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3. Protoplast isolation, culture and fusion
 

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 2C 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 27C 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 45C.

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 25-30C 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 30C.

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 30C 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 protoplasts/ml.

8.      Seal the petri dish with parafilm.  Incubate the culture in humid chamber at 25C 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 4C 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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