DISTANT HYBRIDIZATION

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WELCOME:

WELCOME D B SHARMA

DISTANT HYBRIDIZATION:

DISTANT HYBRIDIZATION SEMINAR ON Presented by: Divya Bhanu Sharma Department of Plant Breeding & Genetics Rajasthan College of Agriculture Maharana Pratap University of Agriculture & Technology Udaipur 313001 D B SHARMA

PowerPoint Presentation:

Hybridization crossing between two genetically dissimilar parents is called hybridization. Wide Hybridization Interspecific Hybridization:- Crosses made between distantly related species Intergeneric Hybridization:- Crosses made between distantly related genera Somatic hybridization (Protoplast fusion) Crosses made between somatic cells   Hybridization (recombination) is the third major evolutionary process with an importance not exceeding that of mutation and natural selection. D B SHARMA

History :  :

History :  Thomas Fairchild (1717): The first authentic record of a distant hybridization for the crop improvement is the production of a hybrid between Carnation (Dianthus caryophyllus) and Sweet willian (Dianthus barbatus). Karpechenko (1928): An interesting intergeneric hybrid, raphanobrassica, was produced.   Rimpu (1890): Produce the first intergeneric hybrid triticale which have greater potential than raphanobrassica. D B SHARMA

Introgressive hybridization :

Introgressive hybridization Transference of genetic material across an incompletely developed interspecific barrier, usually via a partially sterile F 1 hybrid, by means of repeated backcrossing and selection of well adopted backcross types, has been termed by Anderson and Hubricht(1938a) introgressive hybridization or introgression. D B SHARMA

PowerPoint Presentation:

Ex . partially sterile F 1 hybrid between fulva and HGC Species A Species B Hybrids D B SHARMA

Inter-specific hybridization: :

Inter-specific hybridization: Ex. Nerica, an upland rice for Africa Oryza sativa (Asian upland rice): non-shattering, resistant to lodging, high yield potential Oryza glaberrima (African rice): drought tolerant, disease resistant, weed-suppressing Nerica rice combines the best of both species.   D B SHARMA

Triticale (intergeneric cross):

Triticale ( intergeneric cross) Triticale, a new cereal created in the lab. Triticale, a cross(intergeneric cross) between wheat and rye, was produced by embryo rescue of the product of fertilization and a chemically induced doubling of the chromosomes.  Embryo rescue becomes necessary when fertile offspring is never produced by an interspecific cross. D B SHARMA

PowerPoint Presentation:

Interspecific combinations of wheat and rye that produces hexaploid and octaploid triticales. Hexaploid triticale ABR genome Triticum taschii D genome Triticum turgidum var. durum Durum wheat AB genome Seacle cereal Rye R genome Triticum aestivum common wheat ABD genome octaploid triticale ABDR genome D B SHARMA

Requirement of distant hybridization ::

Requirement of distant hybridization : Diseases and insect resistance Quality Wider adaptation Mode of reproduction yield Development of new varieties Production of new crop species (e.g; Triticale hexaploid) Transfer of cytoplasm D B SHARMA

Difficulties encountered in interspecific hybrids   :

Difficulties encountered in interspecific hybrids   Failure of zygote formation Failure of zygote development Failure of F 1 seedling development D B SHARMA

Major interspecific crossability barriers:

Major interspecific crossability barriers I. Temporal and spatial isolation of species II. Pre-fertilization barriers On the surface of the stigma before pollen tube entry Inside the tissues of he stigma and style Inside the ovary and embryo sac III. Post fertilization barriers Non viability of hybrid embryos Failure of hybrid to flower Hybrid sterility Lack of recombinant Hybrid breakdown in F2 or later generation D B SHARMA

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 D B SHARMA

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 D B SHARMA

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 D B SHARMA

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 D B SHARMA

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 D B SHARMA

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 D B SHARMA

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 D B SHARMA

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 D B SHARMA

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 D B SHARMA

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 D B SHARMA

Some other techniques for production of distant hybrids :

Some other techniques for production of distant hybrids The species with shorter style should be used as a female parent A part of style may be cut off to make it shorter, eg. Part of stigma of maize is cut off when it is crossed with tripsacum. Autopolyploidy: B. oleracea(cabbage) and B. campestris (turnip rape) do not cross with each other at the diploid level, but they produce embryos, when tetraploid species forms of two species are crossed; embryo culture has to be used to recover embryos. D B SHARMA

