In vitro fertilization as a tool for investigating sexual reproduction of angiosperms

13 Pages·2006·0.5 MB·English

SexPlantReprod(2006)
DOI10.1007/s00497-006-0029-1
REVIEW
Ya Ying Wang Æ Anxiu Kuang Æ Scott D. Russell
Hui Qiao Tian
In vitro fertilization as a tool for investigating sexual reproduction
of angiosperms
Received:30September2005/Accepted:1May2006
(cid:1)Springer-Verlag2006
Abstract In vitro fertilization (IVF) of isolated male Keywords Angiosperm Æ Gamete isolation Æ In vitro
and female gametes of flowering plants was first fertilization Æ Zygote culture
accomplished in the last decade. Successful isolation of
male and female gametes, and culturing of in vitro
zygotes to form new plants, is a prelude to the use of Introduction
IVF for research into the cellular and molecular con-
trol of fertilization in higher plants and its application Fertilizationisacriticaleventinvolvingthecombination
as a tool in biotechnology. Genes unique to male and ofmaleandfemalegametecellsandnucleiduringsexual
female gametes and zygotes of higher plants, although reproductionofangiosperms.Thisunionoccursthrough
currently incompletely characterized, are expected to thefusionofspermandeggcells,contributingtogenetic
permit direct molecular dissection of fertilization. By diversity and providing a basis for crop improvement
applying IVF and microculture to zygotes and endo- during plant breeding. However, the protected location
sperm obtained by both in vivo and in vitro methods, of the gametes, particularly the female egg and central
newly activated fusion products may be observed and cells, restricts our ability to explore the fertilization
manipulated in media where they are directly accessi- process of higher plants, and has thus led to a reason-
ble to the techniques of molecular cell biology. IVF ably limited understanding of fertilization in these
and zygote culture may also offer potential for creating plants. The technique of in vitro fertilization (IVF), in
new hybrid plants by fusing isolated gametes from which isolated sperm and egg cells are induced to fuse
different species to produce unique zygotes and ulti- under controlled conditions, removes much of the
mately plants that would be impossible to obtain using interfering presence of somatic tissues, as well as inter-
typical crossing techniques. Transformation and ference of immediately surrounding maternal gameto-
regeneration frequencies using IVF may also be high phyticcells.Thus,IVFprovidesopportunitiestodirectly
enough to avoid the necessity of adding controversial test hypotheses about gamete recognition and zygote
antibiotic and herbicide resistant genes to screen activation. The use of IVF in higher plants is therefore
transformed products. This review describes advances an important contemporary research area in plant
using IVF in plant sexual reproduction and discusses developmental and reproductive biology with poten-
its potential in the genetic improvement of flowering tially significant scientific applications.
plants. Thefirstisolationsoflivingspermcellswerereported
by Cass (1973), in which he described cellular charac-
teristics of male gametes of barley (Hordeum vulgare)
isolated by bursting anthesis pollen grains in a 20%
sucrose solution. Following the first mass isolation of
CommunicatedbyTsuneyoshiKuroiwa
spermcellsin1986,successinmalegameteisolationwas
Y.Y.WangÆA.KuangÆH.Q.Tian(&) repeated with modification in numerous angiosperms
SchoolofLifeSciences,XiamenUniversity,Xiamen, (reviewed in Russell 1991). Figure 1 shows isolated
Fujian361005,China
sperm cells of two flowering plants representative of the
E-mail:[email protected]
sizeandmorphologyofmalegametes.Isolationofliving
Tel.:[phone]
Fax:[phone] eggandcentralcellswasfirstreportedintobaccobyHu
et al. (1985) and in Plumbago by Huang and Russell
S.D.Russell (1989). An often-employed method of female gamete
DepartmentofBotanyandMicrobiology,
isolation is to use a dilute enzyme treatment to loosen
UniversityofOklahoma,Norman,OK73019,USA
Fig.1 Sperm cell isolation viewed using scanning (a–c) and theseimagesofmalegametesofPlumbagozeylanica.dTEMview
transmission electron microscopy (d). a Nicotiana tabacum sperm of isolated rapidly frozen freeze substituted sperm cells of P.
cells initially possess cellular projections (arrowheads) following zeylanica. Bars in a, b=5lm; bars in c, d=1lm. Images a–c
isolation.b,cIsolatedspermcellsrapidlybecomespheroidal,asin arecourtesyofZhaojieZhang;imagediscourtesyofStrout
surrounding cell walls, followed or accompanied by display G fusion and there are a myriad of patterns
2
micromanipulation to separate remaining ovule cells expressed(Friedman1999).Pollenmaybereleasedprior
from embryo sacs. Cells of the embryo sac—consisting to sperm cell formation (bicellular pollen species), but
of the egg cell, two synergids, central cell and three even in tricellularpollen, sperm cells may be in G , S or
1
variablypresentantipodals—canthenbeisolatedasfree G phaseandthusnotnecessarilyinsynchronywiththe
2
cells.Kranzetal.(1991)reportedthefirstsuccessfulIVF femalegametophyte.Unfortunately,theconditionofthe
experiments by using electrofusion to combine sperm cell cycle during fertilization is described for only a
and egg cells of maize, producing an in vitro zygote handful of plants but these have displayed remarkable
(Fig. 2). Such products were cultured to form multicel- variability (Friedman 1999). According to these studies,
lular structures. Within 2 years, this research group was grassesareadmirablysuitedtoIVFasthemalegametes
able to use in vitro-produced zygotes to achieve regen- are generally released in G phase and this is the phase
1
eration of fertile plants by modifying this procedure of the cell cycle in which they fuse in nature (Mogensen
(Kranz and Lo¨rz 1993). and Holm 1995; Mogensen et al. 1995).
