Introduction
Exploitation of genetic variability is an essential strategy in plant
breeding programme for the genetic improvement of crop plants. Improvements
of crop plants by conventional breeding is time consuming, expensive and
labour intensive. Unconventional techniques can shorten the process. One of
these techniques is somatic hybridization. Somatic hybridization through
protoplast fusion has been envisaged for the production of parasexual
hybrids to overcome sexual incompatibility. The ultimate objective to
transfer desirable traits from distantly related taxa. This para-sexual
hybridization is a means of increasing genetic variability of several crops
gene pool, not only by overcoming sexual incompatibility but it can produce
nuclear – cytoplasmic combinations that are difficult or impossible to
obtain by conventional sexual crossing.
Klercker (1892) first isolated plant protoplasts of
Stratiotes aloides
by plasmolysing and subsequently slicing the tissue. Protoplasts obtained
by this procedure were used mainly in physiological studies. However,
limited yields, excessive damage, and restriction of the technique to
specific tissues, were the major drawbacks. Large scale isolation of plant
protoplasts, using cell wall degrading enzymes (Cocking, 1960), gave impetus
to research in protoplasts. Initially, a two step procedure was adopted,
first producing single cells and then protoplasts. However, with further
experimentation, a mixed enzyme procedure was developed, involving
maceration and protoplast isolation in a single step from intact tissues.
The major activity of the cell wall degrading enzymes is in the digestion of pectins, cellulase and hemicellulases (xylans) that largely constitute the
plant cell wall. Pectinases degrade the galacturonic acid residues of
pectins that confer the cell to cell adhesion, and apparently macerate the
tissue to single cells; cellulases digest the cellulose component,
conferring the spherical shape to the protoplasts, while hemicellulases
assist in the breakdown of xylans. Incubation in a mixture of the
three types of enzymes in suitable ratios facilitates release of a large
number of protoplasts. The impurities present in the commercially supplied
crude enzymes reduce the protoplast viability and enzyme purification by gel
filtration can be
beneficial for sustained division of protoplasts. The pH of the enzyme
mixture and temperature of incubation are some of the factors that influence
the release of protoplasts. In certain cases, vacuum assists initial
penetration of the enzyme and subsequent digestion.
Following protoplast release, filtering through an autoclavable mesh removes
undigested tissue, while repeated washing by centrifugation, removes enzymes
and finer debris. Separation of protoplasts from debris is also achieved
using sucrose floatation. A two-phase system using dextran and PEG or
sodium metrizoate or a discontinuous density gradient system incorporating
percoll are some of the other methods used to purify plant protoplasts from
debris.
Protoplast isolation
Growing plants for protoplast isolation from mesophyll tissues
Mesophyll tissues of leaves are a convenient source for a large number of
uniform cells for protoplast isolation. The following points require close
attention prior to selecting leaves for protoplast isolation:
· Growth
conditions in terms of nutrition, humidity, light and temperature are
critical in determining the yield of stable protoplasts. Over-nourished or
undernourished plants are not suitable.
· Age
of the plants from which the leaves are excised for protoplast isolation
determines their stability. Generally plants between 6-7 weeks are suitable
in case of
Hyoscyamus muticus
and
Nicotiana tabacum
· Position
of the leaf on the source plant also influence the yield and quality of the
protoplasts. A well expanded 3rd and 4th leaf in case of both
H.muticus
or
N.tabacum
are suitable.
· In
vitro
raised axenic cultures are favoured over the field/glass-house grown plants
for obtaining leaves for protoplast isolation.
Growth of plants under in vitro conditions
· Soak
seeds of
H. muticus
for
2hrs
· Surface
sterilize in 0.1% mercuric chloride for 2-3 minutes and rinse with sterile
water 5-6 times
· Transfer
seeds aseptically to culture vessel containing half strength MS medium
without plant growth regulator with 0.8% agar and 1.5% sucrose. After 10
days cut out the root from seedlings and transfer shoots, one in each flask
(250 ml) containing medium (half strength MS salts with Nitsch Vitamins,
0.8% agar and 3% sucrose).
