Green Biotechnology Flashcards
Progress in plant biotechnology depends on:
1) Knowledge and understanding (of plants)
2) Development of new technologies
3) Commercialisation of plant biotechnologies
How can Plant Biotechnology deliver sustainable industry?
1) Inputs - sustainable sources, minimise fossil fuels, carbon friendly
2) Processing/manufacture - low energy, minimal waste, efficient use of all inputs
3) Product usage - sustainable consumption, efficient use of all product, minimise waste
4) Post usage - maximal waste recycling
Future Directions of plant GMO through metabolic engineering and synthetic biology
1) Energy/ Biofuels
2) Pharmaceuticals and High Value Chemicals
3) High nutritional value food
4) Resistant Crops
Argued Benefits of Plant GMO
1) Increase crop productivity
2) Conserve biodiversity (save hectares from cultivation)
3) Provide better environment (reduce herbicide/insecticide)
4) Reduce CO2 emissions
5) Help alleviate poverty and hunger
Plant Engineering Methods (Unit Coverage)
1) Nuclear Genome Engineering and Editing (e.g. over-expression, gene silencing, CRIPSR etc)
2) Chloroplast transformation and engineering
Applications of plant genetic engineering (Unit coverage)
1) Biotic stress tolerant crops
2) Improving nutrition of plants by GM e.g. biofortification
3) Use plants to express and produce high value products (e.g. vaccines)
4) Generating and testing superfoods
Recent GM approval updates (there are others)
Sept 14 - The Philippines approved the canola event LBFLFK for food, feed, and processing.
Aug 24 - The Philippines approved the cotton event GFM cry1A for commercial cultivation.
Mar 3 - Brazil approved the wheat event HB4 for commercial cultivation.
2022
Dec 12 - The Philippines approved the soybean event GMB 151 for food, feed, and processing.
Oct 18 - The Philippines approved the eggplant event EE-1 for cultivation
Oct 4 - USA approved the canola event MON94100 for food and feed.
Jul 18 - Nigeria approved the wheat event HB4 for food and feed.
Jul 7 - The USA approved the corn event MON87429 for food and feed.
June 30 - Ghana approved the cowpea event AAT709A for food, feed, and cultivation.
June 22 - The USA approved the wheat event HB4 for food and feed.
May 31 - Turkey approved the maize event MON87427 for feed.
Genetic Technology (Precision Breeding) Act UK
Allows the creation and marketing of ‘precision bred’
or genome-edited plants and vertebrate animals in England, removing them from the regulatory system for the release of genetically modified organisms (GMOs).
Genetically Modified Organism (EU directive 2001)
an organism, with the exception of human beings, in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination;
Are excluded: crosses, graft hybrids, mutagenesis, some somatic hybrids, cells infected with recombinant virus, in vitro fertilisation
CRISPR mutated are not necessarily GM
DNA Insertion - altering genetic material in a way that does not occur naturally by mating and/or natural recombination
Transfection - definition
Introduction of a DNA molecule into a cell, usually followed by expression of 1 or more genes from the introduced DNA
Usually up to 50% Cell Population transfected due to DNA being destroyed by DNAase
Transfection GM?
Transfection is not GM if no integration
If transfection leads to Transformation it is GM?
Transformation Definition:
When a DNA molecule has been introduced in a cell and has become heritable. In eukaryotic cells, means DNA molecule has integrated in one of three genomes = GM
How do you select for transformation?
Select for transformation using a chemical marker
Three ‘Modifiable’ Genomes in Eukaryotic Cells (e.g. Plants) - CHATGPT
Nuclear Genome: This is the primary genome of the cell, contains the majority of the cell’s genetic information. In the context of genetic engineering, introducing foreign DNA into the nuclear genome is often the main goal to bring about desired traits or characteristics. This integration can lead to the expression of new genes or the alteration of existing ones.
