SEED DEVELOPMENT IN CITRUS
L. K. JACKSON; Revisedby F. G. GMITTER, JR.
ProfessorEmeritusand AssociateProfessor,Departmentof Horticultural Sciences,University of
Florida, Citrus Researchand EducationCenter,Lake Alfred, FL 33850
Becauseseeddevelopmentis necessarilya part of the developmentof fruit. it is important
to considerthe processes
responsiblefor seeddevelopmentand someof the reasonsfor the lack of
seedsin somecases.We will then turn attentionto the actualgrowth and developmentof the seed
within the fruit. Lastly, we needto considerthe horticultural significanceof seedproduction and
especiallysomeof the uniquefeaturesof citrus seedswhich makethem both an assetand a liability
for growersand researchscientists.
Citrus Flower and Fruit MoQ1holo~. There are 2 main types of bloom in citrus. Leafy
bloom occurswhen flowers form on and with new vegetativegrowth in the spring. The vegetative
growth of the elongatingshoot is transformedinto a tenninal inflorescenceof 1 or more flowers
about the time lateral shootsemergefrom bud scales.Other flowers are produceddirectly in leaf
axils of previousgrowth flushesand aretermedbouquetbloom.
As a vegetative growth point is transfonned into a flower bud, it becomes broadened and
flattened, forming a floral apical meristem. Each flower part is fonned from the outside in
(acropetally). The sepal (button) primordia are fonned first, then those of the petals, the anthers and
finally the carpels which will form the segments.The female flower parts (pistil) are composed by
fusion of a whorl of approximately 10 carpels. The cavities fonned by fusion of the carpels are called
locules. Each locule contains 2 vertical rows of ovules (Fig. 1), that can develop into seeds.
Fig. 1. Diagrammaticportrayal of opencitrus flower.
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The ovules are arrangedin rows near the central axis (axile placentation). The inner
integumentis differentiatedfirst. The outer integumentis developedlater. Both integumentstake
part in the fonnation of the seedcoatandthe micropylewhich providesthe openingthrough which
the pollen tube entersthe ovule.
The First Stel2sto Seed Formation: Me~asl2oro~enesis
and Embrxo Sac Develol2ment.
Within the ovule, the archesporia!cell forms nearthe apexof the nucellusbeforethe integuments
arecompletelydeveloped.The cell is distinguishedfrom thosesurroundingit by its larger size and
larger nucleus.The archesporia!cell divides to form a tapetalcell and an embryo sacmother cell
(megasporocyte).The tapetal cell divides rapidly and the megasporocyteis soon surroundedby
tissuenearthe centerof the nucellus.The megasporocyteundergoesreductiondivision, producing
4 haploidcells with only onesetof chromosomes
per cell. Thesecells,the megaspores,
arearranged
in a longitudinal row in the nucellus. Three of the 4 megasporesdegenerateand the remaining
megasporedevelopsinto the megasporemothercell and later, the embryo sac.(Fig. 2).
MJcellus
inner in~ment
outer integument
outer ntegument
inner integument
archesporial cell
nucellus
~megasporocyte
~II
-:;
'lis.2a.
Developing ovule
with archesporial
cell.
Fig.2b.
Developing ovule
with
megasporocyte.
ooter in~ment
integument
in~ment
inner integUment
tetrad
3 degenerated
m~ores
-7
'1a.2c..
Developing ovule
vi th -a.spore
tetrad.
functional
megaspore
Fig.2d.
Developing ovule
vith'.eg&spore
lIother
cell.
Megasporogenesis.
34
micropyle
outer integument
inner integument
developing
embryo sac
I
First
stage of embryo
sac developaent
in
ovule - aegaspore
.other
Fig.
3~.
Pig.3b.
cell.
Four-celled stage of
eabryo sac developaent
in ovule.
/
Two-celled stage of
embryo sac developD8Dt
in ovule.
Eight-celled
stage
of e8bryo sac
developaent in ovule.
Fig. 3. Embryo sacdevelopment.