· The
leaves from these shoot cultures can be had after 5-6 wk.
Isolation of leaf mesophyll protoplasts of H. muticus
Plant Material Glass house grown plants or
in vitro
raised shoot cultures as is outlined earlier.
Media Solutions Enzyme solution-1, CPW 13M, CPW 21S, PCM (for details
see Appendix).
Other Requirements Cotton plugged pasteur pipettes, sterilized petri
dishes, sieve (60-75 m ), screw capped centrifuge tubes, ceramic tile,
jeweler's forceps, scalpel blade, haemocytometer (Fuchs Rosenthal marking)
cell counter, centrifuge.
Procedure
All
operations are to be carried out in a laminar flow bench.
1.
Select
in vitro
plants of 5-6 weeks of age and excise well expanded leaves.
2.
Remove the lower epidermis by peeling or slice the leaf into thin pieces
with a scalpel blade.
3.
Transfer the leaf pieces exposing the peeled surface to CPW 13M (10 ml)
solution in a 9 cm petridish for a minimum of 30 min.
4.
Pipette off the CPW 13M solution from beneath the leaf pieces and replace by
10 ml of the enzyme solution.
5.
Incubate overnight (16 hrs.) in the dark at 25° C.
6.
Pass the protoplast suspension in the enzyme solution through a 75 micron (
sieve/muslin cloth) into a fresh petri dish.
7.
Transfer the protoplast suspension to centrifuge tubes and spin at 80 g for
5 min. so as to sediment the protoplasts.
8.
With the help of a pipette, remove the enzyme solution (supernatant) and
replace with 12 ml of CPW 21S solution in each tube.
9.
Spin at 100 g for 10 min. The protoplasts will collect in the form of a
green band at the surface of the CPW 21S solution in the centrifuge tube.
10.
Carefully transfer the protoplasts to a fresh centrifuge tube with CPW 13M
solution.
11.
Count the protoplasts using a haemocytometer
12.
Spin at 80 g for 5 min. and replace CPW 13M by PCM.
13.
Bring the protoplast suspension to a final density of 1x10
4
protoplasts/ml and culture the protoplasts in 90 mm petridishes pouring 10
ml of the protoplast suspension in each petri dish. Protoplasts can be
cultured at densities of 1x10
5
protoplasts /ml and 0.5x10
5
protoplasts /ml for optimal response.
14.
Seal the petridishes with parafilm and place them in
containers (plastic or glass dishes) to maintain humidity and temperature.
15.
Protoplasts are incubated at 25 ± 2°C initially under dark conditions.
Isolation of pollen protoplasts
1.
Aseptically excise anthers and squeeze the pollen tetrads directly to a 5ml
enzyme solution in a petridish.
2.
Incubate statically for 1 hr at 27°C in dark.
3.
Pass through 40m sieve and wash with PCM solution.
4.
Purify by centrifugation (60 g for10 min) 2 times and suspend in 10 ml of
PCM to count. Use them in fusion experiments.
Counting of protoplasts
Counting of protoplasts is important to adjust suitable density for various
objectives such as culture, transformations, electroporation etc. For this
purpose a haemocytometer (Mod-Fuchs – Rosenthal) marking with chamber depth:
0.2 mm and volume of each small sub units 1/16 mm is used.
Procedure
1.
Protoplasts that are isolated are adjusted in 10 ml (or X= known volume) of
medium.
2.
Moisten edges of the haemocytometer and place the cover slip firmly.
3.
Add a drop of a well mixed protoplast suspension to the counting chamber of
the haemocytometer (any delay will cause the cells to settle in the
pipette).
4.
Count the number of protoplasts in 5 triple ruled squares (each with 16
single ruled small squares) (=n).
5.
Calculate the yield (Y) as follows: Y = n x 103
x X
n =
number of protoplasts counted in 5 triple ruled squares.
