Cell Physiology and Genetics of Higher Plants

Volume II

A. Rashid

• Publisher: CRC Press
• Year: 2018
• ISBN:
  9781351087377
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Botany
• Index: Biological Sciences

This book is devoted to fundamental aspects concerning cell physiology that are basic to the theme of biotechnology of higher plants. It highlights the regeneration potential of higher plant cells, for micropropagation of plants. The book is concerned with the genetic engineering of plants.

Chapter 1

Protoplast — Isolation and Cell Regeneration

I. Definition and Significance

A plant protoplast is a cell without a wall. Due to the absence of a cell wall and consequent exposure of the plasma membrane, the protoplast becomes a very fragile structure. It is disrupted easily, unless it is maintained in an osmoticum. In an osmoticum, due to partial plasmolysis, the protoplast is a perfect circular structure (Figure 1A). The delicate nature of a protoplast makes it a relatively difficult material to handle as compared to a cell. However, a plant protoplast readily regenerates a wall and is a fully totipotent structure, with all the attributes of a cell.

The absence of a cell wall around the protoplast makes it a suitable system for many studies — fundamental as well as applied — which are not possible with an intact cell, within the confines of a cell wall. The more significant of the potential applications of plant protoplasts is hybridization of two protoplasts for crop improvement. It is a novel method, often described as parasexual hybridization, to expand the pool of genetic variability needed for crop improvement. In parasexual fusion it is possible to combine distant genes due to the absence of crossability barriers, often encountered in gametic fusion, particularly when distant hybridization is programed. It can also bring about selective gene transfer such as incorporation of cytoplasmic genes. Further, protoplasts can be useful in plant improvement by way of cell transformation, i.e., incorporation into a protoplast of purified foreign genetic material, an organelle, or even a microorganism.

The protoplast is an ideal free-cell system suitable for fundamental studies such as cell wall regeneration and isolation of mutants, especially auxotrophs where intercell feeding is undesirable and is a hindrance. Haploid protoplasts are particularly well suited for the raising of mutants. A haploid protoplast is a higher plant-equivalent of a microbe. Other uses of protoplasts are the study of membrane (transport and cotransport of molecules across a membrane) and studies on hormone action, mechanism of fungal and bacterial infection, and viral multiplication, which can ultimately find application in testing for drug and disease resistance. Due to the absence of a cell wall a protoplast can be easily disrupted and this property can be employed for the isolation of organelles and macromolecules without the damaging effects of shearing forces required to break the cell wall.

For the use of protoplasts for these diverse purposes a basic requirement is the isolation of viable protoplasts in high frequency which are capable of regeneration into cells and ultimately into plants. This chapter concerns these fundamental aspects of protoplast technology.

II. Isolation

The key operation in the release of a plant protoplast is the removal of cell wall in a way that it does not damage the protoplast and impair its ability to regenerate into a cell.

A prerequisite for the release of a protoplast from the confines of the cell wall is a suitable osmoticum of proper osmolality. In the absence of an osmoticum, an immediate lysis of a protoplast is a certainty due to the lack of wall pressure usually exerted by the cell wall. Use of an osmoticum of either too high or too low osmotic potential results in irreversible damage to the protoplast. Deviation of the isolation medium from the isotonic concentration can cause the protoplast to shrink or swell. This process may influence membrane physiology and viability of the protoplast.

FIGURE 1. Protoplasts from cell suspension culture of Nicotiana tabacum, stained with fluorescein isothiocyanate: (A) bright field illumination and (B) epifluorescence illumination. (From Galbraith, D. W., Afonso, C. L., and Harkins, K. R., Plant Cell Rep., 3, 151, 1984. With permission.)

A. Methods of Isolation

The isolation of plant protoplasts — removal of the cell wall in the presence of a suitable osmoticum — can be accomplished by the following two methods.

1. Mechanical Isolation

The pioneering¹⁵⁴ attempt to isolate protoplasts from a higher plant was by a mechanical method. The method involved a preplasmolysis of tissue followed by its random sectioning which resulted in the release of a few undamaged protoplasts along with several broken cells. Using this method protoplasts could be obtained only from highly vacuolated cells of tissues such as onion bulb scale, mesocarp of cucumber, and roots of radish and beet. In this method it is essential that a protoplast contracts away from cell wall. Therefore, it cannot be applied to meristematic cells. This method is not recommended for routine operations because of (1) poor yield, (2) restricted applicability to tissues which are vacuolated and easy to plasmolyze, and (3) tedium of the operation.

More recently, in a new approach²³ the material (cross sections of petiole of Saintpaulia ionantha) was grown on a medium enriched with a relatively high level of auxin, 2,4-D. The thin-walled cells so formed when teased apart with dissecting needles readily resulted into protoplasts, in an osmotic medium.

2. Enzymatic Isolation

Large-scale isolation of protoplasts⁵⁹ was possible when enzymatic digestion of cell wall was demonstrated from root cells of tomato. To digest the cell wall the enzyme (cellulase) source was a culture filtrate of the fungus Myrothecium verucaria.

Following this, commercial preparations of enzymes^244,276 employed for the isolation of plant protoplasts are macerozyme and cellulase. The former is rich in pectinase and brings about cell maceration and the latter digests the cell wall, releasing protoplasts.

a. Steps in Enzymatic Isolation

In an enzymatic isolation of protoplasts the tissue is subjected to pectin- and cellulose-dissolving enzymes (macerozyme and cellulase) either in two steps (sequentially) or in one step (simultaneously).

In the two-step or sequential method,²⁷⁶ macerozyme and cellulase are employed one after another. To begin with, there is cell maceration by macerozyme which results in the separation of cells; this is followed by the transformation of cells into protoplasts, effected by cellulase. In this method the cells remain in contact with the enzymes for a shorter period than in the one-step process.

The one-step method²²⁷ is a simplification of the two-step process. Here the tissue is subjected to an enzyme mixture comprising pectinase as well as cellulase. This method minimizes the chances of microbial contamination, as the operation is reduced to a minimum. Higher yields are possible with this method, particularly from leaf tissue because mesophyll as well as palisade cells are transformed into protoplasts. However, in other tissues, such as pods of soybean,³²⁶ no difference either in protoplast yield or viability was recorded with either the one- or two-step method.

A modification of the isolation procedure is an i...

Table of contents

• Cover
• Title Page
• Copyright Page
• Preface
• The Author
• Cell Physiology and Genetics of Higher Plants
• Contents
• Chapter 1 Protoplast — Isolation and Cell Regeneration
• 2. Cell Modification
• 3. Cell Fusion
• 4. Cell Transformation
• 5. Cell Preservation
• Index