Todate,IVFofangiospermshashadlimitedsuccess. Tobacco in contrast has proven to be a particularly
Tobacco would seem to be an obvious model for IVF refractory model for IVF. Although sperm cells are
givenitspotentialtoregenerateplants;howevertobacco formed in the pollen tube at G , they ultimately pass
1
gametesdidnotfuseeasilyanddidnotproduceembryos. through S phase and fuse at G phase in nature (Tian
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Sperm and egg cells exposed to a polyethylene glycol etal.2005).Theexactcircumstancesoffusion,however,
(PEG)solutionstimulatedfusion,butfusedproductsdid are even more challenging. Sperm cells do not enter S
notcontinuetheirdevelopment(TianandRussell1997a, phase until the very last stages of their passage in the
b;Sunetal.2000a,b).Instead,theIVFzygotearrested. pollen tube, and they complete S phase within the syn-
Thegrassescontinuetobethemostsuccessfulgroup,but ergid, fusing immediately upon entry into G ; thus the
2
success has still been limited. Kova´cs et al. (1995) were receptive sperm cells are essentially inaccessible (Tian
abletofusespermandeggcellsofwheatusingthesame etal.2005).Eggcellprogressionthroughthecellcycleis
IVFtechnique,butinvitrozygotesinthisplantaborted, synchronized with the sperm cells through a short dis-
producing a multicellular structure. tancecommunicationsignal,soobtainingfullyreceptive
Inadditiontoisolationofhighqualitylivinggametes, eggcellsmaybeequallydifficult.Matchingthecellcycle
cell cycle appears to be a critical factor in successfully of gametes with their normal fusion condition thus
combining gametes. Unlike most eukaryotes, in which represents a potentially grave pitfall in meeting IVF
fertilizationoccursduringG ,fusion,manyangiosperms congruity requirements (Friedman 1999).
1
cellsinpollenofPlumbagozeylanicaexhibitedstructural
dimorphism, differing in both morphology (Russell and
Cass 1981) and internal cellular organelle complements
(Russell 1984). Further observations revealed that the
sperm cells from a single pollen tube of P. zeylanica
exhibitedpreferentialfertilization:onespermcontaining
mostly mitochondria fused with the central cell, and the
other cell containing mostly plastids fused with the egg
cell (Russell 1985). Sperm dimorphism and preferential
fertilization in P. zeylanica suggested the presence of a
recognition mechanism between the two gametes, where
each had a preferred female target cell. Although pref-
erential fertilization remains best described in P. zeyla-
nica and a nuclear-based counterpart in populations of
maize, a number of other species display differences in
sperm organization that indicate dimorphism in other
angiosperms also occurs (Mogensen 1992). These plants
withdimorphicspermcellsmayalsopossesspreferential
fertilization. To the extent that all angiosperms exhibit
double fertilization, it also remains plausible that many
if not all angiosperms exhibit some preferentiality of
fertilization in vivo. Recently, Saito et al. (2002) ob-
served organellar DNA distributions of sperm or gen-
erative cells in 115 species (104 genera, 56 families) and
found distinctly polarized organellar DNA within gen-
erative cells or between sperm cells in six plants (six
genera in four families). Since each pollen tube ulti-
Fig.2 Schematic diagram of electrically induced in vitro fertiliza-
matelyproducestwospermcellsandthesecellshavetwo
tion. Procedure is initiated by gamete isolation, followed by
targets, it seems conceivable that there is targeting in a
alignment of sperm and egg cell next to electrode, initially
dikaryoticartificialzygoteafterinvitrofusionusingelectrofusion, number.Competitionfortheabilitytofusewiththeegg
andthenmicrocultureofartificialzygotesinamillicellwithfeeder cell may have important genetic consequences. Occur-
cells.ReproducedwithpermissionfromKranzetal.(1991) rence of dimorphic sperm cells also suggests that, in
addition to being dimorphic, the two sperm cells may
Successes in obtaining products of IVF in grasses, also differ at the molecular level, and that these differ-
however, are a hopeful sign that this technique is encesarelikelyreflectedonthecellsurfaceforsuccessful
applicable to some of our most important crop plants targeting to occur.
