Tissue culture and molecular markers

Tissue culture methods and applications

Tissue culture refers to a method in which fragments of a tissue (plant or animal tissue) are introduced into a new, artificial environment, where they continue to function or grow. While fragments of a tissue are often used, it is important to note that entire organs are also used for tissue culture purposes. Here, such growth media as broth and agar are used to facilitate the process.

Methods of Tissue Culture

Seed culture

Seed culture is the type of tissue culture that is primarily used for plants such as orchids. For this method, explants (tissue from the plant) are obtained from an in-vitro derived plant and introduced in to an artificial environment, where they get to proliferate. In the event that a plant material is used directly for this process, then it has to be sterilized to prevent tissue damage and ensure optimum regeneration.

Embryo Culture 

Embryo culture is the type of tissue culture that involves the isolation of an embryo from a given organism for in vitro growth.  *Note, the term embryo culture is used to refer to sexually produced zygotic embryo culture.  Embryo culture may involve the use of a mature of immature embryo. Whereas mature embryos for culture are essentially obtained from ripe seeds, immature embryo (embryo rescue) involves the use of immature embryos from unripe/hybrid seeds that failed to germinate. In doing so, the embryo is ultimately able to produce a viable plant.

For embryo culture, the ovule, seed or fruit from which the embryo is to be obtained is sterilized, and therefore the embryo does not have to be sterilized again. Salt sucrose may be used to provide the embryo with nutrients. The culture is enriched with organic or inorganic compounds, inorganic salts as well as growth regulators.

 

 

Callus Culture

Callus is the term used to refer to unspecialized, unorganized and a dividing mass of cells. A callus is produced when explants (cells) are cultured in an appropriate medium – A good example of this is the tumor tissue that grows out of the wounds of differentiated tissues/organs.

In practice, callus culture involves the growth of a callus (composed of differentiated and non- differentiated cells), which is the followed by a procedure that induces organ differentiation.

For this type of tissue culture, the culture is often sustained on a gel medium, which is composed of agar and a mixture of given macro and micronutrients depending on the type of cells. Different types of basal salt mixtures such as murashige and skoog medium are also used in addition to vitamins to enhance growth.

Organ Culture

Organ culture is a type of tissue culture that involves isolating an organ for in vitro growth. Here, any organ plant can be used as an explant for the culture process (Shoot, root, leaf, and flower).

With organ culture, or as is with their various tissue components, the method is used for preserve their structure or functions, which allows the organ to still resemble and retain the characteristics they would have in vivo. Here, new growth (differentiated structures) continues given that the organ retains its physiological features. As such, an organ helps provide information on patterns of growth, differentiation as well as development.

Major Steps of Tissue Culture method (Plants)

Initiation Phase (Stage 1)

The initiation phase is the first phase of tissue culture. Here, the tissue of interest is obtained and introduced and sterilized in order to prevent any microorganism from negatively affecting the process. It is during this stage that the tissue is initiated in to culture.

Multiplication Phase (Stage 2)

The multiplication phase is the second step of tissue culture where the in vitro plant material is re- divided and then introduced in to the medium. Here, the medium is composed of appropriate components for growth including regulators and nutrients. These are responsible for the proliferation of the tissue and the production of multiple shoots.

*This step is often repeated several times in order to obtain the desired number of plants

Root formation (Stage 3)

It is at this phase that roots are formed. Here, hormones are required in order to induce rooting, and consequently complete plantlets.

Applications of tissue culture

Rapid Clonal Propagation

A clone is a group of individuals or cells derived from a single parent individual or cell through asexual reproduction. All the cells in callus or suspension culture are derived from a single explants by mitotic division. Therefore, all plantlets regenerated from a callus/suspension culture generally have the same genotype and constitute a clone. These plantlets are used for rapid clonal propagation.

Soma-clonal Variation

Genetic variation present among plant cells of a culture is called soma-clonal variation. The term soma-clonal variation is also used for the genetic variation present in plants regenerated from a single culture. This variation has been used to develop several useful varieties.

Transgenic Plants

A gene that is transferred into an organism by genetic engineering is known as transgene. An organism that contains and expresses a transgene is called transgenic organism. The transgenes can be introduced into individual plant cells. The plantlets can be regenerated from these cells. These plantlets give rise to the highly valuable transgenic plants.

Induction and Selection of Mutations

Mutagens are added to single cell liquid cultures for induction of mutations. The cells are washed and transferred to solid culture for raising mu ant plants. Useful mutants are selected for further breeding. Tolerance to stress like pollutants, toxins, salts, drought, flooding etc. can also be obtained by providing them in culture medium in increasing dosage. The surviving healthy cells are taken to solid medium for raising resistant plants.

Resistance to Weedicides

It is similar to induction of mutations. Weedicides are added to culture initially in very small concentrations. Dosage is increased in subsequent cultures till die desired level of resistance is obtained. The resistant cells are then regenerated to form plantlets and plants.

Molecular breeding and marker assisted selection

Molecular breeding

Molecular Breeding or Marker assisted breeding (MAB) is the process of using the results of DNA tests to assist in the selection of individuals to become the parents in the next generation of a genetic improvement program. The choice among various methods of MAB depends on the complexity of the trait and a prior knowledge on the gene (s) or segments of chromosomes (known as quantitative trait loci (QTL). Molecular markers facilitate conventional breeding, improve selection efficiency, reduce cost for developing new varieties, and/or quality control (ensuring line purity and genetic identity).

