3. INTRODUCTION
Hanstein introduced the term ’protoplast’ .
The isolation of protoplasts form was first achieved
through by klercker [1892] on plasmolysed cells.
Cooking [1960] for the first time isolated the protoplasts
of plant tissues by using cell wall degrading enzymes viz.,
cellulase, hemicellulase, pectinase , and protease extracted
from a saprophytic fungus trichoderma viride and
myrothecium verrucaria.
First achievement in protoplast fusion by power[1970].
4. SOMATIC HYBRIDIZATION
Development of hybrid plants through
the fusion of somatic protoplasts of two
different plant species/varieties is called
somatic hybridization
5. SOMATIC HYBRIDIZATION
TECHNIQUE
1. PROTOPLAST ISOLATION.
2. FUSION OF PROTOPLAST.
3. IDENTIFICATION AND SELECTION OF HYBRIDS .
4. CULTURE OF THE HYBRID CELLS.
5. REGENERATION OF HYBRID PLANTS.
10. INDUCED FUSION
Mechanical Fusion:
Physical fusion of protoplasts under microscope by
using micromanipulator and
perfusion micropipette .
11. CHEMOFUSION
CHEMOFUSION- FUSION INDUCED BY CHEMICALS
TYPES OF FUSOGENS :
1. High pH – High Ca+ treatment
2. Polyethylene glycol (PEG)
3. Electro fusion technique.
HIGH PH – HIGH CA+ TREATMENT:
protoplasts of desired species are mixed in equal proportions.
The mixture is then subjected to the high pH (10.5) and high
Ca+ concentration (50 m mol l-1) at 37 0C for about 30
minutes.
12. POLYETHYLENE GLYCOL (PEG)
INDUCED PROTOPLAST FUSION
This technique is most commonly used.
The protoplast mixture is treated with 28 -50 % PEG for
15 -30 min followed by gradual washing to remove the
PEG.
The washing medium is usually alkaline (pH 9 -10) with
high concentration of Ca+ ion concentration.
During washing process, the PEG molecules pull out the
plasma molecules bound to them which disturbs the
membrane and leads to the fusion of protoplasts.
13.
14. ELECTROFUSION TECHNIQUE
Protoplasts are placed into a small culture vessel
containing electrodes .
A potential difference is applied.
The protoplasts will line up between the electrodes.
Short, electric shock is applied for few micro seconds
and protoplasts can be induced to fuse.
15.
16. ELECTROPORATION
It is the introduction of DNA into cells by exposing them
for a very brief periods to high voltage electrical impulses.
There are basically two systems of electroporation .
1) Low voltage – long pulses method .
2) High voltage – short pulse approach.
17.
18. BIOLISTIC
This method was first introduced by Klein and coworkers
in onion epidermis.
This device injects cells with genetic information using a
heavy metal element bullet coated with plasmid DNA.
The particles are placed inside the gene gun and a partial
vacuum is created between the target tissue and the gun.
The particles are then fired at the target and the DNA is
effectively introduced to the cells. .
19.
20. 4. Identification and Selection of somatic
hybrid cells;
Based on difference between the parental cells and hybrid
cell.
5.Culture of the hybrid cells;
Hybrid cells are cultured on suitable medium provided
with the appropriate culture conditions.
21. REGENERATION OF HYBRID PLANTS;
Plant are induced to regenerate from hybrid cell.
These hybrid plants must be at least partially fertile ,
In addition to having some useful property , to be of
any use in breeding schemes.
22. BULK CROSS METHOD
Bulk method was first used by Nilsson Ehle in 1908. F2
and the subsequent generations are harvested as bulks
to raise the next generation. At the end of bulking
period individual plants are selected and evaluated in
a similar manner as in the pedigree method. The
duration of bulking may vary from 7-30 generation
artificial selection may seldom be practiced .
23. PROCEDURE OF BULK
METHOD:
1) Hybridization: Parents are selected according to the objective of the
breeding programme and crossed.
2) F1 Generation: The F1 generation (10 to 25 F1) is space planted and
harvested in bulk.
3 F2 to F6 generations are planted at commercial seed rate and spacing.
These generations are harvested in bulk. During these generations
the population size should be as possible, preferably 30 to 50
thousand plants should be grown in each generation.
4) F7 Generation: About 30-50 thousand plants are space planted and
out of this only 1000 to 5000 plants with superior phenotypes are
selected and their seeds harvested separately. Selection is made on
the basis of phenotypes of plants, grain characteristics etc.
24. 5) F8 Generation: Individual plant progenies are grown in single
or multi row plots Most of the progenies would be homozygous
and are harvested in bulk. Weak and inferior progenies are
rejected and only 100-300 individual plant progenies with
desirable characters are selected.
6) F9 Generation: Preliminary yield trial is conducted along with
standard variety as check. The evaluation of progeny is done
for important desirable characteristics. Quality test may be
conducted to reject the undesirable progenies
7) F10-F12 Generation: Replicated yield trails are conducted at
several locations using standard commercial varieties as check.
The lines are evaluated for important agronomic
characteristics. If lines are superior to the standard check,
released as new varieties.
8) F13 Generation: Seed multiplication of the newly released
variety for distribution to the farmers.
25.
26. MERITS OF BULK METHOD:
1) This method simple, convenient and inexpensive.
2) Little work and attention is required in F2 and
subsequent generation.
DEMERITS OF BULK METHOD:
1) It takes much longer to develop a new variety.
2) It provides little opportunity for the breeder to exercise
his skill in selection.
27. BACK CROSS METHOD
A Crossing between a F1 hybrid or its segregating
generation with one of its parents is known as Back
cross. The hybrid and its progenies in the subsequent
generations are repeatedly back crossed to one of their
parents. As a result, the genotype of back cross
progeny becomes increasingly similar to that parent to
whom the back crosses are made. At the end of 6-8
back crosses, the progeny would be almost identical
with the parent involved in back crossing .
28. APPLICATION OF BACK CROSS
METHOD
This method is commonly used to transfer disease
resistance from one varity to another. But it is also useful for transfer of
other characteristics .
1)Intervarietal transfer of simply inhireted charecters.
Ex:- disease resistance , seed coat colour.
2)Intervarietal transfer of quantitative characters.
Ex :-plant hight , seed size , seed shape.
3)Interspecific transfer of simply inherited characters.
Ex :-transfer of disease resistance from related species to cultivated
species.
4)Transfer of cytoplasm.
Ex :-this is employed to transfer male sterility.
the female parent will be having the sterile cytoplasm and recurrent
parent will be used as male parent
29. Ex :- sesamum malabaricum sesamum idicum
female parent recurrent parent
5)Transgressive segregation.
Back cross method may be modified to produce
transgressive segregants . the F1 is backcrossed to
recurrent parent for 2 to 3 times for getting
transgressive segregants .
6)Production of isogenic lines.
7)Germ plasm conversion.
This was done in the case of sorghum .
30. Transfer of a dominant gene(R)for resistance to a disease
through backcross method
31. Transfer of a recessive gene(rr)for resistance to a disease
through backcross method
32. CONCLUSION
PROTOPLAST fusion provides a method of combining
the different genomes of different genera and species
with the potential to overcome sexual incompatibility
barrier between the parents.
In bulk cross method it takes generations to form a new
variety.
