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SOMATIC HYBRIDIZATION. Fundamentals of Biotechnology. This is a non conventional genetic procedure involving fusion b.w isolated protoplast under in vitro condition and subsequent development of their product ( heterokaryon ) to a hybrid plant Or

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somatic hybridization

SOMATIC HYBRIDIZATION

Fundamentals of Biotechnology

slide2
This is a non conventional genetic procedure involving fusion b.w isolated protoplast under in vitro condition and subsequent development of their product (heterokaryon) to a hybrid plant

Or

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

slide3

Heterokaryon: binucleate cell (heterocyte)

Hybrid: nuclei are fused (synkaryocyte)

Hybridization

Cybrid: fusion of nucleated and enucleated different somatic cell. (Cybridization)

somatic hybridization technique
Somatic hybridization technique

1. isolation of protoplast from suitable plants

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

slide6

Isolation of Protoplast

(Separartion of protoplasts from plant tissue)

1. Mechanical Method

2. Enzymatic Method

A. Sequential (two step)

B. Mixed (simultaneous)

1 mechanical method
1. Mechanical Method

Plant Tissue

CellsPlasmolysis

MicroscopeObservation of cells

Release of protoplasm

Cutting cell wall with knife

Collection of protoplasm

1 mechanical method8
1. Mechanical Method
  • Used for vacuolated cells like onion bulb scale, radish and beet root tissues (storage tissues)
  • Low yield of protoplast
  • Laborious and tedious process
  • Low protoplast viability
enzymatic method
Enzymatic Method

Leaf sterlization, removal of

epidermis

Mixed (simultaneous)

Sequential (two step)

Plasmolysed

cells

Plasmolysed

cells

Pectinase +cellulase

Pectinase

Protoplasm

released

Release of

isolated cells

Protoplasm released

cellulase

Isolated

Protoplasm

enzymatic method10
Enzymatic Method
  • Used forvariety of tissues and organs including leaves, petioles, fruits, roots, coleoptiles, hypocotyls, stem, shoot apices, embryo microspores
  • Mesophyll tissue - most suitable source
  • High yield of protoplast
  • Easy to perform
  • More protoplast viability
slide11

Protoplast Fusion

(Fusion of protoplasts of two different genomes)

1. Spontaneous Fusion

2. Induced Fusion

Intraspecific

Intergeneric

Chemofusion

Mechanical

Fusion

Electrofusion

spontaneous fusion
Spontaneous Fusion
  • Protoplast fuse spontaneously during isolation process mainly due to physical contact
      • Intraspecific produce homokaryones
induced fusion
Induced Fusion
  • Chemofusion- fusion induced by chemicals
    • Types of fusogens
      • PEG
      • NaNo3
      • High pH/Ca 2+ ions
      • Polyvinyl alcohal
induced fusion14
Induced Fusion
  • Mechanical Fusion- Physical fusion of protoplasts under microscope by using micromanipulator and perfusion micropipette
induced fusion15
Induced Fusion
  • Electrofusion- Fusion induced by electrical stimulation
      • Pearl chain of protoplasts is formed by low strength electric field (10kv m-1)
      • Fusion of protoplasts of pearl chain is induced by the application of high strength electric field (100kv m-1) for few microseco
  • High voltage pulse induce a reversible breakdown of plasma membrane at the sit of cell contact, leading to fusion and consequently membrane reorganization.
  • Simple, quicker and more efficient than chemical induced fusion.
cell wall regeneration
Cell Wall Regeneration

May be complete in two to several days

Although protoplast in culture generally start regenerating a cell wall within a few hours after isolation.

Protoplast lost their characteristic spherical shape once the wall formation is complete.

Regeration of cell wall can be demonstrated using Calcalfluor White ST fluoresecent Stain (USA) or Tinapol solution (UK)

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 (fluorosceinisothiocyanate) and RITC (Rhodamineisothiocyanate) are used for labelling of hybrid cells
      • Presence of chloroplast
      • Nuclear staining
        • Heterokaryon is stained by carbol-fuschin, aceto-carmine or aceto-orcein stain
slide18
Hybrid Selection

(Several markers are used )

  • Genetic complementation
  • Phytotoxins
  • Specific amino acid
  • Auxin autotrophy
  • Antibiotics
  • Auxotrophic and metabolic mutants
  • Chromosomal analysis
  • Herbicides
culture of the hybrid cells
Culture of the hybrid cells

Hybrid cells are cultured on suitable medium provided with the appropriate culture conditions.

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 hybridization
Advantages of somatic hybridization
  • Production of novel interspecific and intergenic hybrid
    • Pomato (Hybrid of potato and tomato)
  • Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids
  • Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters
advantages of somatic hybridization24
Advantages of somatic hybridization
  • Production of heterozygous lines in the single species which cannot be propagated by vegetative means
  • Studies on the fate of plasma genes
  • Production of unique hybrids of nucleus and cytoplasm
limitations of somatic hybridization
Limitations of Somatic hybridization
  • 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
introduction
Introduction

In nature plants propagate either

Sexually (seeds generation) results heterogeneity

Or

Asexually (vegetative multiplication) produce genetically identical plants.

