![Protoplast fusion](https://upload.wikimedia.org/wikipedia/commons/f/f8/Protoplast_fusion.jpg)
Somatic fusion, also called protoplast fusion, is a type of
genetic modification
Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including t ...
in plants by which two distinct species of plants are fused together to form a new
hybrid
Hybrid may refer to:
Science
* Hybrid (biology), an offspring resulting from cross-breeding
** Hybrid grape, grape varieties produced by cross-breeding two ''Vitis'' species
** Hybridity, the property of a hybrid plant which is a union of two dif ...
plant with the characteristics of both, a somatic hybrid. Hybrids have been produced either between different varieties of the same species (e.g. between non-flowering
potato
The potato is a starchy food, a tuber of the plant ''Solanum tuberosum'' and is a root vegetable native to the Americas. The plant is a perennial in the nightshade family Solanaceae.
Wild potato species can be found from the southern Unit ...
plants and flowering potato plants) or between two different species (e.g. between wheat ''
Triticum
Wheat is a grass widely cultivated for its seed, a cereal grain that is a worldwide staple food. The many species of wheat together make up the genus ''Triticum'' ; the most widely grown is common wheat (''T. aestivum''). The archaeologica ...
'' and rye ''
Secale
''Secale'' is a genus of the grass tribe Triticeae, which is related to barley (''Hordeum'') and wheat (''Triticum''). The genus includes cultivated species such as rye (''Secale cereale'') as well as weedy and wild rye species. The most well kn ...
'' to produce
Triticale
Triticale (; × ''Triticosecale'') is a hybrid of wheat (''Triticum'') and rye (''Secale'') first bred in laboratories during the late 19th century in Scotland and Germany. Commercially available triticale is almost always a second-generation h ...
).
Uses of somatic fusion include making potato plants resistant to
potato leaf roll disease. Through somatic fusion, the crop potato plant ''Solanum tuberosum'' – the yield of which is severely reduced by a viral disease transmitted on by the aphid
vector
Vector most often refers to:
*Euclidean vector, a quantity with a magnitude and a direction
*Vector (epidemiology), an agent that carries and transmits an infectious pathogen into another living organism
Vector may also refer to:
Mathematic ...
– is fused with the wild, non-tuber-bearing potato ''Solanum brevidens'', which is resistant to the disease. The resulting hybrid has the chromosomes of both plants and is thus similar to
polyploid
Polyploidy is a condition in which the cells of an organism have more than one pair of ( homologous) chromosomes. Most species whose cells have nuclei ( eukaryotes) are diploid, meaning they have two sets of chromosomes, where each set contain ...
plants.
Somatic hybridization was first introduced by Carlson et al. in ''Nicotiana glauca''.
Process for plant cells
The somatic fusion process occurs in four steps:
# The removal of the cell wall of one cell of each type of plant using
cellulase
Cellulase (EC 3.2.1.4; systematic name 4-β-D-glucan 4-glucanohydrolase) is any of several enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze cellulolysis, the decomposition of cellulose and of some related polysaccharide ...
enzyme to produce a
somatic cell
A somatic cell (from Ancient Greek σῶμα ''sôma'', meaning "body"), or vegetal cell, is any biological cell forming the body of a multicellular organism other than a gamete, germ cell, gametocyte or undifferentiated stem cell. Such cells compo ...
called a
protoplast
Protoplast (), is a biological term coined by Hanstein in 1880 to refer to the entire cell, excluding the cell wall. Protoplasts can be generated by stripping the cell wall from plant, bacterial, or fungal cells by mechanical, chemical or enzy ...
# The cells are then fused using electric shock (electrofusion) or chemical treatment to join the cells and fuse together the nuclei. The resulting fused
nucleus
Nucleus ( : nuclei) is a Latin word for the seed inside a fruit. It most often refers to:
*Atomic nucleus, the very dense central region of an atom
*Cell nucleus, a central organelle of a eukaryotic cell, containing most of the cell's DNA
Nucle ...
is called
heterokaryon
A heterokaryon is a multinucleate cell that contains genetically different nuclei. Heterokaryotic and heterokaryosis are derived terms. This is a special type of syncytium. This can occur naturally, such as in the mycelium of fungi during sexual ...
