Elementary plant Biochemistry and Biotechnology (2+1)

Protoplast culture

Purification of protoplasts

• After the material has been incubated in enzymes solution for an adequate period the incubation vessel is gently swirled or the leaf pieces are gently squeezed to release the protoplasts held in the original tissue. The digestion mixture consist of subcellular debris, especially chloroplasts, vascular elements, undigested cells and broken protoplasts besides intact and healthy protoplasts. The large debris is removed by passing through a metal or nylon filter (30-100 µm) and further purified by centrifugation.

Viability of the protoplasts
Viability of the freshly isolated protoplasts can be checked by a number of methods:

1. Observation of cyclosis or cytoplasmic streaming as an indication of active metabolism.
2. Oxygen uptake measured by an oxygen electrode which indicated respiratory metabolism.
3. Photosynthetic activity
4. Exclusion of Evan’s blue dye by intact membranes
5. Staining with fluroscein diacetate- which is most commonly used.
 
Protoplast culture
Protoplasts may be cultured in agar plates. An advantage in using semi-solid medium is that the protoplasts remain stationary which makes it convenient to follow the development of specific individuals (Figure). However, liquid medium has been generally preferred for the following reasons:

1. the osmotic pressure of the medium can be effectively reduced after a few days of culture
2. protoplasts of some species would not divide if plated in agarified medium.
3. if the degenerating component of the protoplast population produces some toxic substances which could kill the healthy cells it is possible to change the medium.
4. the density of cells can be reduced or cells of special interest may be isolated after culturing them for a few days at a high density.
 




• The protoplasts suspension is plated as a thin layer in petriplates, or incubated as static cultures in flasks or distributed in 50-100 µl drops in petri plates and stored in a humidified chamber. Embedding protoplasts in agarose beads or discs is reported to improve plating and regeneration efficiency in many species. Alginate is another gelling agent used for culture of protoplasts, particularly of the species which are heat sensitive such as Arabidopsis thaliana. After 2-4 days in culture, protoplasts lose their characteristic spherical shape and this has been taken as an indication of new wall regeneration. While the presence of a proper wall is essential for regular division, not all such cells regenerated from protoplasts embark upon division. In protoplast cultures, the cell divisions are asynchronous. The first division may be equal or unequal. Mitosis is normal. Continuous cell division leads to callus formation and the plant will be regenerated through normal developmental process.

Applications

1. Virus uptake: Studies on the mechanism of infection and host parasite relationships
2. 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.
3. Incorporation of Cyanobacterial cells (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.
4. 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.
 
Table: Some commonly used commercially available enzymes for protoplast isolation

Enzymes	Source
Cellulases	Trichoderma viride T. viride
Onozuka RS Cellulase R-10
Hemicellulase	Aspergillus niger A. niger
Hemicellulase Rhozyme HP150
Pectinase	Rhizopus spp. Rhizopus spp.
Macerozyme R-10 Macerase


Protoplast fusion

• With the development of techniques for enzymatic isolation of protoplasts and subsequent regeneration, a new tool of genetic manipulation of plants has now become available. Moreover, the fusion of protoplasts of genetically different lines or species has also been possible. For example, some plants that show physical or chemical incompatibility in normal sexual crosses may be produced by the fusion of protoplasts obtained from two cultures of different species. (Somatic hybridization of crop plants represents a new challenge to plant breeding and crop improvement. In the field of pest and disease resistance and transfer of C3 photosystems into C4 crop plants somatic crosses show most interesting promises