Protoplast fusion and cybrids in plants

1. CYBRIDS
IN PLANT IMPROVEMENT
PROGRAMME

2. CONTENTS
• What is cybrid?
• Advantages of cybrids
• Need for cybrids
• Protoplast isolation
• Techniques involved
• Cybrids in plant breeding

3. CYBRID
• Cybrid – cytoplasmic hybrid.
Cybrids are cells or plants containing nucleus of one
species but cytoplasm from both the parental species.
• Cybridization – production of somatic cybrid.
The process of protoplast fusion resulting in the
development of cybrid is known as cybridization.

4. • In 1892 Klercker was the first to isolate protoplast from
Stratiotes aloides using mechanical method.
• Cocking in 1960 was the first to report the isolation of
protoplast from tomato root tips using concentrated
solutions of cellulase from the fungus Myrothecium
verrucosa.
• Enzymes for protoplast isolation was first employed by
Takebe and his co workers in 1968.
• Kao and Michayluk in 1974 first proposed PEG for
fusion of protoplast.
• Gleba 1979 fused tobacco protoplast which produced a
cybrid.

5. • Melchers and Labib in 1974 fused protoplast of two haploid
light sensitive lines of Nicotiana tabacum.
• Kao and Wetter in 1976 isolated cell cybrids of Glycine max
and Nicotiana glauca .
• Pental and Cocking proposed that triploids could be
produced by fusing protoplast isolated from microspores at
the tetrad stage (n) of a species with protoplast isolated from
the somatic cells of other species.
• Pirrie and Power synthesized triploids by fusing microspore
protoplast of Nicotiana glutinosa with somatic cell protoplast
of Nicotiana tabacum .

6. • Production of full hybrids through protoplasts fusion of
distantly related plants – unrealistic- wide spread instability of
the two disimilar genomes in common cytoplasm.
• Undesirable –exhibit structural and developmental
abnormalities.
• Partial genome transfer – fusion of normal protoplasts of the
recipient with enucleated protoplasts of the donar-cybridization.

7. • Two different parental genomes that cannot reproduce
sexually (asexual or sterile) are recombined.
• Overcomes sexual incompatibility barriers.
• Used in study of cytoplasmic genes & their activities- plant
breeding experiments.
• To transfer cytoplasmic male sterility (tomato,tobacco).
• To transfer antibiotic resistance character(tobacco)
• To transfer herbicide resistance (brassica)
• Used in mitochondrial research.

9. PROPERTIES OF PROTOPLAST
• Lack cell wall and have plasma membrane
• Semi permeable and selectively permeable
• Fusion property
• Regenerate the cell wall (totipotent)
• Phagocytises and phenocytises
• Spherical in shape
• Sensitive to osmotic presser

11. • Cybrids are produced during fusion of protoplast from two
phylogenetically distant species.
• Regeneration from phylogenetically distant species will have
plastomes from both parental species but the functional
genome of only one species through chromosomal elimination.
• The extranuclear genes which control agronomically important
characters are of considerable interest.

12. • Isolation of protoplasts
• Culture of protoplasts
• Fusion of protoplasts
• Identification and selection of hybrid cells and their
subsequent regeneration of protoplast into whole
plants.

13.  Mechanical method (non enzymatic)
Cells are plasmolysed in iso-osmotic solution-tissues cut into thin
strips –protoplast realeased from cut ends.
 Sequential enzymatic method (two step)
Lower epidermis is peeled-leaf cut into pieces-macerated with
macerozyme –release of mesophyll cell-cellulase to digest cell wall.
 Mixed enzymatic (simultaneous)
Mesophyll tissue subjected simultaneously to macerozyme and
cellulase treatment-macerates the tissue by breaking the middle lamella
and release protoplast by digesting cell wall.

14. • Cellulases
cellulysin ,driselase ,meicelase p-1
• Hemicellulases
helicase ,hemicellulase ,rhozyme HP 150
• Pectinases
macerase,pectinol,pectolyse y-23 , zymolyase ,macerozyme
R-10

15. • Source material
• Pre-enzyme treatment
• Enzyme treatment
• Osmoticum
PROTOPLAST VIABILITY AND DENSITY
Flourescein diacetate method
Evan’s blue staining
Phenolsafranine staining

16. CULTURE OF PROTOPLAST
• B5 and MS media
• Reduction in ammonia
• Two – four times increased calcium concentrations – membrane
stability and integrity.
• Osmotica – sorbitol, mannitol,glucose and sucrose

17. • Protoplast in culture show rapid cytoplasmic streaming ,increasing
in size and chloroplast aggregate conspicuously around the
nucleus.
• Cell wall formation begins within few hours after isolation and
may be completed in two to several days.
• Characteristic spherical shape is lost once the wall formation is
complete.
• The freshly formed cell wall is composed of loosely arranged
microfibrils – requires readily metabolised carbon sourse(sucrose)
in nutrient medium.

