HYBRIDIZATIONHYBRIDIZATION

TaryonoTaryono

Faculty of AgricultureFaculty of Agriculture

Gadjah Mada UniversityGadjah Mada University

HybridizationHybridizationThe formation of a new organism by normal The formation of a new organism by normal sexual processes or by protoplast fusionsexual processes or by protoplast fusion

Wide Hybridization (Interspecific Wide Hybridization (Interspecific Hybridization)Hybridization)

Crosses made between distantly related species Crosses made between distantly related species or generaor genera

Somatic hybridization (Protoplast fusion)Somatic hybridization (Protoplast fusion)

Crosses made between somatic cellsCrosses made between somatic cells

► ► One of the most effective methods of One of the most effective methods of crops improvement programscrops improvement programs

► ► Most the hybridization work carried out Most the hybridization work carried out has used genetic variability within specieshas used genetic variability within species

What is pollination?What is pollination?

• PollinationPollination: :

The transfer of The transfer of pollen from the pollen from the male male antheranther to to the female the female stigmastigma

Why is pollination important?Why is pollination important?

Sexual reproductionSexual reproduction is important for is important for evolution:evolution:

Sexual reproductionSexual reproduction produces produces variable variable offspring,offspring, creating diversity and variation creating diversity and variation among populations (shuffling of genes)among populations (shuffling of genes)

You need variation for Natural Selection to occurYou need variation for Natural Selection to occurSexual reproduction is advantageous to an Sexual reproduction is advantageous to an

organism only if it happens with someone other organism only if it happens with someone other than itself! than itself!

Out-breeding = good! (inbreeding = bad…)Out-breeding = good! (inbreeding = bad…)

Sexual reproductionSexual reproductionIn animals: It’s easy because you have In animals: It’s easy because you have

separate male and female individuals.separate male and female individuals.In flowering plants: Not so easy, In flowering plants: Not so easy,

because most flowers have because most flowers have bothboth male male and female parts in them, called and female parts in them, called perfect flowersperfect flowers..

So flowering plants have evolved So flowering plants have evolved special ways to insure special ways to insure out-breeding/out-crossing – and to out-breeding/out-crossing – and to prevent inbreeding.prevent inbreeding.

Function of flowerFunction of flowerTo attract pollinators with colorful petals, To attract pollinators with colorful petals,

scent, nectar and pollenscent, nectar and pollen

Carpel/

Overview of floral organsOverview of floral organs

Reproductive floral organs: femaleReproductive floral organs: femaleCarpel Carpel or or pistilpistil – female – female

reproductive organs; reproductive organs; contains:contains:

• Stigma – Stigma – is where pollen sticks is where pollen sticks toto

• StyleStyle – is the long tube that – is the long tube that connects stigma to ovaryconnects stigma to ovary

• Ovary Ovary – enlarged structure at – enlarged structure at the base of carpel/pistil where the base of carpel/pistil where the ovules are located; it will the ovules are located; it will become the fruit.become the fruit.

• Ovules Ovules – contains female – contains female gametophyte, becomes gametophyte, becomes the seedthe seed

• Plants have style!Plants have style!

carpel or pistil

ovary

Reproductive floral organs: maleReproductive floral organs: male

StamenStamen – male floral – male floral organ, consists of:organ, consists of:

Anther Anther – part of the – part of the stamen that produces stamen that produces pollenpollen

FilamentFilament – stalk-like – stalk-like structure that holds antherstructure that holds anther

PollenPollen – immature male – immature male gametophytegametophyte

Non-reproductive floral organsNon-reproductive floral organsPetalsPetals – whorl of flower organs – whorl of flower organs

that are often brightly colored to that are often brightly colored to attract pollinatorsattract pollinators

CorollaCorolla – whorl of petals – whorl of petals in a flowerin a flower

Sepals – whorl of leaf-like organs outside the corolla; help protect the unopened flower bud.

Calyx – whorl of sepals in a flower

Tepals – when sepals and petals look the same

Pollination and FertilizationPollination and Fertilization

• Pollen contains TWO nuclei: a Pollen contains TWO nuclei: a spermsperm nucleus and nucleus and tubetube nucleus nucleus

• Sperm nucleus is protected in gametophyte tissue Sperm nucleus is protected in gametophyte tissue (pollen can travel in the air)(pollen can travel in the air)

Pollination and FertilizationPollination and Fertilization For pollen sperm to successfully fertilize the egg, there must For pollen sperm to successfully fertilize the egg, there must

be be pollinationpollination: a method to get the pollen from the male : a method to get the pollen from the male anther to the stigma.anther to the stigma.

Pollen sticks to the stigma, starts growing a pollen tubePollen sticks to the stigma, starts growing a pollen tube FertilizationFertilization begins when begins when

tube begins to grow tube begins to grow toward the eggtoward the egg

Double FertilizationDouble Fertilization• Double fertilizationDouble fertilization occurs: One sperm nucleus occurs: One sperm nucleus

(1n) fertilizes the egg, producing a (1n) fertilizes the egg, producing a zygotezygote (2n) (2n) which becomes the plant which becomes the plant embryo embryo inside the seedinside the seed

• Another sperm nucleus fuses with the polar nuclei, Another sperm nucleus fuses with the polar nuclei, resulting in a triploid resulting in a triploid endospermendosperm (3n) (3n)

• Endosperm is a source of food for the young embryo.Endosperm is a source of food for the young embryo.

