distant hybridization

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Presentation Transcript

Slide 1: 

Distant Hybridization in Crop Plants

Slide 2: 

Hybridization The formation of a new genotype 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

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.

Slide 4: 

Inter-specific hybridization: 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. African rice Nerica rice Asian rice

Slide 5: 

Triticale, a new cereal created in the lab.Triticale, a 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 Triticale is now grown all over the world Wheat, triticale and Rye


Requirement of distant hybridization : 

Requirement of distant hybridization Diseases and insect resistance Quality Wider adaptation Mode of reproduction Development of new varieties. Production of new crop species (e.g; Triticale hexaploid)

Major interspecific crossability barriers : 

Major interspecific crossability barriers

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

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.

Embryo Rescue : 

Embryo Rescue

Embryo rescue in barley : 

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

Slide 13: 

‘’WIDE’’ CROSSING OF WHEAT AND RYE REQUIRES EMBRYO RESCUE AND CHEMICAL TREATMENTS TO DOUBLE THE NO. OF CHROMOSOMES TRITICALE

Selected examples of distant hybrids obtained by embryo rescue : 

Selected examples of distant hybrids obtained by embryo rescue

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.

SOMATIC HYBRIDIZATION : 

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

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

Slide 18: 

Protoplasts It is the cell with out cell wall created by degrading the cell wall using enzymes

Slide 19: 

N CELL WALL PECTINASE (0.1-1%)+ CELLULASE(1-2%)+ PLASMA MEMBRANE 500-800 m mol/l SORBITOL+ 50-100 m mol/l CaCl2 PLANT CELL N PROTOPLAST FIG. Production of protoplasts by enzyme treatment (enzymes are depicted above the arrows ).Osmoticum (shown below the arrow) is added to stabilize the protoplasts and prevent them from bursting.N,nucleus. PLASMA MEMBRANE

Slide 20: 

Isolation of Protoplast (Separartion of protoplasts from plant tissue) 1. Mechanical Method 2. Enzymatic Method

1. Mechanical Method : 

1. Mechanical Method Plant Tissue Collection of protoplasm Cells Plasmolysis Microscope Observation of cells Cutting cell wall with knife Release of protoplasm

Enzymatic Method : 

Enzymatic Method Leaf sterlization, removal of epidermis Plasmolysed cells Plasmolysed cells Pectinase +cellulase Pectinase Protoplasm released Release of isolated cells cellulase Protoplasm released Isolated Protoplasm

Slide 23: 

Protoplast Fusion (Fusion of protoplasts of two different genomes) 1. Spontaneous Fusion 2. Induced Fusion Intraspecific Intergeneric Electrofusion Mechanical Fusion Chemofusion

Slide 24: 

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

Slide 25: 

PROTOPLASTS OF SPECIES(A) PROTOPLASTS OF SPECIES(B) (SUSPENDED IN ENZYME MIXTURE) HIGH Ca +,HIGH pH TREATMENT 2 Ca Ca2+ 50 m mol 1 -1 Ph 10.5,Temp.37OC 30 min PEG- INDUCED FUSION PEG 28-50% (MW 1,500-6,000) 15 -30 min PROTOPLAST AGGREGATION WASHING MEDIUM Ph 9-10 Ca 50 m mol 1 -1 2+ ELECTROFUSION LOW VOLTAGE HIGH VOLTAGE (FEW MILLI-SECONDS) PROTOPLAST FUSION A schematic representation of the 3 most successful protoplast fusion strategies. PROTOPLAST CHAIN FORMED (DESIRED PROTOPLAST PAIR ALINGED WITH A MICRO –MANIPULATOR)

Cont… : 

Cont… PROTOPLASTS (SPECIES A) PROTOPLASTS (SPECIES B) FUSOGEN TREATMENT A B A A + B B A B + + UNFUSED PROTOPLASTS HOMOKARYONS HETEROKARYON USEFUL ONE PRODUCTS THAT INVOLVE TWO PROTOPLASTS ARE DEPICTED HERE

Possible Result of Fusion of Two Genetically Different Protoplasts : 

Possible Result of Fusion of Two Genetically Different Protoplasts = chloroplast = nucleus Fusion heterokaryon cybrid cybrid hybrid hybrid = mitochondria

Slide 28: 

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 Uses for protoplast fusion

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

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.

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 Advantages of somatic hybridisation

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

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 Limitations of somatic hybridizations

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.

Cont… : 

Cont… 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.

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.

Slide 37: 

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