53)Genetic engineering. Methods of genetic transformation

Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome using biotechnology. (Indirect genetic modification through artificial selection has been practiced for centuries.) New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism. Genes may be removed, or "knocked out", using a nuclease. Gene targeting is a different technique that uses homologous recombinationto change an endogenous gene, and can be used to delete a gene, remove exons, add a gene, or introduce point mutations.

55 The methods of cultivation of plant protoplast. Protoplasts are plant cells without walls. Protoplasts are most easily isolated by enzymatically digesting the cell wall. The general procedure is: (1) surface sterilize the leaf; (2) rinse the tissue in the proper osmotic agent such as sorbitol or mannitol. Protoplasts must be maintained in an isotonic medium so they don't burst; (3) cut the leaf into strips or peel the epidermis to expose the tissue for enzymatic digestion; (4) treat with enzymes (sequentially or mixed). Typically, a combination of cellulase, pectinase and hemicellulase, is used. The specific amount and type of enzyme used is generally a result of trial and error. Cellulysin and Meicelase are trade names for cellulase and pectinase, respectively; (5) rinse the preparation to remove the enzymes; and (6) isolate and purify the protoplasts. An alternative method to isolate protoplasts is to mechanically chop up the plant tissue in isotonic medium. Protoplasts can be ruptured in a variety of ways to release the cell contents. Three commonly used methods include: (1) mechanical shear; (2) osmotic shock; and (3) mild detergent. Each method has specific advantages and disadvantages and the method selected depends upon the application. Protoplasts are cultured in either liquid or agar solidified nutrient media. The culture media are very similar in composition to those required for the in vitro culture of cells, but with the addition of an osmotic stabilizer to prevent bursting. Early stages of wall synthesis are preceded by extensive infoldings of the plasmalemma together with an accumulation of pectin-like substances in vesicles found in the peripheral layer of cytoplasm. These early stages of wall synthesis, detected after 18 hours, are unaffected by the presence, in the culture medium, of protein synthesis inhibitors, suggesting that synthesis of new RNA or protein is not required for wall initiation and that residual protein and endogenous hormone levels are sufficient. Structurally, the first formed envelope is amorphous and consists of pectins, but after a few days a second inner layer of cellulose fibrils is progressively laid down on the protoplast surface, eventually producing a near normal cellulose matrix after four or five days. Nuclear division and cytokinesis is concomitant with cell wall formation. Occasionally cytokinesis does not occur, perhaps due to the presence of an incomplete cell wall, and gives rise to binucleate cells which may not be capable of further division. The nutritional and hormonal requirements of cultured protoplasts are constantly varying depending upon the stage in the regeneration process. The photosynthetic capacity and respiration rate of the protoplast is suppressed by the plasmolysing conditions. Following cell wall regeneration and division, the requirement for exogenous growth regulator supply is lost.