Western blotting:- Detect Protein.
> Western blot is performed by using polypropylene gel electrophoresis. SDS-PAGE allows protein samples to be separated and transferred to a solid support, such as nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membrane.
Hup gene (Hydrogen uptake gene):- It is a group of genes found in some nitrogen-fixing bacteria, particularly Rhizobium, Bradyrhizobium, and some cyanobacteria. These genes encode the uptake hydrogenase enzyme, which recycles hydrogen gas (H₂) produced during nitrogen fixation.
Transgenic Plant:-
• Definition:- Stable transformation is achieved when a desired gene is integrated into the genome of a plant, this plant is called transgenic plant.
• These transgenic plants are developed for the following purposes:-
1. Insect Resistance
2. Virus Resistance
3. Seed Protein Quality
4. Gene Silencing
5. Male Sterility
6. Biochemical Production
Secondary Metabolites:- These are organic compounds produced by any lifeform, e.g. bacteria, fungi, animals, or plants, which are not directly involved in the metabolism for normal growth, development, or reproduction of the organism.
Classification:-
1. Terpenes
2. Phenolics
3. Alkaloids
Male sterility:- It is the inability of a plant to produce viable or functional pollen grains while the female reproductive organs remain fertile. It enables efficient hybrid seed production, promotes heterosis, reduces labour costs, and helps develop high-yielding, disease-resistant, and superior-quality crop varieties.
Patent:- A patent is a legal right granted by the government to an inventor, giving them the exclusive right to make, use, sell, or license an invention for a limited period (generally 20 years from the filing date), in exchange for publicly disclosing the details of the invention.
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Concept of Gene:-
> A gene can be described as a polynucleotide chain, which is a segment of DNA.
> It is a functional unit controlling a particular trait such as eye colour.
> Beadle and Tatum concluded by various experiments that gene is a segment of DNA that codes for one enzyme. They proposed one gene-one enzyme hypothesis.
> But as some genes code for proteins that are not enzymes, the definition of gene was changed to one gene-one protein hypothesis.
Protein Hypothesis:-
> Since proteins are polypeptide chains of amino acids translated by mRNA, gene was defined as one gene-one polypeptide relationship.
Some proteins have two or more different kinds of polypeptide chains, each with a different amino acid sequence. They are products of different genes.
> For example, haemoglobin has two kinds of chains a andβ chains, which differ in amino acid sequence and length. They are encoded by different genes.
> Thus, gene is defined as one gene-one polypeptide relationship.
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Transcription in prokaryotes:-
Introduction:-
• It is the process of copying genetic information from one strand of the DNA into RNA.
• Here, adenine pairs with uracil instead of thymine.
• Both strands are not copied during transcription, because:-
- The code for proteins is different in both strands. This complicates the translation.
- If 2 RNA molecules are produced simultaneously this would be complimentary to each other, hence form a double stranded RNA. This prevents translation.
Transcription Unit:-
• It is the segment of DNA between the sites of initiation and termination of transcription. It consists of 3 regions:-
- A promoter (Transcription start site):- Binding site for RNA polymerase.
- Structural gene:- The region between promoter and terminator where transcription takes place.
- A terminator:- The site where transcription stops.
• The DNA- dependent RNA polymerase catalyzes the polymerization only in 5’→3’direction.
• 3’→5’ acts as template strand and 5’→3’ acts as coding strand.
3’-ATGCATGCATGCATGCATGCATGC-5’ template strand.
5’-TACGTACGTACGTACGTACGTACG-3’ coding strand.
Transcription unit and gene:-
• Gene:- Functional unit of inheritance. It is the DNA sequence coding for RNA molecule.
• Cistron:- A segment of DNA coding for a polypeptide.
• Structural gene in a transcription unit is of 2 types:-
i. Monocistronic structural genes (split genes):- It is seen in eukaryotes. Here, the coding sequences exons are interrupted by introns.
ii. Polycistronic structural genes:- It is seen in prokaryotes. Here, there are no split genes.
Reverse Transcription:- It is the process in cells by which an enzyme makes a copy of DNA from RNA. The enzyme that makes the DNA copy is called reverse transcriptase and is found in retroviruses, such as the human immunodeficiency virus (HIV).