and may provide unique insight into zygote activation. We know from a number of works using isolated
Since a recently published review by Okamoto and male gametes that specific proteins are present on the
Kranz (2005) focuses on cellular differentiation in IVF cell surface. Southworth and Kwiatkowski (1996) tested
products and specifically on cellular determination of sperm cells of Brassica and Lilium using antibodies to
higher plant zygotes, we provide little coverage on this arabinogalactan proteins and found two monoclonal
topicinourcurrentreview.Nonetheless,regenerationof antibodies, JIM8 and JIM13, bound to the sperm cell
IVF fusion products into fertile plants may be particu- surface. They proposed that antibodies to arabinoga-
larlyhelpfulforexaminingepigeneticeffectsandnucleo- lactan proteins may prove useful for discriminating
cytoplasmic interactions inherent to gametes, as well as male gametes from other cells. Xu and Tsao (1997)
experimentally separating endosperm, embryo and isolated proteins from the plasma membrane of maize
ovular influences from the post-fusion development of sperm cells and found some proteins were different
IVF products. Transgenic gametes and fusion products from those of the somatic cell when peroxidase-conju-
areexpectedtobeparticularlyvaluableforexperimental gated Con A was used to label the proteins. Using
modification of plant development and functional dis- FITC-conjugated lectins as probes, Sun et al. (2002)
section of critical molecular events. localized sugar moieties of glycoconjugates on the cell
surface of isolated maize sperm cells, egg cells, central
cells, antipodal cells, synergids and in vitro and in vivo
Molecular studies using isolated gametic cells fertilized zygotes of maize. The fluorescence signal of
FITC-Con A was stronger on egg and central cells but
Molecular biology of the generative cell and sperm weaker on sperm cells. In contrast, the signal of FITC-
WGA was present on egg cells but not on sperm cells;
In the 1980s, electron microscopy combined with three- however, the signal for FITC-PHA-E was found on
dimensionalreconstructionindicatedthatthetwosperm neither egg nor sperm cells (Sun et al. 2002). These
findings reflect surface-based distinctions in glycosyla- associated with the vegetative nucleus (that which pref-
tion sites that remain poorly characterized despite their erentially fuses with the central cell and forms endo-
potential importance in cell binding and recognition; sperm)displaysan8–16foldincreaseinexpression.Such
this topic remains an attractive target for molecular differential expression presumably contributes to
characterization. molecular differences between the two male gametes
Sperm cells are expected to fulfill the functions of during development, potentially related to networks of
recognition, adherence and fusion with the female sperm-specific or sperm-enhanced genes. It is suspected
gamete during fertilization. Each of these functions of that among these genes may be some that are related to
the male gamete may be controlled by activation of a male and female gamete recognition during the fertil-
unique gene or network of genes. The isolation of such ization process. Early work in this area arose from ex-
fertilization-related genes in sperm cells seems increas- pressed sequence tag (EST) analysis, which reflects
ingly likely as more male germ cells are isolated and transcript production. Microarray and suppression
characterized using molecular biology. Xu et al. (1999a) subtractive hybridization (SSH) may further contribute
isolated and characterized two cDNA clones of the lily to understanding gamete gene expression as it ap-
precursor generative cell that encode generative cell- proaches the transcriptome and proteome level of
specific histone genes, gcH2A and gcH3. This paralleled analysis.
the prior discovery of specific generative cell-expressed Since sperm cell dimorphism and preferential fertil-
histone proteins (Ueda and Tanaka 1995). In situ izationwerefirstreportedinP.zeylanica(Russell1985),
hybridization analysis revealed that transcripts of sub- the phenomena of sperm dimorphism have been re-
stitution histone genes gcH2A and gcH3 increased in ported independently in numerous plants (Mogensen
abundance during gametic cell maturation, which was 1992; Hu and Tian 2002). Some dimorphic sperm cells
the first report of male germ lineage specific expression differ in cell size, whereas others differ in organelle
in a higher plant. Xu et al. (1999b) also isolated a male content. Relatively few plants have been examined that,
gametic cell-specific gene, LGC1, and confirmed its when examined in detail, reveal no statistical difference
expression, demonstrating that the gene product was between the two sperm cells. One particularly well-
localizedonthesurfaceofmalegameticcells.TheLGC1 studied example is tobacco, in which many develop-
protein is anticipated to be related to cellular interac- mental stages from the time of cell division (Yu and
tions between themale andfemale gametes, presumably Russell 1993) to sperm cell maturation (Yu and Russell
related to recognition or signaling. Ueda et al. (2000) 1994) have been examined, up the time of pollen tube
havesinceisolatedgenesencodingthreehistoneproteins entry into the ovary. Examination of later progamic
from Lilium longiflorum. The highly heterochromatic stages (near the time of fertilization), however, indicate
appearance of lily male gametic nuclei may link directly that size differences in tobacco become more evident
to the abundance of these histones, which are suspected during development (Tian et al. 1998, 2001). Dimor-
tocontrolcondensationofchromatin.Heterochromatin phism between the two sperm cells may play a role in
is recognized as one of the most conspicuous forms of gametic recognition during double fertilization. Inter-
epigenetic control and is implicated in modeling nuclear estingly, there is also evidence that the sperm cells differ
organization that affects expression of multiple genes even more extremely in size within the synergid, just
and gene networks. priortotransmission.Althoughthisobservationwasthe
In the Mariani lab, Xu et al. (2002) constructed a result of only one serially reconstructed cell pair (Yu
cDNA library of mRNA transcripts isolated from to- et al. 1994), it is tempting to speculate that this may
bacco male germ cells derived from in vitro culture, relate todistinctionsingene expression,whichmayalso
distinguishing two cDNAs believed to be uniquely ex- reflect molecular differences in the cells. With this goal,
pressed in sperm cells. These products, NtS1 and NtS2, the Tian lab has recently isolated populations of over a
apparently represent sperm cell-expressed transcripts thousand sperm cells each of the two dimorphic sperm
that were isolated from an initial screen of 396 clones. celltypes,S (vegetativenucleus-associatedspermcells)
vn
The products of these two clones accumulated in both and S (sperm cells unassociated with the vegetative
ua
the generative and sperm cells. NtS1 codes for a polyg- nucleus) from pollen tubes of tobacco using microma-
alacturonase, suggesting a role for this enzyme in wall nipulator-based collections (Fig. 3), as fluorescence-
modification during differentiation of sperm cells in to- activated cell sorting is not well adapted to the small
bacco. number of gametes that can be isolated in most flower-
Singh et al. (2002) isolated polyubiquitin-encoding ing plants (Yang et al. 2005). Interestingly, isolated
cDNAclonesingenerativecellsofL.longiflorumandin sperm cells of tobacco display significant differences in
sperm cells of P. zeylanica that are differentially ex- electrophoretic mobility that may relate to potential
pressed. They suggested that ubiquitin differentially al- differential expression (Yang et al. 2005). Once dimor-
ters patterns of protein degradation in the germline and phic sperm cells are collected into populations of suffi-
is likely involved in determining lifespan of some pro- cient quantity (e.g., a thousand or more sperm cells of
teins during male reproductive maturation. In Plum- each type), differences between the two sperm cells may
bago,thegene-encodingpolyubiquitinisup-regulatedin beidentifiedusingsensitivemolecularmethods.Thetwo
one of the two sperm cell types. The sperm cell that is purified populations of sperm cells can be used to ad-
Fig.3 Isolationofdimorphic
spermcellsofNicotiana
tabacum.aPopulationoflarger
spermcells,representingthe
S .bPopulationofsmaller
ua
spermcellsrepresentingtheS .
