PROTOPLASTS- ISOLATION, CULTURE AND REGENERATION

PROTOPLASTS- ISOLATION, CULTURE AND REGENERATION

Protoplasts are naked cells that can be obtained through mechanical/enzymatic degradation of cell walls. They are plant cells with a plasma membrane but without cell wall.

Sources: Young leaves, roots stems, petals, reproductive organs, friable callus, tissues/cells, fast growing cell suspensions. Coleoptiles, aleuronic layers, Plant cell tumors/galls.

Commercial enzyme preparations

  Onozuka R10 Cellulase (Japan)

  Cellulase R 10 (Japan)

  Macerozyme R 10, Pectinase (USA)

  Hemicellulase (USA)

  Driselase, Cellulase (Japan)

  Celulysin, Cellulase (USA)

  Pectinase (USA, Sigma)

  Macerase (USA)

  Rhozyme HP 150, Hemicellulase (USA).

Enzyme mixture is normally dissolved in culture media together with an osmotic stabilizer. The osmotic agents may be sugar, alcohols, sorbitol or mannitol (13% w/v). Sucrose can be used for this purpose.

Isolation Methods

Mechanical

e.g. Onion bulbs, scales, radish roots. This method gives poor yield of protoplasts

and is not suitable for isolating protoplasts from meristematic and less vacuolated

cells. This method is tedious and not often in use.

Enzymatic method

Preplasmolysis –Enzyme digestion-Washing- Purification-Culture

Advantages

  Protoplasts can be obtained in large quantities.

  The cells are not broken like in mechanical isolation.

  Osmotic shrinkage is much less.

Direct (one step) method

Treatment with Macrozyme or Pectinase and Cellulase is done simultaneously. The enzyme mixture in direct method consists of 0.5% Macerozyme + 2% cellulase in 13% sorbitol or mannitol at pH 5.4.

Sequential (two step) method: In two step method, leaf segments with mixture A (0.5% macerozyme+ 0.3% potassium dextran sulphate in 13% Mannitol at pH 5.8) are vacuum infiltrated (in a desiccators) for about 5 min and washed cells are then, incubated with enzyme mixture B (2% Cellulase in a 13% solution of Mannitol at pH 5.4) for above 90 min at 30°C. After incubation, the mixture is centrifuged at 100g for 1 minute, so that protoplasts form a pellet, which is cleaned three times as in one step method.

Protoplast culture and Regeneration of plants

Like the totipotency of plant cells, isolated protoplasts regenerate a cell wall around themselves to reconstitute a cell and undergo repeated division to form callus. Protoplasts can be cultured following 'Bergman’s cell plating technique', hanging drops or in micro chambers or following multiple drop array method Isolated protoplasts is mixed with 1.0% agar culture medium and maintained at 40-45°C. Small amount of agar (liquid) protoplasts mixture is then poured into sterile plates. The parameters to be calculated before culture are:

1. Viability and Plating density of protoplasts

2. Minimum Plating density (MPD) :In general, protoplast density within a range of 5 x 104 to 105/ml seems to be suitable. Normally, for the induction of division in the protoplasts, they have to be plated at final densities higher than 104/ml of the medium.

Protoplasts in culture start to regenerate a wall within a few hours and may take two to several days to complete. After three weeks, colonies are visible. Once small colonies are formed, their further growth is slowed down/inhibited altogether if they are allowed to remain on original high osmotic medium. The colonies therefore should be transferred to Mannitol free medium.

Applications

Virus uptake : Studies on the mechanism of infection and host parasite relationships

Bacterial uptake: Symbiotic nitrogen fixing bacterium (Rhizobium, Azotobacter) can be introduced into legume. Direct DNA transfer and expression of a bacterial gene in protoplasts of exogenous DNA by cells or protoplasts of T. Monococcum and N. tabucum are reported.

Incorporation of Cyanobacterial (e.g.): Cyanobacteria or BGA Co incubate algal preparation with isolated protoplasts with 25% PEG and high planting density. Protoplasts begin engulf algal cells.

Incorporation of exogenous DNA

Exogenous DNA can be taken up by higher plant cells/protoplasts and this is known as Trasngenosis.

Transplantation of nuclei: Organelles such a large nuclei can be introduced through plasma lemma into protoplasts. Both intra and inter specific nuclear transplantations have been observed in Petunia hybrida, Nicotiana tabacum and zea mays.

Chloroplast implantation: This has been done in Petunia and carrot.

Mutation and Genetic variability: Like callus cells, isolated protoplasts especially haploid ones would make an ideal system for studying the effect of irradiation and for the induction of mutations by planting them in media supplemented with various chemical mutagens.