Steps of transcription in prokaryotes:-
i. Initiation:- Here, the enzyme RNA polymerase binds at the promoter site of DNA. This causes the local unwinding of the DNA double helix. An initiation factor (σ factor) present in RNA polymerase initiates the RNA synthesis.
ii. Elongation:- The RNA chain is synthesized in the 5’-3’ direction. In this process, activated ribonucleoside triphosphates (ATP, GTP, UTP & CTP) are added. This is complementary to the base sequence in the DNA template.
iii. Termination:- A termination factor (ρ factor) binds to the RNA polymerase and terminates the transcription.
NOTE:- In bacteria (Prokaryotes) transcription and translation can be coupled (Translation can begin before mRNA is fully transcribed) because:-
- mRNA requires no processing to become active.
- Transcription and translation take place in the same compartment (no separation of cytosol and nucleus).
Polysome or Polyribosome or Ergosome:- It is a group of ribosomes bound to an mRNA molecule like “beads” on a “thread”. It consists of a complex of an mRNA molecule and two or more ribosomes that act to translate mRNA instructions into polypeptides.
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Molecular farming:- It describes the production of recombinant proteins and other secondary metabolites in plants. This technology depends on a genetic transformation of plants.
The strategies of plant transformation:- Molecular farming depending on the production of transgenic plants. It has been operated by two general methods:
1. Stable or permanent expression systems:-
a. Stable nuclear transformation:-
> It refers to the integration of foreign genes into the nuclear genome of plants, which results in the change of genetic structures and consequent expression of transgenes after integration with the host genomes.
> The largest amount of recombinant proteins has been produced by one of the most common methods of stable nuclear transformation.
> A method exploited for aggregating proteins in dried beans of maize culminates in a long-term storage in the beans at the room temperature without decomposition of proteins.
> In addition, it has a considerable potential for producing crops like cereals that actually grow everywhere.
> Limitations:-
i. A long production cycle for some crops.
ii. Potential collisions of some crops with natural species or food products.
b. Stable plastid transformation:-
> Numerous advantages:-
i. Preventing transgene escape through amphimixis, because plastids are inherited through the maternal tissue in the majority of species.
ii. Absence of chloroplasts in pollen and consequent improbability of their transfer, which reduces environmental concerns.
> The transformed transgenic plants with homoplasmic chloroplasts (all chloroplasts carry transgenes) were selected after several generations of plant regeneration from bombarded leaf explants.
> Selection was conducted on a medium containing spectinomycin or combined with streptomycin.
> The researchers have already extracted a human pharmaceutical protein, more than 3% to 6%, from the total soluble proteins in the chloroplasts of tobacco.
> Recently, Oey reported a very high level (70% of an entire soluble protein) for a protein antibiotic with the chloroplast system, which, till today, has been the highest concentration of recombinant proteins.
> Functional limitation:-
i. Chloroplast transformation only in tobacco is practically possible, but unfortunately this plant is inedible and full of poisonous alkaloids.
ii. Long lasting storage in refrigerators will bring about changes in protein stability.
c. Plant cell suspension culture:-
> This method involves the removal of cell walls and gene transfer to the obtained protoplasts and suspension culture.
> The purification system and its downstream processing are cheaper and easier.
> In addition, the use of suspension culture can decrease heterogeneity in proteins and sugar (N-glycans) regarding the uniformity of the type and size of cells.
> Furthermore, as a fast system there is no need for the production of transgenic plants.
> Some samples of plant suspension cultures can be used for producing biomedicines, including:-
i. Vaccines of Newcastle disease virus of chicks
ii. Recombinant human glucocerebrosidase for treating diabetes.
> Though this method is cheaper, safer, and easier in comparison to the other methods of genetic manipulation, cell suspension has not yet been suggested as an optimal production choice of production in plant systems.
> Limitation:- It is believed that the ultimate products and their usability are constrained by reducing the level of recombinant proteins during the stationary phase because of the enhanced proteolytic activity.