vn
Reproducedwithpermission
fromYangetal.(2005)
vance research on sperm cell biology of higher plants This early screen revealed some up-regulated cDNA
from the cell to the molecular level by using such sen- library members in maize egg cells and some differen-
sitive techniques as immunoblotting, elicitation of tially expressed genes.
monoclonal antibodies, transcript analysis and work on To uncover expressional differences relating to zy-
expressed proteins. The utility of these techniques will gote activation, Dresselhaus et al. (1996) constructed
enhance our understanding of sperm cell biology in another library from 104 maize IVF-generated zygotes
higher plants, allowing greater appreciation of the un- at 18 h after IVF to distinguish and isolate newly ex-
ique properties of angiosperm male gametes and con- pressed and highly up-regulated genes in zygotes.
tributing to our knowledge of double fertilization. Among the products, they isolated a full-sized cDNA
Ironically, the sperm cells occupy a diminishing cellular clone encoding calreticulin from a differential screening
volume during development (Russell and Strout 2005), of egg cell and zygote cDNA libraries. The expression
so their molecular components may represent a dimin- of this isolated gene is enhanced after fertilization and
ishingbutimportantresourceduringmaturation.Sperm strongly correlated with cell division, constituting the
cells, containing a complex and largely unique network first report of an isolated zygote gene. From further
of genes during development that are distinct compared differential screening of cDNA libraries of unfertilized
to those of other somatic and reproductive cell types egg cells and in vitro fertilized zygotes of maize, 50
(Engel et al. 2003) remain somewhat of an enigma in additional transcripts were sequenced and reported
light of their outwardly simple appearance and behav- (Dresselhaus et al. 1999a). From these products, a
ioral repertoire. cDNA library member encoding the eukaryotic trans-
lation initiation factor eIF-5A was isolated—a protein
thought to be necessary for selective mRNA stabiliza-
Molecular biology of the egg cell and zygote tion and translation (Dresselhaus et al. 1999b). This
protein is stored in the egg cell in large amounts, but is
Egg cell isolation has been considerably more difficult, relatively quickly inactivated. After fertilization, selec-
yielding several orders of magnitude fewer cells com- tive mRNA translation was quickly triggered by spe-
pared to sperm cells. Major factors limiting isolation of cific developmental changes of the zygote. In addition,
female gametophytes include that they occur singly among the isolated cDNAs they found, seven encoded
within ovules, the egg and central cells are deeply proteins related to translation and two related to DNA
embedded, and female gametophyte cells are integral to replication, both of which are strongly induced upon
the ovules, displaying extensive common interfaces fertilization. Four other novel genes (S21A, S21B, L39,
between central cell, synergids and egg cells. Despite P0) involved in ribosome biosynthesis and translation
these difficulties, female gametophytic cells have been were also identified. The expression of all four genes
isolated through enzyme maceration, micromanipula- was correlated with cell division activity and was
tion and a combination of these techniques. Their lar- strongly induced during the G phase of the zygotic cell
1
ger size compared with male gametes allows greater cycle (Dresselhaus et al. 1999a). Sauter et al. (1998)
yield per cell (Fig. 4). With the successful isolation of examined the expression of cell cycle regulatory genes
female gametic cells, a comparison of expressed genes to explore the mechanisms controlling zygotic cell
in egg and somatic cells through cDNA library char- division of maize. After analyzing transcripts of cdc2
acterization is possible, which may be useful in exam- (cdc2ZmA/B) and cyclin (Zeama;CycB1;2, Zeama;
ining expressional differences of specific egg cell and CycA1;1, Zeama;CycB2;1) genes of sperm cells, egg
zygote genes. The first molecular biology report was cells, synergids, the central cell and antipodal cells, they
that of Dresselhaus et al. (1994), who constructed a found that the cdc2 gene is expressed constitutively in
cDNA library from 128 isolated maize egg cells using a all cells prior to fertilization and throughout zygote
reverse transcriptase/polymerase chain reaction tech- development, whereas the cyclin genes showed cell-
nique. This first egg cDNA library was then compared specific expression in the cells of the embryo sac and
with similarly constructed somatic cDNA libraries. differential expression during zygote development.
Fig.4 IsolatedeggcellsandzygotesofToreniafournieri.aEggcell cence. c Isolated zygote from an ovule 14h after pollination. d
isolated from an embryo sac 2days after anthesis. b Viability of Viability of isolated zygote evaluated by FDA fluorescence. e
isolatedegg cellevaluated byfluorescein diacetate (FDA) fluores- Collectionoffiveeggcells.fCollectionofzygotes
Recently, Okamoto et al. (2004) identified major zygotes, interference of genes from somatic tissue is
protein components expressed in maize egg cells, which avoided, making it possible to locate the genes specifi-
included three cytosolic enzymes, two mitochondrial cally controlling or contributing to the development of
proteins and annexin p35. They identified some genes these reproductive cells. Recent research has provided
thatareup-ordown-regulatedintheapicalorbasalcell details about expression of female gametophyte-specific
of maize two-celled embryos. Genes were categorized elementsinvolvedinpollentubeguidance(Martonetal.