Endosperm

Hermaphroditic FlowersHermaphroditic Flowers

• Self-compatible (SC)Self-compatible (SC)– Capable of self-Capable of self-

fertilization or cross-fertilization or cross-fertilizationfertilization

• Self-incompatible (SI)Self-incompatible (SI)– Only capable of cross-Only capable of cross-

fertilizationfertilization– Inability of Inability of

hermaphroditic plant to hermaphroditic plant to produce zygotes w/ self produce zygotes w/ self pollenpollen

Autogamy Autogamy

• Self-fertilizationSelf-fertilization

• Pollen transfer Pollen transfer within or among within or among flowers of same flowers of same individualindividual

• ~25% of plant taxa~25% of plant taxa

Advantages of AutogamyAdvantages of Autogamy

• Insures seed set in absence of Insures seed set in absence of pollinators.pollinators.

• Overcomes sterility.Overcomes sterility.

• Selectively advantageous by Selectively advantageous by transmitting both sets of genes to transmitting both sets of genes to offspring.offspring.– Well-adapted genotypes preserved.Well-adapted genotypes preserved.

• Only single colonizing individual needed.Only single colonizing individual needed.

Disadvantages of AutogamyDisadvantages of Autogamy

• Decreases genetic variability.Decreases genetic variability.

• Inability to adapt to changing Inability to adapt to changing conditions.conditions.

• Increases inbreeding depression.Increases inbreeding depression.– Reduces heterozygosity and increases Reduces heterozygosity and increases

homozygosity of deleterious alleles.homozygosity of deleterious alleles.– More uniform populations.More uniform populations.

Cleistogamy

• Flowers never open and Flowers never open and only capable of self-only capable of self-fertilization in bud.fertilization in bud.

• Inconspicuous, bud-like Inconspicuous, bud-like apetalous flowers that apetalous flowers that form directly into seed form directly into seed capsules.capsules.

• Has evolved Has evolved independently multiple independently multiple timestimes– throughout the throughout the

angiosperms, including angiosperms, including some basal lineages.some basal lineages.

• 488 species, across 212 488 species, across 212 genera and 49 families.genera and 49 families.– Violaceae, Fabaceae, Violaceae, Fabaceae,

PoaceaePoaceae

Cleistogamy

• Mixed mating systems -Mixed mating systems -can produce both CL can produce both CL and CH on an and CH on an individual.individual.

• CL fls are a “back-up” in CL fls are a “back-up” in case pollinators scarce.case pollinators scarce.

• CL occur after normal CL occur after normal flowering period. flowering period. – CH fls early spring and CL CH fls early spring and CL

fls rest of season.fls rest of season.

• CL fls occur through CL fls occur through mutations with loss of mutations with loss of SISI..

How do plants get pollen from one How do plants get pollen from one plant to another?plant to another?

• Because plants are rooted in Because plants are rooted in the ground, they must use the ground, they must use different strategies:different strategies:

WIND POLLINATIONWIND POLLINATION: : Gymnosperms and some Gymnosperms and some

flowering plants (grasses, flowering plants (grasses, trees) trees) use wind pollination.use wind pollination.

Flowers are small, grouped Flowers are small, grouped togethertogether

Not a very efficient methodNot a very efficient method(too chancy and wasteful)(too chancy and wasteful)

ANIMALSANIMALSMany flowering plants rely on animals for Many flowering plants rely on animals for cross-pollination:cross-pollination:

InsectsInsects – bees, wasps, flies, butterflies, moths – bees, wasps, flies, butterflies, mothsBirdsBirds – hummingbirds, honey creepers – hummingbirds, honey creepersMammalsMammals – bats, mice, monkeys – bats, mice, monkeys

Even some reptiles and amphibiansEven some reptiles and amphibians!!

CoevolutionCoevolution• CoevolutionCoevolution

interactions between two different species interactions between two different species as selective forces on each other, resulting as selective forces on each other, resulting in adaptations that increase their in adaptations that increase their interdependency.interdependency.

• Animal-flowering plant interaction is a Animal-flowering plant interaction is a classic example of coevolution:classic example of coevolution:1. Plants evolve elaborate methods to attract 1. Plants evolve elaborate methods to attract animal pollinatorsanimal pollinators

2. Animals evolved specialized body parts and 2. Animals evolved specialized body parts and behaviors that aid plant pollinationbehaviors that aid plant pollination

A word about pollen…A word about pollen…• The shape and form of The shape and form of

pollen is related to its pollen is related to its method of pollination…method of pollination…

Insect-pollinated Insect-pollinated speciesspecieshave sticky of barbedhave sticky of barbedpollen grainspollen grains

Wind-pollinated speciesWind-pollinated speciesis lightweight, small is lightweight, small andandsmooth (corn pollen)smooth (corn pollen)

Palynology: the study of pollenPalynology: the study of pollen

• Palynology is useful in many fields:Palynology is useful in many fields:Petroleum geologyPetroleum geology – fossil pollen can – fossil pollen can

determine if a field will have oil-rich depositsdetermine if a field will have oil-rich depositsArcheologyArcheology – studying ancient pollen – studying ancient pollen

samples, archeologists can determine samples, archeologists can determine agricultural practices, diet, etc.agricultural practices, diet, etc.

