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.

Protoplast can be used to study

wall synthesis and deposition

Protoplasts can be studied as

single cell systems

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.

•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

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,

•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.

haemocytometer

The optimum plating efficiency (tobacco protoplasts) 5 x 104

protoplasts/cm3. Protoplasts fail to divide when plated at one tenth of

this concentration.

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