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.

54. Methods of receiving plant materials without viruses

Production of virus Free Plants

„ Tissue culture is an excellent tool for multiplying, maintaining, storing and distributing plants. Maintaining unique plants for breeding, propagation, or distribution can be expensive in terms of the time, space and labor required. In--vitro propagation of apical meristems is an important part of virus--elimination therapy for improving the health of plant collections. „ Distribution of tissue cultured germplasm often assists breeders and nurseries in meeting quarantine regulations.

Germplasm may be a source of genes for improving such traits as insect and disease resistance, improved quality, drought or cold tolerance, increased yields, low chilling requirements, adaptation to mechanical harvesting or longer shelf life in distribution and retail channels. Foreign pathogens continue to pose a considerable threat to crops. Prevent introduction of quarantine pathogens in imported prohibited germplasm using a range of diagnostic techniques to intercept them.Develop improved methods of detecting quarantine pathogens and investigate the etiology of poorly described diseases and pathogens of quarantine significance. Eliminate quarantine pathogens from valuable plant germplasm. Complete therapy on sweet potatoes infected with geminiviruses. Provide tissues from in vitro therapy for PCR detection of geminiviruses in sweet potatoes.

Mechanism of virus Elimination in meristems

„ Failure to invade meristem is due to: 1. High auxin concentration in meristematic cells 2. Competition for nutrients enzymes for virus replication 3. Active metabolic process which is not suitable for virus multiplication 4. Action of growth regulators (Cytokinins) 5. Presence of inhibitors (Phenolamines) 6. Metabolic disruption of enzymes necessary for viral replication, RNA degradation

Virus elimination through heat treatment

Preparation for therapy: surface sterilization & culture establishment

Temperature treatment: Three weeks after subculture in vitro shoots are transferred to a special growth chamber and grown at an increased temperature regime for 14 to 21 days.

Regeneration and indexing: Regeneration of small plantlets occurs within a few weeks. After indexing virus free plants selected and multiplied further.

Acclimatization: Plantlets are rooted in vitro and transferred to the greenhouse. After careful hardening potted plants are re-tested for their pathogen-free status.

Multiplication of Virus Free Plants:For budwood production plants are transferred to an insect proof screenhouse.

Thermotherapy High temperature treatment has been widely used in production of

virus free plants (30 to 40C).

Chemotherapy The use of chemicals to suppress virus symptoms and multiplication in infected plants

„ Use of antiviral compounds-- Ribavirin/Virazole, DTHGrowth promoting chemicals--cytokinins„ Antimetabolite chemicals--Azaguanine, Thiouracil.

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.