Biotechnology

Why are genetically engineered bacteria used in biotechnology?

• Single-celled organisms: Bacteria are simple and efficient for genetic manipulation.

• Easy to manipulate: Bacteria can be easily modified to produce specific products.

• Quick expression of products: Engineered bacteria can rapidly produce the desired product once modified.

• Rapid cell division: Bacteria divide quickly, allowing for large-scale production of products in a short time.

What are the applications for genetically engineered bacteria?

• Fuels

• Chemicals

• Drugs

• Human hormones

What are the main biotechnology approaches in producing products with microorganisms?

• Products made by microorganisms: Microorganisms are used to produce a variety of products, such as antibiotics, enzymes, and biofuels.

• Genetic manipulation: The genetic makeup of microorganisms is altered to improve their ability to produce specific compounds or perform certain functions.

• Genetic modification of a pathway: Entire biochemical pathways can be engineered in microorganisms to enhance the production of desired products, like hormones, biofuels, or pharmaceuticals.

How is acetic acid (vinegar) produced by microorganisms?

Acetic acid bacteria (e.g., Acetobacter) are responsible for producing acetic acid (vinegar).

• They convert sugars or ethanol into acetic acid through fermentation.

• Obligate aerobes: They require oxygen to carry out the oxidation process.

• These bacteria grow well at pH < 5, which is optimal for vinegar production.

How is Vitamin C produced by acetic acid bacteria?

Acetic acid bacteria can carry out incomplete oxidation of certain higher alcohols and sugars. The metabolic products of this process are then used to produce Vitamin C.

How are antibiotics produced by Streptomyces?

Streptomyces are a genus of bacteria known for their ability to produce over 500 different types of antibiotics.

• Streptomyces species produce the antibiotic streptomycin, which is commonly used to treat bacterial infections.

• Streptomyces are particularly important in the production of antibiotics, with some species capable of producing more than one antibiotic.

What is an antibiotic assay and how is it performed?

An antibiotic assay is a laboratory method used to test the effectiveness of antibiotics against specific microorganisms.

• Purpose: To determine the activity of an antibiotic and its minimum inhibitory concentration (MIC), which is the lowest concentration that inhibits microbial growth.

• Method:

  1. A microorganism (often bacteria) is cultured on an agar plate.

  2. Antibiotic disks or samples are placed on the agar surface.

  3. The zone of inhibition (area around the disk where bacteria cannot grow) is measured.

  4. The size of the zone indicates the antibiotic's effectiveness—larger zones suggest stronger activity.

How is Swiss cheese made and what causes its characteristic holes and taste?

Swiss cheese is produced with the help of Propionic acid bacteria.

• These bacteria ferment lactic acid, producing CO₂ and propionic acid.

• The CO₂ gas builds up during fermentation, forming the characteristic holes (also called eyes) in the cheese.

• Propionic acid contributes to the distinctive taste of Swiss cheese.

How is insulin commercially produced using genetically engineered bacteria?

Insulin was the first human protein to be commercially produced by bacteria.

• It went on sale in 1982 under the brand name ‘Humulin’.

• Insulin is a small peptide hormone.

• The most efficient method involves constructing an artificial gene that encodes the final hormone, rather than producing the large precursor protein that insulin naturally derives from.

• The insulin gene is inserted into a plasmid vector to create a recombinant plasmid.

• This plasmid is introduced into transgenic bacteria, which are then grown in culture to produce the hormone.

• The insulin is then extracted from the bacterial culture for use in medical treatments.

How do COVID-19 vaccines work to elicit an immune response?

There are two main types of COVID-19 vaccines:

1. mRNA vaccines (e.g., Pfizer-BioNTech, Moderna):

   • The mRNA for the spike protein antigen of the virus is encapsulated in a lipid nanoparticle.

   • The lipid nanoparticle mimics the structure of human cells, allowing it to enter the cells.

   • Once inside the cells, the mRNA instructs the cells to produce the spike protein.

   • The immune system recognizes this protein as foreign and triggers an immune response, preparing the body to fight the actual virus.

2. Viral vector vaccines (e.g., AstraZeneca, Johnson & Johnson):

   • The mRNA for the spike protein antigen is inserted into a modified, harmless chimpanzee virus.

