HYBRIDIZATION: HYBRIDIZATION Taryono Faculty of Agriculture Gadjah Mada University


Slide2: Hybridization The formation of a new organism by normal sexual processes or by protoplast fusion Wide Hybridization (Interspecific Hybridization) Crosses made between distantly related species or genera Somatic hybridization (Protoplast fusion) Crosses made between somatic cells ► One of the most effective methods of crops improvement programs ► Most the hybridization work carried out has used genetic variability within species


What is pollination?: What is pollination? Pollination: The transfer of pollen from the male anther to the female stigma


Why is pollination important?: Why is pollination important? Sexual reproduction is important for evolution: Sexual reproduction produces variable offspring, creating diversity and variation among populations (shuffling of genes) You need variation for Natural Selection to occur Sexual reproduction is advantageous to an organism only if it happens with someone other than itself! Out-breeding = good! (inbreeding = bad…)


Sexual reproduction: Sexual reproduction In animals: It’s easy because you have separate male and female individuals. In flowering plants: Not so easy, because most flowers have both male and female parts in them, called perfect flowers. So flowering plants have evolved special ways to insure out-breeding/out-crossing – and to prevent inbreeding.


Function of flower: Function of flower To attract pollinators with colorful petals, scent, nectar and pollen Carpel/


Overview of floral organs: Overview of floral organs


Reproductive floral organs: female: Reproductive floral organs: female Carpel or pistil – female reproductive organs; contains: Stigma – is where pollen sticks to Style – is the long tube that connects stigma to ovary Ovary – enlarged structure at the base of carpel/pistil where the ovules are located; it will become the fruit. Ovules – contains female gametophyte, becomes the seed Plants have style! carpel or pistil ovary


Reproductive floral organs: male: Reproductive floral organs: male Stamen – male floral organ, consists of: Anther – part of the stamen that produces pollen Filament – stalk-like structure that holds anther Pollen – immature male gametophyte


Non-reproductive floral organs: Non-reproductive floral organs Petals – whorl of flower organs that are often brightly colored to attract pollinators Corolla – whorl of petals in 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 Fertilization: Pollination and Fertilization Pollen contains TWO nuclei: a sperm nucleus and tube nucleus Sperm nucleus is protected in gametophyte tissue (pollen can travel in the air)


Pollination and Fertilization: Pollination and Fertilization For pollen sperm to successfully fertilize the egg, there must be pollination: a method to get the pollen from the male anther to the stigma. Pollen sticks to the stigma, starts growing a pollen tube Fertilization begins when tube begins to grow toward the egg


Double Fertilization: Double Fertilization Double fertilization occurs: One sperm nucleus (1n) fertilizes the egg, producing a zygote (2n)  which becomes the plant embryo inside the seed Another sperm nucleus fuses with the polar nuclei, resulting in a triploid endosperm (3n) Endosperm is a source of food for the young embryo. Endosperm


Hermaphroditic Flowers: Hermaphroditic Flowers Self-compatible (SC) Capable of self-fertilization or cross-fertilization Self-incompatible (SI) Only capable of cross-fertilization Inability of hermaphroditic plant to produce zygotes w/ self pollen


Autogamy : Autogamy Self-fertilization Pollen transfer within or among flowers of same individual ~25% of plant taxa


Advantages of Autogamy: Advantages of Autogamy Insures seed set in absence of pollinators. Overcomes sterility. Selectively advantageous by transmitting both sets of genes to offspring. Well-adapted genotypes preserved. Only single colonizing individual needed.


Disadvantages of Autogamy: Disadvantages of Autogamy Decreases genetic variability. Inability to adapt to changing conditions. Increases inbreeding depression. Reduces heterozygosity and increases homozygosity of deleterious alleles. More uniform populations.


Cleistogamy : Cleistogamy Flowers never open and only capable of self-fertilization in bud. Inconspicuous, bud-like apetalous flowers that form directly into seed capsules. Has evolved independently multiple times throughout the angiosperms, including some basal lineages. 488 species, across 212 genera and 49 families. Violaceae, Fabaceae, Poaceae


Cleistogamy : Cleistogamy Mixed mating systems -can produce both CL and CH on an individual. CL fls are a “back-up” in case pollinators scarce. CL occur after normal flowering period. CH fls early spring and CL fls rest of season. CL fls occur through mutations with loss of SI.