2. Temporary or transient expression systems:-
a. Agrobacterium transformation method:-
> Infiltration of recombinant agrobacterium suspension into tobacco leaf tissue is achieved without stable gene transfer, which facilitates the transfer of T-DNA to a very high percentage of cells, where the transgenes are expressed at a high level without a stable transfer of genes.
> Presently, this method has been very efficient for the production of clinical biomedicines with a fast expansion.
b. Viral infection methods:- The viral infection method depends on the capability of plant viruses, such as tobacco mosaic virus and X potato virus, which functions as a vector to convey foreign genes into plant genomes without fusing with the genome of that plant.
c. Magnifection system:-
> Expression systems based on viral vectors and agrobacterium methods suffer from some constraints for the co-expression of two or more polypeptides required for the production of hetero-oligomeric proteins.
> Thus, a new transient expression system known as MagnICON technology has been developed by Icon Genetics Company.
> This method includes removing coat proteins (responsible for systemic movement) of non-competitive virus stains and systemic delivery of the derived viral vectors to all of the plants using agrobacterium as the medium of primary infection.
> This method not only optimizes the infection but also significantly increases proliferation, and finally results in the co-expression of several polypeptides and the rise of functional protein production more than 100 times.
The advantages of utilizing transgenic plants as bioreactors:- Comparison of different expression systems reveals the advantages of plants in comparison with other expression systems as follows:
i. The healthiness of derived products (plants cannot be the host of human pathogens and bacterial toxins).
ii. Capability of post-translational processing (respecting the features of eukaryotic cells).
iii. The possibility of using breeding methods and sexual crosses to obtain active recombinant multi-chain proteins (therefore, there is the possibility of producing antibodies without application of a double transformation).
iv. Reducing the costs of production (plants can produce biological materials by the use of carbon dioxide, solar energy, and inorganic materials. Moreover, the scale of production can be manipulated regarding scalability).
v. Reducing the costs of storage and transportation of recombinant proteins (when they are produced in dry textures like grains).
vi. Removing the purification step (when the plant tissues containing recombinant protein are edible).
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Alkaloids:-
i. Morphine:- Papaver somniferum – Latex – Analgesic
ii. Quinine:- Cinchona officinalis – Bark – Anti-malarial
iii. Atropine:- Atropa belladonna – Leaves & roots – Pupil dilator
iv. Reserpine:- Rauvolfia serpentina – Roots – Antihypertensive
v. Vincristine:- Catharanthus roseus – Leaves – Anticancer
vi. Caffeine:- Coffea arabica – Seeds – CNS stimulant
vii. Nicotine:- Nicotiana tabacum – Leaves – Insecticide
viii. Colchicine:- Colchicum autumnale – Corms & seeds – Polyploidy induction
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Uses of Cloned Genes in Industry:- Cloned genes are genes that have been isolated and copied using recombinant DNA technology. They are widely used in various industries for the large-scale production of valuable products.
Uses of Cloned Genes in Industry:-
Production of Industrial Enzymes:-
> Cloned genes are inserted into microorganisms such as Escherichia coli or yeast to produce enzymes like amylase, protease, cellulase, and lipase.
> These enzymes are used in the food, detergent, paper, textile, and leather industries.
Manufacture of Pharmaceuticals:-
> Cloned genes enable the production of therapeutic proteins such as human insulin, human growth hormone, erythropoietin, interferons, and clotting factors.
> These products are safer, more effective, and available in large quantities.
Vaccine Production:-
> Cloned genes encoding pathogen proteins are used to develop recombinant vaccines.
> Example: Hepatitis B vaccine, produced using cloned genes in yeast.
Production of Biochemicals:- Cloned microorganisms produce amino acids (lysine, glutamic acid), vitamins (Vitamin B12), organic acids (citric acid), and biofuels (ethanol), reducing production costs.
Agricultural Biotechnology:-
> Cloned genes are used to develop insect-resistant, herbicide-tolerant, and disease-resistant crops.
> Example: Bt cotton containing the cry gene from Bacillus thuringiensis.
Food and Dairy Industry:-
> Recombinant chymosin (rennin), produced using cloned genes, is widely used in cheese manufacturing.
> Cloned enzymes also improve food processing and shelf life.