35
The functionalmegasporedividesto fonD2 daughternuclei which migrateto oppositeends
of the sacandremainthereuntil the next nucleardivision. Meanwhile,the cytoplasmis reducedto
a thin layer in the periphery of the embryo sac,exceptaroundthe nuclei. The 2 daughternuclei
divide again,producing4 nuclei which fonD 2 pairs at oppositeendsof the sac.The nuclei divide
againto form the final 8 nuclei.The 4 nuclei in the micropylar end of the sacdevelopinto the egg,
2 synergids,and upperpolar nucleus. The 4 nuclei in the chalaza!end fonD the 3 antipodalsand
lower polar nucleusto completeembryo sacdevelopment(Fig. 3).
The time requiredfor embryosacdevelopmentvarieswith the cultivar. The embryosacsare
reportedto be in the 2- or 4-nucleatestageat anthesisin most ovules of 'Foster'grapefruit, while
'Pineapple'sweetorangeembryo sacsaremostly in the 4- or 8-nucleatestageat anthesis.
Maturation of the embryo sacreportedly occurs several days prior to petal opening (anthesis)
in 'Foster' grapefruit, at anthesis in 'Pineapple' sweet orange and several days after anthesis in
'Washington' navel sweet orange and 'Satsuma' mandarin. The process of embryo sac formation
varies from ovule to ovule within the same fruit. Formation of the mature embryo sac with respect
to floral development is apparently cultivar-dependent and may also be influenced by other external
and internal factors. Megaspore or embryo sac development at anthesis ranges from the megaspore
tetrad stage through the fully developed embryo sac. 'Pineapple' sweet orange embryo sacs
apparently undergo a rapid development. Thirty-four percent of the ovules of this cultivar contain
mature embryo sacsat anthesis,28% were in various stagesof development, and the remaining 38%
aborted at some stage. Embryo sac formation is reportedly complete in some ovules several days
prior to anthesis in 'Foster' grapefruit, although many ovules contain embryo sacs in the 1-, 2- or
4-nucleate stages at anthesis.
PollenTubeGrowth.Fertilization.and Endos~rm Develol2ment.Pollen grainsdevelopin
the anthers,following reduction division of microsporocytesto produce microsporeswith the
haploid chromosomenumber. Insectsvector the pollen to the stigma, where they germinateto
producea pollen tubethat growsthroughthe styleto the ovary. This tube penetratesthe micropyle
of the developingovules,andthe 2 spermnucleiaredischargedinto the embryosac.Onefuseswith
the eggnucleusandthe secondnucleusfuseswith the 2 polar nuclei andforms a triploid endosperm
nucleus.This doublefertilization apparentlyoccurssimultaneously.The zygote goesinto a resting
stageafter fertilization for severalweeksbeforedividing (Fig. 4).
The endospermnucleusdividessuccessivelymanytimes,without cell wall development,to
produce a multinucleate endosperm. Free nuclear endospermcan be seenup to 67 days after
pollination.This endospermmaterialbecomescellular,with 3 layersof cells on the micropylar end
and a single layer of cells further down the chalazalend. The endospermprovides a sourceof
nutrition that is absorbedasthe embryodevelops;it is consumedby 100daysafter pollination.
Sterilitx. Gameticsterility is the inability to producefunctionalpollen or embryosacs.Male
sterility may resultfrom imperfectdevelopmentof stamensbut usuallyis causedby defectivepollen
development.Pollensterility is found in varying degreesin manycultivarsof the genusC.i.tms.The
'Washington'sweetorangeis essentiallyvoid of pollen. Satsumamandarinpollen also degenerates
36
degenerated
synergids
multinucleate
endospenn
de~netated
antipodals
Fig. 4. Fertilization and endospermdevelopmentwithin the embryo sac.
during formation,leavingonly a few nonnal grainsat anthesis.Cross-pollinationslightly increases
seedsetof commerciallyseedlesscitruscultivars;their relative seedlessness
is associatedwith low
pollen viability and meiotic anomaliesresulting from chromosomalstructuralchanges.