How do plants get pollen from one plant to another?: How do plants get pollen from one plant to another? Because plants are rooted in the ground, they must use different strategies: WIND POLLINATION: Gymnosperms and some flowering plants (grasses, trees) use wind pollination. Flowers are small, grouped together Not a very efficient method (too chancy and wasteful)


ANIMALS: ANIMALS Many flowering plants rely on animals for cross-pollination: Insects – bees, wasps, flies, butterflies, moths Birds – hummingbirds, honey creepers Mammals – bats, mice, monkeys Even some reptiles and amphibians!


Coevolution: Coevolution Coevolution interactions between two different species as selective forces on each other, resulting in adaptations that increase their interdependency. Animal-flowering plant interaction is a classic example of coevolution: 1. Plants evolve elaborate methods to attract animal pollinators 2. Animals evolved specialized body parts and behaviors that aid plant pollination


A word about pollen…: A word about pollen… The shape and form of pollen is related to its method of pollination… Insect-pollinated species have sticky of barbed pollen grains Wind-pollinated species is lightweight, small and smooth (corn pollen)


Palynology: the study of pollen: Palynology: the study of pollen Palynology is useful in many fields: Petroleum geology – fossil pollen can determine if a field will have oil-rich deposits Archeology – studying ancient pollen samples, archeologists can determine agricultural practices, diet, etc. Anthropology – uses of pollen in rituals Criminology – to determine the whereabouts of an individual, examine pollen clinging to clothes Aerobiology – to determine what plants cause hay fever and allergic reactions – in landscaping


Animal pollinators: Bees: Animal pollinators: Bees Bees – the most important group of flower pollinators They live on the nectar and feed larvae, also eat the pollen. Bees are guided by sight and smell See yellow and blue colors, also ultraviolet light (not red) Flowers have “honey guides” and bee landing platforms..


Butterflies and moths: Butterflies and moths Also guided by sight and smell Butterflies can see red and orange flowers Usually shaped as a long tube because of insect’s proboscis – to get nectar Moth-pollinated flowers are usually white or pale, with sweet, strong odor – for night pollination.


Flies and beetles: Flies and beetles Flies like flowers that smell like dung or rotten meat. Lay their eggs there, but larvae die due to lack of food Beetles pollinate flowers that are dull in color, but have very strong odor


Birds: Birds Birds have a good sense of color, they like yellow or red flowers… But birds do not have a good sense of smell, so bird-pollinated flowers usually have little odor. Flowers provide fluid nectar in greater quantities than insects Hummingbird-pollinated flowers usually have long, tubular corolla Pollen is large and sticky


Mammals: bats and mice: Mammals: bats and mice Bats pollinate at night, so flowers are white Mouse-pollinated flowers are usually inconspicuous, they open at night


Why do animals pollinate plants?: Why do animals pollinate plants? They get a REWARD: food! In exchange for moving their pollen to another flower Nectar – a sugary solution produced in special flower glands called nectaries Nectar concentration matches energy requirements of the pollinator: bird- and bee-pollinated flowers have different sugar conc. Pollen – is high in protein, some bees and beetles eat it. Flowers can produce two kinds of pollen: a normal and a sterile, but tasty, kind, for the insect.


Getting the pollinator’s attention: Getting the pollinator’s attention Plants advertise their pollen and nectar rewards with Colors – bees see blue, yellow, UV; while birds see red. Bats don’t see well, so flowers are white. Nectar or honey guides – a visual guide for pollinator to locate the reward (pansy flower) Aromas – for insects, nectar. Can also be carrion or dung smell


Plant Mimicry: Plant Mimicry Some plants take advantage of the sex drive of certain insects… Certain orchids look like female wasps, and even smell like them! Males try to mate with them, and in the process they pollinate the plant The orchid gets pollinated, but the male wasp only gets frustrated!


Selfers vs. Outcrossers: Selfers vs. Outcrossers SC Small flowers (few) Unscented flowers Nectaries & nectar guides absent Maturation of reproductive parts Anthers near stigma Style included All fruits mature Low pollen/ovule ratio SI or SC Large showy flowers (many) Scented flowers Nectaries & nectar guides present Differential maturation of reproductive parts Anthers far from stigma Stigma well-exserted Only some fruits mature High pollen/ovule ratio


Strategies to Prevent Self-fertilization: Strategies to Prevent Self-fertilization


Strategies to avoid self-pollination: Strategies to avoid self-pollination Perfect flowers have both male and female organs, so plants have strategies to avoid self-pollination: 1. Timing – male and female structures mature at different times 2. Morphological – structure of male and female organs prevents self-pollination (imperfect flower) 3. Biochemical – chemical on surface of pollen and stigma/style that prevent pollen tube germination on the same flower (incompatible)