.
# The formation of the cell wall is then induced using
hormone
A hormone (from the Greek participle , "setting in motion") is a class of signaling molecules in multicellular organisms that are sent to distant organs by complex biological processes to regulate physiology and behavior. Hormones are required ...
s
# The cells are then grown into
calluses
A callus is an area of thickened and sometimes hardened skin that forms as a response to repeated friction, pressure, or other irritation. Since repeated contact is required, calluses are most often found on the feet and hands, but they may o ...
which then are further grown to
plantlet
A plantlet is a young or small plant."Plantlet", Merriam-Webster'
Many plants such as spider plants naturally create stolons with plantlets on the ends as a form of asexual reproduction. Vegetative propagules or clippings of mature plants may ...
s and finally to a full plant, known as a somatic hybrid.
The procedure for seed plants describe above, fusion of
moss
Mosses are small, non-vascular flowerless plants in the taxonomic division Bryophyta (, ) '' sensu stricto''. Bryophyta (''sensu lato'', Schimp. 1879) may also refer to the parent group bryophytes, which comprise liverworts, mosses, and hor ...
protoplasts can be initiated without electric shock but by the use of
polyethylene glycol
Polyethylene glycol (PEG; ) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular we ...
(PEG). Further, moss protoplasts do not need
phytohormone
Plant hormone (or phytohormones) are signal molecules, produced within plants, that occur in extremely low concentrations. Plant hormones control all aspects of plant growth and development, from embryogenesis, the regulation of organ size, pat ...
s for
regeneration
Regeneration may refer to:
Science and technology
* Regeneration (biology), the ability to recreate lost or damaged cells, tissues, organs and limbs
* Regeneration (ecology), the ability of ecosystems to regenerate biomass, using photosynthesis
...
, and they do not form a
callus
A callus is an area of thickened and sometimes hardened skin that forms as a response to repeated friction, pressure, or other irritation. Since repeated contact is required, calluses are most often found on the feet and hands, but they may o ...
. Instead, regenerating moss protoplasts behave like
germinating
Germination is the process by which an organism grows from a seed or spore. The term is applied to the sprouting of a seedling from a seed of an angiosperm or gymnosperm, the growth of a sporeling from a spore, such as the spores of fungi, fern ...
moss
spore
In biology, a spore is a unit of sexual or asexual reproduction that may be adapted for dispersal and for survival, often for extended periods of time, in unfavourable conditions. Spores form part of the life cycles of many plants, algae, f ...
s. Of further note
sodium nitrate
Sodium nitrate is the chemical compound with the formula . This alkali metal nitrate salt is also known as Chile saltpeter (large deposits of which were historically mined in Chile) to distinguish it from ordinary saltpeter, potassium nitrate. T ...
and calcium ion at high pH can be used, although results are variable depending on the organism.
[Mahesh. Plant Molecular biotechnology. 2009. Book.]
Applications of hybrid cells
Somatic cell
A somatic cell (from Ancient Greek σῶμα ''sôma'', meaning "body"), or vegetal cell, is any biological cell forming the body of a multicellular organism other than a gamete, germ cell, gametocyte or undifferentiated stem cell. Such cells compo ...
s of different types can be fused to obtain hybrid cells. Hybrid cells are useful in a variety of ways, e.g.,
:(i) to study the control of
cell division
Cell division is the process by which a parent cell (biology), cell divides into two daughter cells. Cell division usually occurs as part of a larger cell cycle in which the cell grows and replicates its chromosome(s) before dividing. In eukar ...
and
gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...
,
:(ii) to investigate
malignant transformation
Malignant transformation is the process by which cells acquire the properties of cancer. This may occur as a primary process in normal tissue, or secondarily as ''malignant degeneration'' of a previously existing benign tumor.
Causes
There are ...
s,
:(iii) to obtain
viral replication
Viral replication is the formation of biological viruses during the infection process in the target host cells. Viruses must first get into the cell before viral replication can occur. Through the generation of abundant copies of its genome an ...
