Protoplast Culture and Regeneration of Plants, Bergmanns cell Plating Technique, Semi solid Medium, Hanging Drops, Multidrop Array, Microchambers

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Protoplast Culture Regeneration and Somatic Hybridization Protoplast Culture Regeneration and Somatic Hybridization - Introduction Isolation of Proplasts Mechanical Method Enzymatic Method Direct One Step Method Sequential Two Step Method Purification of Isolated Protoplasts Sedimentation and Washing Flotation Viability and Plating Density of Protoplasts Testing Protoplast Viability Minimum Plating Density MPD and Culturing Individual Protaplasts Protaplast Culture and Regeneration of Plants Isolation of Subprotoplasts Protoplast Fusion and Somatic Hybridization Techniques of Protoplast Fusion

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Protoplast Culture and Regeneration of Plants

The culture methods for isolate protoplasts are similar to those for single cells earlier discussed in. The culture method selected also depends upon the objective of the experiment.

For instance, protoplasts can be cultured following ‘Bergmann’s cell plating technique’. Both semi-solid medium and liquid medium can be used, although the liquid medium is preferred.

Different culture methods (a) agar embedding, (b) liquid culture poured over a feeder layer or over a layer of inactivated protoplasts of another species, (c) microchamber constructed of a small plastic ring with oil and a cover slip, (d) hanging drop cultures

A. Agar Embedding	B. Liquid Culture Poured Over a feeder layer

C. Microchamber of Plastic ring Sealed with oil and cover slip	D. Hanging drop Cultures

Protoplasts can be suspended in a liquid medium in Erlenmeyer flasks without shaking and can be cultured in small quantities in ‘hanging drops’ or in ‘microchambers’. ‘Multidrop array (MDA) techniques may also be used (consult ‘Plant Protoplasts’ by T. Bengochea and J.H. Dodds, 1986 for details).

Within 2-4 days, protoplasts start developing cell walls, which can be detected by staining with 0.1% calcofluor white (CFW) fluorescent stain.While the presence of a proper wall is essential for a regular division, it is not always a prerequisite for nuclear division; not all protoplasts with cell wall formed, embark upon division.

The protoplasts, which are capable of dividing, undergo first division within 2-7 days and form multicellular colonies after 2-3 weeks. After another two weeks, these colonies can be treated as standard cultured tissues.

From these colonies or tissues in culture described as callus, plants can be regenerated, although success in this effort has been achieved only in a few species.

The first step in regeneration of plants involves the transfer of callus to a medium capable of initiating differentiation. It behaves just like the callus derived from cells. For instance, calli differentiate into shoots within 3-4 Weeks after transfer of protoplasts to a medium containing IAA (4mg/1) and cytokinin (2mg/1).

In this medium, roots may also be formed, failing which they can be induced by transfer of shoots to a basic White’s medium. Subsequently, the plantlets may be transferred to pots. Different steps from isolated protoplasts to regeneration of plants are depicted.

Regeneration of Petunia Parodii Plants from Isolated Mesophyll Protoplasts

However, the variation in regenerated plants derived from protoplasts is much greater than that observed in plants regenerated from a meristem, a stem segment or a leaf disc.

Diagrammatic Rerpesentation of Different Regeneration Methods with Potato

1. Meristem (Stable)	2. Stem Segment (soem variation)
3. Protoplast (great variation)	4. Leaf Disc (some variation)

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Induced Fusion NaNo₃-treatment Treatment with Calcium Ions ca⁺⁺ at High pH Polyethylene Glycol PEG Treatment Electrical Fusion Selection of Fused Protaplasts Chromosome Status of Somatic Hybrids Interspecific Hybrids Produced Through Protoplast Fusion Intergeneric Hybrids Produced Through Protaplast Fusion Use of Protoplast Fusion and Somatic Hybrids Somatic Hybrids for Gene Transfer Asymmetry in Somatic Hybrids for Gene Transfer Somatic Hybrids for Cytoplasmic Male Sterility Cytoplasmic Hybrids or Cybrids Genetic Modification Transformation of Protoplasts