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Plant Tissue Culture

Plant Tissue Culture

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Published by: rednri on Jan 11, 2011
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Plant Cells
A plant cell is bounded by a cell wall and the living portion of the cell is within the walls and is divided into two portions: the nucleus, or central control center; and the cytoplasm, a fluid in which membrane bound organelles arefound. Between the primary cell walls of adjacent plant cells, lies a pectic middle lamella. There can be a secondary cellwall which would be located just to the inside of the primary wall. Both walls consist mainly of cellulose, but thesecondary cell wall may contain lignin and other substances. The outer boundary of the protoplasm (cytoplasm andnucleus) is a sandwich-like, flexible plasma membrane. This membrane regulates what enters and leaves the plant cell.Pla nt cell organelles include: endoplasmic reticulum, with and without ribosomes attached; Golgi bodies, mitochondria,and plastids. Plastids are chloroplasts, chromoplasts or leucoplasts—depending on the color and likewise the function.Chloroplasts are of specific interest to those studying plants. A plant cell also, obviously, contains a nucleus which is bounded by a nuclear envelope with pores. The pores in the nuclear envelope allow for movement of substances in andout of the nucleus. Within the nucleus is a number of chromosomes. The number present is specific to the organism andit will be later noted how sex cells contain one-half the number of chromosomes, and restore chromosome number uponfertilization. All of these organelles and the nucleus are suspended in the cytoplasm. The cytoplasm has movements thatare referred to as cytoplasmic streaming or cyclosis. The particular function of the other organelles contained in plantcells can be reviewed below:1. The
nucleus
is in the center of most cells. Some cells contain multiple nuclei, such as skeletal muscle, whilesome do not have any, such as red blood cells. The nucleus is the largest membrane-bound organelle.Specifically, it is responsible for storing and transmitting genetic information. The nucleus is surrounded by aselective nuclear envelope. The nuclear envelope is composed of two membranes joined at regular intervals toform circular openings called nuclear pores. The pores allow RNA molecules and proteins modulating DNAexpression tomove through the pores and into the cytosol. The selection process is controlled by an energy-dependent processthat alters the diameter of the pores in response to signals. Inside the nucleus, DNA and proteins associate toform a network of threads called chromatin. The chromatin becomes vital at the time of cell division as it becomes tightly condensed thus forming the rodlike chromosomes with the enmeshed DNA. Inside the nucleusis a filamentous region called the nucleolus. This serves as a site where the RNA and protein components of ribosomes are assembled. The nucleolus is not membrane bound, but rather just a region.2.
Ribosomes
are the sites where protein molecules are synthesized from amino acids. They are composed of  proteins and RNA. Some ribosomes are found bound to granular endoplasmic reticulum, while others are freein the cytoplasm. The proteins synthesized on ribosomes bound to granular endoplasmic reticulum aretransferred from the lumen (open space inside endoplasmic reticulum) to the golgi apparatus for secretionoutside the cell or distribution to other organelles. The proteins that are synthesized of free ribosomes arereleased into the cytosol.3. The
endoplasmic reticulum
(ER) is collectively a network of membranes enclosing a singular continuous space.As mentioned earlier, granular endoplasmic reticulum is associated with ribosomes (giving the exterior surface arough, or granular appearance). Sometimes granular endoplasmic reticulum is referred to as rough ER. Thegranular ER is involved in packaging proteins for the golgi apparatus. The agranular, or smooth, ER lacksribosomes and is the site of lipid synthesis. In addition, the agranular ER stores and releases calcium ions (Ca
2+
).4. The
golgi apparatus
is a membranous sac that serves to modify and sort proteins into secretory/transport vesicles.The vesicles are then delivered to other cell organelles and the plasma membrane. Most cells have at leastone golgi apparatus, although some may have multiple. The apparatus is usually located near the nucleus.5.
Endosomes
are membrane-bound tubular and vesicular structures located between the plasma membrane andthe golgi apparatus. They serve to sort and direct vesicular traffic by pinching off vesicles or fusing with them.6.
Mitochondria
are some of the most important structures in the cell. They are they site of various chemical processes involved in the synthesis of energy packets called ATP (adenosine triphosphate). Each mitochondrionis surrounded by two membranes. The outer membrane is smooth, while the inner one is folded into tubulestructures called cristae. Mitochondria are unique in that they contain small amounts of DNA containing the genesfor the synthesis of some mitochondrial proteins. The DNA is inherited solely from the mother. Cells withgreater activity have more mitochondria, while those that are less active have less need for energy producingmitochondria.
 