Physical Separation of Reproductive Parts (Herkogamy): Physical Separation of Reproductive Parts (Herkogamy) Within flowers Among flowers


Heterostyly: Heterostyly Flowers in different individuals of the same species having 2 or 3 different style lengths With stamen lengths varying inversely Distyly Tristyly


Distyly: Distyly 2 floral morphs. “Thrum” flower long filaments with short styles “Pin” flower short filaments with long styles Only pollinations between different floral morphs are successful. E.g.: Primula


Tristyly: Tristyly 3 floral morphs Style long, stamens short and medium Style medium, stamens short and long Style short, stamens medium and long


Slide40: Physical Separation of Reproductive Parts Unisexual flowers Staminate and carpellate flowers Monoecy Dioecy


Slide41: Monoecy Common in wind-pollinated plants. Common in temperate regions. Self-pollination possible but less likely.


Slide42: Dioecy 4% of angiosperms Scattered throughout Common in tropical regions and oceanic islands Gen small fl size 100% out-crossing, but inefficient Often controlled by sex chromosomes Silene


Slide43: Polygamous Flowers Both bisexual and unisexual flowers on the same plant. Androdioecy = bisexual and staminate individuals in a population. Andromonoecy = bisexual and staminate flowers on same individual. Euphorbia, Solanum Gynodioecy = bisexual and carpellate individuals in a population. Sidalcea hendersonii, Silene Gynomonoecy = bisexual and carpellate flowers on same individual. Silene, Solidago Polygamodioecy = some plants with bisexual and staminate flowers & some plants with bisexual and carpellate flowers in a population. Polygamomonoecy = bisexual, staminate, and carpellate flowers on same individual.


Evolution of Dioecy: Evolution of Dioecy From hermaphroditism Vestigial sex organs Few families entirely dioecious From monoecy From SC Within groups that have lost original GSI system From distyly Unequal pollen flow & gender function Change in pollinator frequency Non-functional anthers at low level in female flowers Non-functional pistil in male flowers


Temporal Separation of Reproductive Parts (Dichogamy): Temporal Separation of Reproductive Parts (Dichogamy) Protandry Anthers release pollen before stigma receptive Common in insect-pollinated plants Geranium maculatum 1st day flower 2nd day flower


Temporal Separation of Reproductive Parts (Dichogamy): Temporal Separation of Reproductive Parts (Dichogamy) Protogyny Stigma receptive before pollen release Less common than protandry Magnolia grandiflora 1st day flower 2nd day flower


Geitonogamy: Geitonogamy Self pollination between different flowers on same plant.


Evolution of Breeding Systems: Evolution of Breeding Systems Evolutionary trends go both ways and in a variety of ways. Ancestral angiosperms were SC, hermaphroditic. SI has evolved many times. SC has evolved from SI plants as well.


Crossability barriers: Crossability barriers prevent the fusion of male and female gametes originating from individuals of different species/genera and/or the development of a fertilized ovule into viable seed Include the limit effective utilization of the hybrids for gene introgression Incompatibility (self) ? Very frequent in interspecific and intergeneric hybridization program


Major interspecific crossability barriers: Major interspecific crossability barriers


I. Temporal and spatial isolation of parental species: I. Temporal and spatial isolation of parental species Non synchronous flowering of the parental species due to different agro-ecological or geographical background Early/staggered sowing Suitable photoperiodic treatment


II. Pre-fertilization barriers: II. Pre-fertilization barriers A. Unilateral incompatibility (UI) Prevent fertilization by arresting post pollination events at one or many levels Incompatibility operates in one direction, whereas the reciprocal cross is successful (unilateral incompatibility = UI) UI is more common when cross includes a self-compatible (SC) and a self incompatible (SI) The crosses show incompatible when an SI species is used as a female parent (SI x SC) Self-incompatibility inhibition is the result of active recognition of the pollen. Self pollen is positively recognized as a result of the interaction of S allele product in the pollen and the pistil


II. Pre-fertilization barriers: II. Pre-fertilization barriers B. Active versus passive inhibition Self-incompatibility inhibition is the result of active recognition of the pollen. Self pollen is positively recognized as a result of the interaction of S allele product in the pollen and the pistil Positive recognition results in the activation of metabolic processes in the pollen and/or the pistil to bring about pollen inhibition The arrest of post pollination events seems to be passive (not a result of active recognition of pollen) and a result of lack of co-adaptation between the pollen and the pistil It is like a “lock and key” mechanism (absent of suitable key(s) with the pollen for the lock(s) present in the pistil results in incompatibility