Environmental Applications:- Cloned genes are introduced into microorganisms for bioremediation, enabling them to degrade pollutants such as oil spills, pesticides, and toxic chemicals.
Research and Diagnostic Industries:- Cloned genes are used to produce DNA probes, molecular markers, diagnostic kits, and recombinant proteins for disease detection and genetic research.
Biotechnology Intellectual Property Rights:-
> Biotechnology intellectual property rights are the legal ownership of an interest in a patent, trademark or trade secret. This means that another company cannot use those assets without permission from the company established as the official owner. In health care, intellectual property rights give their owners exclusive use of pharmaceuticals, brand names and more. Intellectual property rights are often the primary driver of value for these companies, particularly in biotech.
Key Points:-
> Intellectual property rights, as they pertain to the Biotechnology sector, concern the legal ownership and exclusive rights to patents, trademarks, and trade secrets.
> Just like with other industries, intellectual property rights allow biotech firms to establish ownership and protect their products from the threat of competitors.
> A company might own the patent to a specific drug and the exclusive right to market it under a certain name, for example, because it holds the intellectual property rights.
Understanding Biotechnology IPRs:- Biotechnology intellectual property rights provide health care companies with a means to protect their claim to and ownership of these assets through common law, state law or federal law. There is some controversy over intellectual property rights in biotechnology. Those in favor argue that they provide a key incentive for developers to innovate because these protections will allow them to be financially rewarded for successful innovations. Those opposed to the strict enforcement of these protections argue that broader sharing of information would reduce prices and increase access to care, especially in developing countries.
Biotechnology IPRs Examples:-
> Here is one example of how intellectual property rights work in the health care industry. Federal protection allows companies to use the ® symbol with a trade name to indicate that it has a registered trademark and that no one else can use that name. More than one company may sell the same chemical compound, which means the same drug, but only one company can legally use the trademarked name to market that drug.
> For example, while many companies sell the antidepressant drug fluoxetine hydrochloride, only Eli Lilly can call it Prozac. Likewise, only Hoffmann-La Roche can use the trademarked name Tamiflu to market a drug called Oseltamivir that is designed to prevent and treat influenza. Trademarks aren’t just used with drugs, however; they’re also used with hospital names, physician practice names, and other entities with distinct branding. This is of major importance to companies in this business environment, where branding, marketing, and image are central components of business operations and strategic positioning. Some studies estimate that pharmaceutical companies spend as much as $30 billion on marketing annually to raise brand awareness for their drugs.
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Nitrogen fixing genes and their genetic manipulation:-
Nif genes:-
1. Introduction:-
> Nif means:-
Ni = Nitrogen
f = fixation
> These are genes encoding enzymes involved in the fixation of atmospheric nitrogen.
> Nif genes also encode a number of regulatory proteins involved in nitrogen fixation.
> The nif genes are found in both free-living and symbiotic nitrogen-fixing bacteria.
> The primary enzyme encoded by the nif genes is the nitrogenase.
2. Regulation of Nif genes:- In most bacteria, regulation is done by NifA protein.
i. When there is not enough fixed nitrogen, NtrC triggers NifA expression, and NifA activates the nif genes.
ii. When there is not enough fixed nitrogen, NifL inhibit NifA expression, and nif genes remain inactive.
Reversible ADP-ribosylation:- It is an additional regulation mechanism found in Rhodospirillum rubrum. Reversible ADP-ribosylation of a specific arginine residue in the nitrogenase complex. When reduced nitrogen is present, DraG and DraT catalyze the ribosylation of arginine residue in the nitrogenase. It causes a barrier in the electron transfer flow and thereby inactivates nitrogenase activity.
3. Expression of Nif genes:-
> There are total 20 nif genes.
> nifH, nifD, and nifK:- They encode the nitrogenase subunits.
> nifE, nifN, nifU, nifS, nifV, nifW, nifX, nifB, and nifQ:- They encode proteins involved the assembly and incorporation of Fe and Mo atoms into the nitrogenase subunits.
> nifF and nifJ:- They encode proteins related to electron transfer taking place in the reduction process.