   • This virus acts as a carrier, delivering the mRNA into human cells.

   • The cells then produce the spike protein, stimulating an immune response without causing disease.

What are the issues with expressing mammalian genes in bacteria, and how can they be addressed?

• Eukaryotic genes must be put under the control of a bacterial promoter:

  • Solution: Design special expression vectors with bacterial promoters and ribosome binding sites to ensure proper transcription and translation in bacteria.

• Bacterial genes don’t have introns:

  • Solution: Clone the gene using mRNA and generate cDNA to avoid introns and ensure proper gene expression in bacteria.

• Codon bias:

  • The genetic code is degenerate, meaning most amino acids can be encoded by multiple codons. However, codon usage varies between organisms.

  • Solution: You may need to edit the gene sequence to adjust the codons to those more commonly recognized by your bacterial species, often through gene synthesis.

• Post-translational modifications:

  • Many mammalian proteins require post-translational modifications (e.g., glycosylation), which bacteria may not be able to perform.

  • The protein may be degraded in the bacterial host, or the eukaryotic protein may be toxic to the bacteria.

  • Solution: If this happens, further engineering of the host or vector may be required to improve protein stability and functionality.

How is genetic modification of bacteria used to accumulate a desired metabolite?

• If the goal is to produce metabolites (e.g., food additives, dyes, antibiotics, biofuels), a single gene is typically insufficient.

• Building a metabolic pathway is necessary, as it involves:

  • Multiple genes that encode enzymes involved in the metabolic process.

  • Proper regulation and coordination of their expression to ensure the efficient production of the desired metabolite.

What is pathway engineering and what is its goal in metabolic engineering?

• Pathway engineering is the process of assembling new or improved biochemical pathways using genes from one or more organisms.

• Aim: The goal is to produce large amounts of a particular metabolite (such as biofuels, drugs, or food additives).

• So far, most efforts in pathway engineering have focused on improving existing pathways rather than creating entirely new ones.

How is indigo production achieved in E. coli using biotechnology?

Indigo production in E. coli can be achieved by adding just one gene to enable the necessary biochemical pathway.

• This gene enables the bacteria to produce indigo, a blue dye, by activating the appropriate metabolic pathway.

What is bioremediation?

Bioremediation is the process of using microorganisms to clean up environmental pollutants.

• This involves the microbial breakdown or transformation of harmful substances into less toxic forms.

What types of pollutants can be cleaned up by bioremediation?

Pollutants that can be remediated include:

• Oil

• Radionuclides (e.g., uranium)

• Pesticides

• Plastics

What is the plastic problem and why is it significant?

The plastic problem refers to the widespread pollution caused by plastic waste in the environment.

• Plastics take hundreds of years to decompose, leading to long-term environmental damage.

• Plastics can harm wildlife, as animals may ingest or become entangled in plastic debris.

• Microplastics are also present in the environment and food chain, posing risks to human health and ecosystems.

What are microbial plastics and how are they produced?

Microbial plastics are biodegradable plastics made by bacteria.

• Polyhydroxyalkanoates (PHAs) are a type of bacterial storage polymer used to produce these plastics.

What is PETase and what are the challenges associated with microbial plastic production?

• PETase is an enzyme produced by certain bacteria that can break down PET (polyethylene terephthalate) plastics, a common synthetic plastic. It helps in biodegrading plastics like PET in the environment.

• Challenges with microbial plastic production:

  • High cost of production compared to synthetic plastics.

  • Competition for carbon substrates with biofuels, as both require similar resources.

How do microorganisms break down plastics using biodeterioration, biofragmentation, assimilation, and mineralization?

• Biodeterioration – Microorganisms begin degrading the plastic surface, causing physical and chemical damage.

• Biofragmentation – Enzymes like PETase break down long plastic polymers into smaller molecules (e.g., oligomers and monomers).

• Assimilation – Microbes absorb the fragmented molecules and use them for growth and energy.

• Mineralization – The remaining organic matter is fully converted into CO₂, water, and minerals, completing the biodegradation process.

What is microbial leaching and how is it used in mining?