Anthropology Anthropology – uses of pollen in rituals– uses of pollen in rituals Criminology Criminology – to determine the whereabouts – to determine the whereabouts

of an individual, examine pollen clinging to of an individual, examine pollen clinging to clothesclothes

Aerobiology Aerobiology – to determine what plants – to determine what plants cause hay fever and allergic reactions – in cause hay fever and allergic reactions – in landscapinglandscaping

Animal pollinators: BeesAnimal pollinators: Bees• BeesBees – the most – the most

important group of important group of flower pollinatorsflower pollinators

They live on the nectar They live on the nectar and feed and feed larvae, also eat the larvae, also eat the pollen.pollen.

Bees are guided by sight Bees are guided by sight andandsmellsmell

See See yellowyellow and and blueblue colors,colors,also ultraviolet light (not also ultraviolet light (not red)red)

Flowers have Flowers have “honey guides” “honey guides” and bee landing and bee landing platforms..platforms..

Butterflies and mothsButterflies and moths• Also guided by sight and Also guided by sight and

smellsmell

• Butterflies can see Butterflies can see redred and and orangeorangeflowersflowers

• Usually shaped as a long tube Usually shaped as a long tube

because of insect’sbecause of insect’sproboscis – to get nectarproboscis – to get nectar

• Moth-pollinated flowersMoth-pollinated flowersare usually white or pale,are usually white or pale,with sweet, strong odor – with sweet, strong odor – for night pollination.for night pollination.

Flies and beetlesFlies and beetles• Flies like flowers that Flies like flowers that

smellsmelllike dung or rotten meat.like dung or rotten meat.

• Lay their eggs there, but Lay their eggs there, but larvae die due to lack of foodlarvae die due to lack of food

• Beetles pollinate flowersthat are dull in color, buthave very strong odor

BirdsBirds• Birds have a good senseBirds have a good sense

of color, they like yellow orof color, they like yellow orredred flowers…flowers…

• But birds do not have a goodBut birds do not have a goodsense of smell, so bird-pollinatedsense of smell, so bird-pollinatedflowers usually have little odor.flowers usually have little odor.

• Flowers provide fluid nectar inFlowers provide fluid nectar ingreater quantities than insectsgreater quantities than insects

• Hummingbird-pollinated flowersHummingbird-pollinated flowersusually have long, tubular corollausually have long, tubular corolla

• Pollen is large and stickyPollen is large and sticky

Mammals: bats and miceMammals: bats and mice

• Bats pollinate at night,Bats pollinate at night,so flowers are whiteso flowers are white

• Mouse-pollinated flowersMouse-pollinated flowersare usually are usually inconspicuous,inconspicuous,they open at nightthey open at night

Why do animals pollinate plants?Why do animals pollinate plants?• They get a REWARD: food! In They get a REWARD: food! In

exchange for moving their pollen exchange for moving their pollen to another flowerto another flower

• NectarNectar – a sugary solution produced – a sugary solution produced in special flower glands called nectariesin special flower glands called nectaries

• Nectar concentration matches energy Nectar concentration matches energy requirements of the pollinator: bird- requirements of the pollinator: bird- and bee-pollinated flowers have and bee-pollinated flowers have different sugar conc.different sugar conc.

• PollenPollen – is high in protein, some bees – is high in protein, some bees and beetles eat it. and beetles eat it.

• Flowers can produce two kinds of Flowers can produce two kinds of pollen: a normal and a sterile, but pollen: a normal and a sterile, but tasty, kind, for the insect.tasty, kind, for the insect.

Getting the pollinator’s attentionGetting the pollinator’s attention

• Plants advertise their pollen and Plants advertise their pollen and nectar rewards withnectar rewards with

• Colors – bees see Colors – bees see blueblue, , yellowyellow, , UV; while birds see UV; while birds see redred. Bats don’t . Bats don’t see well, so flowers are white.see well, so flowers are white.

• Nectar or honey guides – Nectar or honey guides – a visual guide for pollinator a visual guide for pollinator to locate the reward (pansy flower)to locate the reward (pansy flower)

• Aromas – for insects, nectar.Aromas – for insects, nectar.Can also be carrion or dung smellCan also be carrion or dung smell

Plant MimicryPlant Mimicry• Some plants take advantage Some plants take advantage

of the sex drive of certain of the sex drive of certain insects…insects…

• Certain orchids look like Certain orchids look like female wasps, and even female wasps, and even smell like them!smell like them!

• Males try to mate with them,Males try to mate with them,and in the process theyand in the process theypollinate the plantpollinate the plant

• The orchid gets pollinated,The orchid gets pollinated,but the male wasp only getsbut the male wasp only getsfrustrated!frustrated!

Selfers vs. Outcrossers

• SCSC• Small flowers (few)Small flowers (few)• Unscented flowersUnscented flowers• Nectaries & nectar Nectaries & nectar

guides absentguides absent• Maturation of Maturation of

reproductive partsreproductive parts– Anthers near stigmaAnthers near stigma– Style includedStyle included

• All fruits matureAll fruits mature• Low pollen/ovule ratioLow pollen/ovule ratio

• SI or SCSI or SC• Large showy flowers Large showy flowers

(many)(many)• Scented flowersScented flowers• Nectaries & nectar Nectaries & nectar

guides presentguides present• Differential maturation Differential maturation

of reproductive partsof reproductive parts– Anthers far from stigmaAnthers far from stigma– Stigma well-exsertedStigma well-exserted

• Only some fruits matureOnly some fruits mature• High pollen/ovule ratioHigh pollen/ovule ratio

