Molecular Techniques and Methods

Protoplast Preparation for
Transformation and Regeneration to Plant

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Protoplasts are isolated, single, and naked plant cells. Some have the potential to (1) regenerate a cell wall, (2) dedifferentiate, (3) divide mitotically and proliferate to form unlimited growing cell clones, and (4) differentiate shoot and root meristems (or embryos) which grow out to regenerate complete plants. Protoplasts are potentially totipotent individuals at the single cell level. Their freely accessible plasma membrane makes them ideal experimental systems for many kinds of genetic manipulation.




MATERIALS AND SOLUTIONS

0.01% (w/v) Mercuric Chloride (1 liter)
HgCl2 --------------------------------------------- 0.1 g
Distilled H2O -------------------------------------- 1 liter


Enzyme Solution (100 ml)
1.5% Cellulase ------------------------------------ 1.5 g
0.5% Pectinase ------------------------------------ 0.5 g
Distilled H2O -------------------------------------- 100 ml
  • Make fresh solution every time.


    Regeneration Medium (1 liter)
    MS Salts (Gibco) ----------------------------------- 4.3 g
    B5 Vitamin Stock ---------------------------------- 1 ml
    3% Sucrose ---------------------------------------- 30 g
    0.6% Agar ------------------------------------------ 6 g
    Add deionized H2O to make a final volume of ----- 1 liter
  • Adjust pH to 5.7 with NaOH.


    B5 Vitamin Stock (100 ml)
    myo-Inositol ---------------------------------------- 10 g
    Thiamine-HCl --------------------------------------- 1 g
    Nicotinic acid --------------------------------------- 0.1 g
    Pyridoxine-HCl ------------------------------------- 0.1 g




    PROCEDURES

    1. Grow plants of a competent genotype of a herbaceous dicot in potting compost in clay pots in a growth chamber (12 hour-light, 5000 lux, 27oC/20oC day/night temperature, 50/70% relative humidity) and water with a 0.1%, commercial fertilizer solution at 8.00 am and 4.00 p.m.

    2. Harvest leaves expanded to two-thirds their final size from plants at the 7-9 leaf stage.

    3. Wash leaves with tap water, and surface sterilize as follow:
  • Rinse in 70% ethanol three times.
  • Sterilize in 0.01% (w/v) Mercuric Chloride for 10 minutes.
  • Rinse leaves with sterile distilled water five times.


  • 4. Arrange the leaves in stacks of 6, and carefully cut into fine cross sections.

    5. Transfer 500 mg sectioned leaves to 10 ml Enzyme Solution and vacuum infiltrate to replace the intercellular air with Enzyme Solution.

    6. Incubate at 28oC for 3 hours.

    7. Observe the process of protoplasting from time to time under the inverted microscope.
    Gentle shaking helps to release protoplasts from the digested tissue toward the end of the incubation period.

    8. Filter the total digest through a 100-um stainless-steel sieve, mixed with an equal volume of osmoticum (mannitol), and distribute into capped centrifuge tubes.

    9. Centrifuge for 5 minutes at 60g to sediment the protoplasts without sticking them too tightly to the bottom of the tube.

    10. Discard the supernatant by carefully pipetting, and resuspend the sediment in the residual 0.5 ml of liquid, and fill the tube with fresh osmoticum. Adjust the population density to 2 x 104/ml.

    11. Pipet aliquots into petri dishes to give a thin liquid layer which just covers the bottom.

    12. Seal the dishes with parafilm and incubate at a constant temperature of 26oC in the dark.

    13. Cell wall regeneration and dedifferentiation are visible after one day and the first divisions after three days. The division frequency can be estimated 7-10 days after isolation by counting dividing and non-dividing protoplasts in representative fields and calculating the percentage of the total surviving population which has divided at least once.

    14. Dilute the suspension with 1/3 volume of culture medium with reduced osmotic pressure and growth regulator concentration.

    15. Repeat step 14 weekly and after a total of 4 weeks protoplast-derived colonies become visible to the naked eye. When these have reached a size of 1-2 mm in diameter, the plating efficiency (colony-forming efficiency) is established by calculating how many proliferating cell clones have developed per 100 protoplasts originally plated.

    16. Transfer cell colonies onto the surface of agar-solidified medium for further proliferation at low osmotic pressure.

    17. After a total of 2 months the colonies are large enough (5 mm in diameter) to be transferred to Regeneration Media which promote the development of meristems and the outgrowth of shoots and roots.

    18. After a further month a fraction of the clones will have regenerated multiple shoots (percentage regeneration efficiency) which are rooted as cuttings on a medium with a low auxin concentration.

    19. Two weeks later wash the rooted shoots carefully free of agar, pot rooted shoots into a fine potting compost, and adjust slowly to a low relative humidity. The plants can then grow further without any special care.