• Microbial leaching is the process of using microorganisms to extract valuable metals from low-grade ores.

• Certain bacteria, such as Acidithiobacillus ferrooxidans, can break down minerals and release metals like copper, gold, and uranium.

• This method is more environmentally friendly and cost-effective compared to traditional mining techniques.

How are microorganisms used as tools in biotechnology?

• Bacteria are essential for nearly all biotechnology applications.

• They are widely used in cloning and DNA manipulation techniques.

• Key tools include:

  • Restriction enzymes – Cut DNA at specific sequences for genetic engineering.

  • CRISPR-Cas9 – A powerful gene-editing tool derived from bacterial defense systems.

What is a metagenome?

A metagenome is the collective genome of all microorganisms present in a specific environment.

What is gene mining, and why is it important?

• Gene mining is the process of isolating useful genes from environmental DNA without the need to culture the organism.

• It allows scientists to discover novel enzymes for applications in medicine, industry, and environmental science.

• Metagenomics has identified genes encoding:

  • Pollutant-degrading enzymes

  • Antibiotic-producing enzymes

  • Lipases, chitinases, esterases

  • Enzymes with enhanced industrial stability

How is metagenomics used in gene mining?

• Identify an environment rich in the target compound (e.g., polluted site).

• Isolate and clone DNA from the microbial community.

• Introduce cloned DNA into host bacteria.

• Screen for bacteria that grow on the target compound.

• Test bacterial extracts in vitro to identify the enzyme of interest.

How did fungi contribute to the start of industrial microbiology?

• 1851 – Pasteur discovered that alcoholic fermentation is caused by microbial activity.

• 1883 – Pure strain brewing (Saccharomyces carlsbergensis) began at Carlsberg Brewery.

• 1923 – Pfizer established the first citric acid production plant using Aspergillus niger.

Why are microorganisms (including fungi) ideal for industrial applications?

• Ease of mass cultivation

• Grow rapidly

• Grow on cheap substrates (e.g.

agricultural waste)

• Diversity of potential products

• Can be genetically manipulated

What are the pharmaceutical uses of fungi?

Antibiotics

Steroids

Statins

Other drugs

What is the history of Penicillin as a wonder drug?

• 1928: Alexander Fleming discovered the antibacterial effects of Penicillium notatum at St Mary’s Hospital.

• 1941: Florey and Chain purified penicillin, driven by the need to treat war casualties.

• Impact: Penicillin became a "wonder drug", significantly increasing life expectancy by a decade.

What antibiotics are produced by fungi and their spectrum?

• Penicillins – Penicillium chrysogenum – Gram-positive bacteria

• Cephalosporins – Cephalosporium acremonium – Gram-positive & negative bacteria

• Griseofulvin – Penicillium griseofulvum – Antifungal

• Fusidic acid – Fusidium coccineum – Gram-positive bacteria

What is the use of Cortisone and how was it produced?

• Uses: Cortisone is used to treat conditions like rheumatoid arthritis, asthma, and eczema.

• Production: Originally extracted from animal adrenal glands, making the process costly.

How did the Upjohn Chemical Company reduce the cost of Cortisone production?

• Rhizopus arrhizus was found to convert diosgenin (a steroid from Mexican yams) into an intermediate.

• This intermediate could then be converted to cortisone, dramatically reducing production costs.

What is 1-Ephedrine, and how is it used?

• Source: Produced by certain yeast species.

• Uses:

  • Treatment for asthma

  • Ingredient in nose drops and inhalants.

What are ergot alkaloids, and how are they used?

• Source: From Claviceps species.

• Uses:

  • Vasoconstrictors (though highly toxic)

  • Induce uterus contractions

  • Treatment for migraines

• Lysergic acid (LSD), first made by Swiss scientists Hofmann and Stoll.

What are statins used for?

• Statins are used to control cholesterol levels.

• They are the most widely used pharmaceutical in the Western world.

What is Ganoderma lucidum (Reishi/Lingzhi), and what are its medicinal properties?

• Ganoderma lucidum (also known as Ganoderma ling-zhi, Reishi, or Lingzhi in China).

• Medicinal Properties:

  • Contains bioactive triterpenoids and polysaccharides.

