Download Protoplast Culture: definition

Protoplast Culture: definition
Isolated protoplasts have been described as "naked" cells
because the cell wall has been removed by either a mechanical
or an enzymatic process. In the isolated protoplast the outer
plasma membrane is fully exposed
Protoplasts can be induced to fuse to
produce a hybrid plant, which cannot
be produced by conventional plant
breeding due to incompatibility.
Isolated protoplast are capable of
ingesting "foreign" material into the
cytoplasm. This material includes the
introduction of nuclei, chloroplasts,
mitochondria, DNA, plasmids, bacteria
and viruses.
Protoplasts can be studied as
single cell systems
Protoplast can be used to study
wall synthesis and deposition
The chief function of the cell wall is to exert wall pressure on
the protoplast preventing excessive water uptake and bursting of
the cell. Before the cell wall is removed, the cell must be bathed
in an isotonic plasmolyticum (mannitol or sorbitol 13 %, these
sugar alcohols are less readily metabolised by plant cells). It
may be advantageous to test a range of mannitol concentrations
varying from 8 -15% (w/v)
Cut plasmolyzed tissue and subsequent
deplasmolysis results in expansion and release of
the protoplasts from the cut ends of the cell.
In practice this technique is difficult and the
yield of viable protoplasts is meager. One
advantage, however, is that the deleterious
effects of the wall-degrading enzymes on the
metabolism of the protoplasts are eliminated.
Use of enzymes results in a high yield of uniform protoplasts after
removal of cellular debris Protoplasts can originate from different sources:
greenhouse or field material, micropropagated plants, calli,
•obtain sterile plant
material
•rinsing in a suitable
osmoticum
•facilitating enzyme
penetration
•sequential or enzim
karışımı
•purification of the
isolated protoplasts
(removal of enzymes
and cellular debris)
•transfer to a suitable
medium
•Healthy leaves, removed from the plant and washed, sterilized and
rewashed in sterile distilled water (subsequent procedures are
conducted under aseptic conditions).
•When leaves are in the final rinse, lower epidermis is peeled from
the leaves or the lower epidermis is scored several times.
•Cut the leaves into small sections, and transfer to filter sterilized
enzyme solution.
•Seal the dishes wrap them with aluminum foil (leave overnight).
•Teased gently with forceps to release the protoplasts.
•Purify protoplasts (filtration, centrifugation, and washing)
• Protoplasts are filtered through a nylon mesh
(64micrometer) to remove undigested tissue, cell
clumps, and cell wall debris.
• Transfer filtrate to centrifuge tube and spin at + 75
x g (5 min).
• Debris (in supernatant) is carefully removed
(protoplasts have formed a pellet at the base of the
tube).
• Protoplasts are carefully resuspended in culture
medium (plus 13% mannitol), and the process is
repeated three times.
• Protoplasts are examined for density and viability.
•Fluorescein diacetate:
accumulates only inside
the plasmalemma of viable
protoplasts, can be detected
with fluorescence/UV
microscopy
•Evans blue: Intact viable
protoplasts, exclude the
Evans blue stain.
Impermeability of the cell
to Evans blue indicates
a living cell.
•Cyclosis or protoplasmic
streaming can be a measure
of viability.
The optimum plating efficiency (tobacco protoplasts) 5 x 104
protoplasts/cm3. Protoplasts fail to divide when plated at one tenth of
this concentration.
haemocytometer
Protoplasts can been cultured in several ways:
•Hanging-drop cultures
•Microculture chambers
•Soft agar (0.75 % w/v) matrix.
This is one of the better methods
as it ensures support for the protoplast.
The first division
of a rice protoplast
four days after
isolation
Once the protoplasts have regenerated a cell
wall, they undergo cell division and form a
callus.This callus can be subcultured. The
callus may undergo embryogenesis or
organogenesis after about 3-4 weeks, in the
correct culture conditions. The
embryoids/organs
can be grown up in the same manner as for
most cultured plantlets .
Protoplast derived
plantlet of rice
growing in a test tube
SWEET ORANGE SUSPENSION CULTURE PROTOPLASTS
LEAF-DERIVED CITRUS PROTOPLASTS
TYPICAL SUSPENSION PROTOPLAST + LEAF
PROTOPLAST PEG-INDUCED FUSION
RMAN
NEW SOMATIC HYBRID PLANT
NOVA + SUCCARI SOMATIC HYBRID FRUIT
(father of several hundred triploid progeny)
SOMATIC CYBRIDIZATION
- Unfused leaf protoplasts not capable of plant
regeneration
- Diploid plants often regenerated from symmetric
fusions of embryogenic callus + leaf that are
morphologically identical to the leaf parent (from
more than 50 parental combinations).
- RFLP analyses indicates that these plants are always
cybrids containing the mitochondrial genome of the
callus parent! The chloroplast genome inheritance is
random.
Cybrid of “Murcott” (the Honey tangerine) containing
the mtDNA CMS of G1 Satsuma mandarin