Plant Biotechnology - Genetic Improvements in Agriculture
Introduction to Crop Improvement
Crop Plant Domestication and Beyond:
Breeding technologies are continuously advancing.
Modern molecular plant breeding is practiced now and will continue into the future.
Transgenic plants are part of modern molecular plant breeding.
Emerging technologies are addressing concerns related to transgenics.
Applications of Breeding & Innovations:
The Second Green Revolution aims to improve agriculture sustainably.
Metabolic engineering is used for:
Molecular farming of carbohydrates, lipids, and protein.
Producing fine chemicals and plant-derived compounds as drugs.
Exploring plants as alternative fuels due to current demand.
Genetic Improvements in Agriculture: From Hunter-Gatherer to Green Revolution
Timeline:
Life on Earth is approximately four billion years old.
Homo sapiens emerged around 300,000 years ago.
Human activities have significantly altered the planet's physical, chemical, geological, atmospheric, and biological aspects.
Impact of Human Activities
Environmental Changes:
Humans have removed mountains.
Rivers have been dammed, exemplified by the Three Gorges Dam, the world's largest hydroelectric power generator, creating a reservoir over 2 miles (3 km) wide.
Extinctions:
Human arrival led to the extinction of species like the dodo by the end of the 17th century.
Species Modification:
Plant genomes have been modified for thousands of years.
Global Challenges
Population Growth and Resource Depletion:
The atmospheric CO_2 levels and global population have increased significantly since 1800.
The central question is how to feed more people without further damaging the planet and with fewer resources.
Role of Plant Breeding
Addressing Global Challenges:
Plant breeding is crucial in addressing global challenges.
Genetic Improvements in Agriculture: A Historical Perspective
Timeline:
The Distant Past: Crop plant domestication and early agriculture.
The Recent Past: Hybrid seed development and the First Green Revolution.
Now and Into The Future: Breeding for improved human health, drought tolerance, agricultural innovation, and the Second Green Revolution.
The Distant Past (10,000 Years Ago to 1900)
Early Agriculture:
Homo sapiens originated 400,000 – 250,000 years ago.
Major crops were domesticated ~10,000 – 5,000 years ago.
The development of human civilizations is correlated with the development of agriculture.
Plant domestication followed the end of the most recent glacial period.
Transition to Agriculture
Gradual Change:
A gradual shift from seeking and following food sources to semi-settled migration and, finally, permanent settlements.
Plant Domestication
Parallel Development:
Plants were domesticated in parallel in several regions.
Examples:
Wheat, barley, pea, lentil ~ 13,000 years ago
Rice, soybean ~ 9000 years ago
Rice, bean ~ 8500 years ago
Corn, squash, bean, potato ~ 10,000 years ago
Genetic Modification Through Cultivation
Natural Variation:
Genetic modification arose as a consequence of cultivation, utilizing natural variation within populations.
Selective Planting:
Planting seeds from “good” plants increased their representation in subsequent generations.
Example: During maize domestication, cob size increased.
Selected Traits During Domestication
Grain Characteristics:
The hard casings around many grains were eliminated.
Humans selected against hard coverings in maize domestication.
Decrease in branching and increase in seed size were also selected for.
Seed Dispersal:
Seeds that don’t break off easily were selected.
Wild grains have a brittle rachis for seed dispersal, while domesticated grains have a tough rachis to facilitate harvesting.
Genomic Rearrangements
Polyploidy:
Many crops are products of extensive genomic rearrangements.
Common wheat resulted from interspecific hybridization between three ancestors.
Polyploid (multi-genome) plants are often bigger and were selected for propagation.
Brassicas share three genomes recombined in various ways.
Domestication and Modern Crops
Genome Modification:
Domestication through genome modification gave us modern crops.
Rice Domestication and Modification
Extensive Genomic Modification:
Indica rice IR64, one of the most widely grown crops, is the product of a complex breeding program that has caused extensive genomic modification, mutation, deletion, and rearrangement.
The Myth of Natural Food
Extensive Modification:
The food we eat comes from plants already extensively modified from their original form.