  • Antioxidant effects.

  • Immunoregulatory properties.

  • Helps lower cholesterol.

What are some examples of organic acids produced by microorganisms?

• Citric acid

• Itaconic acid

• Gluconic acid

• Ascorbic acid

How is citric acid produced and what are its uses?

• Production: Produced through Aspergillus niger fermentation.

• Uses:

  • Food industry: Found in soft drinks, jams, and confectionery.

  • Preservative: Prevents the loss of vitamin C in canned fruit and vegetables.

What are the uses and production sources of Itaconic and Gluconic acid?

• Itaconic acid:

  • Uses: Improves properties of vinyl polymers, such as in emulsion paint.

  • Production: Produced by Aspergillus terreus in stainless steel aerated tanks.

• Gluconic acid:

  • Uses: Used in toothpaste manufacture.

  • Production: Produced by Aspergillus niger.

What are some examples of alcohols produced by microorganisms?

• Ethyl alcohol

• Glycerol

How is industrial ethanol produced, and what are its uses?

• Production:

  • Can be made chemically from petroleum or through fermentation using yeasts.

• Uses:

  • Used as a solvent in various industrial processes.

  • Used as a fuel.

• Alternative to petroleum: The rising cost and decreasing supply of petroleum make fermentation a more attractive alternative.

What are the uses and production source of glycerol?

• Production: Produced by Saccharomyces cerevisiae.

• Uses:

  • Solvent, plasticizer, and sweetener.

  • Used in the manufacture of explosives, cosmetic soaps, and anti-freeze.

  • Also used in the printing industry.

What are some examples of fine chemicals produced by microorganisms?

• Enzymes

• Vitamins

• Plant growth factors

What are some enzymes produced by fungi?

• Amylase – Breaks down starch into sugars.

• Cellulase – Degrades cellulose into simpler sugars.

• Laccase – Involved in the oxidation of phenolic compounds.

• Protease – Breaks down proteins into amino acids.

• Lipase – Breaks down lipids (fats).

• Xylanase – Degrades xylan, a component of plant cell walls.

What vitamins and pigments are produced by fungi?

• B vitamins

• Beta-carotene

• Ergosterol

• Riboflavin

What plant growth factors are produced by fungi?

Gibberellic Acid – Produced by Fusarium moniliforme, a growth-promoting plant hormone.

• Functions: Regulates fruit setting, prevents fruit drop, and increases yield.

How are fungi used in the food industry beyond enzymes?

• Fungi as Food

• Use of fungi in the food industry

What is the significance of fungi as food, specifically in mushroom production?

Mushroom Production:

• The major cultivated mushroom is Agaricus bisporus, which makes up 37% of global production and is worth approximately $4.7 billion per year worldwide.

• Other commercial species include:

  • Oyster Mushroom: Pleurotus ostreatus

  • Shiitake Mushroom: Lentinula edodes, typically grown on logs.

What is Mycoprotein (Quorn) and how is it produced?

Mycoprotein (Quorn):

• Developed by Marlow Foods (subsidiary of ICI).

• Produced using Fusarium venenatum, isolated from soil in Buckinghamshire.

• Grown on glucose from maize.

• Spun to resemble the texture of chicken.

• Annual sales of Quorn exceed £15 million in the UK.

What are the uses of fungi in the food industry?

• Beverages

• Bakers yeast

• Dairy produce

• Food additives

• Asian foods (Natto, Temph and Miso)

• Amino acids

What is the historical significance of fungi in bread and beverage production?

• Bread and alcoholic beverages are among the most widely consumed foods processed with the help of fungi.

• Origins:

  • The Sumerians produced a beer from moist fermented bread before 7000 BCE.

  • The Egyptians also made this "bread-beer."

  • Both bread-making and brewing were refined by the Greeks and Romans, becoming a normal part of household activities.

How are yeasts used in the production of bread and alcoholic beverages?

• The production of both bread and alcoholic beverages uses yeasts, primarily strains of Saccharomyces cerevisiae.

• Yeasts ferment glucose to yield ethyl alcohol and carbon dioxide.