Female sterility may result from aborted female flower parts or defective embryo sac
development. Studies of'Satsuma' mandarin and 'Washington' sweet orange provide classic
examples of female sterility. The megaspore mother cell of 'Washington' often aborts before
division. The cell becomeslong and narrow and is compressedupward by the surrounding nucellar
tissue. Generally, the megaspore mother cells pass through nomIal divisions, producing the linear
tetrad of megaspores.Soon after the nomIal degenerationof the 3 megasporesnearestthe micropyle,
the functional megaspore shrivels and aborts. Embryo sacs often abort in other stages of
development. Many of the embryo sacsexhibit delayed development which indicates the egg is not
mature and viable for fertilization at the proper time. Embryo abortion is another form of sterility.
Functional gametes(male and female parts) may be produced and fertilization occur, yet the embryo
may fail to develop. The occurrence of empty seed coats in fruit suggestsembryo abortion.
Citrus nonnally has 18 chromosomesbut various other multiples of chromosome levels. The
Tahiti, or Persian, lime for example is a triploid citrus cultivar (i.e. 2n = 3x = 27). When triploid
citrus attempt to undergo meiosis, gametes(eggs and pollen) are producted with unbalancednumbers
of chromosomes because, quite simply, 27 is not evenly divisible by 2. Citrus breeders are
attempting to exploit this fonn of sterility to develop seedlesscultivars. Unlike other cultivars such
as 'Clementine' which are seedlessby virtue of self-incompatibility, but which will bear seededfruit
when cross-pollinated by other citrus, triploids should be seedlessregardlessof pollination by other
cultivars. One method to develop triploids is to induce embryogenesisin endospenn cultural in vitro;
31;
although it has been accomplished, the limited number of successesmakes this method unattractive.
The most commonly used approachhas been to cross normal monoembryonic diploids with pollen
from tetraploids(2x X 4x
--+-
3x). Although embryosare producedfrom such interploid
hybridizations, seed development is halted becausethe normal embryo to endosperm ratio is not
achieved. (i.e. 3 :4, rather than 2:3). Tissue culture of rescuedembryos is necessaryto recover plants
from these crosses. This is now done routinely by many breeding programs.
Incom~atibilit1:. Sterility prior to fertilization is characterizedby non-functionalgametes,
but with gameticincompatibility the pollen and ovules arefunctional and failure to producefruit
with seedresultsfrom a physiologicalhindranceto fertilization.This is usually manifestedin citrus
by slow growth of the pollen tube down the style.
There are 2 basic types of gametic incompatibility mechanisms, sporophytic and
gametophytic. The sporophytic type is characterizedby the inhibition of pollen germination.
Gametophyticincompatibility is geneticallycontrolledand pollen tube growth down the styles is
retarded.
Zx~oteDevelonment.Developmentof the zygotic (sexual)embryoin dicotyledonsfollows
a somewhatregular order. The first division of the zygoteis usually transversewith respectto the
long axis. This is usuallyfollowed by furthertransversedivisions,andin somepartsof the embryo,
vertical divisions. Gradually,the embryoassumesa club-like form. The distal part of the embryo
thenbecomesthe centerof active cell division and developsinto a sphericalstructure.This sphere
is called the embryoproper, and the stalk at the proximal end,the suspensor.Subsequentchanges
producea distally flattenedstructurewith bilateralsymmetry.This flatteningprecedesthe initiation
of the 2 cotyledons.The embryoassumesthe shapeof a heartasthe cotyledonsare formed. Further
divisions organizeroot and shootmeristemsand rudimentsof other organs(Fig. 5).
The fertilized zygote in citrus undergoes a resting period of several weeks before division.
This period is 21 to 28 days in trifoliate orange and 50 days in 'Foster' grapefruit. First signs of
zygote division in 'Orlando' tangelo occur 40 days after pollination. A 4-celled suspensorcan be seen
5 days later and future growth of the embryo follows the classical pattern. The sexual embryo
remains attached by the suspensorat the micropylar tip of the developing seed. The terminal end of
the embryo flattens 9 weeks after pollination and a fork-like embryo develops 12 weeks after
pollination as the cotyledons are differentiated. The endosperm develops as the sexual embryo is
forming and later deteriorates. The nucellus also shrinks away, leaving only vestiges to contribute
to seed coat formation.