Strategies to Prevent Self-Strategies to Prevent Self-fertilizationfertilization

Strategies to avoid self-pollinationStrategies to avoid self-pollination

Perfect flowers have both male and Perfect flowers have both male and female organs, so plants have female organs, so plants have strategies to avoid self-pollination:strategies to avoid self-pollination:

1. 1. TimingTiming – male and female – male and female structures mature at different timesstructures mature at different times

2. 2. MorphologicalMorphological – structure of– structure ofmale and female organs preventsmale and female organs preventsself-pollination (imperfect flower)self-pollination (imperfect flower)

3. 3. BiochemicalBiochemical – chemical on – chemical on surface of pollen and stigma/stylesurface of pollen and stigma/stylethat prevent pollen tube that prevent pollen tube germinationgermination

on the same flower (incompatibleon the same flower (incompatible))

Physical Separation of Physical Separation of Reproductive Parts (Herkogamy)Reproductive Parts (Herkogamy)

• Within flowersWithin flowers • Among flowersAmong flowers

HeterostylyHeterostyly

• Flowers in different individuals of the Flowers in different individuals of the same species having 2 or 3 different style same species having 2 or 3 different style lengthslengths– With stamen lengths varying inverselyWith stamen lengths varying inversely

• DistylyDistyly

• TristylyTristyly

DistylyDistyly

• 2 floral morphs.2 floral morphs.

• ““Thrum” flower Thrum” flower – long filaments with long filaments with

short stylesshort styles

• ““Pin” flowerPin” flower– short filaments with short filaments with

long styleslong styles

• Only pollinations Only pollinations between different between different floral morphs are floral morphs are successful.successful.

• E.g.: E.g.: PrimulaPrimula

TristylyTristyly

• 3 floral morphs3 floral morphs

• Style long, stamens Style long, stamens short and mediumshort and medium

• Style medium, Style medium, stamens short and stamens short and longlong

• Style short, Style short, stamens medium stamens medium and longand long

Physical Separation of Reproductive PartsPhysical Separation of Reproductive Parts

• Unisexual flowersUnisexual flowers– Staminate and Staminate and

carpellate flowerscarpellate flowers

• MonoecyMonoecy

• DioecyDioecy

MonoecyMonoecy

• Common in wind-Common in wind-pollinated plants.pollinated plants.

• Common in Common in temperate regions.temperate regions.

• Self-pollination Self-pollination possible but less possible but less likely.likely.

DioecyDioecy

• 4% of angiosperms4% of angiosperms– Scattered throughoutScattered throughout

• Common in tropical Common in tropical regions and oceanic regions and oceanic islandsislands

• Gen small fl sizeGen small fl size

• 100% out-crossing, 100% out-crossing, but inefficientbut inefficient

• Often controlled by Often controlled by sex chromosomessex chromosomes– SileneSilene

Polygamous FlowersPolygamous Flowers

• Both bisexual and unisexual flowers on the same Both bisexual and unisexual flowers on the same plant.plant.– Androdioecy = bisexual and staminate individuals in a population.Androdioecy = bisexual and staminate individuals in a population.– Andromonoecy = bisexual and staminate flowers on same individual.Andromonoecy = bisexual and staminate flowers on same individual.

• Euphorbia, SolanumEuphorbia, Solanum

– Gynodioecy = bisexual and carpellate individuals in a population.Gynodioecy = bisexual and carpellate individuals in a population.• Sidalcea hendersonii, SileneSidalcea hendersonii, Silene

– Gynomonoecy = bisexual and carpellate flowers on same individual.Gynomonoecy = bisexual and carpellate flowers on same individual.• Silene, SolidagoSilene, Solidago

– Polygamodioecy = some plants with bisexual and staminate flowers & Polygamodioecy = some plants with bisexual and staminate flowers & some plants with bisexual and carpellate flowers in a population.some plants with bisexual and carpellate flowers in a population.

– PolygamomonoecyPolygamomonoecy = bisexual, staminate, and carpellate flowers on = bisexual, staminate, and carpellate flowers on same individual.same individual.

Evolution of DioecyEvolution of Dioecy• From hermaphroditismFrom hermaphroditism

– Vestigial sex organsVestigial sex organs– Few families entirely dioeciousFew families entirely dioecious

• From monoecyFrom monoecy• From SCFrom SC

– Within groups that have lost Within groups that have lost original GSI systemoriginal GSI system

• From distylyFrom distyly– Unequal pollen flow & gender Unequal pollen flow & gender

functionfunction– Change in pollinator frequencyChange in pollinator frequency– Non-functional anthers at low Non-functional anthers at low

level in female flowerslevel in female flowers– Non-functional pistil in male Non-functional pistil in male

flowersflowers

Temporal Separation of Reproductive Parts Temporal Separation of Reproductive Parts (Dichogamy)(Dichogamy)

• ProtandryProtandry– Anthers release Anthers release

pollen before stigma pollen before stigma receptivereceptive

– Common in insect-Common in insect-pollinated plantspollinated plants

• Geranium Geranium maculatummaculatum– 1st day flower1st day flower– 2nd day flower2nd day flower

Temporal Separation of Reproductive Parts Temporal Separation of Reproductive Parts (Dichogamy)(Dichogamy)

• ProtogynyProtogyny– Stigma receptive Stigma receptive

before pollen before pollen releaserelease

– Less common than Less common than protandryprotandry

• Magnolia Magnolia grandifloragrandiflora– 1st day flower1st day flower– 2nd day flower2nd day flower

GeitonogamyGeitonogamy

• Self pollination between different flowers Self pollination between different flowers on same plant.on same plant.