  • Reaction:
    C6H12O6→2C2H5OH+2CO2C6​H12​O6​→2C2​H5​OH+2CO2​

How do fungi contribute to the flavour and texture of cheese?

• Chimosin (rennet), an enzyme used in cheese-making, can be obtained from fungi like Cryphonectria parasitica, instead of from calf's stomachs.

• Penicillium camemberti: Contributes to the texture and flavour of Camembert cheese.

• Penicillium roqueforti: Responsible for the flavour and aroma of Roquefort cheese (made from sheep's milk).

How do fungi contribute to the flavour of chocolate?

Fungi play a key role in the fermentation of cocoa beans, which is a crucial step in the production of chocolate. During fermentation, the naturally occurring yeasts and molds (such as Aspergillus and Penicillium) break down the beans' sugars and other compounds. This process enhances the flavour development, contributing to the unique taste of chocolate.

What is the role of fungi in the production of Asian foods like tempeh and shoyu?

• Tempeh: Made using Rhizopus spp., which ferments soybeans to produce a protein-rich food staple in Indonesia.

• Shoyu (Soy Sauce): Produced in East and Southeast Asia by fermenting a mixture of soybeans, wheat, and flour using Aspergillus oryzae. Japan is the largest producer with over 27,000 manufacturers.

What are the agricultural uses of fungi, particularly in biological control and mycorrhizal fungi?

• Biological Control Agents

• Mycorrhizal Fungi

What is biological control?

use of one organism to control another. It is an attractive alternative to chemical pesticides.

How are microbial biological control agents used in agriculture?

• Microbes have been used as biological

control agents in the USA for nearly 40 years,

but their use has failed to keep pace with

chemical pesticides.

• The success has been limited by their ability

to control a narrow range of pests, by their

slow action and short field life.

What is Paecilomyces lilacinus and how is it used in agriculture?

• Paecilomyces lilacinus is a soil-borne fungus.

• It parasitizes the eggs and larvae of nematodes that damage plant roots.

• Registered as Biocon® in the Philippines, it is used as a biological control agent to reduce nematode damage in crops.

What is Green Muscle®, and how is it used?

• Green Muscle® is a mycopesticide developed from the spores of Metarhizium anisopliae, an insect pathogen fungus.

• It is available as a spore powder or an oil miscible concentrate.

• Green Muscle® is applied using normal spraying equipment to control insect pests.

How does Phlebia gigantea help in the control of Heterobasidium annosum?

• Phlebia gigantea is a fungal biological control agent used to control Heterobasidium annosum, a pathogen that causes root rot in coniferous trees.

• It works by outcompeting Heterobasidium annosum for space and nutrients on the tree stump surfaces, preventing its infection.

• This biocontrol method reduces the need for chemical treatments in forest management and promotes healthier forests.

What are mycorrhizal inoculants and how are they used in agriculture?

• Mycorrhizal inoculants are fungal spore tablets used to enhance plant growth.

• They are applied to plant roots during transplanting, helping establish a beneficial symbiotic relationship between plants and mycorrhizal fungi.

• These products often contain additional ingredients, like organic matter to encourage microbial activity and water-absorbing gels to help adhere the fungal spores to the root system.

• Mycorrhizal fungi improve plant nutrient uptake, especially phosphorus, and can enhance plant stress tolerance.

What is bioremediation and how does it work?

• Bioremediation is the use of living organisms (like bacteria, fungi, or plants) or their products to metabolize or detoxify harmful environmental pollutants.

• It is a process used to clean up polluted environments such as soil, ground water, surface water, and air by breaking down hazardous chemicals and waste.

• This method is used in industrial and developing countries to address environmental contamination, offering a sustainable alternative to traditional cleanup methods.

What is the focus of most research on fungal bioremediation?

Most research centers on the lignin-degrading enzyme system of white rot fungi, which helps break down pollutants, particularly lignin, and other toxic compounds in the environment.

What is mycofiltration and how does it work?

• Mycofiltration involves the impregnation of fungal spores and hyphae into fabric landscaping cloth.

• The fabric is placed over contaminated ground.

• The fungal mycelium acts as a filter, trapping and degrading contaminants.

• It is a new technology and is currently used on a small scale.

• Widespread adoption depends on public acceptance and regulatory approval.

