Somatic fusion
Encyclopedia
Somatic fusion, also called protoplast fusion, is a type of genetic modification in plants by which two distinct species of plants are fused together to form a new hybrid plant with the characteristics of both, a somatic hybrid. Hybrids have been produced either between the different varieties of the same species (e.g. between non-flowering potato
plants and flowering potato plants) or between two different species (e.g. between wheat triticum and rye secale
to produce Triticale
).
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
– 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 plants.
Different from the procedure for seed plants describe above, fusion of moss
protoplasts can be initiated without electric shock but by the use of polyethylene glycol
(PEG). Further, moss protoplasts do not need phytohormones for regeneration
, and they do not form a callus
. Instead, regenerating moss protoplasts behave like germinating
moss spore
s . Of further note sodium nitrate and calcium ion at high pH can be used, although results are variable depending on the organism .
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
and gene expression
,
(ii) to investigate malignant transformation
s,
(iii) to obtain viral replication
,
(iv) for gene
or chromosome mapping and for
(v) production of monoclonal antibodies
by producing hybridoma (hybrid cells between an immortalised cell and an antibody
producing lymphocyte
), etc.
Chromosome mapping through somatic cell hybridization is essentially based on fusion of human and mouse
somatic cells. Generally, human fibrocyte
s or leucocytes are fused with mouse continuous cell lines.
When human and mouse cells (or cells of any two mammal
ian species or of the same species) are mixed, spontaneous cell fusion
occurs at a very low rate (10-6). Cell fusion is enhanced 100 to 1000 times by the addition of ultraviolet
inactivated Sendai
(parainfluenza) virus or polyethylene glycol
(PEG).
These agents adhere to the plasma membranes 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, e.g., HAT medium
, which allows the multiplication
of hybrid cells only.
Several clone
s (each derived from a single hybrid cell) of the hybrid cells are thus isolated and subjected to both cytogenetic
and appropriate biochemical analyses for the detection of enzyme
/ protein
/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 in the hybrid clones.
If there is a perfect correlation
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
, aminopterin
and thymidine
, hence the name HAT medium. Antimetabolite
aminopterin blocks the cellular biosynthesis
of purine
s and pyrimidine
s from simple sugars and amino acid
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
, which ordinarily recycles the purines and pyrimidines produced from degradation of nucleic acid
s.
Hypoxanthine is converted into guanine
by the enzyme hypoxanthine-guanine phosphoribosyltransferase
(HGPRT), while thymidine is phosphorylated
by thymidine kinase
(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 cells 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
) 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.
s can be recombined
among plants that cannot reproduce sexually
(asexual or sterile).
2. Protoplasts of sexually sterile (haploid, triploid, and aneuploid) plants can be fused to produce fertile diploids and polyploids.
3. Somatic cell fusion overcomes sexual incompatibility barriers. In some cases somatic hybrids between two incompatible plants have also found application in industry
or agriculture
.
4. Somatic cell fusion is useful in the study of cytoplasmic genes and their activities and this information can be applied in plant breeding
experiments.
Table: Reference #5
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.
Potato
The potato is a starchy, tuberous crop from the perennial Solanum tuberosum of the Solanaceae family . The word potato may refer to the plant itself as well as the edible tuber. In the region of the Andes, there are some other closely related cultivated potato species...
plants and flowering potato plants) or between two different species (e.g. between wheat triticum and rye secale
Secale
Secale is a genus of grasses in the Triticeae tribe. The most known member is rye .Other species include:*Secale montanum Guss.*Secale sylvestre Host....
to produce Triticale
Triticale
Triticale is a hybrid of wheat and rye first bred in laboratories during the late 19th century. The grain was originally bred in Scotland and Sweden. Commercially available triticale is almost always a second generation hybrid, i.e., a cross between two kinds of primary triticales...
).
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 (molecular biology)
In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes...
– 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 plants.
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 cellulaseCellulase400px|thumb|right|alt = Colored dice with checkered background|Ribbon representation of the Streptomyces lividans beta-1,4-endoglucanase catalytic domain - an example from the family 12 glycoside hydrolases...
enzyme to produce a somatic cellSomatic cellA somatic cell is any biological cell forming the body of an organism; that is, in a multicellular organism, any cell other than a gamete, germ cell, gametocyte or undifferentiated stem cell...
called a protoplastProtoplastProtoplast, from the ancient Greek πρῶτον + verb πλάθω or πλάττω , initially referred to the first organized body of a species.Protoplast has several biological definitions:... - The cells are then fused using electric shock (electrofusion) or chemical treatment to join the cells and fuse togethor the nuclei. The resulting fused nucleusCell nucleusIn cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
is called heterokaryonHeterokaryonA heterokaryon is a cell that contains multiple, genetically different nuclei. This can occur naturally, such as in the mycelium of fungi during sexual reproduction, or artificially as formed by the experimental fusion of two genetically different cells. A medical example is a heterokaryon composed...