Evolution of Breeding SystemsEvolution of Breeding Systems

• Evolutionary trends go both ways and in a Evolutionary trends go both ways and in a variety of ways.variety of ways.

• Ancestral angiosperms were SC, Ancestral angiosperms were SC, hermaphroditic.hermaphroditic.

• SI has evolved many times.SI has evolved many times.– SC has evolved from SI plants as well.SC has evolved from SI plants as well.

Crossability barriersCrossability barriers

prevent the fusion of male and female prevent the fusion of male and female gametes originating from individuals of gametes originating from individuals of different species/genera and/or the different species/genera and/or the development of a fertilized ovule into development of a fertilized ovule into viable seedviable seed

Include the limit effective utilization of the Include the limit effective utilization of the hybrids for gene introgressionhybrids for gene introgression

Incompatibility (self) ?Incompatibility (self) ?Very frequent in interspecific and Very frequent in interspecific and

intergeneric hybridization programintergeneric hybridization program

Major interspecific crossability barriersMajor interspecific crossability barriers

I.I. Temporal and spatial isolation of speciesTemporal and spatial isolation of species

II.II. Pre-fertilization barriersPre-fertilization barriers

On the surface of the stigma before pollen tube entryOn the surface of the stigma before pollen tube entry

Inside the tissues of he stigma and style Inside the tissues of he stigma and style

Inside the ovary and embryo sacInside the ovary and embryo sac

III.III. Post fertilization barriersPost fertilization barriers

Non viability of hybrid embryosNon viability of hybrid embryos

Failure of hybrid to flowerFailure of hybrid to flower

Hybrid sterilityHybrid sterility

Lack of recombinantLack of recombinant

Hybrid breakdown in F2 or later generationHybrid breakdown in F2 or later generation

I. Temporal and spatial isolation of parental I. Temporal and spatial isolation of parental speciesspecies

Non synchronous flowering of the parental species Non synchronous flowering of the parental species due to different agro-ecological or geographical due to different agro-ecological or geographical backgroundbackground

1.1. Early/staggered sowingEarly/staggered sowing

2.2. Suitable photoperiodic treatmentSuitable photoperiodic treatment

II. Pre-fertilization barriersII. Pre-fertilization barriers

A. Unilateral incompatibility (UI)A. Unilateral incompatibility (UI) Prevent fertilization by arresting post pollination Prevent fertilization by arresting post pollination

events at one or many levelsevents at one or many levels Incompatibility operates in one direction, whereas the Incompatibility operates in one direction, whereas the

reciprocal cross is successful (unilateral reciprocal cross is successful (unilateral incompatibility = UI)incompatibility = UI)

UI is more common when cross includes a self-UI is more common when cross includes a self-compatible (SC) and a self incompatible (SI)compatible (SC) and a self incompatible (SI)

The crosses show incompatible when an SI species is The crosses show incompatible when an SI species is used as a female parent (SI x SC)used as a female parent (SI x SC)

Self-incompatibility inhibition is the result of active Self-incompatibility inhibition is the result of active recognition of the pollen.recognition of the pollen.

Self pollen is positively recognized as a result of the Self pollen is positively recognized as a result of the interaction of S allele product in the pollen and the interaction of S allele product in the pollen and the pistilpistil

II. Pre-fertilization barriersII. Pre-fertilization barriers

B.B. Active versus passive inhibitionActive versus passive inhibition Self-incompatibility inhibition is the result of active Self-incompatibility inhibition is the result of active

recognition of the pollen.recognition of the pollen. Self pollen is positively recognized as a result of the Self pollen is positively recognized as a result of the

interaction of S allele product in the pollen and the interaction of S allele product in the pollen and the pistilpistil

Positive recognition results in the activation of Positive recognition results in the activation of metabolic processes in the pollen and/or the pistil to metabolic processes in the pollen and/or the pistil to bring about pollen inhibitionbring about pollen inhibition

The arrest of post pollination events seems to be The arrest of post pollination events seems to be passive (not a result of active recognition of pollen) passive (not a result of active recognition of pollen) and a result of lack of co-adaptation between the and a result of lack of co-adaptation between the pollen and the pistilpollen and the pistil

It is like a “lock and key” mechanism (absent of It is like a “lock and key” mechanism (absent of suitable key(s) with the pollen for the lock(s) suitable key(s) with the pollen for the lock(s) present in the pistil results in incompatibilitypresent in the pistil results in incompatibility

II. Pre-fertilization barriersII. Pre-fertilization barriers

C. C. Inhibition on the stigma surfaceInhibition on the stigma surface Result in the arrest of pollen germination or Result in the arrest of pollen germination or

pollen tube entry into the stigmapollen tube entry into the stigma One of frequent barriers, particularly in One of frequent barriers, particularly in

distantly related speciesdistantly related species The causative factors for the failure of pollen The causative factors for the failure of pollen

germination:germination:1.1. Lack of effective adhesionLack of effective adhesion2.2. Lack of full hydrationLack of full hydration3.3. Absence of pollen germination factors on the stigmaAbsence of pollen germination factors on the stigma Pollen adhesion and hydration are Pollen adhesion and hydration are

prerequisites for germinationprerequisites for germination

II. Pre-fertilization barriersII. Pre-fertilization barriers

Pollen adhesionPollen adhesion Largely depends on the nature and extent of the Largely depends on the nature and extent of the

surface component of the pollen and the stigmasurface component of the pollen and the stigma It is not a constraint in species having wet stigmaIt is not a constraint in species having wet stigma