into six groups: (1) up-regulated only in the apical cell, 2005) and anther maturation (Dresselhaus et al. 2005);
(2)up-regulatedonlyinthebasalcell,(3)up-regulatedin both findings are quite promising in extending our
both the apical and basal cells after gamete fusion, (4) knowledge of sperm cell attraction. The ZmEA1 gene
down-regulated only in the apical cell, (5) down-regu- attracts maize pollen tubes to the micropyle and if it is
latedonlyinbasalcelland(6)constitutivelyexpressedin down-regulated, short distance signaling by the egg
the egg cell and embryos. The genes up-regulated in the apparatus to pollen tubes fails and fertilization is
apical or basal cell were already expressed in the early unsuccessful. This gene has orthologs in other mono-
zygote (Okamoto et al. 2005). cots, including rice, but not in dicots, indicating that
The use of SSH to produce essentially pre-screened such signaling molecules likely diverged after their evo-
gamete libraries has also been exploited to examine lutionary split. The successful isolation of male and fe-
ESTsthatareuniquelyexpressedinspecificcelltypes.In male germ cells of higher plants has stimulated
a continuation of their early work on maize egg cell considerableresearchonthemolecularbiologyofsperm
ESTs, Le et al. (2005) produced SSH-EST libraries of andeggcelldevelopmentandthisapproachhasbecome
both isolated egg cells and isolated central cells to fur- a focal point in the study of sexual reproduction of
ther discriminate the unique transcriptional products higher plants, with increasingly important papers being
that may contribute to embryo versus endosperm dif- published every year.
ferentiation.Theeggcelldisplayedincreasedabundance
oftranscriptsinvolvingcellcommunication,cellgrowth/
division, DNA synthesis, signal transduction and stress Exploring egg cell activation
related factors, whereas the central cell displayed in-
creased abundance of transcripts involving metabolism The isolation of sperm and egg cells is considerably
and energy, protein synthesis and protein modification, easiertoachieveinanimalsandlowerplants,soitisnot
with many products in the ‘‘no hit’’ category. In each surprising that more is known about the mechanism of
case, these products seemed to reflect functional cate- egg cell activation in these organisms. Prior to sperm
goriesrepresentedintherespectivedevelopmentalfuture andegg cell isolation in angiosperms, egg cellactivation
of the embryo and endosperm (Le et al. 2005). was examined principally through ultrastructural
The above-mentioned results from molecular studies observations; however, physiological understanding of
ofmaleandfemalegermcellsareallbasedontheuseof the processes involved was quite limited. Now, using in
isolated cells. By using isolated sperm, egg cells and vitro techniques, direct investigations of living cells
during syngamy and early stages of zygote development by fluorescence returned to the same level as that ob-
are possible using a wealth of microscopic techniques served in the unfertilized egg cell (Fig. 5). This research
and biochemical manipulations. Egg cell activation of was the first direct evidence of a transient elevation of
higher plants may also be examined using specialized free cytosolic Ca2+ within in vitro zygotes of flowering
probes. plants, opening the way toward identification of early
The unfertilized mature egg cell is known to be a signals induced by fertilization, the role of cytosolic
relativelyquiescentcellasisevidencedbyultrastructural Ca2+ during egg activation, and early zygote develop-
observationsfrommanydifferentspecies,anditrequires ment in flowering plants.
fusion with the male gamete for reactivation. Since the Antoine et al. (2000) used an extracellular Ca2+-
zygote is the first cell in the ontogeny of a plant, selective vibrating probe to measure influx of extracel-
understanding egg and zygote activation is a very lular Ca2+ induced by gamete fusion during maize IVF
interesting research topic. The process of egg reactiva- and found small and stable Ca2+ fluxes in egg cells be-
tioninvolvesthefusionofaspermcellandaneggcellto fore gamete fusion. Adhesion of a male gamete to the
form a zygote in vivo. Sperm and egg cells do not fuse egg cell did not induce flux changes. After male and
spontaneously in nonfusigenic media in vitro; however, female gametes fused, however, a long-lasting Ca2+ in-
an observation that has been reported for a number of flux took place at the site of fusion with a peak average
plant species (Kranz and Lo¨rz 1993; Tian and Russell of 14.9±1.83 pmol cm(cid:1)2 s(cid:1)1 and an average duration
1997b; Cao and Russell 1997). These observations sug- of 24.4±3.13 min. The Ca2+ influx spread from the
gest that there are special requirements to ensure male position of fusion throughout the egg cell plasma
and female gamete fusion in vivo. Kranz and Lo¨rz membrane as a wavefront with an average rate of
(1994) fused maize egg and sperm cell in a solution 1.13 lm s(cid:1)1.Theyalsofoundthatthecalciuminfluxisa
containing 50 mmol/L CaCl at pH 11.0, which is a necessary condition for cell wall deposition and is
2
strongly fusigenic solution, and the fusion products of mediated by gadolinium-sensitive channels. This result
the egg and sperm cell divided and developed into mi- alsoindicatesthatsimilarCa2+signalingeventsoccurin
crocalli consisting of approximately 30–50 cells. They higher plants, as reported in animals and lower plants,
observed the early events of cell wall completion, first and that the sperm triggers a transient rise of intracel-
nuclear division and the unequal division of the zygote lularfreeCa2+levelsinthefertilizedegg.Aneffectseen
following in vitro gametic fusions using both chemical-
and electroporation-based methods. They found that
newly formed cell wall material began to accumulate
within 30 s, and most of the organelles became densely
grouped around the nucleus of the zygote within 15 h
aftergametefusion.Organellesandthenucleusofthein
vitro zygote also displayed a polarized distribution be-
fore zygote division (Kranz et al. 1995).