18. • Cell increase in size and first division occurs between 2-7 days.
• Direct relationship between wall formation and cell division.
• After 1-3 weeks ,macroscopic colonies –transferred to osmotic
free medium to develop callus.
• Embryogenesis or organogenesis.
• Embryogenesis- cells from protoplast culture differentiate into
embroids then into whole plants.
• Organogenesis – callus transferred to a cytokinin free medium
for shoot formation and medium containing auxin for root
formation.

19.  Facilitates mixing of two whole genomes and could be exploited in
crosses at interspecic, intergeneric or interkingdom level.
Plasmodesmata of adjoining cells expand to form
homokaryons.
Rare and non reproducible.
External agent is used to fuse. Reproducible and sufficient.
Mechanical fusion - micropipette
Chemical fusion - fusogen(PEG,NaNO3,lysozyme)
Electrical fusion - electric charge

24. • Protoplast population consists of unfused parental types ,
homokaryons and heterokaryons.
• Fusion is random and uncontrolled.
Differential growth characteristics and nutritional
requirementsof unfused and hybrid mesophyll protoplast.
Nicotiana glauca and Nicotiana langsdorfii
 Petunia parodii and Petunia hybrida - drug actinomycin D
 Nicotiana sylvestris and Nicotiana knightiana –drug resistance
cell lines.

25. • Complementation by auxotrophic mutants.
• Auxotrophs are mutants requiring specific compounds for thier
growth.
• Only hybrid lines are expected to survive in minimal medium.
Chlorophyll deficient and light sensitive protoplasts of Nicotiana
tobacum under high intensities of light produced green colonies.
Visual identification of heterokaryons at the light microscope
level in fusion of chlorophyll deficient of one parent and green
chloroplast of one parent.

26. • Selection based on abnormal morphology.
• In regeneration of intergeneric somatic hybrids such as
pomatoes and topatoes.
• Intermediate morphology of callus determined the
intergeneric somatic hybrids between Vicia faba and Petunia
hybrida.
Flow cytometry and flurescent activated cell sorting while
maintaing viability.
Electronic sorting of heterokaryons formed with flurescent
dyes (rhodomine isothiocynate and floreceine isothiocynate)- in
cell sorter machine.

28. • Nuclear genome of one parent “donar” is inactivated
chemically or with irradiation before protoplast fusion.
• Irradiation with x-rays or gamma rays ,in doses of 50 to
300 Gy, is effective in partial or complete inactivation of
donar cells.
• Fusion of untreated “recipient” protoplast with “donar”
and culturing result in cybrid plants possessing the
nucleus of the “recipient” and the cytoplasm of both
parental species.
• Phylogenetically distant species produce cybrids during
protoplast fusion.

29.  The cybrid produced by electrofusion between Citrus unshiu
and C.sinensis.
 Restoration of male fertile Nicotiana by fusion of protoplast
derived from two different cytoplasmic male sterile cybrids.
 Correct deficiencies of nitrate reductase and xanthium
dehydrogenase activities in tobacco by fusing its normal
protoplast with protoplast of Physalis and Datura
inactivated by x-ray treatments.
 Streptomycin resistance has been transferred from
Nicotiana tabacum to three other species of tobacco.

30. • Chloroplast and mitochondria possess many genes- phenotypic
expression.
• The chloroplast trait, RuBPcase activity was exploited for
indentifying the somatic hybrids of Nicotiana langsdorffii and
N.glauca.
• cytoplasmic male sterility(CMS) is strictly maternally inherited
character having control from mitochondria.
• CMS is controlled by nuclear/mitochondrial interaction as in
Nicotiana- organelle marker.
• CMS was used to sort out the somatic cybrids/hybrids in Nicotiana
debneyi(CMS line) and N.tabacum cross.

31. CMS-ASSOCIATED PHENOTYPES IN CYBRIDS
• Cybrids of Nicotiana tabacum and Hyoscyamus niger.
• A protoplast fusion –based alloplasmic cytoplasm transfer by
conventional backcrosses- useful for generating alternative CMS
sources with novel nucleo cytoplasmic compositions.
• These alterations resulted in modified floral and vegetative
phenotypes.

32. • Biparental inheritance of cytoplasm during sexual reproduction
occurs in only a few genera .
• Plant regeneration from protoplasts is often a difficult, or even
impossible task.
• Instability of transferred genes in somatic hybrids.
• Recovering controlled asymmetric hybrids due to factors like cell
fusion, nuclear fusion, nuclear genes segregation and
recombination.

33. • S.Ignaciumuthu.S , PLANT BIOTECHNOLOGY
• Chawla, H.S. Introduction to plant biotechnooogy
• Agricultural biotechnology