suggests that meiotic abnormalities rarely affect
the genetic constitution of the functional megaspore and
the megagametophytic cell lineage in the genotypes
analyzed. The use of molecular markers to assess genetic
variability in full-sib progeny from selfed and cross-
pollinated genotypes indicates that seeds are sexually
derived, confirming that the meiotic abnormalities ob-
served do not influence the genetic constitution of female
gametes or the normal mechanism of double fertilization.
The kinetics of pollen tube growth in self-compatible
diploid genotypes of wild Solanum species has rarely been
documented (Johri et al. 1992). In most self-incompatible
species reported to date the progression of pollen tubes
after self-pollination is abruptly arrested in the mid-portion
of the stylar length, several millimeters before reaching the
ovary (de Nettancourt 2001). Early reports indicate that,
after fertile pollinations of Solanum spp., fertilization
occurs 24–48 hap, and that the primary endosperm nucleus
divides only 48 hap (Lee and Cooper 1958). Compared to
these reports, pollen tube growth in diploid genotypes of
S. cardiophyllum is relatively slow and appears to be
equivalent after cross- or self-pollination. In both cases a
large proportion of pollen tubes arrested their growth in
the mid-portion of the style 40–60 hap. In our experi-
ments, the distance at which most pollen tubes abort is
variable (comprised between 4.2 and 7.2 mm with respect
to the base of the stigma) and is probably influenced by
environmental and nutritional factors affecting pollination
(Lush and Clarke 1997). Nevertheless, our results show
that in self-pollinated pistils at least 1–2% of all
germinating pollen tubes are able to reach the ovary and
penetrate the female gametophyte some time between 60
and 84 hap. This small proportion of compatible pollen
tubes is sufficient to ensure double fertilization and normal
seed formation in the diploid genotypes analyzed. We
could observe the initiation of endosperm development
only 84 hap. In S. cardiophyllum the primary endosperm
nucleus is often adjacent to the central cell wall and not in
direct contact with the plasma membrane of the egg cell,
suggesting that the movement of the sperm nucleus within
the cytoplasm of the central cell could in many cases delay
double fertilization and subsequent endosperm develop-
ment. In Solanum spp. developing a cellular type of
endosperm (Johri et al. 1992), the division of the zygote is
reported to occur when the endosperm is composed of at
least 40 cells (Lee and Cooper 1958). Since we did not
observe the first division of the zygote in ovules fixed
84 hap, we suggest that formation of the embryo is also
subsequent to the initiation of endosperm development in
Fig. 4 Genetic variability in full-sib seedlings of S. cardiophyllum
Lindl. AFLP fingerprints were generated for 13 full-sib seedlings
corresponding to progeny resulting from cross-pollination of a
single maternal genotype (P)ofS. cardiophyllum. Conspicuous
polymorphic fragments are indicated by asterisks
123
S. cardiophyllum. Determination of the moment of endo-
sperm cellularization and occurrence of the first zygotic
division will require further cytological investigation.
The reproductive characteristics of diploid genotypes of
S. cardiophyllum offer new alternatives in the study of the
genetic basis and molecular mechanisms that control
megagametophyte development and true seed formation in
a tuber-bearing Solanum. The establishment of insertional
mutagenesis and gene trapping approaches in these genetic
backgrounds will dramatically simplify the identification
of genes important for embryo and endosperm develop-
ment (Estrada-Luna et al. 2002). These new approaches,
combined with the use of meiotic mutants to solve genetic
problems associated with 2n-gamete formation, promise to
accelerate progress in the understanding and establishment
of true potato seed as a more efficient strategy for potato
production.
Acknowledgements We thank Daphné Autran for critically
reading the manuscript, Ana Laura Aguirre for help during the
preparation of semi-thin sections, and Emigdia Alfaro and June
Simpson for assistance with AFLP analysis. Mario Luna-Cavazos
kindly provided some additional germplasm. This research was
supported by grants from CINVESTAV (JIRA-2001) and CON-
ACyT (B-34324 and Z-029). J.-Ph.V.-C. is an International Scholar
of the Howard Hughes Medical Institute.
References
Almekinders CJM, Chilver AS, Renia HM (1996) Current status of
the TPS technology in the world. Potato Res 39:2898–2303
Arnason TJ (1948) Female sterility in potatoes. Can J Bot 21:41–56
Cipar MS (1964) Self-compatibility in hybrids between Phureja and
haploid Andigena clones of Solanum tuberosum. Eur Potato J
7:152–160
Cipar MS, Peloquin SJ, Hougas RW (1964) Inheritance of
incompatibility in hybrids between Solanum tuberosum hap-
loids and diploid species. Euphytica 13:163–172
Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for
small quantities of fresh leaf tissue. Phytochem Bull 19:11–15
Estrada-Luna AA, Huanca-Mamani W, Acosta-García G, León-
Martínez G, Becerra-Flora A, Pérez-Ruíz R, Vielle-Calzada J-
Ph (2002) Beyond promiscuity: manipulating sexuality and
apomixis in flowering plants. In Vitro Cell Dev Biol Plant
38:146–155
Galindo-Alonso J (1982) La papita güera. Naturaleza 13:175–180
Hanneman RE Jr (1989) Potato germplasm resources. Am Potato J
66:655–668
Hawkes JG (1990) The potato: evolution, biodiversity and genetic
resources. Belhaven Press, Oxford
Hermsen JGTh (1984) Haploids as a tool in breeding polyploids.