Faure et al. (1994) induced sperm and egg cell fusion
of maize using a solution containing 5 mmol/L CaCl ,
2
pH 6–6.5, in which 79.7% of the sperm and egg cells
fused after adhering for 4 min. In this medium, the fu-
sion process was completed in less than 10 s. Although
this method of fusing sperm and egg cells used a rela-
tivelylowconcentrationofcalciumsolution,thereisstill
the possibility of calcium stimulation, since fusion of
isolated gametes in other species, such as rice, also dis-
play fusion at these and higher calcium concentrations
(Khalequzzaman and Haq 2005; Uchiumi et al. 2006).
Using the in vitro procedure described above, Digonnet
et al. (1997) used the fluorescent acetoxymethyl ester
form of fluo-3 to investigate free calcium change during
egg cell activation. They observed a small amount of
fluorescence of free calcium in the egg cell prior to fer-
tilization—about the same amount as when the sperm
cell adhered to it, and no fluorescence in the sperm cell.
Whenthespermcellandeggcellbegantofuse,however,
the adhering sperm cell first displayed some cytosolic
Ca2+. The fusion of both cells triggered a transient Fig.5 Evidence for intracellular Ca2+ elevation triggered by IVF
cytosolicCa2+increaseinthefertilizedeggcell,withthe of Zea mays egg cell. Time sequence of pseudo-color images
illustratingtemporalandspatialdistributionoftheCa2+transient
increase in fluorescence lasting about 2 min and then
triggered by sperm–egg fusion. Reproduced from Digonnet et al.
subsiding. After 29 min, the calcium level as measured (1997)withpermissionofDumas
in plants but not in other systems is the requirement of Recently, Hoshino et al. (2004) examined fertiliza-
cell wall completion for further development, under a tion-inducedchangesinthemicrotubulararchitectureof
requirement of calcium oscillations, as well. Recently, the maize egg and zygote and described that relatively
Po´nya et al. (2004) fused isolated sperm and egg cells of few cortical microtubules occur in the unfertilized, ma-
wheatandfoundthatwithin3 minofinvitrosperm–egg ture egg cell. However, in the zygote, cortical microtu-
fusion,atransientfragmentationofpolaraccumulations bules clearly increased, and microtubules remained
of endoplasmic reticulum occurred. They hypothesized visibleupto7 hafterIVF.Followingthisphase,strands
thatthisERfragmentationserves asacalcium reservoir of microtubules radiating from the nucleus into the cell
that contributes to increased capacity of fertilized eggs periphery were formed and persisted throughout the
to release calcium by increasing the curvature of the remainder of zygote development. Based on the above-
membranes, from which calcium transport is facilitated mentioned results, an understanding of egg cell activa-
by calcium transport proteins. tionisemerging.Webelievethatcontinuingresearchon
Han et al. (2002) examined Ca2+ changes in the egg cell activation in higher plants using IVF and
central cell of Torenia fournieri using another method. modern techniques could provide crucial insights into
They microinjected a soluble sperm extract, along with understanding the unique biology ofthe flowering plant
calcium green-1 conjugated 10 kDa-dextran (CG-1) zygote.
into the mature central cell, which induced a significant Patterns of paternal contribution to transcription
rise in cytosolic free calcium concentration ([Ca2+]). between eggs, sperm cells and zygotes have been further
i
The rise reached a maximum 20 min after injection and exploredbyNingetal.(2006),providingevidenceof(1)
then steadily declined. A relatively high level of [Ca2+] clustersofgenespresentineggcellspersistentinzygotes,
i
was maintained even 40 min after injection. They also (2) clusters of genes represented in sperm cells and per-
injected caged inositol 1,4,5-triphosphate (InsP ) into sistent in zygotes, as well as (3) a large cluster of genes
3
Torenia central cells to compare the pattern of Ca2+ produced by apparently new transcripts within the zy-
rise induced by the sperm extract. The Ca2+ elevation gote. Their report includes one histone gene present in
triggered by the release of InsP was much faster than both the egg cell and zygote, which may represent one
3
that induced by sperm extract, but the increase in potential mechanism by which gene networks are con-
[Ca2+] reached a maximum at 70–80 s and then de- trolled. Activation of other histone modeling genes in
i
clined to resting levels within 300 s. The authors the male gamete has also been reported that could
hypothesized that sperm extract might contain factors potentially be involved in male germ line gene expres-
triggering the release of Ca2+ into central cell. There sion, as well (Okada et al. 2005a). Cell-specific pro-
are some differences in the results reported by Han motersactinginspermcellsrepresentfurtherevidenceof
et al. (2002), Digonnet et al. (1997) and Antoine et al. gametic control of expressional networks (Okada et al.
(2000). First, the cells used in their assays are differ- 2005b). Taken together, these reports contribute to an
ent—Han et al. (2002) used the central cell and Di- emerging recognition that cellular identity and expres-
gonnet et al. (1997) and Antoine et al. (2000) used egg sion during early embryogenesis in angiosperms may be
cells; the former study also used a different trigger, controlled through a number of multigenic control
sperm extract or IP , rather than sperm cells as in the mechanisms.