Even heritage varieties are extensively genetically modified.
The Recent Past – Scientific Plant Breeding
Population Growth:
The world population quadrupled in just over 100 years.
Advancements:
Improvements in plant propagation and breeding were needed to keep up with population growth.
Key Figures
Mendel and Darwin:
Mendel and Darwin paved the way for scientific plant breeding.
Hybrid Corn
Increased Yields:
The development of hybrid corn led to a significant increase in yields.
The progeny of two genetically different parents often show enhanced growth – this effect is termed “hybrid vigor” (also known as heterosis).
Farmers rapidly adopted hybrid corn because increased yields more than offset increased costs, even though they had to purchase seed every year.
Norman Borlaug and the Green Revolution
Key Accomplishments:
Norman Borlaug (1914-2009), “father of the green revolution,” was a distinguished plant breeder and Nobel Laureate.
One of the most significant accomplishments of 20th-century science was the development of lodging-resistant, high-yielding semi-dwarf grain varieties.
Impact of Green Revolution
Increased Crop Yields:
Improved green-revolution plants dramatically increased crop yields.
The introduction of disease-resistant, semi-dwarf varieties turned countries from grain importers to grain exporters.
CGIAR
CGIAR is an international organization of agricultural research groups which includes:
CIMMYT (International Maize and Wheat Improvement Center)
International Rice Research Institute (IRRI)
Rice Breeding at IRRI
Significant Yield Increases:
Rice breeding at IRRI also brought huge yield increases.
IR8, released in 1966, was known as “miracle rice” because of its high yields.
Crop Productivity and Population
Increased Yields:
Crop productivity has kept pace with the population because of increased yields.
Crop area has not increased as rapidly as crop production because yields (food per hectare) have increased.
Growing more food without using more land helps mitigate climate change and slow the loss of biodiversity.
Modern Plant Breeding Methods
Molecular Methods:
Modern plant breeders use molecular methods, including DNA sequencing and proteomics, as well as field studies.
Advances in Genetic Technologies
Advances in genetic technologies contribute to improved plants:
Marker-assisted selection
Genome-wide association studies
Recombinant DNA technology and transgenic plants
Cisgenics and intragenics
Transgrafting
Precision genome editing
Marker Assisted Selection (MAS)
Selecting for DNA Markers:
Selecting for DNA markers is faster than selecting for phenotype (physical expression of traits).
Genotype refers to the sequence of all the genes in a genome.
How Markers Work
Gene Reassortment:
Each generation, genes reassort or shuffle.
Markers let us “see” which genes each individual has inherited.
Example: Introgression of a Disease Resistance Gene
Process:
Cross an elite tomato with a poor tomato that is disease resistant.
Use markers to look at their DNA and identify those with the resistance gene.
Repeatedly cross back to the elite tomato, using markers to identify plants with the disease resistance gene.
After several generations, achieve an elite, disease-resistant tomato.
Markers greatly accelerate breeding programs.
MAS in Submergence Tolerant Rice (Sub1)
Flooding:
Many rice-growing regions are prone to flooding.
Submergence Tolerance
Survival:
Submergence-tolerant rice can survive floods as long as 17 days.
Sensitive rice cannot survive prolonged flooding.
Sub1 rice growth arrests during flooding, enhancing survival.
Production of Swarna-Sub1
Process:
Cross Swarna (high-yielding but flood-sensitive rice variety) with a Sub1 donor.
After several generations, Swarna-Sub1 is produced.
MAS allowed the Sub-1 trait to be rapidly introgressed into Swarna.
The Swarna-Sub1 rice accounted for over 1/4 of the rice planted in India in 2010.
Advances in Genomics Technologies
Facilitating Breeding:
Genome sequence data are available for more than 20 plant species.
Molecular breeding and mapping tools are developed for many species.
Genome-wide association studies help match genes to traits.
Genome-Wide Association Studies (GWAS)
Identifying Genes:
Genome-wide methods make it possible to identify genes associated with complex traits like yield or water use efficiency.