> nifA and nifL:- They are regulatory proteins in charge of regulating the expression of the other nif genes.
Nitrogen fixation:- It is a chemical process by which molecular nitrogen found in the air is converted into ammonia or related nitrogenous compounds.
1. Types of nitrogen fixation:-
a. Physical Nitrogen Fixation
b. Biological Nitrogen Fixation
a. Physical Nitrogen Fixation:-
i. Natural Nitrogen Fixation:- Under the influence of lightning and thunder, N2 and O2 of the air react to form nitric oxide (NO). The nitric oxides are again oxidized with oxygen to form nitrogen peroxide (NO2).
ii. Industrial Nitrogen Fixation:- Ammonia is produced industrially by direct combination of nitrogen with hydrogen (obtained from water) at high temperature and pressure. Later, it is converted into various kinds of fertilizers, such as urea etc.
b. Biological Nitrogen Fixation:- The conversion of atmospheric nitrogen into the nitrogenous compounds by living organisms is called biological nitrogen fixation. Only prokaryotes can fix nitrogen. Nitrogen fixation require anaerobic conditions because oxygen inactivates nitrogenase enzyme.
Hence for obligate anaerobes nitrogen fixation is easy, but in case of facultative anaerobes the nitrogen fixation occurs only in anaerobic conditions. In case of obligate aerobes the oxygen level inside the cell must be kept low for nitrogen fixation.
2. Nitrogen Fixers (Diazotrophs):- Among the earth’s organisms, only some prokaryotes like bacteria and cyanobacteria can fix atmosphere nitrogen. They are called nitrogen fixers or diazotrophs. They fix about 95% of the total global nitrogen fixed annually by natural process.
a. Asymbionts (Free living)
b. Symbionts
a. Asymbionts (Free living):-
i. Bacteria:- They add up to 10-25 kg, of nitrogen/ha/annum.
> Azotobacter (Aerobic, Saprophytic)
> Beijerinckia (Aerobic, Saprophytic)
> Clostridium (Anaerobic, Saprophytic)
> Desulphovibrio (Chemotrophic)
> Rhodopseudomonas (Photoautotrophic)
> Rhodospirillum (Photoautotrophic)
> Chromatium (Photoautotrophic)
ii. Blue Green Algae (Cyanobacteria):- Heterocysts are the special cells that fix nitrogen. They add 20-30 kg Nitrogen/ha/annum.
> Nostoc
> Anabaena
> Aulosira:- A. fertilissima is the most active nitrogen fixer in Rice fields.
> Cylindrospermum:- It is active in sugarcane and maize fields.
> Trichodesmium
b. Symbionts:- Live in close symbiotic association with other plants.
i. Blue Green Algae (Cyanobacteria):-
> Nostoc and Anabaena:- They are common symbionts in lichens, Anthoceros, Azolla and cycad roots.
> Anabaena azollae:- It is found in fronds of Azolla pinnata (a water fern). It is often inoculated to Rice fields for nitrogen fixation.
ii. Bacteria:-
> Rhizobium:- It is aerobic, gram negative nitrogen fixing bacterial symbionts of legume roots. Sesbania rostrata has Rhizobium in root nodules and Aerorhizobium in stem nodules.
> Frankia:- It is symbiont in root nodules of many non-leguminous plants like Casuarina and Alnus.
> Xanthomonas and Mycobacterium:- They occur as symbiont in the leaves of some members of the families Rubiaceae and Myrsinaceae (e.g., Ardisia).
3. Rhizobium Nitrogen Fixation:-
> Rhizobium bacteria:-
i. Free living
ii. Gram negative
iii. Aerobic
iv. Soil bacteria
> Rhizobium becomes anaerobic upon entry into roots.
> Leghaemoglobin (legHb or symbiotic Hb):-
- It is a pink coloured pigment.
- It occurs in the root nodules of leguminous plants.
- It acts as an oxygen scavenger. It provides anaerobic conditions for the nitrogenase enzyme and protects the enzyme from inactivation.