7.
Lysosomes
are bound by a single membrane and contain highly acidic fluid. The fluid acts as digestingenzymes for breaking down bacteria and cell debris. They play an important from in the cells of the immunesystem.8.
Peroxisomes
are also bound by a single membrane. They consume oxygen and work to drive reactions thatremove hydrogen from various molecules in the form of hydrogen peroxide. They are important inmaintaining the chemical balances within the cell.9. The
cytoskeleton
is a filamentous network of proteins that are associated with the processes that maintain andchange cell shape and produce cell movements in animal and bacteria cells. In plants, it is responsible for maintaining structures within the plant cell, rather then whole cell movement. The cytoskeleton also formstracks along which cell organelles move propelled by contractile proteins attached to their various surfaces.Like a little highway infrastructure inside the cell.
Three types of filaments
make up the cytoskeleton.1.
Microfilaments
are the thinnest and most abundant of the cytoskeleton proteins. They are composed of actin, a contractile protein, and can be assembled and disassembled quickly according to the needs of the cell or organelle structure.2.
Intermediate filaments
are slightly larger in diameter and are found most extensively in regions of cells that are going to be subjected to stress. Once these filaments are assembled they are not capable of rapid disassembly.3.
Microtubules
are hollow tubes composed of a protein called tubulin. They are the thickest and most rigidof the filaments. Microtubules are present in the axons and long dendrite projections of nerve cells.They are capable of rapid assembly and disassembly according to need. Microtubules are structuredaround a cell region called the centrosome, which surrounds two centrioles composed of 9 sets of fusedmicrotubules. These are important in cell division when the centrosome generates the microtubluar spindle fibers necessary for chromosome separation.10.
Chloroplasts
It is necessary to note a bit about the form of chloroplasts, as you will encounter them throughout this tutorial.Inside a chloroplast is a matrix called the stroma. Enzymes are found in the stroma as well as grana—stacksof coin-shaped discs, called thylakoids. It is within the thylakoids that photosynthesis takes place. Note thatchloroplasts, like mitochondria contain their own DNA. They do rely on proteins from the nucleus, and areconsidered semi-autonomous organelles. Photosynthesis will be discussed in greater detail in the PlantMetabolism tutorial.11.
Vacuoles
Plant cells are also notorious for having huge vacuoles. Up to 90% of the volume of a mature cell may be taken up by a single large vacuole or several vacuoles. The vacuole is bound by a special membrane, called thetonoplast, and contains cell sap—which is composed of dissolved substances and may include pigments.
PLANT TISSUE CULTUREINTRODUCTION
Most methods of plant transformation applied to genetically modified crops require that a whole plant is regeneratedfrom isolated plant cells or tissues that have been genetically trans- formed. This regeneration is conducted in vitro sothat the environment and growth medium can be manipulated to ensure a high frequency of regeneration. In addition tothis, the re- generable cells must be accessible to gene transfer by whatever technique is chosen (gene- transfer methods are described in Chapter 3). The primary aim is therefore to produce, as easily and as quickly as possible, alarge number of regenerable cells that are accessible to gene transfer. The subsequent regeneration step is often themost difficult step in plant transformation studies. However, it is important to remember that a high frequency of regeneration does not necessarily correlate with high transformation efficiency.This chapter will consider some basic issues concerned with plant tissue culture in vitro, particularly as applied to plant transformation. It will also look at the basic culture types used for plant transformation and cover some of thetechniques that can be used to regenerate whole transformed plants from transformed cells or tissue.
 
Plant tissue culture
Practically any plant transformation experiment relies at some point on tissue culture. There are some exceptions tothis generalization (some of which will be looked at in Chapter 3), but the ability to regenerate plants from isolatedcells or tissues in vitro underpins most plant transformation systems.
Plasticity and totipotency
Two concepts, plasticity and totipotency, are central to understanding plant cell culture and regeneration. Plants, dueto their sessile nature and long life span, have developed a greater ability to endure extreme conditions and predationthan have animals. Many of the processes involved in plant growth and development adapt to environmentalconditions. This plasticity allows plants to alter their metabolism, growth, and development to best suit their environment. Particularly important aspects of this adaptation, as far as plant tissue culture and regeneration areconcerned, are the abilities to initiate cell division from almost any tissue of the plant and to regenerate lost organs or undergo dif ferent developmental pathways in response to particular stimuli. When plant cells and tissues are cultured invitro they generally exhibit a very high degree of plasticity, which allows one type of tissue or organ to be initiatedfrom another type. In this way, whole plants can be subsequently regenerated.This regeneration of whole organisms depends upon the concept that all plant cells can, given the correct stimuli,express the total genetic potential of the parent plant. This maintenance of genetic potential is called totipotency. Plantcell culture and re- generation do, in fact, provide the most compelling evidence for totipotency. In practical termsthough, identifying the culture conditions and stimuli required to manifest this totipotency can be extremely difficultand it is still a largely empirical process.
Plant cell culture media
Culture media used for the cultivation of plant cells in vitro are composed of three basiccomponents:1. Essential elements, or mineral ions, supplied as a complex mixture of salts;2. An organic supplement supplying vitamins and/or amino acids; and3. A source of fixed carbon; usually supplied as the sugar sucrose.For practical purposes, the essential elements are further divided into the followingcategories:1. Macro elements (or macronutrients);2. Microelements (or micronutrients); and3. An iron source.Complete plant cell culture medium is usually made by combining several different components, as outlined in Table2.2.
Media componentsMacro elements
As is implied by the name, the stock solution supplies macro elements required in large amounts for plant growthand development. Nitrogen, phosphorus, potassium, magnesium, calcium, and sulphur (and carbon, which is addedseparately) are usually regarded as macro elements. These elements usually comprise at least 0.1% of the dry weight of  plants. Nitrogen is most commonly supplied as a mixture of nitrate ions (from KNO3) and ammonium ions (from NH4NO3).Theoretically, there is an advantage in supplying nitrogen in the form of ammonium ions, as nitrogen must be in thereduced form to be incorporated into macromolecules. Nitrate ions therefore need to be reduced beforeincorporation. However, at high concentrations, ammonium ions can be toxic to plant cell cultures and uptake of 

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