Cybrid Production
Cybrids are also called as cytoplasmic hybrids. Cytoplasmic hybrids are obtained when nucleus is
derived from one parent and cytoplasm is derived from both the parents.
Cybrids can be obtained using any one of the following methods:
1. Fusion of normal protoplast from one parent with enucleated protoplast from the other parent.
2. Fusion of normal protoplast from one parent and protoplast containing non-viable nuclei from the
other.
3. Selective elimination of one of the nuclei from the heterokaryon.
4. Selective elimination of chromosome of one parent at a later stage after fusion of nuclei.
Objective of Cybrid Production
Combine cytoplasmic genes of one species with the nuclear and cytoplasmic genes of another species.
Plant Protoplast Isolation, Purifiction and Culture
The potential which protoplasts offer as experimental system has been recognized by workers
from various disciplines of plant biology. Currently, such isolated cells find application in basic
morphological and physiological studies, in the production of somatic hybrids, cybrids, genetic
transformation studies and as a source of cells for the isolation of biochemical mutants.
Fundamental to all these approaches is the requirement to obtain consistently high yields of
viable protoplasts, which, in most long-term investigations should be capable of wall
regeneration, division and preferably plant regeneration.
Protoplast Isolation, Purification and Culture
Source Material
Fully open young leaves of tobacco/tomato/capsicum or callus tissues of these plants can be
used.
Surface Sterilization
1. Leaves of greenhouse-grown plants (5-6 weeks old) are surface sterilized with sodium
hypochlorite 10% (v/v) solution for 10 minutes and subsequently washed with sterile
distilled water.
2. Shoot cultures from greenhouse-grown plants and seedlings from surface-sterilized seed
are raised on suitable media and their leaves are used as source for protoplast isolation.
Enzymatic Digestion and Protoplast Isolation
Protoplast isolation is carried out in a filter-sterilized enzyme mixture with an osmoticum and a
buffer (3mM MES, 2-(N-morpholino) ethanesulphonic acid adjusted to pH 5.7) to stabilize the
pH during digestion. Suitable combination of hydrolytic enzymes e.g., cellulase, hemicellulase
and pectinase are used.
Isolation step may be performed in darkness for 12-14 hours at temperature 28oC.
Osmotic Stabilization
Proper level of osmoticum must be ascertained by light microscopic examination of cells and
protoplasts in solutions of differing osmotic potential. 0.4-0.6 M level of osmoticum is
generally used.
Osmoticum: Sorbitol, Mannitol, MgSO4. Place the source material into a ‘pre-soak’ solution
containing the osmotic stabilizer.
Culture
Purified protoplasts are cultured in appropriate media (liquid). Media are filter sterilized
through a 0.45 micron filter.
Calcium (CaCl2) is added for membrane stability.
The gradual adjustment of the osmoticum as the protoplasts regenerate their walls and
begin to grow is a crucial feature of the entire protoplast culture process. (2.5 ml quantities
of fresh regeneration medium is added to the cultures periodically to gradually lower the
osmoticum)
If petriplates are used, parafilm sealing is done to eliminate evaporation. Cultures are
maintained at 24oC on 14:10 hr light and dark cycle.
Samples are periodically tested for viability, wall regeneration and division.
Cell colonies are picked and given several transfers to obtain microcalli.
Protoplast Concentration and Planting Density
The optimum initial concentration of protoplasts is also an important factor. The final protoplast
concentration should be about 5 x 104 protoplasts per ml.
Plant Protoplast Isolation, Purifiction and Culture Sequence
Source Material
Surface Sterilization
Wash with sterile distilled water
“Pre-soaking”
Replace the “pre-soak” solution with enzyme solution + osmoticum
Incubation for protoplast isolation (12 – 14 hrs)
Filtration through 40 micron stainless steel sieve, or Dendron mesh
Centrifuge (low speed)
Pellet resuspended in washing medium
Floatation on sucrose solution
Resuspend in liquid culture medium + Osmoticum
Repeat the step (washing)
Protoplasts counted and planting density adjusted
Plating and incubation in culture medium
Changes – testing for viability
Cell colonies