Association analysis leads to gene discovery.
GWAS and Complex Traits
Identifying Genes with Small Effects:
This approach allows hundreds of genes with small effects to be identified.
In maize, grain yields are correlated with leaf angle and size. A genome-wide association survey (GWAS) revealed hundreds of single-nucleotide polymorphisms (SNPs) associated with these traits.
GWAS and Disease Resistance
Identifying SNPs:
GWAS reveals SNPs that contribute to disease resistance.
Similar studies have led to the identification of genes contributing to other agronomically important traits, including drought tolerance.
Genetic Modification (GM)
Process:
Recombinant DNA (or GM) allows a single gene to be introduced into a genome.
This method can be faster than conventional breeding.
Gene Introduction
Steps:
Isolate the gene of interest using molecular biology methods.
Recombine it into recipient plant DNA.
Once a gene is introduced into the plant genome, it functions like any other gene.
Molecular Breeding vs. GM
Molecular Breeding:
Desired trait must be present in the population.
Genetic resources must be available.
Plant should be propagated sexually.
GM:
Gene can come from any source.
Genetic resources not required.
Plant can be propagated vegetatively.
GM Example: Disease Resistant Banana
Bacterial Wilt:
Banana bacterial wilt (Xanthomonas campestris pv. musacearum) is destroying plants in eastern Africa.
Transgenic plants carrying a resistance gene from pepper are resistant to the disease.
GM Example: Insect Resistance (Bt Gene)
Bt Toxin:
Bacillus thuringiensis (Bt) bacteria produce insecticidal proteins.
The Bt toxin affects only some insects because, to be effective, it has to be processed and bind to a specific receptor protein in the insect intestine.
After binding, the insecticidal proteins assemble to form a pore in the lining of the insect intestine which kills the insect.
GM Example: Herbicide Resistance
Weed Control:
Many farmers use herbicides to eliminate weeds (undesired plants) from their fields.
Environmental Benefits:
Farmers that plant herbicide-tolerant crop plants use less herbicide, herbicides that are less toxic, and till (plow) less, saving soil and fuel.
Gene Flow
Monitoring and Control:
Gene flow through pollen movement has to be monitored and controlled.
There have been confirmed cases of gene transfer from crops to weeds and vice versa.
Emerging Technologies
Addressing Concerns:
Emerging technologies circumvent some concerns about transgenics.
In the conventional approach to transgenic plant production, a large piece of DNA, derived from several sources, is inserted randomly into the genome.
Cisgenics and Intragenics
Cisgenics: Genes from the same or closely related species.
Might mean that little foreign DNA is introduced.
Intragenics: Bacterial and viral DNA may be included, but no protein-coding regions from other organisms.
Small amounts of DNA from T-DNA borders may be incorporated.
Advantages: Avoids lengthy backcrossing process and Particularly useful for plants propagated vegetatively, such as potato or apple
Disadvantages: Gene must exist in gene pool & Gene silencing can be induced by introduction of antisense or hairpin RNA, or overexpression of an endogenous gene
RNA Silencing
Gene Silencing:
Gene silencing can be induced by the introduction of antisense or hairpin RNA, or overexpression of an endogenous gene.
Transgrafting
Process:
A non-transgenic shoot can be grafted onto a transgenic root, so the food products and pollen don’t carry the transgene.
Precise Genome Engineering
Methods:
Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are proteins that can produce double-strand DNA breaks that, when repaired, introduce site-specific mutations or insertions.
The clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system uses RNAs to target nucleases to specific sites; when repaired, site-specific mutations or insertions are introduced.
Genome Engineering Tools
ZFNs and TALENs Function:
ZFNs recognize 3-basepair sequences. A series of four linked ZFNs has a 12-bp binding specificity.
TALENs recognize DNA via a series of 33 – 35 amino acid domains that each recognizes a single DNA basepair.
The CRISPR / Cas system used RNA for targeting, making it cheaper than proteins.
Mutations and Insertions
Repair of Double-Strand Breaks:
Repair of the ds breaks can lead to small insertions and deletions.