Mitochondrial Genome: mitochondrial DNA (mtDNA) is separate from the nuclear DNA and is inherited maternally in most eukaryotic organisms. Genetic modification of the mitochondrial genome can be used to alter traits related to energy production and metabolism.
Chloroplast Genome: In plant cells, chloroplasts (the organelles responsible for photosynthesis) contain cpDNA. Like mitochondrial DNA, chloroplast DNA is distinct from the nuclear DNA. Genetic modifications in the chloroplast genome can affect traits related to photosynthesis and other processes localised in chloroplasts.
modifying the chloroplast genome is often used in plants to express high levels of foreign proteins, while avoiding certain gene flow issues related to pollen dispersal, as chloroplasts are typically maternally inherited.
Transformation Frequency (success rate)
<0.05% to 1%
What factors affect transformation frequency?
- Frequency of transfection
- amount of DNA entering nucleus
- whether DNA is protected from DNAse
- efficiency of selection protocol for transformants
- regeneration efficiency from one cell to a plant
Methods to transfect cell
- Micro-injection using glass microcapillaries
- electroporation of protoplasts
- Biolistics: particle/gun/DNA gun/micropartivle bombardment
- Agrobacterium tumefacians
Define cisgenic organism
An organism that has stably incorporated one or more genes from the same species, through genetic engineering
Define transgenic organism
an organism that has stably incorporated one or more genes (transgenes) through genetic engineering = GM
Breeding is not regulated by patents but by Plant Variety Rights
Patent v Breeding
Breeding
available to anyone to breed a better plant, must be able to show that plant created is different
Precision Breeding
Interface between breeding and GM
GM Labelling in UK
Any food product sold in UK that contains intentionally GM protein or DNA must be clearly labelled
UK Supermarkets and GM
Legal but too much trouble for them to sell GM products
GM Oil?
Oil from GM/Non Gm is basically identical, contains very little DNA (traces) but still noted as GM
Labelling is not required for
If presence is accidental and below 0.9% for EU-approved
GM. Note that unapproved GM varieties are forbidden.
* For meat, eggs and milk from animals fed on GM.
* When GM food-processing-aids are used but absent
from the final product. such as chymosin (rennin). which is
used to make some hard cheeses ( 90% of UK cheese).
Patents
- DNA transfer protocols are patented
- Regeneration protocols are patented
- DNA sequences are patented
Technology
- Agro is the work horse of GM technology
- Biolistics in the second main approach
- Agro: good example of a spin off from basic
sciences (curiosity driven). - ‘Recalcitrant crops’ may require years of research
to develop a suitable transformation protocol
GM in Plant Biotech
GM technology is a heavily patented field; unlike plant breeding
(see PVR website)
GM approach is only a part of plant Biotech. (your project can be
GM technology may be invisible in the final product, but the product
is still defined as GM, except for Precision Breeding.
GM labelling is to inform customer choice not toxicity
GM definition will need to adapt to changes and faces challenges
Mechanisms of gene silencing
Post-transcriptional silencing by small RNAs
RNA interference (RNAi) mediated by siRNAs, miRNAs, antisense
Directed DNA mutation
Site-specific nucleases (ZFNs, TALENs)
CRISPR/Cas
Define Gene Silencing
— suppression (knockdown) or complete absence (null, knockout) of gene/protein
expression
Can be caused by block in transcription (DNA mutation) or block in translation (RNA
degradation or modification)
Small RNA Gene Silencing - details
- stable inherited in Mendelian Fashion
- can be engineered to be very specific (target specific mRNA, single gene)
- or multiple genes simultaneously
What are small RNAs?
