Genetics Lab Report (Suvekshya Shrestha) (REVISED).docx -...

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Assessment of Expression Patterns of pCNT103 , cig1 , and gapC in Nicotiana tabacum Tissue through Tissue Culture, DNA and RNA Extraction, RT - PCR and Gel Electrophoresis Suvekshya Shrestha April 22, 2020 Genetics Laboratory [BIOL 332L Spring 2020] Professor Jose Mediavilla Ramapo College of New Jersey
Abstract: The main objective of the molecular genetics lab experiments was to study the expression patterns in differentiated tissues by extracting, replicating and examining DNA of Nicotina tabacum callus using various molecular DNA techniques. The cells from which DNA was extracted to be analyzed was prepared using a sterile technique to differentiate a callus tissue of the plant. After extraction of the DNA, its quantity was amplified through PCR and RT-PCR using three different types of primers which were specific for genes expected to be over- expressed in either root or shoot tissue or expressed equally in both tissues after induction with plant hormones, followed by an agarose gel electrophoresis. The migrated fragments of amplified DNA and RNA were captured by an UV image of the gel. The UV image suggested that the tobacco root when treated with pCNT103, which is an auxin induced gene, promotes the growth of roots forming an intense band in the UV image of the gel electrophoresis. Also, as all the samples were tobacco roots, except for cig1 primer which promotes the growth of shoots, gapC primer and pCNT103 primer were almost equally expressed. Introduction: Hormones are chemical signals that coordinate the different parts of an organism. These chemicals are produced in very small amounts in one area of an organism and are then sent to another part, where a response is triggered. Even though plants seem unresponsive as compared to animals, they also have hormones that control their physiological activities. The plant hormones are produced by cells in one area of the plant - such as the leaves, stems or root, and then transported to a different area of the plant to produce a response. There are 5 main plant hormones: auxins, cytokinins, gibberellins, ethylene, and abscisic acid (1). Auxins and cytokinins are plant growth hormones which stimulate the growth of plant roots and shoots respectively, by
encouraging the expression of one gene over another in a plant’s callus cells. A plant callus is a collection of living plant cells that are undifferentiated, analogous to the stem cells in animals. The plant callus cells have the ability to differentiate into different kinds of cells with completely different functions when certain signals are given (2). Auxins or cytokinins can be added to the growth medium of the culture and will cause the callus cells to differentiate into plant roots or shoots (3). In these molecular genetics experiments, cells from the Nicotina tabacum plant, commonly called tobacco, were cultured using media with differing plant hormone concentrations. Tissue culture is a method of breeding cells from an organism in a laboratory and is assisted through the use of a growth medium such as agar. Tissue cultures allow for the

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