> Two main steps:-
a. Nodule formation
b. Nitrogen fixation
a. Nodule formation:- Root nodule formation is initiated, when the soil contains a low level of nitrogen. Steps of nodulation are:
i. Aggregation:- Roots of legumes secrete flavonoids, which attracts rhizobia towards the root. Rhizobia aggregate around root hairs.
ii. Developmental changes:- Rhizobia secrete nod factors, which causes stimulate many developmental changes:
- Membrane depolarization
- Curling of root hairs
- Cell division in the root cortex
- Intracellular calcium movement
iii. Infection thread:- The nod factor attaches to receptors present on the plasma membrane of the root hairs, which leads to the formation of the infection thread.
iv. Entry:- Infection thread provides the passage to bacteria to enter epidermal cells. Rhizobia then enter cortex cells, each bacterium gets surrounded by a plant-derived membrane known as symbiosome.
v. Nodulation:- Nodule formation is initiated by chemicals produced by rhizobia. It is a result of calcium dependent signal transduction pathway, which triggers biochemical changes leading to cell division and nodule formation. Cytokinin also plays an important role in nodules formation.
vi. Bacteroids:- Within nodules, bacteria get differentiated into bacteroids, which fix nitrogen. The Rhizobia stop dividing, loose cell wall and become nitrogen fixing cells as led bacteroids . Vascular tissues are developed for nodules for exchange of nutrients.
b. Nitrogen fixation:-
- The nodule serves as site for N2 fixation.
- Nodule contains nitrogenase and leghaemoglobin.
- The nitrogenase has 2 components:
i. Molybdoferredoxin (Mo-Fe protein)
ii. Azoferredoxin (Fe-protein).
- The free di-nitrogen first bound to MoFe protein and is not released until completely reduced to ammonia.
- In this process ferredoxin serves as an electron donor to Fe-protein (nitrogenase reductase) which in turn hydrolyzes ATP and reduce MoFe protein, the MoFe protein in Turn reduce the substrate N2. The electrons and ATP are provided by photosynthesis and respiration of the host cells.
- Many intermediates are formed to form ammonia (NH3).
Dinitrogen → Hydrazine → Diamine → Ammonia
- Ammonia (NH3) is immediately protonated at physiological pH to form ammonium ion (NH4+). As NH4+ is toxic to plants, it is rapidly used near the site of generation to synthesize amino acids.
Genetic manipulation of nitrogen fixation:-
> The subunits of nitrogenase from different nitrogen fixing microorganisms can be mixed to produce
functional system
> Genes can be manipulated to improve the fixation of dinitrogen.
> The microorganisms are modified in the host so that they are unable to assimilate the fixed nitrogen until the function of the nitrogenase is over and release NH4+ directly to the plants.
> The nitrogen fixation can be regulated by leguminous plants by:-
i. Reducing the number of root nodule formation
ii. Regulating the carbon flow to the microorganisms.
> The well known nitrogen fixing bacteria K. pneumoniae resembles closely to non-nitrogen fixing bacterium E.coli. The genes of these two species could be transferred and expressed in either of the organisms.
- Nif- mutants of K. pneumoniae that are deficient in fixing nitrogen are located between genes for histidine biosynthesis (his) and shikimik acid uptake (shi A).
- The his and nif regions can be actively transferred from a strain of K. pneumoniae to an E.coli
strain which require histidine.
- E.coli cells that do not require histidine anymore have acquired the ability to fix nitrogen.
- The conjugative plasmid pRDI that picked up the nif and his genes was selected and transferred to other bacterial genome.
- The gene, nifL, that serves as the repressor of nitrogen fixation can be deleted thus allowing constitutive expression of nif promoter mediated by nifA and ntrC (nitrogen regulator) gene products.
- The nitrogenase activity has been observed in E.coli that carries the nif plasmid pRDI which was very
much similar to that of K. pneumoniae strain.
> Agrobacterium tumefaciens, an obligate aerobe not resembling Klebsiella did not result in nitrogen fixing recombinants upon transfer of pRDI to itself.
> Mutants of Azotobacter vinelandii that lack either of the components of nitrogenase regained the
nitrogenase activity when pRDI was transferred to them.
> Nitrogenase can be protected from the inhibitory action of oxygen by a protein leghaemoglobin. The genes encoding these proteins can be isolated and transferred to other nitrogen fixing systems so as to
protect the nitrogenase from oxygen activity.