Cont..:

Cont.. When two species say A and C, can not be crossed directly , a third species, e.g. B, may be used as a bridge species. Transfering resistance to eye spot from Ae. ventricosa to T. aestivum, T. turgidum is used as a bridge sp. Use of growth regulator e.g. IAA, 2,4-D, napthelene acetamide N. tabacum does not hybridize with N. rependa, but this cross is possible when IAA applied to pedicle of flowers in a lanoline paste. D B SHARMA

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 D B SHARMA

Techniques to remove the crossability barriers in distant hybridizarion :  :

Techniques to remove the crossability barriers in distant hybridizarion :  Embryo rescue Somatic hybridization Alien Addition lines Alien substitution lines Transfer of small chromosome segments D B SHARMA

Embryo rescue :  :

Embryo rescue :  Embryo rescue When embryos fails to develop due to endosperm degeneration, embryo culture is used to recover hybrid plants; this is called hybrid rescue. e.g; H. vulgare x Secale cereale. Embryo rescue generally used to overcome endosperm degeneration. D B SHARMA

PowerPoint Presentation:

D B SHARMA

Embryo rescue in barley :  :

Embryo rescue in barley :  Embryo Rescue This technique was once more efficient than microspore culture in creating haploid barley Hordeum vulgare Barley 2n = 2X = 14 Hordeum bulbosum Wild relative 2n = 2X = 14 Haploid Barley 2n = X = 7 H. Bulbosum chromosomes eliminated X D B SHARMA

PowerPoint Presentation:

’Wide’’ crossing of wheat and rye requires embryo rescue and chemical treatments to double the no. of chromosomes triticale Triticum durum (4X) x Secale cereale (2X) AABB RR ABR F 1 (3X): EMBRYO RESCUE CHROMOSOME DOUBLING HEXAPLOID TRITICALE (6X) AABBRR D B SHARMA

Limitations of embryo rescue : :

Limitations of embryo rescue :  High cost of obtaining new plantlets Sometimes deleterious mutations may be induced during the in vitro phase. A sophisticated tissue culture laboratory and a dependable greenhouse are essential for success. Specialized skill for carrying out the various operations are required. D B SHARMA

SOMATIC HYBRIDIZATION :  :

SOMATIC HYBRIDIZATION :  Development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties is called somatic hybridization   D B SHARMA

Somatic hybridization technique :  :

Somatic hybridization technique :  1. isolation of protoplast 2. Fusion of the protoplasts of desired species/varieties 3. Identification and Selection of somatic hybrid cells 4. Culture of the hybrid cells 5. Regeneration of hybrid plants D B SHARMA

PowerPoint Presentation:

PLANT CELL PROTOPLAST 500-800 m mol/l SORBITOL+ 50-100 m mol/l CaCl2 PECTINASE (0.1-1%)+ CELLULASE(1-2%) Protoplast It is the cell with out cell wall created by degrading the cell wall using enzymes D B SHARMA

Protoplast Fusion:

Protoplast Fusion (Fusion of protoplasts of two different genomes) 1. Spontaneous Fusion : Intraspecific, Intergeneric 2. Induced Fusion: Electrofusion, Mechanical Fusion, Chemofusion D B SHARMA

Induced Protoplast Fusion:

Induced Protoplast Fusion Electrofusion: A high frequency AC field is applied between 2 electrodes immersed in the suspension of protoplasts- this induces charges on the protoplasts and causes them to arrange themselves in lines between the electrodes. They are then subject to a high voltage discharge which causes them membranes to fuse where they are in contact. Polyethylene glycol (PEG): causes agglutination of many types of small particles, including protoplasts which fuse when centrifuged in its presence D B SHARMA

PowerPoint Presentation:

PROTOPLASTS OF SPECIES(A) PROTOPLASTS OF SPECIES(B) x (SUSPENDED IN ENZYME MIXTURE) HIGH Ca +,HIGH pH TREATMENT PEG- INDUCED FUSION ELECTROFUSION Ca Cl2+ 50 m mol 1 -1, Ph 10.5, Temp.37OC PEG 28-50% (MW 1,500-6,000) LOW VOLTAGE 30 min 15 -30 min PROTOPLAST AGGREGATION PROTOPLAST CHAIN FORMED (DESIRED PROTOPLAST PAIR ALINGED WITH A MICRO –MANIPULATOR) WASHING MEDIUM Ph 9-10 Cacl2 50 m mol 1 -1 HIGH VOLTAGE (FEW MILLI-SECONDS) PROTOPLAST FUSION D B SHARMA