3
latter studies. Thus, it is necessary to determine how
reactions may differ between these two cells. Second,
the origin of increased calcium is different—in the Creating distant hybrids using IVF
Antoine et al. (2000) study the calcium influx came
from external calcium in the medium, but in the Han Another advantage of using IVF is the ability to fuse
et al. (2002) study, the Ca2+ rise is dependent upon sperm and egg cells of phylogenetically separated plants
internal Ca2+ released from internal calcium stores. usingtheirrespectivegametestoproducehybridzygotes
Third, Digonnet et al. (1997) and Antoine et al. (2000) invitro.Sincegametesarenaturallyrecombinantcells,it
demonstrated that sperm-induced transient Ca2+ ele- is presumed that barriers to wide hybridization may be
vation generated signals on the surface of the female reduced at the zygotic level. Such zygotic hybridization
gamete, but injected soluble sperm extract did not in- through IVF would avoid the effects or influence of
duce signals on the plasma membrane of the cell. These somatic tissues of the stigma, style and ovule and may
differences are interesting and worthy of further also avoid ploidy problems occurring during somatic
investigation. Based on their observations, Antoine fusion. To date, the technique of somatic hybridization
et al. (2000) proposed a process of initial fertilization cansuccessfullyfuselivingcellsofanyplantspecies,but
where (1) membrane fusion of both gametes starts a hybridcellsaredifficulttoculture,usuallypolyploidand
calcium influx close to the fusion site; (2) a wave of often unstable in generation of desirable regenerated
contraction, presumably of actomyosin nature, is trig- hybrids. During somatic hybridization, the volume of
gered; and (3) the calcium triggered opening of more sporophytic parent cells differs little and thus mixing of
stretch-activated calcium channels. The rise of calcium the two cells may result in an equal contribution of
contributes to a positive calcium feedback loop of cytoplasmic components, including mitochondria and
oscillation that is related to zygote activation. plastids, which have their own semi-autonomous
heredity. Afterthe twosomaticcells fuse, thecytoplasm comparing a sperm cell of a diameter of approximately
of the hybrid cell is often physiologically incompatible; 3 lm to a diameter of the egg cell of 30 lm. Although
this may be particularly conspicuous immediately after their relative difference in diameter is 1:10, the volume
fusion,withconsequencesthatmaypersistforavariable difference is 1:1,000, meaning that cytoplasmic com-
amount of time thereafter. One phenomenon often ob- ponents of the male gamete are exceeded by three or-
served in hybrid cells of distant species is that the ders of magnitude over the egg cell. In nature, however,
chromosomes of one parent are eliminated. There are the relative diameter ratios of many male:female gam-
many other observed problems associated with distant ete pairings in angiosperms, however, may exceed a
somatic cell hybridization such as absence of cell divi- 1:10 ratio, and be 1:20 or more, resulting in a dramatic
sion in hybrid cells, lack of differentiation in cells of the dilution of any paternal or dual parent effect during
hybrid callus, genetic instability, refractory ability to early zygote development.
regenerate plants and a high level of sterility in regen- Culturing in vitro hybrid zygotes, however, does not
erated hybrid plants. These problems have interfered always overcome the problem of the remote genetic
with success in somatic hybridization of higher plants, relationships. Fusion products of maize egg cells with
resulting in only limited success, such as production of the sperm cells of Brassica did not result in zygotes
potato–tomatohybrids(GlebaandSytnik1984),butno capable of division. Although cultured maize eggs re-
wider crosses. Given the complexity of nuclear-cyto- main viable for about 3 weeks after isolation, cell death
plasmic,inter-cytoplasmicandploidylevelconflictinthe occurred 18–42 h after fusion with Brassica sperm cells,
offspring, it is not surprising that somatic hybridization demonstrating the possible influence of the distant
has not had widespread success in plant genetic engi- relationship (Kranz et al. 1995). Another equally
neering. plausible explanation is simply that the gametes are not
Cultured in vitro zygotes overcome a number of the in the same phase of the cell cycle. Whereas grass ga-
intrinsic limitations of somatic cell hybridization and metes tend to be in G phase at anthesis and gametic
1
provide anumber ofadvantages for wide hybridization. fusion, Arabidopsis is in S phase at anthesis and G
2
First, sperm cells contain little cytoplasm, with most of phase at fusion (Friedman 1999). There is no known
the cytoplasm in the zygote contributed by the egg cell. mechanism by which such cell cycle conflicts can be
The early development of the zygote may be regulated easily resolved in a hybrid cytoplasmic background.
by the resources stored in the egg cytoplasm (Dressel- Thus, both underlying physiological and cell cycle-
haus et al. 1999a), and there is reduced conflict in or- based differences may need to be carefully considered
ganellar DNA. Secondly, some genes of the sperm cells when selecting suitable parents, so that difficulties
are activated only after the zygote undergoes several encountered in subsequent culture of the hybrid zygote
divisionstoformaproembryoof32–46cells,suggesting are minimized.
that genes from the egg cell regulate early development
of the zygote (Vielle-Calzada et al. 2000). Recently,
Okamoto et al. (2005) reported that the genes up-regu- Improving plant transformation using the zygote
lated in the apical or basal cell oftwo-celledembryos of as a recipient cell
maize were expressed in the early zygote, proving zyg-
otically predetermined expression during early embryo- Transgenic tobacco plants were among the first ob-
genesis. Therefore, although the zygote is a product of tained transgenic plants in the early 1980s. Regenera-
the fusion of male and female gamete, the early devel- tion of other such plants, however, has become
opment of the zygote may be controlled by the pre- increasingly common and is a vigorous research area in
dominate volume of the maternal cytoplasm, which modern molecular biology. To date, the most prevalent
reduces cytoplasmic conflict and thus promotes suc- techniques for introducing foreign genes into higher
cessful cell signaling, progression through the cell cycle, plants include electrofusion, electroporation, PEG-
mitotic division and cytokinesis. mediated fusion and microinjection, with incorporation
Kranz et al. (1995) induced maize egg cell fusion of transgenes mediated by Agrobacterium. Techniques
with sperm cells of such genetically remote species as for transformation and regeneration present different
Coix, Sorghum, Hordeum and Triticum. The hybrid advantages and disadvantages, with myriad protocols
zygotes divided at a ratio of 78 (21/27), 50 (17/34), 43 proving to be optimal for different species and specific
(13/30) and 24% (9/38), respectively, to form micro- applications. This combinatorial resource of techniques
calli. The high frequency of hybrid zygotes dividing to for introducing and incorporating genes assures a
form microcalli makes the fusion of egg cells with wealth of potential techniques to ensure success in
sperm cells of different species or genera a potentially transgenic research. A major limitation to introducing
viable method to obtain distant hybridization in an- foreign genes into cells of higher plants is therefore one
giosperms. Successful division of the in vitro zygotes of time to achieve optimal incorporation and rapid
resulting from distant hybridization appears to be re- regeneration.
