Algae, Tree, Herbs, Bush, Shrub, Grasses, Vines, Fern, Moss, Spermatophyta, Bryophyta, Fern Ally, Flower, Photosynthesis, Eukaryote, Prokaryote, carbohydrate, vitamins, amino acids, botany, lipids, proteins, cell, cell wall, biotechnology, metabolities, enzymes, agriculture, horticulture, agronomy, bryology, plaleobotany, phytochemistry, enthnobotany, anatomy, ecology, plant breeding, ecology, genetics, chlorophyll, chloroplast, gymnosperms, sporophytes, spores, seed, pollination, pollen, agriculture, horticulture, taxanomy, fungi, molecular biology, biochemistry, bioinfomatics, microbiology, fertilizers, insecticides, pesticides, herbicides, plant growth regulators, medicinal plants, herbal medicines, chemistry, cytogenetics, bryology, ethnobotany, plant pathology, methodolgy, research institutes, scientific journals, companies, farmer, scientists, plant nutrition
Select Language:
 
 
 
 
Main Menu
Please click the main subject to get the list of sub-categories
 
Services offered
 
 
 
 
  Section: General Biotechnology / Plant Biotechnology
 
 
Please share with your friends:  
 
 

In Vitro Culture Techniques : The Biotechnological Principles

 
     
 
Protoplast Culture
Isolation of protoplasts

Protoplasts (cell minus cell wall) is the biologically active and most significant material of cells. When cell wall is mechanically or enzymatically removed the isolated protoplast is known as "naked plant cell" on which most of recent researches are based.
 

Plant cell wall acts as physical barrier and protects cytoplasm from microbial invasion and environmental stress. It consists of a complex mixture of cellulose, hemicellulose, pectin, lignin, lipids, protein, etc. For dissolution of different components of the cell wall it is essential to have the respective enzymes.
 

Until suitable methods were developed, protoplasts were isolated by cutting the plasmolysed plant tissues and releasing protoplast through deplasmolysis of cells. Cooking (1960) for the first time isolated the protoplasts of plant tissues by using cell wall degrading enzymes viz., cellulase, hemicellulase, pectinase, and protease extracted from a saprophytic fungus Trichoderma viride. Later on protoplasts were cultured in vitro. 

Microorganisms are well equipped with a system to produce substrate specific extracellular enzymes, the extent of which depends on the genetic variability of the specific species and strains. A detailed account of enzyme preparation and their uses in isolation of protoplast has been given by Cooking (1972 ); Bajaj (1977b) and Patnaik et al (1981). However, the basic techniques of isolation and culture of protoplast are given in Figs. 8.5. and 8.6 with a brief description.

(i)

Surface Sterilization of Leaf Samples : Mature leaves are collected from healthy plants which are washed in tap water to remove adhering soil particles and sterilized with sodium hypochlorite solution.

(ii)

Rinsing in Suitable Osmoticum : After 10 min, sample is properly washed with sterile distilled water or MS medium adjusted to a suitable pH and buffer to maintain osmotic pressure. Washing should be done for about 6 times to remove the traces of sodium hypochlorite.

(iii)

Plasmolysis of Cells : The lower epidermis covered by thin wax cuticle is removed with a forcep. Stripping should be done from midrib to margin of lamina. The stripped surface of leaf is kept in mannitol solution (13% W/V) for 3 hours to allow plasmolysis of cells.

(iv)

Peeling of Lower Epidermis : Thereafter, about 1 gm leaves are peeled off and transferred into enzyme mixture already sterilized through a Seitz filter (0.45 mm). This facilitates the penetration of enzyme into tissue within 12-18 hours at 25°C.

(v)

Isolation and Purification of Portoplasts : Leaf debris are removed with forcep, and enzyme solution containing protoplasts are filtered with a nylon mesh (45mm). Filtrate is centrifuged at 75 X g for 5 min and supernatant is decanted. Again a fresh MS medium plus 13% mannitol is added to centrifuge. Repeated washing with nutrient medium, centrifugation and decantation are done for about three time. Finally specific concentration of protoplast suspension is prepared.
 

Content

Totipotency

Historical background

Requirements for cell and Tissue Cultures

 

A tissues culture laboratory

 

Nutrient media

 

 

Inorganic chemicals

 

 

Growth hormones

 

 

Organic constitutents

 

 

Vitamins

 

 

Amino acids

Culture of plant materials

 

Explant culture

 

Callus formation and its culture

 

Organogenesis

 

Root culture

 

Shoot culture and micropropagation

 

Cell culture

 

 

Benefits from cell culture

 

Somatic embryogenesis

 

Somaclonal variation

 

Protoplast culture

 

 

Isolation

 

 

Regeneration

 

Protoplast fusion and somatic hybridization

 

 

Fusion products

 

 

Method of somatic hybridization

 

Anther and pollen Culture

 

 

Culturing techniques

 

In vitro androgenesis (direct and indirect androgenesis)

 

Mentor pollen technology

 

Embryo culture

 

Embryo rescue

 

Protoplast fusion in fungi


Protoplast culture and regeneration

From the protoplast solution of known density (about 105 protoplast/ml) about 1 ml suspension is poured on sterile and cooled down nutrient medium in Petri dishes. The plates are incubated at 25°C in a dim white light.

The protoplasts regenerate a cell wall, undergo cell division and form callus. The callus can also be subcultured. Embryogenesis begins from callus when it is placed on nutrient medium lacking mannitol and auxin. The embryo develops into seedlings and finally mature plants.

 
     
 
 
     



     
 
Copyrights 2012 © Biocyclopedia.com | Disclaimer