  • Protoplast Preparation from Different Sources:






  • NOTES

    FACTORS AFFECTING PROTOPLAST PREPARATION AND REGENERATION

    1. Competence
    The ability of a cell to respond in specific ways to specific stimuli during development. The competence on which the developmental route from protoplast to plant depends describes the capacity of specific plant cells to respond to isolation and in vitro conditions with a self-regulating programme of dedifferentiation, proliferation, pattern formation, differentiation, and plant development.



    2. Plant Species and Genotype
    There are plant species, e.g., Nicotiana tabacum (Nicotiana, Solanaceae), where it has been relatively easy to establish conditions for protoplast proliferation and plant regeneration from numerous genotypes with protoplasts from different source tissues. There are, however, also complete plant families, e.g., the Gramineae (grasses) where intensive efforts with hundreds of genotypes of many species have never produced clear cut evidence for regeneration of a single plant from a protoplast, and where even the induction of protoplast division is still a severe problem.



    3. Source Tissues from Organized Plants and from Cell Culture
    (A) Developmental State:
    For plants grown in soil under natural conditions young plants with expanding leaves before the differentiation of flowers generally give best results. As soon as plants begin to flower, it is difficult, if not impossible, to isolate proliferating protoplasts. The same holds true for organs where senescence has started. One generally uses organs before they have fully matured, e.g., expanding leaves at one-half to two-thirds of their final size.

    (B) Leaf:
    The protoplasts are generally easy to isolate from leaves and to handle experimentally and it is usually no problem to isolate very large populations of protoplasts at a similar state of differentiation which will proliferate at a high plating efficiency and regenerate plants.

    (C) Seedlings:
    These may provide a convenient source of proliferating protoplasts in cases where attempts to culture protoplasts from leaves have failed. Protoplasts from total seedlings, as well as from hypocotyl, cotyledons, and from seedling roots have been successfully cultured.

    (D) Embryos:
    These yield populations of tiny protoplasts as long as they are immature. However, isolation of embryo is so tedious and culture so difficult.

    (E) Shoot Cultures:
    Axenic shoot cultures proliferating under in vitro conditions are the source material of choice in species where they can be easily established and maintained and many successful protoplast laboratories are using them nearly exclusively.

    (F) Cell Cultures:
    Organized cell cultures: Embryogenic cultures are becoming available in an increasing number of plant species, including as interesting groups as the cereals and legumes. The characteristic of these cultures is that small groups of cell have the potential to differentiate to embryos.

    (G) Cell lines:
    A cell line culture type has arisen spontaneously in nearly every plant species kept in culture for long periods of time, including many cereals and grain legumes. Such cell lines are characterized by a short cell cycle, proliferation on a single cell basis, friability, and, unfortunately, a complete lack of morphogenic response.



    4. Environmental Conditions
    (A) Care of plants:
    The care of the plants before the isolation of protoplasts can be a key factor. Plants which have been badly treated rarely yield proliferating protoplast populations. Plants should be watered at regular times with the correct amount of water or fertilizer solution.

    (B) Season:
    It is the experience that season can play a crucial role even with optimized systems. This is not only experienced with greenhouse material, where one can expect it, but also with material which is grown in incubators and growth chambers without any direct interaction with climatic factors. Experiments which do not work between November and February may work well when repeated between March and June.

    (C) Light:
    Protoplasts isolated from plants grown under high light intensity are very sensitive to the isolation procedures and, if they survive, proliferate poorly. Experimental plants have to be kept in low light intensity, i.e., 3,000 lux or lower, for at least 1 day, and preferably longer, before the experiments.

    (D) Temperature and Relative Humidity:
    These contribute to the actual physiological state of the cell at the time of isolation, but again there are no clear data to aid the experimenter. In general it is advisable to grow the plants under conditions close to their natural climate.

    (E) Care of Cell Cultures:
    Cell culture material can be very sensitive to alterations in the culture conditions, e.g., the time interval of subculture, the dilution ratio with fresh culture medium, the material and geometry of the culture vessel, and the aeration and shaking conditions. Isolation of protoplasts is often possible only during a short time period in the exponential growth phase of the culture and quite often cell cultures respond to unknown alterations in the culture conditions in a way which prevents protoplast isolation totally, or reduces their quality.



    5. Endogenous Factors
    (A) Response to Wounding and Pest Treatments:
    Mechanical injury can, within hours, alter the physiology of the whole plant in such a way that protoplast culture is not possible for several days. If leaves are taken from the same plant over a long period of time, it is recommended that 1 week of recovery be allowed between experiments. If pest treatments are necessary, there should be a similar time interval between treatment and isolation of protoplasts.