Why are plants important?

• Textiles/fibres

• Food and fodder

• Pharmaceuticals and cosmetics

• Petroleum substitutes

• Ornamentals/amenity

• Paper/wood products

• Biodiversity

What is plant biotechnology according to the OECD?

Plant biotechnology is the application of science and technology to plants, their parts, products, and models to alter living or inert materials for the development of knowledge, goods, and services.

What are some examples of plant biotechnology applications?

Examples include plant cell factories, plant tissue culture, transgenic plants, molecular breeding, food processing, plant breeding, and wine making.

What is the general trend of world population growth from 1750 to 2150?

The world population grew slowly until the 1800s, then rapidly increased during the 20th century, and is projected to stabilize or decline slightly after 2100.

What are the main categories of food we eat?

Dairy and eggs, meat, grain, sugar and fat, produce (fruits and vegetables), and other (e.g. beverages, spices, processed foods).

What are the main types of nutrients our body needs?

• Micronutrients: Vitamins and minerals

• Macronutrients: Water, proteins, carbohydrates, and fats

What was the Green Revolution and how was it implemented?

• A planned international effort in the 1970s to increase crop yields

• Implemented through:

  • New crop cultivars

  • Irrigation

  • Fertilizers

  • Pesticides

  • Mechanisation

What were the outcomes of the Green Revolution?

• Reduced chronic hunger from 40% to 20% of the global population

• Achieved this while the population doubled

• Saved millions of hectares of land from cultivation

• However: 800 million people are still undernourished

What did plant breeding and the Green Revolution achieve?

• Faster growth: More than one crop cycle per year

• Semi-dwarf habit: Strong stems that prevent lodging (plants don’t fall over)

• High yield:

  • Better soil assimilation

  • Increased biomass

• Disease resistance:

  • Wheat rust

  • Rice blast

• Adaptability to local conditions: Introduction of IR8, the first “miracle rice”

How can plant biotechnology help in plant regeneration?

• Some plants can regenerate from a cutting.

• Many plants can regenerate from a few cells, but only if the right hormones are provided (through plant tissue culture).

Why is plant tissue culture useful?

• Micropropagation allows rapid plant production.

• Eliminating systemic viruses: The virus infects the plant but can't infect the meristem.

• Sterile culture of the meristem eliminates the virus, resulting in a healthy crop.

What is molecular breeding and why is it useful?

• Molecular breeding uses DNA markers linked to desirable traits to select plants early in development.

• It speeds up breeding, especially for slow-growing plants like fruit trees (e.g., apples).

• Example:

  • Apples take 5–10 years to bear fruit.

  • A DNA marker for red fruit helps identify seedlings likely to bear red fruit before maturity.

How do we find DNA markers for molecular breeding?

• Generate a DNA fingerprint of the parent plant using random markers (e.g. SNPs from genome sequencing)

• Identify markers linked to the desired phenotype (e.g. red or yellow fruit)

• Confirm that the marker is closely linked to the trait

• Use the marker to select seedlings with the desired trait early on

What are SNPs in genetics?

SNPs (Single Nucleotide Polymorphisms) are variations at a single base pair in the DNA sequence, commonly used as markers in molecular breeding.

What are some advanced techniques to speed up plant breeding?

• Traditional crosses are limited to the same or closely related species

• Protoplast fusion allows wide crosses:

  • Enzymes remove the cell wall to create protoplasts

  • Protoplasts from different genotypes are fused

  • Fusion is done using polyethylene glycol or an electric pulse

  • Enables crosses between similar but different species (e.g., wheat × rye)

What are pre-fertilisation barriers to reproduction across species in plants?

• Asynchrony of flowering

• Incompatible floral morphology

• Self-incompatibility systems

What are post-fertilisation barriers to reproduction across species in plants?

• Hybrid weakness

• Hybrid sterility

What is embryo rescue and why is it useful?

• A technique to culture embryos that would otherwise die in incompatible crosses

• Helps create new hybrids between different species

• Useful across a wide range of plant species

Can we use plant cells as factories? What can they produce?

