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7/29/2019 Pro Top Last

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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


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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, bacteriaand viruses.

Protoplast can be used to study

wall synthesis and deposition

Protoplasts can be studied as

single cell systems


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The chief function of the cell wall is to exert wall pressure on

the protoplast preventing excessive water uptake and bursting ofthe 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)


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Cut plasmolyzed tissue and subsequentdeplasmolysis 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.


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obtain sterile plant

material

rinsing in a suitable

osmoticum

facilitating enzyme

penetration

sequential or enzimkarm

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,


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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)


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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.

Nylon

mesh

filter


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Fluorescein diacetate:accumulates only inside

the plasmalemma of viable

protoplasts, can be detected

with fluorescence/UV

microscopyEvans 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.


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haemocytometer

The optimum plating efficiency (tobacco protoplasts) 5 x 104 protoplasts/cm3. Protoplasts fail to

divide when plated at one tenth of this concentration


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Protoplasts can been cultured in

several ways:

Hanging-drop cultures

Microculture chambersSoft agar (0.75 % w/v) matrix.

This is one of the better methods

as it ensures support for the

protoplast.


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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 correctculture 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


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SWEET ORANGE SUSPENSION CULTURE PROTOPLASTS


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TYPICAL SUSPENSION PROTOPLAST + LEAF

PROTOPLAST PEG-INDUCED FUSION


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RMAN


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NEW SOMATIC HYBRID PLANT


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NOVA + SUCCARI SOMATIC HYBRID FRUIT

(father of several hundred triploid progeny)


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SOMATIC CYBRIDIZATION

- Unfused leaf protoplasts not capable of plant

regeneration

- Diploid plants often regenerated from symmetric

fusions of embryogenic callus + leaf that aremorphologically identical to the leaf parent (from

more than 50 parental combinations).

- RFLP analyses indicates that these plants are alwayscybrids containing the mitochondrial genome of the

callus parent! The chloroplast genome inheritance is

random.


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Cybrid of Murcott (the Honey tangerine) containing

the mtDNA CMS of G1 Satsuma mandarin

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