T27 - Isolation, culture, and fusion of potato and tobacco protoplasts

onwardhaggardBiotechnology

Dec 12, 2012 (4 years and 10 months ago)

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Cowles
-

M
3

Nonzygotic
embryogenesis


Page
1


T
26

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Use of protoplasts for plant improvement


Protoplasts are plant cells without their cell wall, but only a plasma membrane.

The cell wall normally
prevents the transfer of DNA into plant cells, however transforming these cells into protoplasts removes
this barrier.

Usually this transformation is only temporary and the cells become viable again once they
get their wall back.

Th
e first protoplasts were isolated by Klercker in 1892 by cutting highly vacuolated
onion cells in a plasmolyzing solution.

In the 1960s it became easier to obtain protoplasts using fungal
enzymes to digest the cell wall.

This was discovered by Cocking in 1
960.

The enzymes usually used to isolate protoplasts are cellulase, hemicellulase and pectinase.

The
processes happens at pH of 5.5
-
5.8 and lasts for 3
-
8 hours.

Centrifugation allows whole protoplasts to
be
separated

from broken cells.

Traditionally plants

have been improved by sexual hybridization between plant varieties. However, this
only works with closely related plants. Protoplast culture allows crossing of species that are not closely
related. In addition, it allows foreign genes to be added to plant

cells.

The resulting improved plants can
be cultured further to create new varieties.

I have found it interesting to see the fervor with which some people oppose the genetic engineering of
plants. For the most part I have a hard time seeing where they're
coming from. For one thing we've been
doing the same thing for a long time by crossing plants sexually, although now the removal of barriers
allows for more wide
-
ranging crossing.

To me the biggest factor in favor of genetic engineering is the
huge worldwi
de population explosion. If we're going to have any hope of feeding the number of people
in future generations without destroying the planet we're going to have to use as many methods for
boosting food production as we can.

Anyway, that's my soapbox.

Proto
plasts are often obtained from leaf mesophyll grown in a controlled greenhouse. Leaf mesophyll
works well because the cells are loosely packed which allows the penetration of digestive enzymes.

1E6
-
5E7 protoplasts can be produced per gram of young leaf tis
sue.

The use of leaves grown
in vitro

is
preferred since they don't have to be disinfested.

Protoplast isolation starts with 1
-
24 h of plasmolysis which causes the cell
to draw back from the cell
wall and is often conducted at low temperature. Digestive en
zymes are added to remove the cell walls
and lamellae and this mix is incubate
d with agitation for 3
-
18 hours.

The resulting liquid is swi
r
led to
remove the protoplasts and filtered with 50
-
100 um mesh to remove large debris.

50g centrifugation for
10 min concentrates the protoplasts in the pellet which can then be resuspended in
sucrose medium
topped by mannitol medium.

The protoplasts will gather at the interface, while debris moves to the
pellet during centrifugation.

The p
rotoplasts can then be removed, washed and cultured.

This process doesn't sound as difficult as I would have imagined. It's certainly easier than cutting up
onions in the hope that you happen to cut open a cell wall without damaging the protoplast inside!

Cowles
-

M
3

Nonzygotic
embryogenesis


Page
2


Protoplasts should be cultured at a density of E4
-
E6 cells/ml.

They can be culture in liquid on top of solid
medium or in drops of medium suspended in liquid.

Liquid medium is required for some species and is
often preferable since cells can't divide in so
lid medium.

Viability can be determined by staining with
fluorescein diacetate (FDA). Viable protoplasts will absorb the stain and glow green under UV light.
Evans blue is a stain which doesn't require fluorescence.

Protoplasts start out perfectly spherica
l. However, they redevelop their cell wall within 48
-
96 h which
causes them to become ovoid.
The presence of a cell wall can also be

detected with Calcofluo
r White
which glows bright white under UV light.

Protoplasts that don't regenerate a wall die withou
t dividing.

Plating efficiency (PE

= # dividing/total #
) is used to measure how many of the healthy protoplasts are
dividing.

The first division usually occurs 2
-
10 days after culture and 0.1
-
80% of cells may divide.

Many
different environmental factors af
fect division rates.


After walls have been formed the cells are cultured in medium with lower osmoticum. They form
colonies within 2
-
3 weeks and microscopic callus within 4.