Iowa State J Res 58:449–460
Hosaka K, Hanneman RE (1998) Genetics of self-compatibility in a
self-incompatible wild diploid potato species Solanum cha-
coense. 1. Detection of an S locus inhibitor (Sli) gene.
Euphytica 99:191–197
Jarwosky CA, Phatak SC, Ghate SR, Gitaitis RD (1988) Cultural
practices in use of true seed potato and screening of tuber-
forming Solanum species under hot climatic conditions.
HortScience 23:500–505
Jongedijk E (1985) The pattern of megasporogenesis and mega-
gametogenesis in diploid Solanum species hybrids; its rele-
vance to the origin of 2n-eggs and the induction of apomixis.
Euphytica 34:599–611
Jongedijk E (1987) A rapid methyl salicylate clearing technique for
routine phase contrast observations of female meiosis in
Solanum. J Microsc 146:157–162
Johri BM, Ambegaokar KB, Srivastava PS (1992) Comparative
embryology of angiosperms. Springer, Berlin Heidelberg New
York
Lee JH, Cooper DC (1958) Seed development following hybridiza-
tion between diploid Solanum species from Mexico, Central
and South America. Am J Bot 45:104–110
Luna-Cavazos M (1997) Variación morfológica de poblaciones
silvestres y arvenses de Solanum ehrenbergii (Solanaceae). Bol
Soc Bot Mex 61:85–94
Luna-Cavazos M, Leighton T, García-Moya E (1988) Estudio
biosistemático de papas arvenses (solanum secc. Petota) del
altiplano Potosino-Zacatecano. Agrociencia 71:103–124
Lush WM, Clarke AE (1997) Observations of pollen tube growth in
Nicotiana alata
and their implications for the mechanism of
self-incompatibility. Sex Plant Reprod 10:27–35
Martin FW (1959) Staining and observing pollen tubes in the style
by means of fluorescence. Stain Tech 34:125–128
Martin MW (1988) Cultural practices for using true seed in potato
production under temperate climates. HortScience 23:505–510
McClure B, Mou B, Canevascini S, Bernatzky R (1999) A small
asparagine-rich protein required for S-allele-specific pollen
rejection in Nicotiana. Proc Natl Acad Sci USA 96:13548–
13553
Mendiburu AO, Peloquin SJ, Mok DWS (1974) Potato breeding
with haploids and 2n gametes. In: Kasha K (ed) Haploids in
higher plants. University of Guelph, Guelph, Canada, pp 249–
258
Murashige T, Skoog F (1962) A revised medium for rapid growth
and bioassays with tobacco cultures. Physiol Planta 15:473–
497
Nettancourt D de (2001) Incompatibility and incongruity in wild and
cultivated plants. Springer, Berlin Heidelberg New York
O’Brien M, Kapfer C, Major G, Laurin M, Bertrand C, Kondo K,
Kowyama Y, Matton DP (2002) Molecular analysis of the
stylar-expressed Solanum chacoense small asparagine-rich
protein family related to the HT modifier of gametophytic
self-incompatibility in Nicotiana. Plant J 32:985 –996
Ortiz R (1997) Breeding for potato production from true seed. Plant
Breed Abstr 67:1355–1360
Ortiz R (1998) Potato breeding via ploidy manipulation. Plant Breed
Rev 16:15–86
Peloquin SJ (1983) New approaches to breeding for the potato of the
year 2000. In: Proceedings International Congress “Research
for the potato in the year 2000”. CIP Lima, Peru, pp 32–34
Peloquin SJ, Jansky SH, Yerk GL (1989) Potato cytogenetics and
germplasm utilization. Am Pot J 66:629–638
Peloquin SJ, Yerk GL, Werener JE, Darmo E (1989b) Potato
breeding with haploids and 2n gametes. Genome 31:1000–1004
Peloquin SJ, Boiteux LS, Carputo D (1999) Meiotic mutants of
potato: valuable variants. Genetics 153:1493–1499
Rees-Leonard L (1935) Macrosporogenesis and development of the
macrogametophyte of Solanum tuberosum. Bot Gaz 96:734–
750
Ross H (1986) Potato breeding: problems and perspectives. Parey,
Berlin
Spurr AR (1969) A new viscosity resin embedding medium for
electron microscopy. J Ultrastruct Res 26:31–43
Thompson RD, Uhrig H, Hermsen JG, Salamini F, Kaufmann H
(1991) Investigation of a self-compatible mutation in Solanum
tuberosum clones inhibiting S-allele activity in pollen differ-
entially. Mol Gen Genet 226:283–288
Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M,
Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995)
AFLP: a new technique for DNA fingerprint. Nucleic Acids
Res 23:4405–
4414
Walker RT (1955) Cytological and embryological studies in
Solanum section Tuberarium. Bull Torrey Bot Club 82:87–101
Xu B, Mu J, Nedvinsq DL, Grun P, Kao T-H (1990) Cloning and
sequencing of cDNAs encoding two self-incompatibility
associated proteins of Solanum chacoense. Mol Gen Genet
224:341–346
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