Identification and Selection of somatic hybrid cells ::

Identification and Selection of somatic hybrid cells : Hybrid identification- Based on difference between the parental cells and hybrid cell with respect to – Pigmentation Cytoplasmic markers Fluorochromes like FITC (Fluoroscein Isothiocyanate) and RITC (Rhodamine Isothiocyanate) are used for labelling of hybrid cells. Presence of chloroplast Nuclear staining Heterokaryon is stained by carbol-fuschin, aceto-carmine or aceto-orcein stain D B SHARMA

Regeneration of hybrid plants :  :

Regeneration of hybrid plants :  Regeneration of hybrid plants Plants are induced to regenerate from hybrid calli. These hybrid plants must be at least partially fertile, in addition to having some useful property, to be of any use in breeding schemes. D B SHARMA

Uses for protoplast fusion :

Uses for protoplast fusion Used for combining the two complete genomes Exchange single or few traits between species The transfer of mitochondria or chloroplasts between species Protoplast fusion between male sterile cabbage and normal cabbage was done, and cybrids were selected that contained the radish mitochondria and the cabbage chloroplast D B SHARMA

Advantages of somatic hybridisation :

Advantages of somatic hybridisation Symmetric hybrids can be produced between species which can not be hybridized sexually. Cytoplasm can be transfer in one year ,while backcrossing may take 5-6 years. Mitochondria of one species can be combined with protoplast of another. Production of novel interspecific and intergenic hybrid. i.e. Pomato (Hybrid of potato and tomato). Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids   D B SHARMA

Cont…:

Cont… Production of heterozygous lines in the single species which cannot be propagated by vegetative means Studies on the fate of plasma gene Production of unique hybrids of nucleus and cytoplasm. Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters   D B SHARMA

Limitations of somatic hybridizations :

Limitations of somatic hybridizations Poor regeneration of hybrid plants Non-viability of fused products Not successful in all plants. Production of unfavorable hybrids Lack of an efficient method for selection of hybrids No confirmation of expression of particular trait in somatic hybrids D B SHARMA

Alien addition lines :  :

Alien addition lines :  These lines carries one chromosome pair from a different species in addition to the normal somatic chromosome complement of the parent species When only one chromosome from another species is present, it is known as alien addition monosome. The main purpose of alien addition is the transfer of disease resistance from from related wild species. e.g. transfer of mosaic resistance from Nicotiana glutinosa to N. tabacum. D B SHARMA

PowerPoint Presentation:

The alien addition lines have been developed in case of wheat, oats, tobacco and several other species. Alien addition lines are of still agricultural importance since the alien chromosome generally carries many undesirable genes. D B SHARMA

Alien substitution lines :  :

Alien substitution lines :  This line has one chromosome pair from a different species in place of the chromosome pair of the recipient species. When a single chromosome (not a pair) from different species in place of a single chromosome of the recipient species. Alien –substitution lines have been developed in wheat, cotton, tobbacco, oats. In case of tobbacco, mosaic resistance gene N was transferred from the N. glutinosa to N. tabacum line had 23 pairs of N. tabacum chromosomes and one pair (chromosome H) of N. glutinosa chromosomes. The alien substitution show more undesirable effects than alien additions and more useful in agriculture. D B SHARMA

Transfer of small chromosome segments:

Transfer of small chromosome segments Transfer of small chromosome segments carrying specific desirable gene has been widely used in crop improvement programme. It would be ideal to transfer only the desired gene from the related alien species, since the gene under transfer may be linked with other undesirable genes. Transfer of black arm resistance from G. barbadense(Egyptian cotton) to G. hirsutum (American upland cotton). D B SHARMA

Limitation of distant hybridization:

Limitation of distant hybridization Incompatible crosses F 1 sterility Problems in creating new species Lack of homeology between chromosome of the parental species Undesirable linkage Problems in the transfer of recessive oligogenes and quantitative traits Lack of flowering in F 1 Problems in using improved varieties in distant hybridization Dormancy D B SHARMA

THANK YOU:

THANK YOU D B SHARMA

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