Pollen hydrationPollen hydration The result of the transfer of water from the stigma The result of the transfer of water from the stigma

to the pollen through an osmotic gradientto the pollen through an osmotic gradient Insufficient hydration may result in crosses in which Insufficient hydration may result in crosses in which

the osmotic potential of the pollen does not match the osmotic potential of the pollen does not match that of the stigmathat of the stigma

Rapid hydration that occurs on a wet stigma Rapid hydration that occurs on a wet stigma covered with aqueous exudates may lead to failure covered with aqueous exudates may lead to failure of pollen germinationof pollen germination

II. Pre-fertilization barriersII. Pre-fertilization barriers

D. Inhibition in the stigma and styleD. Inhibition in the stigma and style Failure of the pollen tube to reach the ovary is perhaps the Failure of the pollen tube to reach the ovary is perhaps the

most common interspecific pre-fertilization barriermost common interspecific pre-fertilization barrier Cause:Cause:1.1. The arrest of pollen tubes in the stigmaThe arrest of pollen tubes in the stigma2.2. Just below stigmaJust below stigma3.3. Further down the styleFurther down the style Arrested pollen tubes often show abnormalities in the form:Arrested pollen tubes often show abnormalities in the form:1.1. Thicker tubesThicker tubes2.2. Excessive deposition of calloseExcessive deposition of callose3.3. Swollen tipsSwollen tips4.4. Branching of tubesBranching of tubes Growing pollen tubes utilize stylar nutrients. Arrested pollen Growing pollen tubes utilize stylar nutrients. Arrested pollen

tube growth is the inability of the pollen tubes to utilize stylar tube growth is the inability of the pollen tubes to utilize stylar nutrient (Due to lack of suitable nutrient in the transmitting nutrient (Due to lack of suitable nutrient in the transmitting tissue or lack of suitable enzyme in the pollen tube tissue or lack of suitable enzyme in the pollen tube

II. Pre-fertilization barriersII. Pre-fertilization barriers

E. Technique to overcome barriers in the stigmaE. Technique to overcome barriers in the stigma Effective pollinationEffective pollination

Pollen must be transferred to the correct placePollen must be transferred to the correct place

Pollen should be transferred at the correct timePollen should be transferred at the correct time

Pollen must hydrate properly (rupture to release the Pollen must hydrate properly (rupture to release the stigmatic exudates, rub stigma before or while stigmatic exudates, rub stigma before or while pollen is applied, humid condition, protect pollen is applied, humid condition, protect pollinated stigma by a gelatin capsulepollinated stigma by a gelatin capsule

Mentor pollinationMentor pollinationPollen which is fully compatible with the intended Pollen which is fully compatible with the intended seed parentseed parent

II. Pre-fertilization barriersII. Pre-fertilization barriers

F. Technique to overcome barriers in the stylarsF. Technique to overcome barriers in the stylars Reciprocal crossesReciprocal crosses Mentor pollinationMentor pollination Use of plant growth regulatorsUse of plant growth regulators

PGR sprayed on or near flowers or apllied to pedicel or ovary PGR sprayed on or near flowers or apllied to pedicel or ovary at or after pollinationat or after pollinationAuxin and gibberellins inhibit pollen germination and pollen Auxin and gibberellins inhibit pollen germination and pollen tube growth, but occasionally are stimulatorytube growth, but occasionally are stimulatory

By passing barriers in the styleBy passing barriers in the stylePollen may have to be applied in a medium favoring Pollen may have to be applied in a medium favoring germination to compensate for deficiencies in the immature germination to compensate for deficiencies in the immature stigmastigmaAmputate the style and pollinate the cut stumpAmputate the style and pollinate the cut stumpGraft a compatible-pollinated style and stigma on to an alien Graft a compatible-pollinated style and stigma on to an alien style cut below the zone in which incompatible pollen tubes style cut below the zone in which incompatible pollen tubes would be inhibitedwould be inhibitedBy pass stigma and style completely and apply pollen directly By pass stigma and style completely and apply pollen directly to the ovuleto the ovule

III. Post-fertilization barriersIII. Post-fertilization barriers

Result in the failure of fertilized ovules to develop into mature Result in the failure of fertilized ovules to develop into mature seedsseeds

More prevalent than pre-fertilization barriersMore prevalent than pre-fertilization barriers May operate at different stages of embryo development or May operate at different stages of embryo development or

during germination and subsequent growth of the F1 hybridduring germination and subsequent growth of the F1 hybrid

Factors:Factors: Unbalance of ploidy levelsUnbalance of ploidy levels Abnormalities in the embryo developmentAbnormalities in the embryo development The presence of lethal genesThe presence of lethal genes Genic disharmony in the embryoGenic disharmony in the embryo Failure or early breakdown of endosperm (no cell Failure or early breakdown of endosperm (no cell

walls are formed; short lived, disappearing before walls are formed; short lived, disappearing before seed is matureseed is mature

III. Post-fertilization barriersIII. Post-fertilization barriers

Techniques to overcome:Techniques to overcome: Removed of competing sinksRemoved of competing sinks1.1. Crosses are made using the first flowers to open on the Crosses are made using the first flowers to open on the

maternal parentmaternal parent

2.2. All immature fruits set on the maternal parent are removed All immature fruits set on the maternal parent are removed before the cross is madebefore the cross is made

