![]() A high kinetin/Auxin ratio is preferred for regeneration.A high Auxin/kinetin ratio is preferred to induce cell division.Glucose is the preferred carbon source for protoplast culture.The Calcium concentration is 2 to 4 times higher than the ordinary cell culture.Very fewer quantities of iron and zinc are used.The culture medium should not contain ammonium.It is similar to those used in callus culture EXCEPT: Nutritional Requirements of Culture media Osmotic pressure can be easily changed in a liquid medium.Dilutions can be easily done in liquid culture.Protoplast is generally culture in semi-solid Agar medium or Liquid medium.Īdvantages of liquid culture over Solid culture The development of the cell wall is followed by cell division to form small colonies. Viability test of protoplast: The following are the methods used to test the viability of protoplastĪ) Testing photosynthetic activity of protoplastī) Measurements of cell wall formation can be observed using Calcofluor white (CFW) stain, which bind to the newly formed cell wall and emits fluorescence.Ĭ) Measurement of oxygen uptake by protoplast by using oxygen electrodeĭ) Phenosafranine stain: dead protoplast uptake this stain and turns red, whereas viable protoplast remains unstained.Į) Fluorescein diacetate (FDA) staining: viable cells get stained by the FDA and can be detected by fluorescence microscopy.Ĭell wall development around the protoplast membrane is the prior step of protoplast culture. Purification of protoplast: It is done by filtration to remove cell clumps and undigested tissues, followed by centrifugation and washing of protoplast.Ĥ. One-step method: in this method the tissue is treated simultaneously with cellulose and pectinase.These free cells are treated with cellulose to remove the cell wall, which results in the release of protoplast. Two-step method: in this method the tissue is treated with pectinase to degrade middle lamella, to separate cells from tissue.One of the two methods is used to isolate protoplast by enzymatic method. Enzymes like cellulases, hemicellulases, pectinases, are used to degrade cellulose, hemicellulose, and pectin in the plant cell wall. This process is tedious and laborious.ī) Enzymatic method: This method has an advantage over the mechanical method, which has a high yield of protoplasts with viable cells. The main disadvantage of this method that, the yield and viability of protoplast is very low. Then, the protoplast is released by tissue dissection. Isolation of protoplast: It can be done by two methodsĪ) Mechanical method: In this method, the cells of the epidermis are subjected to plasmolysis, by which protoplast causes to shrink away from the cell wall. Callus and suspension cultures are also good sources for protoplast isolation.Ģ. Source of protoplast: Tissues and organs like leaves, roots, shoot apices, fruits, embryos and microspores are widely used as sources for protoplast. Hence, this protoplast is a functional plant cell without a barrier i.e. This protoplast possesses other cellular components and a plasma membrane. Protoplast is the plant cell without a cell wall. Admission), Pharmacognosy, Pharmacy Exam Questions, Plant biotechnology, Quiz, Study Material, Successfull GPAT Indian, UGC NET JRF Exam applications for Protoplast culture, applications of protoplast, Culture of protoplasts, Isolation of protoplast, MCQ for Protoplast culture for CSIR NET, MCQ of Protoplast culture for GPAT, Protoplast, protoplast culture, protoplast culture MCQ for GATE, Protoplast culture media, protoplast culture methods, Purification of protoplast, regeneration of protoplast, Sources of protoplast, steps involved in protoplast culture, Viability check of protoplasts What is Protoplast? Octojagir.apc Biotechnology, GATE Exam, GPAT Lectures, GPAT Preparation, How to prepare for gpat, MCQ, NEET PG, NEET UG, NIPER JEE Examination (Masters/Ph.D.
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