Read this article to learn about Tissue Culture. After reading article you will learn about: 1. Techniques of Tissue Culture 2. Classification of Tissue Culture Techniques.
Techniques of Tissue Culture:
(i) Surface sterilization:
The explant (the plant or plant part excised for the in-vitro cultivation) is surface sterilized to eliminate contaminants using sodium hypochlorite @ 1-2% or mercuric chloride @ 0.1% solution.
(ii) Nutrient Medium:
It is a medium containing salts, trace elements, vitamins, “carbon sources, growth regulators 2, 4-D @ 0.5-0.2 mg/L is commonly used. Organic supplements like Coconut agent such as agar-agar are used. Generally B-5 medium (Gamborg et al.) or MS medium (Murashige and Skoog) are used.
(iii) Sub culturing:
Transferring of tissue to fresh media after a stipulated time. Usually sub-culturing is done after every 4-6 weeks. However, the suspension cultures are sub-cultured after every 3-14 days.
(iv) Plant regeneration and transfer to soil:
First the transfer of cultured plant is done in small pots and then to green house and finally in soil. Transfer is done when roots and shoots appears.
Classification of Tissue Culture Techniques:
(i) Embryo culture:
Young embryo is removed from developing seeds and planted on a suitable nutrient medium in vitro, with the goal of obtaining a viable seedlings/ plants. It is applied in differentiation studies and somaclonal variations.
(ii) Meristem culture:
It is a culture of isolated mature or immature embryos. It ‘ is applied in micro propagation and production of virus free plants.
(iii) Seed culture:
Culture of seeds in vitro to generate seedlings / plants.
(iv) Anther or Pollen culture:
In this technique, haploid plants are obtained from pollen grains by replacing another or pollens into a suitable medium. This technique is used to obtained haploid plants.
(v) Callus culture:
Callus is basically a more or less non-organized tumour tissue, which usually arises on wounds of differentiated tissue and organs. This culture is used in Cryopreservation of germplasm, in vitro cell selections for resistance to biotic and abiotic stresses.
(vi) Nucellus culture:
Nucellus culture has been utilized to study factors responsible for formation of adventive embryos.
(vii) Cell culture:
The growing of individual cells that have been obtained from an explant tissue or callus. It is applied in production of useful metabolites, synthesis of new chemical substances.
(viii) Protoplast culture:
Protoplasts are the cells without cell wall. Culture of protoplasts and fusion of protoplasts from different strains.
(ix) Organ culture:
Culture of an organ in vitro in a way that allows development and/or preservation of the originally isolated organ.
a. Totipotency:
1. Totipotency is capability of an isolated single cell to multiply and differentiate into multicellular organism.
2. Tissue culture is purely based on the totipotency of cells.
3. ‘Haberlandt’ first demonstrated the totipotency of cells in 1902.
b. Micro propagation is in vitro multiplication of plants from a small tissue explant.
c. Re-culture – Process by which a cell monolayer or a plant explant is transferred without subdivision into a fresh medium.
a. In vitro is a biochemical process or reaction taking place in a test tube (in lab).
b. In vivo is a biological process or reaction taking place in a living cell or organism.
c. Allele is one of a number of different forms of a gene.
d. Codon: A group of three nucleotides coding for an amino acid.
e. Anticodon is triplet of nucleotides in transfer of RNA.
f. Nucleic Acids are family of molecules that includes the DNA and RNA molecules.
g. RNA (ribonucleic acid) is a molecule similar to DNA, helps in process of decoding genetic information carried by DNA Methods used for determination of gene sequences.
h. DNA (deoxyribonucleic acid) molecule encodes genetic information
i. DNA ligase is an enzyme that closes nicks or discontinuities in one strand of double stranded DNA by creating a bond.
j. DNA is a complementary DNA, a fragment of DNA which has been produced from an RNA sequence by reverse transcription. Messenger RNA is commonly used to synthesize cDNA.
k. DNA or Genetic fingerprinting is a technique in which an individual DNA is analyzed to reveal the pattern of repetition of particular nucleotide sequence throughout the genome. The unique pattern of DNA fragments are identified by Southern hybridization or polymerase.
l. Double haploids are Individuals derived from haploid garnets and carry two identical chromosomes.
m. Microsatellite – DNAs consist of repetitions of extremely short units.
n. Electroporation is a technique uses electric discharge to produce pores on cell membrane for intake of recombinant DNA.
o. Electrophoresis is a technique that separates charged molecules (DNA, RNA, proteins) on the basis of relative migration in a appropriate matrix (such as agarose, polyacrylamide) subjected to an electric medium.
p. Embryogenesis is a Process of formation of somatic embryos from callus.
q. Genetic Transformation: Genetic transformation is the heritable change in a cell or organism brought about by the uptake and establishment of introduced DNA.
r. GMO: a Genetically Modified or transgenic plant is a plant that has a novel combination of genetic material obtained through the use of modern biotechnology.
A transgenic crop plant contains a gene or genes which have been artificially introduced instead of the plant acquiring them through pollination these genes are introduced with a view to expressing a novel trait which is not normally found normally in the given species.
s. Hybridization is crossing of two genotypically different plants.
t. Somatic hybridization is crossing of plants through fusion of somatic cell.
u. Chromomere is a serially aligned beads or granules of eukaryotic chromosome from local coiling of continuous DNA thread.
v. Jumping gene or Transposon:
A movable genetic element. A DNA element which has the ability to move from one chromosomal position to another. It can insert at random into plasmids or the bacterial chromosome independently of the host cell recombination system.
w. Genetic engineering:
Change in the genetic constitution of cells by introduction or elimination of specific genes using molecular biology techniques is known as Genetic engineering. This is a non-sexual method of gene transfer.
1. Genetic engineering is the artificial manipulation, modification and recombination of DNA or other nucleic acid molecules in order to modify an organism or population of organisms. Genetic engineering works through recombinant DNA technology.
2. Genetic engineering allows the use of several desirable genes in a single event and reduces the time to introgress novel genes into elite background. Biotechnology has provided several unique opportunities that include: access to novel molecules, ability to change the level of gene expression, capability to change the expression pattern of genes, and develop transgenic plants with novel genes.
3. Father of genetic engineering – Paul Berg
4. Gene cloning is isolating a gene and process of producing identical copies or technique of genetic engineering by which a gene sequence with many identical copies is replicated.
5. Coli is a bacterium used in genetic engineering for its small size i.e. Agrobacterium rhizogenes.
Recombinant DNA technology:
Technology of DNA molecules means the joining or recombining of two pieces of DNA from two different species. The techniques allow an investigator to biological purify (clone) a gene from one species by inserting it into the DNA of another species, where it is replicated along with the host DNA.
By using recombinant DNA technology, Opaque-2 gene is transferred to maize genotype, which is responsible for higher amount of lysine in maize and produce varieties i.e. Ratan, Shakti, Protina.
Gene bank is a group of genes or cloned DNA fragments.
PCR – Polymerase chain reactions (developed by Dr. Karl Mullis in 1980)
PCR technique uses bacterial enzymes for in vitro amplification of DNA. This technique provides several copies of specific DNA sequence.
Molecular markers:
Marker is a sequence of bases at a unique physical location in genome. Markers are the traits of protein or DNA molecules that show easily recognisable differences among different strains or segregants. Molecular markers are DNA fragments used as finger print in identification of any organism or species.