Aspects of Molecular Breeding

Marker assisted breeding

Genotyping and creating molecular maps- genomics The commonly used markers include Simple sequence repeats (or microsatellites), single nucleotide polymorphisms (SNP). The process of identification of plant genotypes is known as genotyping. Development of SNPs has revolutionized the molecular breeding process as it helps to create dense markers. Another area that is developing is genotyping by sequencing.

Phenotyping – phenomics

To identify genes associated with traits, it is important to measure the trait value – known as phenotype. “omics” for measurement of phenotypes is called phenomics. The phenotype can be indicative of the measurement of the trait itself or an indirectly related or correlated trait.

QTL mapping or association mapping

Genes (Quantitative trait loci (abbreviated as QTL) or quantitative trait genes or minor genes or major genes) involved in controlling trait of interest is identified. The process is known as mapping. Mapping of such genes can be done using molecular markers. QTL mapping can involve single large family, unrelated individuals or multiple families (see: Family based QTL mapping). The basic idea is to identify genes or markers associated with genes that correlate to a phenotypic measurement and that can be used in marker assisted breeding / selection.

Marker assisted selection or genetic selection

Once genes or markers are identified, they can be used for genotyping and selection decisions can be made.

Marker-assisted backcrossing (MABC)

Backcross is crossing F1 with its parents to transfer a limited number of loci (e.g. transgene, disease resistance loci, etc.) from one genetic background to another. Usually the recipient of such genes is good adapted cultivars otherwise except the gene that is to be transferred. So we want to keep genetic background of the recipient genotypes, which is done by 4-6 rounds of repeated backcrosses while selecting for the gene of interest. We can use markers from the whole genome to recover the genome quickly in 2-3 rounds of backcrossing might be good enough in such situation.

Marker-assisted recurrent selection (MARS)

MARS include identification and selection of several genomic regions (up to 20 or even more) for complex traits within a single population.

Genomic selection

Genomic selection is novel approach to traditional marker-assisted selection where selection are made based on few markers.[5] Rather than seeking to identify individual loci significantly associated with a trait, genomics uses all marker data as predictors of performance and consequently delivers more accurate predictions. Selection can be based on genomic selection predictions, potentially leading to more rapid and lower cost gains from breeding. Genomic prediction combines marker data with phenotypic and pedigree data (when available) in an attempt to increase the accuracy of the prediction of breeding and genotypic values.

 

Marker-assisted selection

Marker assisted selection or marker aided selection (MAS) is an indirect selection process where a trait of interest is selected based on a marker (morphological, biochemical or DNA/RNA variation) linked to a trait of interest (e.g. productivity, disease resistance, abiotic stress tolerance, and quality), rather than on the trait itself.This process has been extensively researched and proposed for plant and animal breeding, nevertheless, as of 2013 “breeding programs based on DNA markers for improving quantitative traits in plants are rare”.

For example, using MAS to select individuals with disease resistance involves identifying a marker allele that is linked with disease resistance rather than the level of disease resistance. The assumption is that the marker associates at high frequency with the gene or quantitative trait locus (QTL) of interest, due to genetic linkage (close proximity, on the chromosome, of the marker locus and the disease resistance-determining locus). MAS can be useful to select for traits that are difficult or expensive to measure, exhibit low heritability and/or are expressed late in development. At certain points in the breeding process the specimens are examined to ensure that they express the desired trait.

Positive and negative selectable markers

The following terms are generally less relevant to discussions of MAS in plant and animal breeding, but are highly relevant in molecular biology research: 

Positive selectable markers are selectable markers that confer selective advantage to the host organism. An example would be antibiotic resistance, which allows the host organism to survive antibiotic selection.

Negative selectable markers are selectable markers that eliminate or inhibit growth of the host organism upon selection. An example would be thymidine kinase, which makes the host sensitive to ganciclovir selection.

 

Gene vs marker

The gene of interest directly causes production of protein(s) or RNA that produce a desired trait or phenotype, whereas markers (a DNA sequence or the morphological or biochemical markers produced due to that DNA) are genetically linked to the gene of interest. The gene of interest and the marker tend to move together during segregation of gametes due to their proximity on the same chromosome and concomitant reduction in recombination (chromosome crossover events) between the marker and gene of interest. For some traits, the gene of interest has been discovered and the presence of desirable alleles can be directly assayed with a high level of confidence. However, if the gene of interest is not known, markers linked to the gene of interest can still be used to select for individuals with desirable alleles of the gene of interest. When markers are used there may be some inaccurate results due to inaccurate tests for the marker. There also can be false positive results when markers are used, due to recombination between the marker of interest and gene (or QTL). A perfect marker would elicit no false positive results. The term ‘perfect marker’ is sometimes used when tests are performed to detect a SNP or other DNA polymorphism in the gene of interest, if that SNP or other polymorphism is the direct cause of the trait of interest. The term ‘marker’ is still appropriate to use when directly assaying the gene of interest, because the test of genotype is an indirect test of the trait or phenotype of interest.

Selection for major genes linked to markers

‘Major genes’ that are responsible for economically important characteristics are frequent in the plant kingdom. Such characteristics include disease resistance, male sterility,[9] self-incompatibility, and others related to shape, color, and architecture of whole plants and are often of mono- or oligogenic in nature. The marker loci that are tightly linked to major genes can be used for selection and are sometimes more efficient than direct selection for the target gene. Such advantages in efficiency may be due for example, to higher expression of the marker mRNA in such cases that the marker is itself a gene. Alternatively, in such cases that the target gene of interest differs between two alleles by a difficult-to-detect single nucleotide polymorphism, an external marker (be it another gene or a polymorphism that is easier to detect, such as a short tandem repeat) may present as the most realistic option.

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