3.3. Remove all other fruit from the vicinity of a fruit produce by Remove all other fruit from the vicinity of a fruit produce by wide crossingwide crossing

4.4. Pruning the maternal parent to remove all active growing Pruning the maternal parent to remove all active growing pointpoint

Reciprocal crossesReciprocal crosses Manipulation of ploidy levelManipulation of ploidy level Embryo rescueEmbryo rescue Use of plant growth regulatorsUse of plant growth regulators

SELF-INCOMPATIBILITYSELF-INCOMPATIBILITY

TaryonoTaryono

Faculty of AgricultureFaculty of Agriculture

Gadjah Mada UniversityGadjah Mada University

Self-Incompatibility (SI)Self-Incompatibility (SI) A genetic system possessed by many A genetic system possessed by many

hermaphrodite flowering plants when pollen hermaphrodite flowering plants when pollen can’t hybridizes its own ovulecan’t hybridizes its own ovule

Inability of a fertile hermaphrodite seed plant to Inability of a fertile hermaphrodite seed plant to produce zygote after self-pollinationproduce zygote after self-pollination

Crops:Crops: Perennial grasses (Graminae)Perennial grasses (Graminae) Forage legumes (Fabaceae)Forage legumes (Fabaceae) Cabbage (Brassicaceae)Cabbage (Brassicaceae) Sunflower (Asteraceae)Sunflower (Asteraceae) Apples (Rosaceae)Apples (Rosaceae) Tobacco (Solanaceae)Tobacco (Solanaceae)

Self-Incompatibility (SI)Self-Incompatibility (SI)Present contrasting prospect to plant breeder:Present contrasting prospect to plant breeder:→ → It will frustrate efforts to produce homozygous It will frustrate efforts to produce homozygous

lineslines→ → It provides a way to hybridize two lines without It provides a way to hybridize two lines without

emasculation, nuclear or cytoplasmic sterility or emasculation, nuclear or cytoplasmic sterility or restoring to gametocides. Unfortunately, SI restoring to gametocides. Unfortunately, SI systems rarely provide the perfect vehicle for systems rarely provide the perfect vehicle for hybrid seed productionhybrid seed production

SI Systems

1. Heteromorphic

2. Homomorphic

Homomorphic SystemHomomorphic System

Mediated by a single locus (s-locus) which exhibit Mediated by a single locus (s-locus) which exhibit extreme polymorphismextreme polymorphism

GametophyticGametophytic

SI phenotype of the pollen is determined by the genotype SI phenotype of the pollen is determined by the genotype of the gametophyte (pollen)of the gametophyte (pollen)Genotype of the individual microspore determines the Genotype of the individual microspore determines the phenotype of the pollensphenotype of the pollensIt is characterized by very large polyallelic series at the It is characterized by very large polyallelic series at the locus which govern the pollen pistil relationshiplocus which govern the pollen pistil relationship

SporophyticSporophytic

SI phenotype of the pollen is determined by the genotype SI phenotype of the pollen is determined by the genotype of the sporophyte (pollen producing plant= parent plant)of the sporophyte (pollen producing plant= parent plant)

Gametophytic self-incompatibity (GSI)Gametophytic self-incompatibity (GSI)

a common outbreeding mechanism (≥ 60 families of the a common outbreeding mechanism (≥ 60 families of the angiosperms), especially solanaceaeangiosperms), especially solanaceae

Governed by a single, highly polymorphic locusGoverned by a single, highly polymorphic locus Pollen carrying an S-allele identical to one of the two allele Pollen carrying an S-allele identical to one of the two allele

carried by the pistil is prevented from effecting fertilizationcarried by the pistil is prevented from effecting fertilization Incompatible pollen germinates normally on the stigma and Incompatible pollen germinates normally on the stigma and

is able to penetrate the stigma surface. The pollen tubes is able to penetrate the stigma surface. The pollen tubes then enter the stylar transmitting tract, which is composed then enter the stylar transmitting tract, which is composed of files of longitudinally interconnected cells. Initially, of files of longitudinally interconnected cells. Initially, growth appears to be normal, however shortly after growth appears to be normal, however shortly after entering the transmitting tract, incompatible tubes take on entering the transmitting tract, incompatible tubes take on a characteristic appearance.a characteristic appearance.

Incompatible pollen tubes is cytologically an organization of Incompatible pollen tubes is cytologically an organization of the endoplasmic reticulum into concentric whorls and its the endoplasmic reticulum into concentric whorls and its subsequent degradation throughout the cytoplasm of the subsequent degradation throughout the cytoplasm of the pollen tube. This type of whorls is generally associated with pollen tube. This type of whorls is generally associated with the cessation of protein synthesisthe cessation of protein synthesis

The biochemical basis of GSIThe biochemical basis of GSI

Within pistil extracts, certain protein Within pistil extracts, certain protein segregate with their respective S-alleles in segregate with their respective S-alleles in genetic crossesgenetic crosses

These S-proteins are present in high These S-proteins are present in high proportions in style tissue (generally 1-10%) proportions in style tissue (generally 1-10%) and are sufficiently divers to be and are sufficiently divers to be differentiated on SDS-polyacrylamide or differentiated on SDS-polyacrylamide or isoelectric focusing gelsisoelectric focusing gels

S-proteins ranges from 23 – 34 kDaS-proteins ranges from 23 – 34 kDaAll posses high pI value-often higher than All posses high pI value-often higher than