,
:(iv) for
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
or
chromosome map
A karyotype is the general appearance of the complete set of metaphase chromosomes in the cells of a species or in an individual organism, mainly including their sizes, numbers, and shapes. Karyotyping is the process by which a karyotype is disce ...
ping and for
:(v) production of
monoclonal antibodies
A monoclonal antibody (mAb, more rarely called moAb) is an antibody produced from a cell Lineage made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell.
Monoclonal antibodies ca ...
by producing
hybridoma
Hybridoma technology is a method for producing large numbers of identical antibodies (also called monoclonal antibodies). This process starts by injecting a mouse (or other mammal) with an antigen that provokes an immune response. A type of white ...
(hybrid cells between an immortalised cell and an
antibody
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
producing
lymphocyte
A lymphocyte is a type of white blood cell (leukocyte) in the immune system of most vertebrates. Lymphocytes include natural killer cells (which function in cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic ad ...
), etc.
Chromosome
A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
mapping through somatic cell hybridization is essentially based on fusion of human and
mouse
A mouse ( : mice) is a small rodent. Characteristically, mice are known to have a pointed snout, small rounded ears, a body-length scaly tail, and a high breeding rate. The best known mouse species is the common house mouse (''Mus musculus' ...
somatic cells. Generally, human
fibrocyte
A fibrocyte is an inactive mesenchymal cell, that is, a cell showing minimal cytoplasm, limited amounts of rough endoplasmic reticulum and lacks biochemical evidence of protein synthesis.
The term ''fibrocyte'' contrasts with the term ''fibrobla ...
s or
leucocytes
White blood cells, also called leukocytes or leucocytes, are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and derived from mult ...
are fused with mouse continuous
cell line
An immortalised cell line is a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. The cell ...
s.
When human and mouse cells (or cells of any two
mammal
Mammals () are a group of vertebrate animals constituting the class Mammalia (), characterized by the presence of mammary glands which in females produce milk for feeding (nursing) their young, a neocortex (a region of the brain), fur or ...
ian species or of the same species) are mixed, spontaneous
cell fusion Cell fusion is an important cellular process in which several uninucleate cells (cells with a single nucleus) combine to form a multinucleate cell, known as a syncytium. Cell fusion occurs during differentiation of myoblasts, osteoclasts and tropho ...
occurs at a very low rate (10-6). Cell fusion is enhanced 100 to 1000 times by the addition of
ultraviolet
Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nanometer, nm (with a corresponding frequency around 30 Hertz, PHz) to 400 nm (750 Hertz, THz), shorter than that of visible light, but longer than ...
inactivated
Sendai
is the capital Cities of Japan, city of Miyagi Prefecture, the largest city in the Tōhoku region. , the city had a population of 1,091,407 in 525,828 households, and is one of Japan's 20 Cities designated by government ordinance of Japan, desig ...
(parainfluenza) virus or
polyethylene glycol
Polyethylene glycol (PEG; ) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular we ...
(PEG).
These agents adhere to the
plasma membrane
The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment (t ...
s of cells and alter their properties in such a way that facilitates their fusion. Fusion of two cells produces a heterokaryon, i.e., a single hybrid cell with two nuclei, one from each of the cells entering fusion. Subsequently, the two nuclei also fuse to yield a hybrid cell with a single nucleus.
A generalized scheme for somatic cell hybridization may be described as follows. Appropriate human and mouse cells are selected and mixed together in the presence of inactivated Sendai virus or PEG to promote cell fusion. After a period of time, the cells (a mixture of man, mouse and 'hybrid' cells) are plated on a
selective medium
A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss ''Physcomitrella patens''. Different ...
, e.g.,
HAT medium
HAT Medium (hypoxanthine-aminopterin-thymidine medium) is a selection medium for mammalian cell culture, which relies on the combination of aminopterin, a drug that acts as a powerful folate metabolism inhibitor by inhibiting dihydrofolate reducta ...
, which allows the
multiplication
Multiplication (often denoted by the cross symbol , by the mid-line dot operator , by juxtaposition, or, on computers, by an asterisk ) is one of the four elementary mathematical operations of arithmetic, with the other ones being additi ...
of hybrid cells only.