- 21-25 nucleotide RNAs
- contribute to post-transcriptional gene silencing by affecting mRNA stability or translation
- contribute to transcriptional gene silencing through epigenetic modifications to chromatin
RNAi via SiRNAs
1) Trigger: dsRNA
2) Processed into numerous (21-25nt) siRNAs by enzyme called Dicer or Dicer-like (DCL)
3) Incorporated with argonaute (AGO) proteins to form RNA-induced silencing complex
4) Act as specific determinations either:
a) destroy homologous mRNA targets
5) repress translation of mRNAs
RNAi for containment of leafy trees
Silencing gene called LEAFY need for flower development
miRNA = type of siRNA
- encoded by specific miRNA genes but act on other genes (trans-acting regulators)
- plants have small number highly conserved miRNAs, large number non-conserved miRNAs
Generation of inverted repeat RNA silencing plasmids
Invert repeat expression vectors can be designed for the artificial construction of double-stranded hairpin RNA (hpRNA) to induce targeted RNA silencing
miRNA mechanism
1) transcription = -75nt pre-mRNA
2) Transcript processing by DCL1
3) Helicase incorporates miRNA into RISC
4) miRNA-driected negative regulation of mRNA targets
5) Target mRNA Cleavage and reduce translation of uncleaved target mRNA
6) = reduced target protein levels
miRNAs and plant biotechnology
- Arabidopsis has many endogenous miRNAs - function is unknown for most
- Some shown to have gene regulation, development, stress response roles
- Manipulation of endogenous miRNAs could have biotechnological potential
Biotechnological potential miRNA examples
- miRNA
o e.g. miRN156 increased biomass
o reduced pathogen infection on mir393 mutants
Use of artificial miRNAs
engineering via LKR/SDH
RNAi to delay tomato ripening
- 1990s experiment
- ‘antisense’ technology to silence the tomato polygalacturonase gene to delay fruit ripening
altered cell wall properties of fruit = more unmodified pectin in regions of cell wall - increased firmness of modified fruit
Pros and Cons of RNA silencing
- RNAi by siRNA or miRNA is more efficient than ‘older’ methods of plant silencing (antisense)
- silencing can be transmitted from cell-to-cell and sometimes over long distances in the plant
- RNA silencing is dominant - phenotypes seen in subsequent generations
- Silencing level may vary between generations (some cases may be lost after many generations - less problem with miRNA)
- Silencing is variable - may not be complete ‘knock out’ but ‘knock down’
Random DNA Mutagenesis
traditional methods cannot be targeted to a specific gene
Chemical mutagen = nucleotide substitution (may introduce premature STOP codon in gene)
Insertional mutagenesis - insertion of a T-DNA into gene to prevent transcription
Directing ‘random’ DNA mutagenesis
Targeted mutagen = site specific nuclease e.g. ZFN and TALEN
Zinc Finger Nucleases
- composed of 3-6 zinc finger domains, designed to bind 9-18 nucleotides of a specific DNA sequence, linked to a DNA cleavage domain (Foki endonuclease)
- two copies are needed for both sides of DNA strands to yield double strand break
- genes encoding the ZFNS and short mutated repair template are transformed into the plant via a plasmid
TALENs (TALE Nucleases)
- TALE Protein
- Composed of DNA binding domain of a TALE engineered to bind to a specific DNA sequence, linked to a DNA cleavage domain (Foki endonuclease)
- Two copies are needed both sides of DNA strands to yield double strand break
- Genes encoding the TALENs and short mutated repair template are transformed into plant via a plasmid
Pros and Cons of ZFN and TALEN gene silencing
- both methods can provide targeted mutation to specific genes leading to complete loss of expression (knockout rather than knockdown)
- both methods shown to work in numerous plant species : various patents filed for use in both methods in plants/plant biotech
- ZFNs = laborious, time consuming, expensive, high failure rate
- TALENs = easier to design and engineer but resource intensive, some evidence of off-target activity
- both methods need a dimer to function - monomeric nuclease method would be more efficient
Gene mutation by CRISPR/Cas
- clustered regularly interspaced palindromic repeats (CRISPRs) include Cas type II nuclease
- cas9 endonuclease, makes ds breaks after recongising PAM sequence on the DNA
- RuvC and HNH nuclease domains make the cut
- guideRNA made up of CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA)
Potential of CRISPR mutations
- the method of choice in plants for precision genome editing
- works in many plant species including crops
- can generate mutation with high effeciency and specificity
- minimal off target effects reported in plants
- easily designed and requires less components
- shown to be effective at generating large genomic deletions (>200kb), potnetially to delete entire clusters of genes
- other nucleases (e.g. Cas12a, Cas13) alongside Cas9 expanding possibilities.