    (B) Preculture:
    Excision of organs and preculture on a culture medium to induce dedifferentiation in situ prior to protoplast isolation has been helpful with many recalcitrant plants, e.g., legumes. On the other hand, attempts at a similar approach with cereal leaves failed completely because the cell walls were modified, in response to even 1 days preculture, such that protoplast isolation was no longer possible.

    (C) Cell Cycle Phase:
    Fast cycling cells can be sensitive to the isolation procedure but cell culture protoplasts are normally isolated from cycling suspension cultures as opposed to resting ones. Protoplasts isolated from fast growing cell suspensions rarely reach the high plating efficiencies of arrested leaf mesophyll protoplasts, but this may be due to other factors than cell cycle phase, such as the state of differentiation.



    6. Sterilization Procedure
    The sterilization procedure has to be adapted to the type of organ or tissue used in order to ensure that the majority of the cells are not already killed or injured by the sterilizing agent. The minimum concentration and the minimum time required for safe sterilization has to be established for every case.



    7. Mechanical Treatments Prior to Enzyme Application
    The outer cell layer of plants has evolved to prevent access to the inner cell layers. The cuticle, a complex layer of polymers excreted to protect the cells, is not penetrable by the enzymes used for protoplast isolation. The routine procedure is therefore to mechanically bypass the epidermal cell layer by either peeling off the epidermis (where possible), brushing with an abrasive, or slicing the organ into cross sections. These methods require some care and practice to avoid excessive damage of the inner cell layers.



    8. Plasmolysing Conditions
    Plant cells have an internal pressure (turgor pressure) which is contained by the rigid cell wall. Digestion of the wall in a hypotonic environment would cause the protoplasts to burst. Isolation is therefore carried out in hypertonic conditions which leads to plasmolysis and frees the cell wall from its structural role.



    9. Enzyme Treatment
    Plant cell walls are complex chemical and physical structures of celluloses, hemicelluloses, pectins, proteins, and other polymers. The enzymes applied are mixtures of cellulases, hemicellulases, and pectinases. The standard treatment is with a mixture of a cellulose and a pectinase at concentrations around 0.5-2% w/v dissolved in an osmoticum (giving 400-800 mOsm/kg H20) at pH 5.2-5.6 for a few hours. Enzyme concentrations higher than 2% (w/v) are often deleterious although they reduce the time of incubation.



    10. Protoplast Harvest and Purification
    Protoplasts are easily separated from incomplete digested tissues by sieving through meshes with pore sizes adjusted to the size of the protoplasts. Both washing and purification depend upon differences in the relative buoyant density of protoplasts and washing solution. Sugar and sugar alcohols (e.g., sucrose and mannitol) have relatively high buoyant densities. Salt solutions (e.g., CaCl2 and MgCl2) have relatively low buoyant densities at the same osmotic pressure.




    KIT INFORMATION




    REFERENCES

  • Braun, AC, Wood, HN (1962) PNAS 48: 1776.

  • Cocking, EC (1960) Nature 187: 927.

  • Date, PJ (1983) in "Protoplasts 1983: Lecture Proceedings" (I. Potrykus, C. T. Harms, A. Hinnen, R. Hutter, P. J. King, and R. D. Shillito, eds.), p. 31. Birkhauser, Basel.

  • Gamborg, OL, Miller, RA, Ohyama, K (1968) Exp. Cell Res. 50: 151.

  • Halperin, W (1969) Annu. Rev. Plant Physiol. 20: 395.

  • Harins, CT, Loerz, H, Potrykus, I (1979) Plant Sci. Lett. 14: 237.

  • Hess, D (1963) Planta 59: 567.

  • Kao, KN (1977) Mol. Gen. Genet. 150: 225.

  • Kao, KN, Michayluk, MR (1975) Planta 126: 105.

  • Li, L, Kohlenbach, HW (1982) Plant Cell Rep. 1: 209.

  • Linsmaier, EM, Skoog, F (1965) Physiol. Plant. 18: 100.

  • Murashige, T, Skoog, F (1962) Physiol. Plant. 15: 473.

  • Nitsch, JP, Nitsch, C (1969) Science 163: 85.

  • Potrykus, I (1980) in "Advances in Protoplast Research (Ferenczy, L., and Farkas, G.L., eds.)", p. 243. Hung. Acad. Sci., Budapest.

  • Potrykus, I, Hamis, CT, Loerz, H (1979) Plant Sci. Lett. 14: 231.

  • Raveh, D, Huberman, E, Galun, E (1973) In Vitro 9, 216.

  • Shepard, JF (1980) in "Genetic Improvement of Crops: Emergent Techniques" (Rubenstein, I., Regenbach, B., Phillips, R.L., and Green, C.E., eds.), p. 185. Univ. of Minnesota Press, Minneapolis.

  • Wernicke, W, Loerz, H, Thomas, E (1979) Plant Sci. Lett. 15: 239.


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