Repair in the presence of template DNA can lead to insertion of new sequences.
Importance of New Approaches
These and other new approaches are becoming increasingly important.
Future Challenges in Plant Biotechnology
Breeders can use more than one technology to address a challenge.
Public – private partnerships MAS breeding Gene pyramiding Improved agronomic practices GM technology Genome-wide association
Plants, Food, and Human Health
Why We Eat:
Sensorial / physiological reasons.
Social / emotional reasons.
Economic reasons.
Media and marketing.
*Unlike plants, we can’t make our own food, so we must eat; we are heterotrophs
What is Food?
Food comes from plants: leaves, fruits, shoots, roots, and seeds.
*The diary/meat in our diet comes from animals that eat plants
How Diet Affects Human Health
Dietary deficiencies lead to disability and death.
Diet is a factor in some chronic diseases:
Metabolic syndrome
Type 2 diabetes
Cardiovascular disease
Cancer
Global Dietary Deficiencies
More than one billion people are chronically hungry, and more than two billion people do not get adequate vitamins or minerals in their diet.
Vitamin A Deficiency
*Vitamin A deficiency is a leading cause of blindness
*β-carotene (pro-vitamin A)
Vulnerability to Dietary Deficiencies
*Children are particularly vulnerable to dietary deficiencies
Other Vitamin and Mineral Deficiencies
Many people suffer deficiencies of other vitamins and minerals.
Dietary Deficiencies in the Developed World
*Dietary deficiencies are also widespread in the developed world
Promoting Health and Good Nutrition
Basic research to make healthy food available and affordable.
Promote education about health and nutrition.
Contribute to discussions about government’s roles
Basic Research
*Basic research contributes to food affordability
Breeding Plants
Breeding plants for β-carotene (pro-vitamin A) enrichment.
Benefits of Enhanced β-Carotene
*Vitamin A deficiency is a leading cause of blindness
*β-carotene (pro-vitamin A)
β-carotene is converted to vitamin A in the human body.
Many staple foods are poor sources of β-carotene, so many people do not get adequate vitamin A in their diet.
Factors Affecting β-Carotene Content
*Synthesis, storage and breakdown all affect β-carotene content
*β-carotene makes the rice look golden
To increase beta-carotene levels in plants, you need more synthesis, more storage, or less catabolism
Anthocyanin-Enriched Food
*Blood oranges are a naturally anthocyanin-enriched food
High Anthocyanin Tomatoes
*High anthocyanin tomatoes are also being developed
Breeding Better Broccoli
*Breeding better broccoli
Elevated Levels of Glucoraphanin
Beneforté® is broccoli that produces elevated levels of glucoraphanin
Omega-3 Fatty Acid
*Omega-3 fatty acid enriched soy oil
Increasing Mineral Content
*Increasing mineral content of foods
Biofortified Plants
*Biofortified plants are improving nutrition for many
Breeding for Drought Tolerance
*Breeding for drought tolerance
*Food production for one person for one day requires 3000 liters of water
*In the next 50 years, we will have to produce as much food as we have yet produced in human history
*Breeding crops for a second green revolution
Risk Assessments on GM Crops
Before release into the environment, GM crops are subject to risk-assessment and risk-management measures to evaluate:
*Risks to human health (including toxicity and allergenicity)
*Risks of evolution of resistance in target pathogens or pests
*Risks to non-target organisms
*Risks from movement of transgenes
Seeds GMOs
will genes from GMOs contaminate wild populations?
Concerns Regarding Anti-Insecticidal Genes
Will anti-insecticidal genes harm unintended targets?
GMOs and Small Farmers
Will GMOs take away choice and exploit small farmers?
Safety of GM Crops
*Are GM crops safe to eat?