X =
volume of protoplast suspension.
6.
After knowing the yield/fixed volume then we can adjust our protoplast
suspension to desired density of culture, i.e., 1 x 10
5
or 0.5 x 10
5
by either dilution (adding more medium ) or concentration of existing
suspension (by centrifugation and removal of supernatant).
e.g. yield of protoplasts in 10 ml suspension is 10 x 10
6
, we require a density of 1 x 10
5
for plating.
Then 10 x 10
6
=
100 ml
1 x 10
i.e., each 1 ml of protoplast suspension has to be diluted to 10 ml for
plating at a final density of 1 x 10
5
.
Determining the viability of protoplasts by FDA staining
Flourescein diacetate (FDA) is relatively nonpolar, and passes freely across
the plasma membrane. In association with viable protoplasts/cells, the
molecules are hydrolyzed by the action of esterases to release polar
fluorescien molecules which cannot cross the membrane and consequently
accumulate in the cell and stain it greenish white under UV illumination.
Thus, metabolically active protoplasts are visualized.
Plant Material Protoplasts in culture medium.
Media solution Primary stock of 5 mg/ml of FDA dissolved in acetone,
PCM/CPW 1 3M.
Other Requirements Fluorescence Phase contrast microscope, Haemocytometer,
Cell counter.
Procedure
1.
Add 1 drop of FDA stain to 10 ml of protoplast culture medium or CPW 13M.
2.
Mix equal volumes of a dense protoplast suspension to FDA solution (as in
Step 1).
3.
Place a drop of this mixture onto the chamber of the haemocytometer.
4.
View under UV illumination within 10 min. of staining.
Protoplast culture
A
range of basal media for protoplast culture have been used with several
modifications of hormones within each formulation. These media have been
based mainly on those devised by Murashige and Skoog (1962) and Gamborg
(1968), with an addition of an osmoticum, generally a sugar, such as glucose
or a sucrose or a sugar alcohol, for example mannitol or sorbitol. These
sugar alcohol are used for their nearly metabolically inactive property. Kao
and Michayluk (1975) developed a complex medium, wherein single protoplasts
of
Vicia hajastana
were capable of colony formation. This medium, together with minor
modifications, has proved successful in obtaining sustained divisions in
many of the plant systems (Kao and Michayluk, 1980; Santos et al., 1980; Lu
et al., 1982; Xu et al., 1982). Caboche (1980) constituted a fully defined
medium for growth of pre - cultured protoplasts at low densities. Efficient
growth of pre-cultured protoplasts has also been achieved by the use of
"nurse" cultures of albino protoplasts (Patnaik et al., 1983), irradiated
protoplasts (Cella and Galun, 1980) or protoplasts from an auxotrophic
mutant (Hein et al., 1983).
A
multiple drop array (MDA) technique has been useful in analyzing the effects
of different combinations and concentrations of growth regulators in a
particular medium on protoplast development (Potrykus et al., 1979).
Protoplasts have been cultured in sitting or hanging droplets (Kao et al.,
1971), thin, liquid layers embedded in agar medium (Nagata and Takebe 1971),
and liquid over an agar layer (Power et al., 1976). Partanen et al. (1980)
and Santos et al. (1980) employed a filter paper layer at the agar liquid
interface of liquid over agar media for successful culture of fern and
Medicago mesophyll protoplasts, respectively.
Other than agar gelling agent, alginate, which enables protoplasts to be
plated without a temperature shock. The aligmate can be liquified by
addition of a chelating agent, making possible the recovery of developing
colonies (Giri and Reddy 1994; Giri and Reddy 1998 ). In addition, other
factors that influence division and subsequent colony formation, include the
temperature of incubation (Zapata et al., 1977), the initial light intensity
and the type of culture vessel.
Methods of protoplast culture
Consequent to isolation and adjusting of culture density, protoplasts are
plated by any one of the following methods that have been usually employed.