> Nitrogenase activity is correlated with the hydrogenase activity that evolve hydrogen hence it
requires more energy. The energy can be saved if the evolved hydrogen is further reduced to water
releasing electrons. Many nitrogen fixing bacteria possess ‘uptake hydrogenases’ which consists of two subunits HupS and HupL and it is advantageous to introduce it together with the nif genes into hosts that do not possess uptake hydrogenase system.
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Stratagies to increase production of secondary metabolites in tissue culture:-
1. Origin of plant tissue:- The genetics of the plants have a high influence on the production of secondary metabolites. For example, high fielding cultivars produce high nicotine content compared to low fielding cultivars.
2. Culture Media:- The composition of the culture medium influences both, biomass production and the synthesis of secondary metabolites. So, ideal conditions are required to be maintained to keep a balance between these two.
3. Growth Regulators:- They have a significant effect on the production of secondary metabolites, as they can either be synergistic or antagonists in their actions. For example, the addition of gibberellic acid to the Taxus cuspidate’s medium increases the taxol production.
4. Carbon source in the medium:- Sucrose is one the most widely used carbon source in the culture media. However, there are also some others, which include glucose, fructose, and galactose.
5. Temperature:- A higher temperature is reported to enhance the production of secondary metabolites.
6. pH:- A perfect pH between 5-6 is required to be maintained for the production of secondary metabolites.
7. Light intensity:- Apart from stimulating the production of secondary metabolites, light intensity also has a role in determining the amount of the production.
8. High-cell Density culture:- A high-density culture increases the production of secondary metabolites. The case is reported in Japonic cells where they have grown up to 759 \ L of cell mass to increase productivity.
9. Elicitation:- Elicitors are compounds of biological origin involved in plant microbe interaction. Elicitors are considered as mediator compounds which induce secondary metabolites formation in cells cultures. Varieties of elicitors have been used for production of secondary metabolites. Elicitation improves the efficiency of Sec. Product accumulation in plant cell culture by:
i. Minimizing up on time
ii. Avoiding change of media
iii. Induction of enzymes involved in biosynthetic pathway
iv. Inducing excretion of metabolites into the medium
Some of the biotic elicitor- induced products
10. Precursor feeding:- Under in vitro circumstances, often the precursors of metabolite die and can’t carry out the reaction. This can be overcome by supplying enough precursors to cells, which can be done by two methods:
a. Add precussors:- By adding precursors in the medium so that their availability is not limited.
b. Prevent resistant precursor analogs:- By preventing resistant precursor analogs and increasing the intracellular levels of precursors for the metabolite production. Eg.-
i. Production of shikonin increased three-fold after the addition of L-Phenylalanine to the medium in Lithospermum species.
ii. Datura sp. cell suspension cultures are supplemented with hydroquinone, in traces, the arbutin synthesis increase considerably.
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Four areas which have been responsible for the recent advances in biotechnology:- The recent advances in biotechnology are mainly due to progress in the following four areas:
1. Genetic Engineering (Recombinant DNA Technology):-
> Involves isolation, modification, and transfer of genes from one organism to another.
> Enables production of genetically modified organisms (GMOs).
> Applications include insulin production, Bt cotton, and gene therapy.
2. Cell and Tissue Culture Technology:-
> Involves the in vitro culture of plant and animal cells, tissues, and organs under sterile conditions.
> Used for micropropagation, production of disease-free plants, embryo rescue, and haploid production.
> Important in plant breeding and conservation.
3. Microbial Biotechnology (Industrial Fermentation):-
> Uses microorganisms for the production of antibiotics, enzymes, vitamins, organic acids, alcohol, vaccines, and biofuels.
> Modern fermentation technology has greatly improved industrial-scale production.
4. Molecular Biology and Genomics:-
> Studies the structure and function of DNA, RNA, and proteins.
> Includes techniques such as PCR, DNA sequencing, gene cloning, molecular markers, CRISPR, and bioinformatics.
> Widely used in diagnostics, crop improvement, forensic science, and personalized medicine.
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