II. Pre-fertilization barriers: II. Pre-fertilization barriers C. Inhibition on the stigma surface Result in the arrest of pollen germination or pollen tube entry into the stigma One of frequent barriers, particularly in distantly related species The causative factors for the failure of pollen germination: Lack of effective adhesion Lack of full hydration Absence of pollen germination factors on the stigma Pollen adhesion and hydration are prerequisites for germination


II. Pre-fertilization barriers: II. Pre-fertilization barriers Pollen adhesion Largely depends on the nature and extent of the surface component of the pollen and the stigma It is not a constraint in species having wet stigma Pollen hydration The result of the transfer of water from the stigma to the pollen through an osmotic gradient Insufficient hydration may result in crosses in which the osmotic potential of the pollen does not match that of the stigma Rapid hydration that occurs on a wet stigma covered with aqueous exudates may lead to failure of pollen germination


II. Pre-fertilization barriers: II. Pre-fertilization barriers D. Inhibition in the stigma and style Failure of the pollen tube to reach the ovary is perhaps the most common interspecific pre-fertilization barrier Cause: The arrest of pollen tubes in the stigma Just below stigma Further down the style Arrested pollen tubes often show abnormalities in the form: Thicker tubes Excessive deposition of callose Swollen tips Branching of tubes Growing pollen tubes utilize stylar nutrients. Arrested pollen tube growth is the inability of the pollen tubes to utilize stylar nutrient (Due to lack of suitable nutrient in the transmitting tissue or lack of suitable enzyme in the pollen tube


II. Pre-fertilization barriers: II. Pre-fertilization barriers E. Technique to overcome barriers in the stigma Effective pollination Pollen must be transferred to the correct place Pollen should be transferred at the correct time Pollen must hydrate properly (rupture to release the stigmatic exudates, rub stigma before or while pollen is applied, humid condition, protect pollinated stigma by a gelatin capsule Mentor pollination Pollen which is fully compatible with the intended seed parent


II. Pre-fertilization barriers: II. Pre-fertilization barriers F. Technique to overcome barriers in the stylars Reciprocal crosses Mentor pollination Use of plant growth regulators PGR sprayed on or near flowers or apllied to pedicel or ovary at or after pollination Auxin and gibberellins inhibit pollen germination and pollen tube growth, but occasionally are stimulatory By passing barriers in the style Pollen may have to be applied in a medium favoring germination to compensate for deficiencies in the immature stigma Amputate the style and pollinate the cut stump Graft a compatible-pollinated style and stigma on to an alien style cut below the zone in which incompatible pollen tubes would be inhibited By pass stigma and style completely and apply pollen directly to the ovule


III. Post-fertilization barriers: III. Post-fertilization barriers Result in the failure of fertilized ovules to develop into mature seeds More prevalent than pre-fertilization barriers May operate at different stages of embryo development or during germination and subsequent growth of the F1 hybrid Factors: Unbalance of ploidy levels Abnormalities in the embryo development The presence of lethal genes Genic disharmony in the embryo Failure or early breakdown of endosperm (no cell walls are formed; short lived, disappearing before seed is mature


III. Post-fertilization barriers: III. Post-fertilization barriers Techniques to overcome: Removed of competing sinks Crosses are made using the first flowers to open on the maternal parent All immature fruits set on the maternal parent are removed before the cross is made Remove all other fruit from the vicinity of a fruit produce by wide crossing Pruning the maternal parent to remove all active growing point Reciprocal crosses Manipulation of ploidy level Embryo rescue Use of plant growth regulators


SELF-INCOMPATIBILITY: SELF-INCOMPATIBILITY Taryono Faculty of Agriculture Gadjah Mada University


Self-Incompatibility (SI): Self-Incompatibility (SI) A genetic system possessed by many hermaphrodite flowering plants when pollen can’t hybridizes its own ovule Inability of a fertile hermaphrodite seed plant to produce zygote after self-pollination Crops: Perennial grasses (Graminae) Forage legumes (Fabaceae) Cabbage (Brassicaceae) Sunflower (Asteraceae) Apples (Rosaceae) Tobacco (Solanaceae)


Self-Incompatibility (SI): Self-Incompatibility (SI) Present contrasting prospect to plant breeder: → It will frustrate efforts to produce homozygous lines → It provides a way to hybridize two lines without emasculation, nuclear or cytoplasmic sterility or restoring to gametocides. Unfortunately, SI systems rarely provide the perfect vehicle for hybrid seed production SI Systems Heteromorphic Homomorphic