8.08.0 Every S-protein is glycosylatedEvery S-protein is glycosylated

Two locus GSI systems (bifactorial)Two locus GSI systems (bifactorial)

GrassesGrassesTwo loci (S and Z), polyallelicTwo loci (S and Z), polyallelicEach combination gives rise to a distinct Each combination gives rise to a distinct

specificity in the haploid pollenspecificity in the haploid pollenRejection occurs when this specificity is matched Rejection occurs when this specificity is matched

by one of the four possible combination of S- and by one of the four possible combination of S- and Z-alleles in the diploid stigmaZ-alleles in the diploid stigma

It is likely o acquire self-compatible mutant, for It is likely o acquire self-compatible mutant, for the S- and Z- loci act in both a complementary the S- and Z- loci act in both a complementary and an independent mannerand an independent manner

If one locus mutates, the other gives rise to If one locus mutates, the other gives rise to incompatibility incompatibility

Two locus GSI systems (bifactorial)Two locus GSI systems (bifactorial) It differs from other gametophytic not only in having 2 It differs from other gametophytic not only in having 2

locus control, but also in exhibiting many cytological locus control, but also in exhibiting many cytological features that are much more similar to those features that are much more similar to those sporophytic systemssporophytic systems

Gametophytic in grasses has arisen independently Gametophytic in grasses has arisen independently from self compatible plantsfrom self compatible plants

Major morphological different

Although pollen germinate well and the pollen tubes Although pollen germinate well and the pollen tubes start to grown normally. Tube growth ceases as the start to grown normally. Tube growth ceases as the tubes touches the stigma surfacetubes touches the stigma surface

At the tip of the tubes, there is nodules (probably of At the tip of the tubes, there is nodules (probably of microfibrillar pectins), which is responsible for microfibrillar pectins), which is responsible for cessation of tube growthcessation of tube growth

Sporophytic self-incompatibilitySporophytic self-incompatibility

Pollen phenotype is determined by the genotype of Pollen phenotype is determined by the genotype of mother plantmother plant

Dominance interaction occur that determine the Dominance interaction occur that determine the phenotype of the pollenphenotype of the pollen

The number of alleles in S-locus is usually large (22-The number of alleles in S-locus is usually large (22-60)60)

It is associated with floral polymorphism which It is associated with floral polymorphism which reinforces the out-breeding potential of the self-reinforces the out-breeding potential of the self-incompatible plantincompatible plant

Stigma is capped by a layer of papillate cellStigma is capped by a layer of papillate cell The adhesion to the dry stigma surface is poor and The adhesion to the dry stigma surface is poor and

event hydration is absent, but with weaker alleles, event hydration is absent, but with weaker alleles, hydration and germination may occur. Resultant tubes hydration and germination may occur. Resultant tubes succeed in penetrating the stigmatic cuticle but they succeed in penetrating the stigmatic cuticle but they fail to invade the stigma cell wallfail to invade the stigma cell wall

SSI is also developmental regulated and comes into SSI is also developmental regulated and comes into operation 1-2 days pre-anthesis.operation 1-2 days pre-anthesis.

Diallelic SSIDiallelic SSI

Sporophytic system can exist with only two alleles Sporophytic system can exist with only two alleles (dominant (S) and recessive (s)). This is possible (dominant (S) and recessive (s)). This is possible due to the diploid heterozygous male parent due to the diploid heterozygous male parent produces pollen of a single S-phenotype though the produces pollen of a single S-phenotype though the pollen genotype are both S and s.pollen genotype are both S and s.

Almost all diallelic SI systems display floral Almost all diallelic SI systems display floral heteromorphism, usually in the form distyly or heteromorphism, usually in the form distyly or heterostyly, pollen size, cell shape and stigma heterostyly, pollen size, cell shape and stigma morphologymorphology

Heteromorphy is controlled by 2 linkage groups – Heteromorphy is controlled by 2 linkage groups – one comprising genes encoding morph-associated one comprising genes encoding morph-associated characters and the other the S/s incompatibility characters and the other the S/s incompatibility locus – with the two groups themselves closely locus – with the two groups themselves closely linked in a supergene. linked in a supergene.

The operation of diallelic SSIThe operation of diallelic SSI

Within-morph incompatibility can occur at a Within-morph incompatibility can occur at a number of stages in the fertilization processnumber of stages in the fertilization process

a.a. Lodgment, adhesion and germination of pollenLodgment, adhesion and germination of pollen

b.b. Penetration of the stigmatic papillae by the pollen Penetration of the stigmatic papillae by the pollen tubetube

c.c. Growth of the pollen tube in the stigmaGrowth of the pollen tube in the stigma

d.d. Growth of the pollen tube in the styleGrowth of the pollen tube in the style

At any one stage, incompatibility is rarely At any one stage, incompatibility is rarely total and each stage seems to act in a total and each stage seems to act in a quantitative rather than a qualitative quantitative rather than a qualitative mannermanner

Elimination of the self-incompatibility Elimination of the self-incompatibility barriersbarriers

Temporary BreakdownTemporary Breakdown

Physiological inhibitionPhysiological inhibitionPermanent BreakdownPermanent Breakdown

MutationMutation

The generation of new self-incompatibility allelesThe generation of new self-incompatibility alleles

PolyploidyPolyploidy

Overcoming Interspecific IncompatibilityOvercoming Interspecific IncompatibilityInduced mutationsInduced mutationsMentor pollen effectsMentor pollen effectsBud pollinations and inhibitorsBud pollinations and inhibitors