. - The somatic hybrid cell then has its cell wall induced to form using hormoneHormoneA hormone is a chemical released by a cell or a gland in one part of the body that sends out messages that affect cells in other parts of the organism. Only a small amount of hormone is required to alter cell metabolism. In essence, it is a chemical messenger that transports a signal from one...
s - The cells are then grown into callusesCallus (cell biology)Plant callus is a mass of undifferentiated cells derived from plant tissue for use in biological research and biotechnology. In plant biology, callus cells are those cells that cover a plant wound. To induce callus formation, plant tissues are surface sterilized and then plated onto in vitro...
which then are further grown to plantletPlantletPlantlets are young or small plants used as propagules. They are usually grown from clippings of mature plants. Many plants such as spider plants naturally create stolons with plantlets on the ends as a form of asexual reproduction.-External links:...
s and finally to a full plant, known as a somatic hybrid.
Different from the procedure for seed plants describe above, fusion of moss
Moss
Mosses are small, soft plants that are typically 1–10 cm tall, though some species are much larger. They commonly grow close together in clumps or mats in damp or shady locations. They do not have flowers or seeds, and their simple leaves cover the thin wiry stems...
protoplasts can be initiated without electric shock but by the use of polyethylene glycol
Polyethylene glycol
Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...
(PEG). Further, moss protoplasts do not need phytohormones for regeneration
Regeneration (biology)
In biology, regeneration is the process of renewal, restoration, and growth that makes genomes, cells, organs, organisms, and ecosystems resilient to natural fluctuations or events that cause disturbance or damage. Every species is capable of regeneration, from bacteria to humans. At its most...
, and they do not form a callus
Callus (cell biology)
Plant callus is a mass of undifferentiated cells derived from plant tissue for use in biological research and biotechnology. In plant biology, callus cells are those cells that cover a plant wound. To induce callus formation, plant tissues are surface sterilized and then plated onto in vitro...
. Instead, regenerating moss protoplasts behave like germinating
Germination
Germination is the process in which a plant or fungus emerges from a seed or spore, respectively, and begins growth. The most common example of germination is the sprouting of a seedling from a seed of an angiosperm or gymnosperm. However the growth of a sporeling from a spore, for example the...
moss spore
Spore
In biology, a spore is a reproductive structure that is adapted for dispersal and surviving for extended periods of time in unfavorable conditions. Spores form part of the life cycles of many bacteria, plants, algae, fungi and some protozoa. According to scientist Dr...
s . Of further note sodium nitrate and calcium ion at high pH can be used, although results are variable depending on the organism .
Applications in animal cells
Somatic cellSomatic cell
A somatic cell is any biological cell forming the body of an organism; that is, in a multicellular organism, any cell other than a gamete, germ cell, gametocyte or undifferentiated stem cell...
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
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
and gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
,
(ii) to investigate malignant transformation
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....
s,
(iii) to obtain viral replication
Viral replication
Viral replication is the term used by virologists to describe 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. From the perspective of the virus, the purpose of viral replication is...
,
(iv) for gene
Gene mapping
Gene mapping, also called genome mapping, is the creation of a genetic map assigning DNA fragments to chromosomes.When a genome is first investigated, this map is nonexistent. The map improves with the scientific progress and is perfect when the genomic DNA sequencing of the species has been...
or chromosome mapping and for
(v) production of monoclonal antibodies
Monoclonal antibodies
Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....
by producing hybridoma (hybrid cells between an immortalised cell and an antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
producing lymphocyte
Lymphocyte
A lymphocyte is a type of white blood cell in the vertebrate immune system.Under the microscope, lymphocytes can be divided into large lymphocytes and small lymphocytes. Large granular lymphocytes include natural killer cells...
), etc.
Chromosome mapping through somatic cell hybridization is essentially based on fusion of human and mouse
Mouse
A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...
somatic cells. Generally, human fibrocyte
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 fibroblast...
s or leucocytes are fused with mouse continuous cell lines.
When human and mouse cells (or cells of any two mammal
Mammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
ian species or of the same species) are mixed, spontaneous cell fusion
Cell fusion
Cell fusion is an important cellular process that occurs during differentiation of muscle, bone and trophoblast cells, during embryogenesis, and during morphogenesis...
occurs at a very low rate (10-6). Cell fusion is enhanced 100 to 1000 times by the addition of ultraviolet
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...
inactivated Sendai
Sendai virus
Sendai virus , also known as murine parainfluenza virus type 1 or hemagglutinating virus of Japan , is a negative sense, single-stranded RNA virus of the Paramyxoviridae family, a group of viruses featuring, notably, the Morbillivirus and Rubulavirus genera...
(parainfluenza) virus or polyethylene glycol
Polyethylene glycol
Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...
(PEG).
These agents adhere to the plasma membranes 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, e.g., HAT medium
HAT medium
HAT 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 reductase, with hypoxanthine and thymidine which are intermediates in DNA synthesis...
, which allows the multiplication
Multiplication
Multiplication is the mathematical operation of scaling one number by another. It is one of the four basic operations in elementary arithmetic ....
of hybrid cells only.