Homomorphic System: Homomorphic System Mediated by a single locus (s-locus) which exhibit extreme polymorphism Gametophytic SI phenotype of the pollen is determined by the genotype of the gametophyte (pollen) Genotype of the individual microspore determines the phenotype of the pollens It is characterized by very large polyallelic series at the locus which govern the pollen pistil relationship Sporophytic SI phenotype of the pollen is determined by the genotype of the sporophyte (pollen producing plant= parent plant)


Gametophytic self-incompatibity (GSI): Gametophytic self-incompatibity (GSI) a common outbreeding mechanism (≥ 60 families of the angiosperms), especially solanaceae Governed by a single, highly polymorphic locus Pollen carrying an S-allele identical to one of the two allele carried by the pistil is prevented from effecting fertilization Incompatible pollen germinates normally on the stigma and is able to penetrate the stigma surface. The pollen tubes then enter the stylar transmitting tract, which is composed of files of longitudinally interconnected cells. Initially, growth appears to be normal, however shortly after entering the transmitting tract, incompatible tubes take on a characteristic appearance. Incompatible pollen tubes is cytologically an organization of the endoplasmic reticulum into concentric whorls and its subsequent degradation throughout the cytoplasm of the pollen tube. This type of whorls is generally associated with the cessation of protein synthesis


The biochemical basis of GSI: The biochemical basis of GSI Within pistil extracts, certain protein segregate with their respective S-alleles in genetic crosses These S-proteins are present in high proportions in style tissue (generally 1-10%) and are sufficiently divers to be differentiated on SDS-polyacrylamide or isoelectric focusing gels S-proteins ranges from 23 – 34 kDa All posses high pI value-often higher than 8.0 Every S-protein is glycosylated


Two locus GSI systems (bifactorial): Two locus GSI systems (bifactorial) Grasses Two loci (S and Z), polyallelic Each combination gives rise to a distinct specificity in the haploid pollen Rejection occurs when this specificity is matched by one of the four possible combination of S- and Z-alleles in the diploid stigma It is likely o acquire self-compatible mutant, for the S- and Z- loci act in both a complementary and an independent manner If one locus mutates, the other gives rise to incompatibility


Two locus GSI systems (bifactorial): Two locus GSI systems (bifactorial) It differs from other gametophytic not only in having 2 locus control, but also in exhibiting many cytological features that are much more similar to those sporophytic systems Gametophytic in grasses has arisen independently from self compatible plants Major morphological different Although pollen germinate well and the pollen tubes start to grown normally. Tube growth ceases as the tubes touches the stigma surface At the tip of the tubes, there is nodules (probably of microfibrillar pectins), which is responsible for cessation of tube growth


Sporophytic self-incompatibility: Sporophytic self-incompatibility Pollen phenotype is determined by the genotype of mother plant Dominance interaction occur that determine the phenotype of the pollen The number of alleles in S-locus is usually large (22-60) It is associated with floral polymorphism which reinforces the out-breeding potential of the self-incompatible plant Stigma is capped by a layer of papillate cell The adhesion to the dry stigma surface is poor and event hydration is absent, but with weaker alleles, hydration and germination may occur. Resultant tubes succeed in penetrating the stigmatic cuticle but they fail to invade the stigma cell wall SSI is also developmental regulated and comes into operation 1-2 days pre-anthesis.


Diallelic SSI: Diallelic SSI Sporophytic system can exist with only two alleles (dominant (S) and recessive (s)). This is possible due to the diploid heterozygous male parent produces pollen of a single S-phenotype though the pollen genotype are both S and s. Almost all diallelic SI systems display floral heteromorphism, usually in the form distyly or heterostyly, pollen size, cell shape and stigma morphology Heteromorphy is controlled by 2 linkage groups – one comprising genes encoding morph-associated characters and the other the S/s incompatibility locus – with the two groups themselves closely linked in a supergene.


The operation of diallelic SSI: The operation of diallelic SSI Within-morph incompatibility can occur at a number of stages in the fertilization process Lodgment, adhesion and germination of pollen Penetration of the stigmatic papillae by the pollen tube Growth of the pollen tube in the stigma Growth of the pollen tube in the style At any one stage, incompatibility is rarely total and each stage seems to act in a quantitative rather than a qualitative manner


Elimination of the self-incompatibility barriers: Elimination of the self-incompatibility barriers Temporary Breakdown Physiological inhibition Permanent Breakdown Mutation The generation of new self-incompatibility alleles Polyploidy Overcoming Interspecific Incompatibility Induced mutations Mentor pollen effects Bud pollinations and inhibitors