Yes! Plant cells can act as factories to produce:

• Drugs (e.g., vaccines, therapeutic proteins)

• Dyes

• Flavours

• Fragrances

How can we use plant cells as factories to produce valuable compounds?

• Understand the biochemistry behind the desired product

• Identify the genes responsible for its production

• Express the genes in plant cells (via genetic engineering)

• Harvest the chemicals produced by the engineered plant cells

What are some problems to overcome when using plant cells as factories?

De-differentiation of cells in culture

• Changes in gene expression

• Altered enzyme production

• Shifts in metabolite profiles

How are plants used in pharmaceuticals, and what challenges exist?

• 35,000–70,000 plant species have been screened for medicinal use.

• Many compounds are now chemically synthesised, but

• 11% of the 252 drugs deemed basic and essential by the WHO are still exclusively from flowering plants.

What is Taxol and how does it work?

• Taxol is a potent anti-cancer drug.

• It binds to microtubules, stopping cell division by preventing the normal dynamics of the microtubules in the mitotic spindle.

What is the progress in using plant cell cultures for pharmaceuticals, and what is needed?

• Some progress has been made in using plant cell cultures to produce pharmaceuticals.

• To optimize production, transgenic plants are often required, which are genetically modified to enhance or introduce specific traits for pharmaceutical production.

What is conventional or traditional plant breeding?

• Involves crossing plants of the same species

• Selecting phenotypes in the progeny based on desirable traits (e.g., size, color, yield)

What are genetically modified (GM) plants?

• Construct a transgene and transform it into plant cells.

• The transgene can come from any source (e.g., other plants, animals, or microorganisms).

• Regenerate plants with the desired phenotype (e.g., resistance to pests, higher yield).

How is plant transformation done?

• Assemble the transgene in a bacterial plasmid.

• Transfer the plasmid into the plant genome (commonly using bacteria like Agrobacterium).

• Select plants that exhibit the new traits (e.g., resistance, improved yield).

How does Agrobacterium tumefaciens help in plant transformation?

• Agrobacterium tumefaciens is a plant pathogen that causes crown gall, a mass of undifferentiated plant cells.

• The bacterium transfers its own plasmid DNA into the plant genome.

• One gene in the plasmid codes for an enzyme that makes the plant hormone cytokinin, leading to abnormal cell growth (crown gall formation).

How are transgenes inserted into plants using Agrobacterium tumefaciens?

• Replace the genes that cause crown gall disease with the desired transgenes.

• The transgenes are then inserted into the plant chromosomes via the plasmid DNA transferred by the bacterium.

What is the process of Agrobacterium leaf disc transformation?

• Put transgene into Agrobacterium bacteria.

• Incubate Agrobacterium with leaf discs from the plant.

• Move leaf discs onto selective medium to encourage growth.

• Wait for shoots and roots to develop (manipulate growth regulators).

• Grow plants to maturity (e.g., in tobacco, approximately 4 months).

• This method has a good success rate for plant transformation.

What is the process of regenerating plants from tissue culture?

• Incubate leaf discs with Agrobacterium.

• Balance cytokinin and auxin to induce callus growth.

• Add more cytokinin to induce shoot growth.

• Add more auxin to induce root growth.

• Transfer to soil and gradually reduce humidity for acclimatization.

How do plant hormones like cytokinin and auxin affect shoot and root production?

• Cytokinin promotes shoot production.

• Auxin promotes root production.

• The balance between these hormones determines whether the plant forms shoots, roots, or callus during regeneration.

What is a transgene?

A transgene is a gene that has been artificially inserted into the genome of an organism (often from a different species) to impart new traits.

How is a transgene assembled?

• Promoter region: Derived from plant DNA to control gene expression in the plant.

• Coding region: Can be from plant, animal, or bacterial DNA, depending on the desired trait.

• Splice together: The promoter and coding region are joined to form the complete transgene construct.

• Transfer to plant genome: The transgene is introduced into the plant’s genome via transformation techniques (e.g., using Agrobacterium).

How can GM plants help?

• Increased crop yields through protection from pests and pathogens.

• Improvement of nutritional qualities:

  • More vitamins,

  • Better proteins,

  • Improved oils,

  • More antioxidants.

• New products:

  • Vaccines,

  • Plastics.