SeedDevelo~ment.The ovuledevelopsinto the seedfollowing fecundation.The endosperm
enlargesat the expenseof the nucellus.Nucellar embryosmay developon stalks attachedto the
nucellus before the egg cell divides. Theseenlargingembryosand the zygotic embryo gradually
replacethe endosperm,the latter being digestedby the developingcotyledons.An inner seedcoat
or tegmenis formed from the inner integument,the inner parts of the outer integument,and the
remainsof the nucellusandendosperm.The chalazaforms a capover the distal end of the seedand
the brown-to-reddishor purple epidermalcells form the coloredareaadjacentto the nucellus.The
38
Fig. 5. Developmentof the fertilized citrus egg into a zygote and embryo.
testa, or outer seedcoat, is composedprimarily of the outer epidermis of the ovular wall. The
epidermal cells form a secondarywall which makesup a woody, cream-or yellow-colored tough
covering.The testais often quite wrinkled or ridged and extendsbeyondthe rest of the seedat one
or both endsto form a beakof flat plate which may extendalong the length of the seed.
Within the tegmen and the testa are 1 or more embryos. They form a solid, rounded mass in
which the root initials (radicles) normally point toward the micropylar end. A seed with 1 embryo
usually has 2 cotyledons of similar size and shape.The cotyledon size and shapeoften varies greatly
if more than 1 embryo is present. The embryos in polyembryonic cultivars are usually crowded into
the micropylar end. The cotyledons are white, cream or green and constitute most of the mature seed.
39
Horticultural Significance of SeedProduction
Seedproductionin citrus fruit impactsin manyways.The yield or numberof fruit produced
per treeis a function of seednumbersfor many cultivars.The sizeof individual fruits is controlled
to a degreeby the numberof seedsper fruit. Parthenocarpy,or the ability to producefruit without
seeds,is an important quality of many of our citrus cultivars. Polyembryony,the production of
multiple embryoswithin a seed,hasfar-reachingimpactson the entirecitrus industry.Eachof these
are discussedbelow.
Parthen~~.
Many citruscultivarsproduceacceptableyields of fruit with little or no seed
in the fruit. Perhapsthe bestexampleof this is the 'Tahiti'lime which is almostcompletely seedless
andstill producesgoodcrops.Sucha cultivar would be consideredstrongly parthenocarpic.At the
otherextremewould be 'Wilking' mandarin,which setsno fruit whenpollen is excluded. Between
the 2 extremesarethe moderatelyparthenocarpic
cultivars.Often,the yield of citrus cultivars in this
groupwill improveasmoreseedsare setasthe result of betterpollination or cross-pollination.An
exampleof a cultivar in this group would be the 'Orlando' tangelo,which benefits greatly from
cross-pollination.
Seedycultivarsmay alsopossessstrongparthenocarpy
but this characteristicis unimportant
andunnoticed.Therefore,parthenocarpyis not a considerationfor us to study in the production of
normally seedycitrus fruit.
Seedlessfruit are anothermatter.The tendencyto parthenocarpyis quite prevalentamong
citrus, andthe horticulturally successfulparthenocarpiccultivars possessexceptionalability to set
fruit without seeds.If a cultivar incapableof producingseedsis to be successful,it must be strongly
parthenocarpic.Probably many seedlesstypes have arisenin the past which were of little value
becauseof the lack of parthenocarpy.
Cross-pollination is desirableon many moderately parthenocarpiccultivars to increaseyields.
The numerous tangerine hybrids are excellent examples and the value of interplanting is well
established. Cross-pollination increasesseed set by overcoming incompatibilities in these cultivars.
Fruit set may also be increased in weakly or moderately parthenocarpic cultivars by other means.
The use of growth regulators such as gibberellic acid is an established and recommended practice
and girdling has been shown effective, as well.
Fruit Set. Nonnal fruit set is the result of the stimulus of sexual fertilization and the
production of seeds.Parthenocarpiccultivars representan exception to this that is especially
noticeable in incompatible or sterile cultivars where very few seeds are produced.
Non-parthenocarpiccultivars will set few if any fruit if fertilization and seedproduction do not
occur. Therefore,enhancementof seedproductionis often important in maximizing fruit set and
yield.