Several
clones (each derived from a single hybrid cell) of the hybrid cells are thus isolated and subjected to both
cytogenetic
Cytogenetics is essentially a branch of genetics, but is also a part of cell biology/cytology (a subdivision of human anatomy), that is concerned with how the chromosomes relate to cell behaviour, particularly to their behaviour during mitosis an ...
and appropriate
biochemical analyses for the detection of
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
/
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
/
trait under investigation. An attempt is now made to correlate the presence and absence of the trait with the presence and absence of a
human chromosome
A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
in the hybrid clones.
If there is a perfect
correlation
In statistics, correlation or dependence is any statistical relationship, whether causal or not, between two random variables or bivariate data. Although in the broadest sense, "correlation" may indicate any type of association, in statistics ...
between the presence and absence of a human chromosome and that of a trait in the hybrid clones, the gene governing the trait is taken to be located in the concerned chromosome.
The HAT medium is one of the several selective media used for the selection of hybrid cells. This medium is supplemented with
hypoxanthine
Hypoxanthine is a naturally occurring purine derivative. It is occasionally found as a constituent of nucleic acids, where it is present in the anticodon of tRNA in the form of its nucleoside inosine. It has a tautomer known as 6-hydroxypurine. Hyp ...
,
aminopterin
Aminopterin (or 4-aminopteroic acid), the 4-amino derivative of folic acid, is an antineoplastic drug with immunosuppressive properties often used in chemotherapy. Aminopterin is a synthetic derivative of pterin. Aminopterin works as an enzyme inh ...
and
thymidine
Thymidine (symbol dT or dThd), also known as deoxythymidine, deoxyribosylthymine, or thymine deoxyriboside, is a pyrimidine deoxynucleoside. Deoxythymidine is the DNA nucleoside T, which pairs with deoxyadenosine (A) in double-stranded DNA. I ...
, hence the name HAT medium.
Antimetabolite
An antimetabolite is a chemical that inhibits the use of a metabolite, which is another chemical that is part of normal metabolism. Such substances are often similar in structure to the metabolite that they interfere with, such as the antifolate ...
aminopterin blocks the cellular
biosynthesis
Biosynthesis is a multi-step, enzyme-catalyzed process where substrates are converted into more complex products in living organisms. In biosynthesis, simple compounds are modified, converted into other compounds, or joined to form macromolecules. ...
of
purine
Purine is a heterocyclic compound, heterocyclic aromatic organic compound that consists of two rings (pyrimidine and imidazole) fused together. It is water-soluble. Purine also gives its name to the wider class of molecules, purines, which includ ...
s and
pyrimidine
Pyrimidine (; ) is an aromatic, heterocyclic, organic compound similar to pyridine (). One of the three diazines (six-membered heterocyclics with two nitrogen atoms in the ring), it has nitrogen atoms at positions 1 and 3 in the ring. The other ...
s from
simple sugar
Monosaccharides (from Greek ''monos'': single, '' sacchar'': sugar), also called simple sugars, are the simplest forms of sugar and the most basic units (monomers) from which all carbohydrates are built.
They are usually colorless, water-solub ...
s and
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
s.
However, normal human and mouse cells can still multiply as they can utilize hypoxanthine and thymidine present in the medium through a
salvage pathway A salvage pathway is a Metabolic pathway, pathway in which a biological product is produced from intermediates in the degradative pathway of its own or a similar substance. The term often refers to nucleotide salvage in particular, in which nucleoti ...
, which ordinarily recycles the purines and pyrimidines produced from degradation of
nucleic acid
Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...
s.
Hypoxanthine is converted into
guanine
Guanine () ( symbol G or Gua) is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine (uracil in RNA). In DNA, guanine is paired with cytosine. The guanine nucleoside is called ...
by the enzyme
hypoxanthine-guanine phosphoribosyltransferase
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is an enzyme encoded in humans by the ''HPRT1'' gene.
HGPRT is a transferase that catalyzes conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. This r ...
(HGPRT), while thymidine is
phosphorylated
In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, whi ...
by
thymidine kinase
Thymidine kinase is an enzyme, a phosphotransferase (a kinase): 2'-deoxythymidine kinase, ATP-thymidine 5'-phosphotransferase, EC 2.7.1.21. It can be found in most living cells. It is present in two forms in mammalian cells, TK1 and TK2. Cert ...