A.
Liquid layers
B.
Embedded in agar/agarose
C.
Liquid over agar/agarose
D.
Alginate encapsulation
E.
Hanging/sitting drop culture
F.
Filter paper substratum placed on agar
G.
Microdrop array (M.D.A.) technique
In
certain instances agarose has been used in place of agar and this has
improved the culture response.
A.
Liquid layers: 10 ml of the protoplast suspension at requisite density is
plated in 90 mm petridish. Proportionately 3 ml are plated in a 50 mm dish
and 1.5 ml in a 35 mm dish. Seal with parafilm and incubate in moist
environment.
B.
Embedded in Agar/agarose:
1.
Melt double strength agar (1.4%) medium with 9% mannitol and bring to 45°C.
2.
First pour 5 ml of protoplast suspension into a 90 mm petri dish (at double
the required density).
3.
Pour 5 ml of molten double strength agar medium into 5 ml of protoplast
suspension in the 90 mm petri dish.
4.
Mix thoroughly and allow agar to set. Seal dishes with parafilm.
Alternatively, agarose at a final concentration of 1-1.2% can be employed
and blocks of embedded protoplasts can be transferred to liquid media in
petridishes or flasks for slow shaking.
Maintenance of protoplast cultures
v Maintain
the protoplast cultures initially for 10-15 days under dark conditions at
temperatures ranging from 25-30°C depending upon a particular species or
variety.
v Provide
with continuous illumination first at a low intensity of 1,000 lux and
gradually increased to 3,000 lux.
v Maintain
relative humidity in containers during this period to avoid desiccation.
Cell wall formation is evident by the change of shape of the
protoplasts within 24-48 hrs of plating. Divisions occur by 3-7 days of
culture initiation. For sustained growth of protoplasts the osmoticum of
the culture medium has to be lowered by sequential dilutions periodically.
For protoplasts that are embedded in agar or agarose and alginate beads,
small blocks containing the dividing protoplasts are transferred to the
surface of the same medium but with lower osmoticum. The same can further
be shifted again to a medium with a lower osmotic pressure.
Once the protoplasts have grown up to a callus stage, the procedure for
regeneration of plants there from is essentially the same as that employed
in regular tissue culture techniques.
Protoplast fusion
Mechanism of protoplast fusion
Major constituents of the cell membrane are phospholipids and proteins.
Lipid molecules consist of a polar head group that are hydrophilic, to which
is attached one or two long hydrocarbon chains of tails which are
hydrophobic. The phospholipids are arranged in a bilayer into which
peripheral or integral structural proteins are embedded in a mosaic-like
fashion. Exogenous chemical stimuli cause disturbances in the
intra-membranous proteins and glyco-proteins, increasing thereby the
membrane fluidity and resulting in fusion of membranes at the point of
contact. Such fluidity is related to the proportion of saturated
phospholipids in the membrane and is influenced by temperature. The
intra-membranous phosphate groups are postulated to induce a negative
surface charge on the protoplasts. This tends to repel the protoplasts from
one another.
The
underlying mechanism of fusion by chemical methods and also electric field
induced fusion of plant protoplasts, remains similar to that suggested by
Poste and Allison, 1971 for animal cells. According to these workers,
membrane proximity of the fusion partners is an essential pre-requisite
followed by an induction phase, whereby the charges induced in electrostatic
potential of the membrane result in fusion. It was the early part of the
century, that Kuster (1910 ) demonstrated the ability of onion epidermal
protoplasts to fuse upon de-plasmolysis. Also Michel (1937) showed that
protoplasts could be fused by using potassium nitrate as the plasmolyticum.
However, fusions were rare and the culture
of
fusion products was difficult.