Several clone
Clone (cell biology)
A clone is a group of identical cells that share a common ancestry, meaning they are derived from the same mother cell.Clonality implies the state of a cell or a substance being derived from one source or the other...
s (each derived from a single hybrid cell) of the hybrid cells are thus isolated and subjected to both cytogenetic
Cytogenetics
Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell, especially the chromosomes. It includes routine analysis of G-Banded chromosomes, other cytogenetic banding techniques, as well as molecular cytogenetics such as fluorescent in situ...
and appropriate biochemical analyses for the detection of enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
/ protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
/trait
Trait (biology)
A trait is a distinct variant of a phenotypic character of an organism that may be inherited, environmentally determined or be a combination of the two...
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 in the hybrid clones.
If there is a perfect correlation
Correlation
In statistics, dependence refers to any statistical relationship between two random variables or two sets of data. Correlation refers to any of a broad class of statistical relationships involving dependence....
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
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. Hypoxanthine is a necessary additive in certain cell,...
, aminopterin
Aminopterin
Aminopterin , a 4-amino analog of folic acid, is an antineoplastic drug with immunosuppressive properties used in chemotherapy. Aminopterin is a synthetic derivative of pterin. Aminopterin works as an enzyme inhibitor by competing for the folate binding site of the enzyme dihydrofolate reductase...
and thymidine
Thymidine
Thymidine is a chemical compound, more precisely a pyrimidine deoxynucleoside. Deoxythymidine is the DNA nucleoside T, which pairs with deoxyadenosine in double-stranded DNA...
, hence the name HAT medium. Antimetabolite
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 antifolates that interfere with the use of folic acid...
aminopterin blocks the cellular biosynthesis
Biosynthesis
Biosynthesis is an enzyme-catalyzed process in cells of living organisms by which substrates are converted to more complex products. The biosynthesis process often consists of several enzymatic steps in which the product of one step is used as substrate in the following step...
of purine
Purine
A purine is a heterocyclic aromatic organic compound, consisting of a pyrimidine ring fused to an imidazole ring. Purines, including substituted purines and their tautomers, are the most widely distributed kind of nitrogen-containing heterocycle in nature....
s and pyrimidine
Pyrimidine
Pyrimidine is a heterocyclic aromatic organic compound similar to benzene and pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring...
s from simple sugars and amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
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
Salvage pathway
A salvage pathway is a pathway in which nucleotides are synthesized from intermediates in the degradative pathway for nucleotides....
, which ordinarily recycles the purines and pyrimidines produced from degradation of nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
s.
Hypoxanthine is converted into guanine
Guanine
Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with...
by the enzyme hypoxanthine-guanine phosphoribosyltransferase
Hypoxanthine-guanine phosphoribosyltransferase
Hypoxanthine-guanine phosphoribosyltransferase 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 reaction transfers the 5-phosphoribosyl group from...
(HGPRT), while thymidine is phosphorylated
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
by thymidine kinase
Thymidine kinase
Thymidine kinase is an enzyme, a phosphotransferase : 2'-deoxythymidine kinase, ATP-thymidine 5'-phosphotransferase, . It can be found in most living cells. It is present in two forms in mammalian cells, TK1 and TK2...
(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 cells 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
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
) 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
1. Somatic cell fusion appears to be the only means through which two different parental genomeGenome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
s can be recombined
Genetic recombination
Genetic recombination is a process by which a molecule of nucleic acid is broken and then joined to a different one. Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in non-homologous end joining. Recombination is a common method...
among plants that cannot reproduce sexually
Sexual reproduction
Sexual reproduction is the creation of a new organism by combining the genetic material of two organisms. There are two main processes during sexual reproduction; they are: meiosis, involving the halving of the number of chromosomes; and fertilization, involving the fusion of two gametes and the...
(asexual or sterile).
2. Protoplasts of sexually sterile (haploid, triploid, and aneuploid) plants can be fused to produce fertile diploids and polyploids.
3. Somatic cell fusion overcomes sexual incompatibility barriers. In some cases somatic hybrids between two incompatible plants have also found application in industry
Industry
Industry refers to the production of an economic good or service within an economy.-Industrial sectors:There are four key industrial economic sectors: the primary sector, largely raw material extraction industries such as mining and farming; the secondary sector, involving refining, construction,...
or agriculture
Agriculture
Agriculture is the cultivation of animals, plants, fungi and other life forms for food, fiber, and other products used to sustain life. Agriculture was the key implement in the rise of sedentary human civilization, whereby farming of domesticated species created food surpluses that nurtured the...
.
4. 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
Plant breeding is the art and science of changing the genetics of plants in order to produce desired characteristics. Plant breeding can be accomplished through many different techniques ranging from simply selecting plants with desirable characteristics for propagation, to more complex molecular...
experiments.
Inter-specific and inter-generic fusion achievements
| Cross | Crossed with |
|---|---|
| Oat | Maize |
| Brassica sinensis | B. oleracea |
| Torrentia fourneri | T. bailloni |
| Brassica oleracea | B. campestris |
| Datura innoxia | Atropa belladonna |
| Nicotiana tabacum | N. glutinosa |
| Datura innoxia | D. candida |
| Arabidopsis thaliana | Brassica campestris |
| Petunia hybrida | Vicia faba |
Table: Reference #5
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.