(TK); both HGPRT and TK are enzymes of the salvage pathway.
On a HAT medium, only those cells that have active HGPRT (HGPRT+) and TK (TK+) enzymes can proliferate, while those deficient in these enzymes (HGPRr- and/or TK-) can not divide (since they cannot produce purines and pyrimidines due to the aminopterin present in the HAT medium).
For using HAT medium as a selective agent,
human cell
There are many different types of cells in the human body.
Cells derived primarily from endoderm
Exocrine secretory epithelial cells
* Brunner's gland cell in duodenum (enzymes and alkaline mucus)
*Insulated goblet cell of respiratory and ...
s used for fusion must be deficient for either the enzyme HGPRT or TK, while mouse cells must be deficient for the other enzyme of this pair. Thus, one may fuse HGPRT deficient human cells (designated as TK+ HGPRr-) with TK deficient mouse cells (denoted as TK- HGPRT+).
Their fusion products (hybrid cells) will be TK+ (due to the human
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
) and HGPRT+ (due to the mouse gene) and will multiply on the HAT medium, while the man and mouse cells will fail to do so. Experiments with other selective media can be planned in a similar fashion.
Characteristics of somatic hybridization and cybridization
# Somatic cell fusion appears to be the only means through which two different parental
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
s can be
recombined among plants that cannot
reproduce sexually
Sexual reproduction is a type of reproduction that involves a complex life cycle in which a gamete ( haploid reproductive cells, such as a sperm or egg cell) with a single set of chromosomes combines with another gamete to produce a zygote tha ...
(asexual or sterile).
# Protoplasts of sexually sterile (
haploid
Ploidy () is the number of complete sets of chromosomes in a cell, and hence the number of possible alleles for autosomal and pseudoautosomal genes. Sets of chromosomes refer to the number of maternal and paternal chromosome copies, respectively ...
,
triploid
Polyploidy is a condition in which the cells of an organism have more than one pair of ( homologous) chromosomes. Most species whose cells have nuclei ( eukaryotes) are diploid, meaning they have two sets of chromosomes, where each set contain ...
, and
aneuploid
Aneuploidy is the presence of an abnormal number of chromosomes in a cell, for example a human cell having 45 or 47 chromosomes instead of the usual 46. It does not include a difference of one or more complete sets of chromosomes. A cell with any ...
) plants can be fused to produce fertile
diploid
Ploidy () is the number of complete sets of chromosomes in a cell, and hence the number of possible alleles for autosomal and pseudoautosomal genes. Sets of chromosomes refer to the number of maternal and paternal chromosome copies, respectively ...
s and
polyploid
Polyploidy is a condition in which the cells of an organism have more than one pair of ( homologous) chromosomes. Most species whose cells have nuclei ( eukaryotes) are diploid, meaning they have two sets of chromosomes, where each set contain ...
s.
# Somatic cell fusion overcomes sexual incompatibility barriers. In some cases somatic hybrids between two incompatible plants have also found application in
industry
Industry may refer to:
Economics
* Industry (economics), a generally categorized branch of economic activity
* Industry (manufacturing), a specific branch of economic activity, typically in factories with machinery
* The wider industrial sector ...
or
agriculture
Agriculture or farming is the practice of cultivating plants and livestock. Agriculture was the key development in the rise of sedentary human civilization, whereby farming of domesticated species created food surpluses that enabled people to ...
.
# Somatic cell fusion is useful in the study of cytoplasmic genes and their activities and this information can be applied in
plant breeding
Plant breeding is the science of changing the traits of plants in order to produce desired characteristics. It has been used to improve the quality of nutrition in products for humans and animals. The goals of plant breeding are to produce cro ...
experiments.
Inter-specific and inter-generic fusion achievements
Note: The table only lists a few examples, there are many more crosses. The possibilities of this technology are great; however, not all species are easily put into protoplast culture.
Table: Reference #5
References
{{reflist
Genetic engineering
Molecular biology
Biological engineering
Biotechnology