Pros of Using Transgenic Plants as Production Platforms
- low cost of producing proteins
- sustainable: light/nutrients (carbon neutral)
- storage of germplasm as seeds
- rapid scale up from seeds
- no animal pathogens (animal, viruses & pathogenic bacteria ad endotoxins)
- relative low carbon footprint compared to fermentation
Cons of Using Transgenic Plants as Production Platforms
- regulation and containment
- time to isolate stable transgenic plants (>12 months) = transient expression = solution
Gene Transfer Methods
- Agrobacterium tumefaciens = gene transfer to the nucleus
- Particle Bombardment
- Agrobacterium tumefaciens
single clean insertions in single copy DNA chromosomes
random insertion of genes (illegitimate recombination, not targeted)
Stable inheritance of transgenes
No vector sectors (region between T-DNA borders)
More species even cereal amenable to Agrobacterium
Particle Bombardment
- multiple complex insertions into nuclear chromosomes
- ‘random’ insertion of genes (illegitimate recombination - not targeted)
- some insertions unstable
- vector DNA inserted into chromosomes
- efficient in many crops such as cereals and soybean
Particle Bombardment and Tissue Culture in Wheat
First report in 1991, now routine in few labs
Marker Genes and Selection
Plant selectable marker genes (selection in plants)
- nptII most common marker gene (kanamycin resistance)
- extensively tested and gained regulatory acceptance
Transformation methods to remove antibiotic resistance genes are desirable
Plant Expression Hosts
- tobacco
- tomato
- seeds
- cell culture
Tobacco as Plant Expression Host
- high biomass
- 100,000kg per hectare
- non-food
- rapid scale up through seeds
- harvested before flowering
- leaves dried or frozen for transport
Tomato as Plant Expression Host
- high biomass
- 70,000kg per hectare
- edible
- growth in greenhouses (containment)
Maize (corn) as plant expression hosts
High biomass (12,000kg per hectare)
Long term stability in seeds
Hydroponic culture
Well established in commercial horticulture = reduced risk of escape
Prodigene proteins from transgenic plants
First commercial proteins from transgenic maize plants (Avidin 1997)
Avidin = diagnostic agent that binds biotin, normally isolated from chicken egg white
75-80% cost incurred post harvest
Importance of Regulating GM Crops
Enhance containment of transgenic crops
Various requirements exist for growing pharma plants in USA
Plant Expressed Vaccine Epitopes
Hep B (1992)
Rabies and HIV (1997)
Many many more
Cell Culture Expression Hosts
- plant cell cultures required sucrose
- no plant pathogens
Tissue Expressed Vaccines
- Newcastle Disease for use in poultry
Plant based insulin - SemBioSys
- commercial levels of insulin production in safflower
- identical to pharmaceutical grade human insulin
- Should we be directing efforts to reducing need for consumption e.g. through healthy diet and exercise
Stratosome Biologic System
built on two capabilities
1) using GE to attach high-value proteins to oilbodies
2) extraction of oilbodies from seeds in a cost-effective manner
Transgenic Plants v Transient Expression
- for crops can take >12 months to obtain transgenic seeds
- transient expression = alternative faster method, often one week
- transient = stable plant lines not made
Transient Expression Method
Viral Vectors and Agroinfection
a) grow plants and bacteria
b) agroinfilitration
c) plant incubation
d) biomass harvest
e) extraction
f) protein purification
ICON GENETICS GMBH
World leader in developing and using expression systems for production of recombinant proteins in green plants.