Multiplication of genetically identical copies of a cultivar by asexual reproduction is called clonal propagation.

Via tissue culture called micropropagation,

what is micropropagation
What is Micropropagation?

“… the asexual or vegetative propagation (multiplication) of plants in vitro “

Implies - regeneration

- multiplication

- uniformity ??

micropropagation contin
Micropropagation (contin)
  • Positives and negatives of micropropagation
    • positives
      • rapid multiplication rates
      • low space requirement
    • negatives
      • labor costs
      • high overhead (equipment, facilities, supplies)
      • loss by contamination
      • danger of variation
slide31

STAGES

1. Selection of plant material

2. Establish aseptic culture

3. Multiplication

4. Shoot elongation

5. Root induction / formation

6. Acclimatization

slide32

STAGES

1. Selection of plant material

2. Establish aseptic culture

3. Multiplication

4. Shoot elongation

5. Root induction / formation

6. Acclimatization

slide33

Tip bud

Starting material formicropropagation

Leaf

Axillary bud

Internode

Root

slide35

Selection of plant material ...

  • Part of plant
  • Genotype
  • Physiological condition
  • Season
  • Position on plant
  • Size of explant
slide36

Physiological state - of stock plant

  • Vegetative / Floral
  • Juvenile / Mature
  • Dormant / Active
  • Carbohydrates
  • Nutrients
  • Hormones
slide37

STAGES

1. Selection of plant material

2. Establish aseptic culture

3. Multiplication

4. Shoot elongation

5. Root induction / formation

6. Acclimatization

slide39

Disinfestation

  • Stock plant preparation
  • Washing in water
  • Disinfecting solution
  • Internal contaminants
  • Screening
slide40

The medium

  • Minerals
  • Sugar
  • Organic ‘growth factors’
  • Growth regulators
  • Gelling agent
  • Other additives
slide41

Physical Environment

  • Temperature
  • Moisture
  • Light
slide42

STAGES

1. Selection of plant material

2. Establish aseptic culture

3. Multiplication

4. Shoot elongation

5. Root induction / formation

6. Acclimatization

slide43

Origins of new shoots ...

  • Terminal extension
  • Lateral / Axillary buds
  • Adventitious (de novo, re-differentiation)
  • Callus differentiation
slide44

auxins

cytokinins

gibberelic acid

ethylene

abscisic acid

Role of growth regulators ...

  • Cell division
  • Differentiation
  • Cell expansion
  • Apical dominance
slide45

STAGES

1. Selection of plant material

2. Establish aseptic culture

3. Multiplication

4. Shoot elongation

5. Root induction / formation

6. Acclimatization

slide46

Shoot elongation ...

  • Basal ‘hormone free’ medium
  • Gibberellins
  • Carry-over of hormones
slide47

STAGES

1. Selection of plant material

2. Establish aseptic culture

3. Multiplication

4. Elongation

5. Root induction / formation

6. Acclimatization

slide48

Root initiation ...

  • Auxins
  • Co-factors
  • C : N ratio
  • Light / darkness
  • Initiation vs growth
  • Juvenility / rejuvenation
  • Genotype
slide49

STAGES

1. Selection of plant material

2. Establish aseptic culture

3. Multiplication

4. Elongation

5. Root induction / formation

6. Acclimatization

slide50

Acclimatization (hardening)

- survival of the new plant when removed from the in vitro environment

- will be covered later.

micropropagation of almost all the fruit crops and vegetables is possible
Micropropagation of almost all the fruit crops and vegetables is possible
  • Some examples: dwarfing sweet cherry, Shade trees, Ornamental shrubs, Roses, Clematis, Lilacs, Saskatoon berries, Nutraceutical Plants, Rhododendron, Azalea, mustard, corn, soybeans, wheat, rice, cotton, tomato, potato, citrus, turf, legumes
advantages of micropropagation
Advantages of Micropropagation
  • economical in time and space
  • greater output -can produce millions of uniformly flowering and yielding plants
    • African Biotechnologies - fruit crops banana and indoor pot flowers- 6 million pieces per year
  • disease free
  • elite plants with exceptional characteristics
advantages cont d
Advantages Cont’d
  • facilitates safer movements of germplasm across nations - In vitro germplasm assures the exchange of pest and disease free material
  • great for
    • vegetatively reproduced crops
    • crops which produce few seeds or highly heterozygous seeds.
uses of micropropagation
Uses of Micropropagation
  • Used to create transgenic, first generation plants
  • Used in horticulture to produce orchids, African Violets, lilies, and ferns
  • Used in nurseries to grow fruit trees, evergreens, roses, and shade trees
benefits of micropropagation
Benefits of Micropropagation
  • Many genetically identical plants can be created from one parent plant
  • Because plants are clones, the uniformity assures quality
  • Allows many plants to grow in a small place in a short time
  • In some species this method will produce healthier plants