lated to the disproportionately greater abundance of After introduction of foreign genes into the target
egg cell cytoplasm. The typical volume discrepancies cell, DNA must be incorporated meaningfully into a
between the sperm and egg cells can be illustrated by chromosome under the direction of appropriate con-
trollingelements,andinordertobetransmitted,thecell microinjectionexperiments.Transgenictechniquesusing
must divide, thus greatly decreasing transformation in vivo or in vitro zygote cultures convincingly demon-
frequency and it should be stable. The recipient cells, strate the potential of female gametic cells to greatly
regardless of whether they are protoplasts, young em- enhance transgenic efficiency in plant improvement and
bryos or calli, would also need to retain the ability to experimentally dissect the role of genes that control
differentiate, form organs and photosynthesize in order fertilization and early zygote development.
to regenerate into differentiated plantlets. During the Current procedures for obtaining transgenic plants
process of cell growth and differentiation in culture, the use antibiotic or herbicide resistance genes as part of
genes of recipient cultured cells may undergo additional their construct in order to select transformed cells, calli
genetic or epigenetic changes during callus differentia- and plants; however, concerns about the safety of these
tion that inhibit regeneration or result in sterile plants. marker genes in crop transformation have caused sig-
Zygotes exhibit very strong vigor in vivo and have a nificantconcernforscientistsandsociety.Usingzygotes
proven ability for cell division and differentiation; they as receptor cells may be attractive for crop plants be-
serve as the primary founder cell during ‘‘typical’’ cause gene incorporation may be sufficiently effective
embryogenesis to form plantlets; and they execute a that lethal screens using antibiotic and herbicide resis-
pre-established template of pattern formation during tance genes are not needed. When screening tens or
development. Isolation of sperm and egg cells, in vitro hundredsofplants,ratherthanupto100,000plants,the
zygote formation through IVF, and cellular differenti- investmentofusingsinglegameticcellsmayberewarded
ation following IVF have been successful in maize and by eliminating a need for additional reporters (Zhang
wheat. In vivo zygotes of maize, wheat, and barley et al. 1999).
have also been successfully isolated and regenerated.
These in vitro and in vivo zygotes readily divide and
undergo modified embryogenesis to produce fertile Conclusions and prospects
plants during culture (Kranz and Lo¨rz 1993; Holm
et al. 1994; Kumlehn et al. 1998, 1999). Thus, suc- Clearly, difficulties in isolating gametes of higher plants
cessful isolation and culture of zygotes provides a have impeded our understanding of gamete physiology,
strong impetus for using zygotes as recipient cells for activation of development and early embryogenesis in
forming transgenic higher plants. flowering plants. Increasing number of tools, however,
Microinjectingforeigngenesintozygotes,asrecipient are now available to manipulate male and female ga-
cells, is attractive because zygotes lend themselves well metes of higher plants, providing numerous opportuni-
to this technique. Leduc et al. (1996) experimentally ties for scientific and biotechnological progress (Fig. 6).
microinjectedtwotypesofreportergenes(theGUSgene Isolated gametes can be analyzed directly during IVF
and anthocyanin regulatory genes) into 227 maize zy- with modern cellular and physiological probes, while
gotesisolated24 hafterpollination.About3.5%(8/227) means of regulating sexual reproductive development
ofthemicroinjectedzygotesshowedtransientexpression
of both genes. Po´nya et al. (1999) exposed egg and zy-
gote protoplasts of wheat to a high-frequency alternat-
ing-current field to immobilize cells and microinjected
exogenous DNA. Two constructs were used: (1) a GUS
reportergeneunderthecontrolof35Spromoterand(2)
a GFP reporter gene under the control of ubiquitin
promoter. A significantly higher transient expression
rate of both injected genes was displayed in their pro-
toplasts (46 and 52% for 98 egg cells and 77 zygotes,
respectively). Holm et al. (2000) used a rice actin pro-
moter construct, Act1-gusA-nos,asageneconstruct for
microinjectioninto wheatzygotes. Among thehundreds
of injected zygotes, 34% continued development into
embryo-like structures and eventually developed into
plants. PCR screening showed that 21% of the derived
structures contained exogenous DNA; however, only
two green plants were transgenic. It was proposed that
transformationofbarleybymicroinjectionofDNAinto
isolated zygotes was feasible but that gene expression
was rare, possibly due to degradation of the introduced Fig.6 Aschematicsummaryoftechniquesthathavebeenapplied
DNA (Holm et al. 2000). Scholten and Kranz (2001) to deepen our understanding of the events of double fertilization
from early microscopic study, to isolation and physiology, to
attempted transformation of gametes and zygotes using
cellular and molecular biology. Each of these represents accom-
microprojectile bombardment of immature embryos to
plishments that would have been difficult or impossible to study
overcome the problems and time-consuming nature of withinthecontextofinvivosystems