Plant regeneration occurs by shoot formation
or somatic embryogenesis.

There are
several factors that affect protoplast growth and development. One of t
hese is the culture
medium used, which is often similar to that for organ regeneration.

Ammonium nitrate promotes cell
division

but is lowered to prevent toxicity to protoplasts.

Higher

calcium levels help the cells divide
faster and prevent clumping. Both ammonium nitrate and calcium levels are adjusted back to organ
development medium levels when the protoplasts recreate their walls.

Other components may also be
added to promote wall f
ormation and then removed.

It would be interesting to know the cellular processes that these chemicals interact with that helps them
speed up division etc.

Osmotic balance is important to prevent rupture of the cells and is maintained by non
-
metabolic suga
rs.
These are removed once walls have been formed.
Metabolic sugars are also added for food.

Auxins and
cytokinins are added to promote cell division.

Environmental conditions are also important for protoplasts. These vary between species, although all
spe
cies should be kept in the dark until they recreate their wall.

It is easier to recover cells that have been changed via somaclonal variation when using protoplasts
since each cell is viable.

These protoclones are often examined to determine if they have superior
characteristics.

One of the problems with somaclonal variation is the possibility of generating polyploid
or aneuploid plants.

The easiest way to fuse protoplasts is to use a cell lin
e that spontaneously fuses. However, a number of
other techniques can also be used.

Polyethylene glycol (PEG) can be used as a fusogenic agent.

The cells
have to be brought close together before PEG can cause their membranes to start fusing.

They are then
washed with alkaline calcium buffer.

Cowles
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M
3

Nonzygotic
embryogenesis


Page
3


Cells can also be fused with electricity which doesn't used toxic
chemicals such as PEG and gives much
higher yields.

The cells are polarized via dielectrophoresis so they stick together before the fusion pulse
is given
.

It's really cool that a oscillating current can be used to polarize cells so they stick together!

In order to determine if the cells and nuclei fused properly it is useful to combine cell line with 2
different selection systems such as antibiotic resista
nce. That way you can see if the hybrid has both
characteristics. Another way is to stain protoplasts with non
-
harmful stains before fusion to see if the
stains combine in the product.

New genetic information can be added to cells via infection with plasmi
d bearing bacteria.

Another
method is incubation of DNA fragments with PEG which enhances their uptake.

Electric shocks are used
to open pores for DNA movement in electroporation
-
mediated transformation.

Several other methods
for transformation are under d
evelopment.

T
27

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Isolation, culture, and fusion of potato and tobacco protoplasts

Fusion of protoplasts may be used to combine whole genomes (parasexual/somatic hybrids), partial
genomes (asymmetric hybrids) or cybrids with the
nucleus

from one plant and
cytoplasm of another.

Somatic hybrids of potato have been made with many different species.

These may or may not be
fertile, although
asymmetric

hybrids are almost always infertile.

This is not the case of cybrids, which are
generally fertile.

What is the point of a cybrid? I can understand why hybrids could be useful, but I don't understand
cybrids. Perhaps the point is just that it can be done. I suppose another benefit would be the creation of
plants with different mitochondrial and chloropl
ast DNA
than

normal. However, I'm not sure how this
would be helpful.

Both potato and tobacco respond well to protoplast culture. However, it is important to choose the
right cultivar as some respond much better than others.

Five or more different media a
re needed for
potato and tobacco in order to isolate and culture protoplasts.

Tobacco protoplasts can be isolated using techniques mentioned in the last chapter. These can then be
checked for viability using FDA. The resulting protoplasts can be cultured a
nd then checked with
Calcofluor White to determine if walls have been regenerated.

Potato protoplasts can be
fused by the addition of PEG to protoplasts in mineral oi
l on a slide followed
washing by a calcium solution.

Fusion can be observed with an invert
ed microscope or a florescence
microscope of different stains are used.

FITC stains protoplasts green while RITC stains them red. They
will appear yellow when fused.


Cowles
-

M
3

Nonzygotic
embryogenesis


Page
4


Questions

1.

Define protoplast.

2.

Describe electric fusion of protoplasts.

3.

List and describe
several uses for PEG.

4.

What do protoplasts that fluoresce yellow tell you?

5.

What are the advantages of protoplasts?