In
the light of enzymatic procedures for protoplast isolation, interest
developed in protoplast fusion with an aim to genetic manipulation. Power
et al., (1970) reported sodium nitrate induced fusion of root protoplasts
from various species. This method met with success in the production of the
first somatic hybrid. However, fusion frequency following this method was
low and the sodium nitrate treatment also affected the division capability
of the protoplasts. This stimulated interest in other fusogens, and as a
consequence rapid advances in the technique of protoplast fusion have been
made in the last decade (Table
Fusion methods that have gained maximum attention are the use of high pH
combined with high Ca++ in solution, polyethylene glycol (PEG) and more
recently the electric field induced electrofusion. Several other methods
have also been reported to induce protoplast fusion. These include
manipulation with a perfusion micropipette, sea water, lysozyme and
inactivated viruses.
Action of high pH and Ca++
A
calcium solution buffered at high pH induces aggregation of the protoplasts
and their fusion. The addition of Ca++ causes the potential of the surface
negative charge on protoplasts to be reduced, facilitating protoplast
adhesion. The high alkalinity (pH 9.5 - 10.4) induces the formation of
intramembranous lysophospholipids such as lysolecithin and lysophosphatidyl
-ethanolamine that increase membrane fluidity that results in fusion. This
method has been successfully used in the production of numerous somatic
hybrid plants. Protoplasts derived from cell suspension cultures generally
are not as tolerant to the use of high pH Ca++ method as the mesophyll
derived protoplasts.
Action of Polyethyleneglycol (PEG)
PEG
has been most widely used to fuse plant protoplasts. Both, the concentration
and molecular weights of PEG are important in relation to fusion. PEG,
whose general formula is HOCH2-(CH2-O-CH2) -CH2OH is a water soluble
compound whose other linkages make the molecule slightly negative in
charge. Thus, addition of Ca++ (CaCl2 or Ca (NO3)2 links the compound with
membrane surfaces. The high molecular weight of the polymer acts as a
bridge connecting the protoplasts together. A strong affinity of PEG for
water causes local membrane dehydration and increased fluidity. This in
combination with the reduction of an exclusion volume between adjacent
protoplasts causes diminishing mutual membrane electrostatic repulsion. The
redistribution of glycoprotein and glycocalyx macromolecules, causes
fusion. PEG of molecular weight 400 to 6,000 was found to be active in
fusion, whereas PEG 200 and 20,000 was almost inactive. Thus factors, other
than purely chemical structure determine fusogenicity. Compounds
structurally related to PEG, namely, polyvinyl alcohol, polyvinyl
pyrrolidone and polyglycerol are known to induce fusion. Gelatin, and
dextran sulphate also induce fusion to varying degrees.
PEG
has been used in combination with other treatments to enhance fusion
frequency. An increase in the frequency of heterokaryon formation was
observed when protoplasts were pre-incubated in lysozyme. Also, combination
of PEG with high pH Ca++ solution, or the addition of DMSO or concanavalin A
gave rise to higher fusion frequencies in comparison to treatments with PEG
alone. It is generally observed that in fusion experiments involving PEG as
the
fusogen, the elution of PEG subsequent to its application is a critical
step in determining the frequency of stable fusion products. A rapid change
of osmotic pressure subsequent to fusion in the process of transferring to a
culture medium results in excessive damage, particularly when mesophyll
protoplasts are involved in the fusion process.
Protoplast fusion using high pH Ca++ solution
Materials Cell suspension protoplasts of
Hyoscyamus muticus
and mesophyll protoplasts of
Nicotiana tabacum.
Media Solution :
High pH Ca++ solution, CPW stabilizer solution CPW 13M and PCM.
Other Requirements
Water bath at 30°C.
Procedure:
1. Adjust
the density of protoplast suspension to 1x106
protoplasts / ml and mix 1 ml suspension of each of cell suspension and
mesophyll protoplasts in a centrifuge tube.
2. Centrifuge
at 30 g for 5 min. so as to settle the protoplast mixture and remove the
supernatant.
3. Re-suspend
the protoplasts in 5ml solution of high pH Ca++ and centrifuge at very slow
speed so as to settle the protoplasts.
4. Incubate
in a water bath at 30°C for 20 min.