40
Citrus flowers in self-fertile cultivars are usually self-pollinating since the structure of the
flower allows this to happen.Increasedbee activity assiststhis processand is known to help increase
fruit set, especially in self-incompatible cultivars where cross-pollination is necessary.
Fruit Size. In somecultivars, con-elationsbetweenseednumberand fruit size have been
reportedfor at least8 cultivars. With cultivarsplaguedby smallsizes,cross-pollinationis important
to producefruit of an acceptablecommercialsize.
Pol~emb~on~. The fonnation of multiple embryos is quite common in many citrus
cultivars. These "extra" embryos may be the result of multiple zygotic embryos or twinning of a
single zygotic embryo. The predominant causeof multiple embryo fonnation, however, is nucellar
embryony, the development of vegetative embryos from the nucellus. Development of adventive
embryos from nucellar tissue is extremely interesting since they are not the product of sexual
fertilization. They are outgrowths of the nucellus and are therefore the product of vegetative
reproduction, not sexual reproduction as is the casewith zygotic embryos. This asexualreproduction
has very important consequencesin citrus.
Cells destinedto develop into nucellar embryoscan be seenin nucelli even before floral
anthesis. Apparently pollination is essentialto stimulatethe developmentof nucellar embryos.It
is not clear whether actual fertilization is essentialbut certainly excluding pollen will result in a
greatly reducedfruit set. Perhapsthe stimulus of pollen tube activity and the releaseof growth
regulators accompanyingsuch activity is all that is necessaryto promote the growth of nucellar
embryosin many citrus types.
Exceptin cultivarswhich rarely or neverproducenucellarembryos,the zygotic embryowill
have to competewith one or more nucellar embryosfor spacewithin the seed.The result of this
competitiondependsuponthe numberof embryoswithin the seed,their time of development,their
location and vigor. The lower the numberof embryosper seed,the larger the averagesize of the
embryosandthe greaterprobability of survival.Whenzygotic embryosfail to survivein all or most
of the seeds,the resultantseedlingswill largely be from nucellarmaterialand thereforegenetically
identical to the female parent plant. Such cultivars would be referred to as "highly nucellar,"
producinga large proportion of nucellarplantscomparedto zygotic ones.
Threefactorsarementionedwhich contributeto the developmentof nucellarembryosat the
expenseof zygotic: (1) nucellar embryosgrow off more rapidly within the seed,therebygetting a
"headstart" on the zygotic embryo,(2) the zygoticembryois locatedunfavomblyin the apexof the
embryo sac and may receive fewer nutrients and is more subject to crowding pressure,and
(3) zygotic embryos are usually genetically weaker than their nucellar counterpartsbecauseof
inbreedingdepression.
Polyembryony is extremely important for 3 reasons.These are of great horticultural
significanceand will be discussedindividually in the paragraphsthat follow.
4
Virus diseasesare a serious concern for citrus production. The processof nucellar embryony
cleanses plant material of the citrus viruses. The mechanism of the process is unclear but well
documented. Plant material arising from nucellar seedlings will be free of citrus viruses, even if the
parent plant is infected. The benefits ofnucellar, virus-free budwood are well known. In cultivars
where nucellar embryos are not produced, freedom from viruses must be attained in other ways such
as searching for disease-freematerial, or heat treatment of infected sources.
Nursery operations are very dependentupon nucellar embryony becausethe process allows
for vegetative seedreproduction. It is unique to be able to grow plant material in this way, and citrus
nurserymen are lucky to be able to capitalize on this phenomenon.Eliminating off-size and off-type
variants in a seedling population ensuresa very high percentageof nucellar seedling material which
is genetically identical to the desired female parent plant.
Plant breedersare often frustratedby nucellar embryonysinceit greatly complicatestheir
work. The high degreeof nucellar embryony common in many citrus cultivars meansit is very
difficult to obtainhybrid seedlings.This is why all the early breedersusedmonoembryonicfemale
parentplantsor utilized readily-observedgeneticmarkerssuchasthe trifoliate leaf characteristicof
Poncimsto able to discernhybrid seedlings.
42