*Scientists worldwide endorse GM as an important tool for breeding
Molecular farming - Carbohydrate production
Plants produce a range of commercially valuable carbohydrates. The two most important carbohydrates are cellulose (used for paper and polymer production) and starch (for feed and food). Other examples include trehalose, cyclodextrins and oligofructans. Carbohydrates are stored in a number of cellular compartments. So starch is synthesized in plastid while fructans are synthesized in cytosol and stored in vacuole. The major starch-producing-crops are cereals and potatoes and are widely grown in Europe and USA. But mostly they are used for industrial purposes with 30% left for human consumption. Starch is made up of amylose and amylopectin. Starch synthesis begins with triose- phosphate in Calvin cycle which is converted to ADP-glucose and then to starch. This is accumulated in amyloplasts which are specialized plastids to store starch. There are 2 classes of starch synthesis molecules namely starch synthase (SS) which forms the α(1-4) linkage and the other one is starch branching enzyme (SBE) which creates α(1-6) linkage and they together help in synthesis of starch.
Poyfructans
They are soluble polymers of fructose that are synthesized and stored in vacuoles. They have a typical structure of glucose-fructose-fructose (G-F-Fn). Use of fructans as food reserve is widely known in plant kingdom. Between fructan residues, there are glycosidic linkages which give different linear and branched chain structures. The inulins are a major storage carbohydrate found in bulbs like onion and storage roots like chicory and artichoke. The synthesis of fructans is a 2 step process: In first step, there is transfer of fructose from donor sucrose molecule to acceptor sucrose molecule to form ketose via surose-sucrose fructyl transferase (SST) G-F + G-F = G-F-F + G In the second step, the ketose acts as fructose donor to the growing fructan chain via fructose-fructose fructyltransferase (FFT) and sucrose molecule is recycled G-F-Fn + G-F-F = G-F-F(n+1) + G-F In certain bacteria, like B. subtilis, very high molecular weight induced in which sucrose directly acts as fructose donor to the growing chain (sucrose-fructan fructosyltransferase)
*Carbohydrate production – Oligofructans, cyclodextrins and trehalose
Carbohydrate production – Oligofructans
A number of transgenic plants producing polyfructans have now been developed. More recently, short oligofructans (GF2, GF3 and GF4) have been produced in sugar beer using gene encoding the 1-SST enzyme from jerusalem artichoke. This catalyses the production of all 3 oligofructans, these were found to be stored in the roots and almost all of them got converted into oligofructans and so they have been renamed as “fructan beer”. They have applications in ‘neutraceuticals’ and can also be used as an alterative to artificial sweetener. They can be marketed as low calorie sweetener as it cannot bee digested in the gut
Carbohydrate production – cyclodextrins
Cyclodextrins: it is a 6 – 7 to 8 member rings having glucopyranose subunits attached to α(1-4) linkage of starch and form a cone shape with hydrophilic residues on exterior and hydrophobic pockets in the centre of the ring. They are produced by bacterial fermentation of maize starch. Due to this unique structure, it can solubilize hydrophobic pharmaceuticals like steroids. It is released into blood stream after injection. Cyclodextrin gene from Klebsiella pneumoniae was fused to plastid targeting sequence placed under control of promoter from patatin gene. It is a protein that accumulates in potato tubers and increases its expression. But the transfromation failed and no other attempt has been made.
*Carbohydrate production – cyclodextrins and trehalose
Trehalose: Produced by plants in response to osmotic stress. It is therefore a potential target for genetic manipulation of tolerance to abiotic stresses that create water deficiency. It is also a valuable commodity for food processing, dehydration and flavour retention. Genes for trehalose from yeast and E. coli have been used to manipulate drought resistance in tobacco plants
Metabolic Engineering of Lipids
Strategies to increase production of lipids Production of short chain fatty acids : Coconut oil, palm oil are all short chain fatty acids of length C4-C8. The production of these chains can be terminated by the action of thioesterases . A gene is constructed to produce these short chain fatty acids. Production of long chain fatty acids : To increase the chain length, an enzyme called elongase is used. This extends the chain length more than C14 to give long chain fatty acids. Common example is brassica oil Production of saturated fatty acids : This can be done by increasing the degree of saturation by the increasing the action of desaturase enzyme. Common examples are stearic acid, oleic acid etc Plants store oils as triacylglycerols. Oils differ in the length and degree of saturation of their fatty acids. FA are synthesized in plastid but modified in the cytoplasm and ER
Metabolic Engineering of Lipids.