5. The
supernatant is gently removed without disturbing the pellet of protoplasts.
6. Gently
add 8-10 ml of stabilizer solution without disturbing the pellet and
incubate at room temperature for 30-45 min.
7. Remove
the stabilizer solution without disturbing the protoplasts and replace with
PCM (8-10 ml). Incubate at room temperature for 5-10 min.
8. Remove
PCM and repeat washing as above.
9. Finally
suspend the fusion mixture at a final plating density of 1x105
protoplasts/ml and plate 8-10 ml of the suspension in a 90 mm petri dish.
Procedure for protoplast fusion using single step PEG-high pH Ca++
Materials:
Cell
suspension protoplasts of
Hyoscyamus muticus,
mesophyll protoplasts of
Nicotiana tabacum.
Media Solutions:
PCM, PEG-high pH Ca++ solution, and stabilizer solution
Caution:
Vibrations of laminar flow bench need to be
avoided, as this will affect the fusion procedure. A vibration free laminar
bench is desirable.
Procedure
1. 0.5
ml protoplasts suspension at a density of 1 x 106
/ml from each partner are mixed in a centrifuge tube. The protoplast
mixture is centrifuged at 50 g for 5 min. Remove most of the supernatant
leaving 0.5 ml of protoplast suspension.
2. Take
out the protoplast suspension and place it in 90mm sterile petri dish in the
form of 0.1 ml droplets (5-6 droplets/dish).
3. Agitate
the dish gently to accumulate the protoplasts in the center of the drops and
allow the protoplast mixture to settle for 5 min.
4. Add
PEG-high pH Ca++ solution ( ( 0.2 ml/drop) around the drops containing
protoplasts. Incubate at room temperature for 10-12 min.
5. Add
stabilizer solution ( 0.5 ml/drop) slowly and elute the same with PEG - high
pH Ca++ which was initially added. Add fresh stabilizer. Incubate for 5
min., remove the stabilizer solution along with some fusogen. Repeat this
process 2-3 times without further incubation. (This whole process should
take 15-20 min.)
6. After
2-3 washes with stabilizer, 4-5 washes are made with either CPW 13M or PCM
in the same manner as stated above. Repeat to remove the traces of PEG-high
pH Ca++ and stabilizer.
7. The
final volume of the PCM should be such that the overall plating density
remains at 1x10
5
protoplasts/ml.
8. Seal
the petri dish with parafilm. Incubate the culture in humid chamber at 25°C
in dark.
APPENDIX II: FUSOGEN SOLUTIONS
i) High pH Calcium solution (High/pH/Ca++):
0.74% (w/v) CaCl2.6H2O
0.375% (w/v) Glycine
9% (w/v) Mannitol
The
above were dissolved in a distilled water, pH adjusted to 10.6, filter
sterilised and used immediately.
ii) Polyethylene glycol solution (PEG):
25, 30 or 35% (w/v) Polyethylene glycol (M.W. 6000)
9% (w/v) Mannitol
2.36% (w/v) Ca(NO3)2.4H2O
Constituents were dissolved in distilled water, the pH
adjusted to 5.8 and autoclaved. Storage was in the dark at 4°C for up to 8
weeks.
iii) High Calcium solution (Stabilizer)
The
stabilizing solution consisted of an aquous solution of CaCl2.2H2O (3.5%
(w/v)), autoclaved and stored at room temperature. Alternatively, CPW 13M
solution (Appendix I) was modified by the addition of 0.74% (w/v) CaCl2.2H2O
(CPW/Ca).
iv) PEG solution for the single-step fusion method
190 mg/l KNO3 SOLUTION A
44
mg/l CaCl2 2H2O
37
mg/l MgSO4 7H2O
17
mg/l KH2PO4
pH
5.6
20%
PEG 8000 SOLUTION B
35
g/l CaCl2.2H2O
4
g/l glycine
Adjust to pH 10.5 with 10N KOH.
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