BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 3. CELL ENGINEERING

Plant Cell protoplasts as an object of biological engineering

A protoplast is a cell stripped of its Cell wall. Enclosed by The Plasma Membrane, it retains all the essential properties of a plant cell.

Plant protoplasts were first isolated mechanically in 1892 by J. Klercker, who cut open The Cell wall. In 1952, Salton pioneered the enzymatic method of cell wall degradation by using the enzyme Lysozyme to break down bacterial cell walls. In 1960, E. Cocking successfully obtained protoplasts of higher plants for the first time using fungal hydrolytic Enzymes from Myrothecium verrucaria that cleave cell wall components.

Modern research employs three MAIN TYPES OF enzymes to remove the cell wall: cellulases, hemicellulases, and pectinases. The specific combination and ratio of these enzymes depend on the cell type. Compared to mechanical isolation, the enzymatic method offers several distinct advantages: high yield (up to 10 million protoplasts can be isolated from 1 g of tissue or Cells), speed, and minimal cell damage.

Propotlasts serve as a unique model for studying plant physiological processes. Isolated protoplasts are utilized for the following purposes:

- investigating the chemistry and Structure OF THE cell wall, along with the mechanisms of its repair following disruption;

- examining The properties of the Plasmalemma under normal conditions and under METABOLISM/18.html">The Influence of Hormones, phytotoxins, and inhibitors;

- studying transmembrane transport;

- analyzing protoplast interactions within a population;

- introducing foreign Organelles or genes into plant cells;

- exploring Nucleus-Cytoplasm interactions in hybrid cells;

- generating somatic hybrids.

Protoplasts enable the creation of cell hybrids, thereby overcoming incompatibility barriers between distantly related plant species and genera. The fusion of protoplasts from two different plant species produces a heterokaryon, which subsequently develops into a somatic hybrid. The first interspecific hybrid was produced in 1972 by Carlson through the protoplast Hybridization of two tobacco varieties, Nicotiana glauca (24 Chromosomes) and Nicotiana langsdorfii (18 chromosomes). Protoplast hybridization between cultivated and wild potato (Solanum tuberosum and Solanum chacoense) has yielded virus-resistant hybrids. Numerous viable intraspecific, interspecific, and intergeneric hybrids have since been successfully obtained. In 1977, Kao and Wetter generated a soybean-tobacco hybrid. In 1978, G. Melchers produced a potato-tomato hybrid, although it proved to be sterile. In 1997, Japanese scientist H. Kisaka created an intergeneric hybrid between barley and rice.

Protoplast fusion has also enabled the creation of plant-animal cell hybrids. For instance, Edward Cocking obtained a heterokaryon formed by an amphibian cell and a carrot protoplast. Researchers have likewise succeeded in producing hybrid cells from tobacco and Drosophila. However, these hybrid cells lacked the capacity for division. Protoplasts are also widely used as recipients for cellular organelles. Organelles can be transplanted using Liposomes or microinjection. In 1973, I. Potrykus and F. Hoffmann transplanted isolated petunia nuclei into tobacco protoplasts. Alongside nuclei, Mitochondria and Chloroplasts can also be transplanted. Chloroplast transplantation can potentially enhance Photosynthesis and increase plant productivity, whereas mitochondrial transplantation can stimulate tissue Respiration.