Most of the crop oils are C16 or C18. C12:0 – Lauric acid is widely used in soap and detergents Long chain FA: Erucic acid is a valuable industrial oleochemical. HEAR /LEAR – High/low erucic acid rape – Brassica oil High stearic acid (C18:0) oil is an important goal for Industrial feed stock. High oleic acid (C18:1) oil is of value for food purpose. Petroselenic acid from coriander (C18:1) rare FA high value for Industry
Omega-3 fatty acid enriched soy oil
α-Linolenic acid (ALA) Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA) are an n−3, or omega-3, essential fatty acid. Stearidonic acid (SDA) is an ω-3 fatty acid
Vegetable oils (canola, soybean and flaxseed); nuts Fish and fish oil, terrestrial meats, eggs Type of Omega-3 PUFAs ∆6 desaturase (slow step)
ALA (18:3n-3) SDA (18:4n-3) EPA (20:5n-3) DHA (22:6n-3)
Plant-derived omega-3 fatty acids (e.g. ALA) are converted to long-chain PUFAs (e.g. EPA and DHA), but with low efficiency. Soybeans have been engineered to produce elevated levels of SDA, which is more effectively converted into long chain PUFAs SOYMEGATM
Primary dietary source
Biosynthesis of PHBs
Enzymes involved : Ketothiolase , Acetyl CoA- reductase and PHB synthase Gene involoved : phaA -> codes for ketothiolase phaB -> codes for Acetyl CoA- reductase PhaC -> PHB synthase Strategies to increase production of PHB : Strategy 1 : Increase production in cytosol In this, PHB would be synthesized in cytosol. But the limitation in this strategy is that there is low accumulation of PHB in the cytosol and thus not much can be extracted Strategy 2: Increase production in plastid PHB synthesis was directed in the plastid which improved the yield. A plastid transit peptide sequence was used to increase production.
PLastics have been a major source of soil and water pollution. They are non-biodegradable and remain in the environment for a very long time. Their interactions with the ecosystem causes disturbances which leads to varied consequences and leads to loss of species population. So alternate methods are being researched to produce plastics that are organic and less harmful to the environment. One such source are plants. There are 2 types of bioplastics, Polyhydroxy Alkanoates (PHA) and polyhydroxy Butyrate (PHB). The most common of these 2 is PHB. It is produced in the cytosol and then stored in inclusion bodies. These can then be extracted and used for bioplastics production. In Alcaligenes eutrophus, (Bacteria) PHB accumulates as molecular weight polymer up to 80% of total dry weight. Metabolic Engineering of Lipids - Bioplastics
Metabolic Engineering of Proteins
Proteins are biomolecules involved in every aspect of cellular life. Various examples of proteins include enzymes, antibodies, receptors etc. Hence it is essential to procure much valuable proteins fit for human consumption from a variety of sources. One such source is plants. Hence a variety of strategies have been used to ensure the production of mammalian proteins from plants using molecular techniques. The size of the market for pharmaceutical proteins is a major attraction for plant scientists Plastic like material: Polymerizing certain amino acids Like poly – γ – glutamate and poly ε – lysine, they are hydro gel. Poly aspartate - hydro gel Cyanophycin – Cyanophycin synthases
Metabolic Engineering of Proteins -
Production of enzymes for industrial uses Bẞ-Glucuronidase
Stable integration and transient expression systems Hirudin (7kDa – anticoagulant peptide) and insulin production Hirudin and Insulin
Pharmaceutials , wound care
Plant-derived antibodies
There are five types based on their physical, chemical, and immunological properties: Ig A, IgE, IgG, IgM, and IgD. They have 2 H chains and 2 L chains They can be synthesized in the plants, wherein the genes for heavy chain (H- α, δ, ε, γ, and µ) can be coded in a plant and the gene for light chain (κ and λ) in another plant. These plants are crossed and the antibody is produced in plants.