High & Rapid Yields of Recombinant Proteins
Magnifection
- transient amplification of viral vectors delivered to entire plant using Agrobacterium
- marker genes are not required
- containment is enhanced
Ebola Virus Treatment example
Zmapp
Zmapp method
- comprised three ‘humanised’ monoclonal antibodies (mAbs) recognising Ebola virus epitopes on the virus that prevents entry into cells
- effective in monkeys, not tested in humans
- produced in tobacco using magnICON method
Transient Expression
Ebola antibodies
Flu vaccine
Covid Vaccine - Medicago
Covid Antibody - Medicago
cGMP
current good manufacturing practice
cGMP for recombinant protein production in plants
1) transgenic banking system and validation
2) good practice compliant procedures for cultivation and plant management
3) Post-harvest processing of plant material
4) Filtration
5) Purification
Molecular Pharming - key steps for commercialisation
- product yield (>1% total plant protein)
- protein stability
- protein quality
- extraction and downstream processing
- quality assurance and quality control
- regulatory approval
- clinical trials
Commercialisation of plant-dereived pharmaecutical proteins example
Eleyso, by Protalix (israeli) = treat Gaucher disease, where a deficiency in taliglucerase alpha prevents breakdown of fats resulting in organ damage, gene defect common is Ashekanzi Jews.
Strengths of nuclear transgenic planta
variety of crops, accumulation in different parts, different subcellular components or secreted into medium (rhizosecretion), highly scalable from seeds
Negatives of Nuclear Transgenic Plants
recombinant protein accumulation concentration can be low (<1% total protein)
slow
Plant Cell Vaccine System
1) Plasmid transferred into agrobacterium tumefaciens
2) infect plant cells with A. tumefaciens containing plasmid
3) Select for transformed cells
4) Identify and prepare master cell
5) Produce bulk antien
6) Harvest
7) Lyse cells
8) Remove cell debris, bulk fluids, quantitate antigen, assemble, vial, test
Transient expression
Transformation method Agoinfection viral and non-viral infection
Leaf based expression system
Very high concentrations of recombinant proteins
Fast 5-10 days
suitable for toxic proteins
scalable
Negative of Transient Expression
- Leaves contain agrobacterium and associated endotoxins (lipopolysaccharides) - immune response -> septic shock
- each cycle requires re-infection with Agrobacterium (adds costs)
Chloroplast (plastid) transformation main method
main = particle bombardment
Pros of stable chloroplast transformation
Very high accumulation of recombinant proteins, highest in leaves, subcellular compartmentalisation to chloroplasts, highly scalable, gene flow through pollen is restricted
Cons of chloroplast (plastid) transformation
- slow to get stable transgenic plants 6-12 months
- not suitable for glycosylated proteins
Delivering DNA into Plasmids
Bio-Rad PDS 1000/He particle delivery system
Fires microprojectiles coated with a plastid transformation vector at cells
PlyGBS (phage lytic protein)
Protein accumulation does not decline with leaf age, but plastid protein biosynthetic capacity does, indicating the PlyGBS is extremely stable inside chloroplast
Molecular Pharming: key steps for commercialisation
1) Expression Platform (plant species; location of expression: organ, intracellular or secreted; transient; stable; nuclear or chloroplast)
2) Plant Growth conditions
Product yield
3) Protein stability
4) Protein quality
5) Biomass production and harvesting
6) protein extraction and downstream processing (purification)
7) quality assurance and quality control
8) regualatory approval
clinical trials
Diamante
exploits plants as bioreactors for the sustainable production of nanoparticles based on modified plant viruses that are used in developing new tools in autoimmune disease diagnosis.
Plant viruses are exploited as a scaffold in order to stabilize and display high number of
functional portions of a protein (peptides). Through a fast and environmental-friendly
production process
ExpressTec
simple and elegant solution to the need for recombinant proteins in medicine and biotechnology. Instead of a stainless-steel bioreactor, our production process harnesses the humble rice plant’s natural ability to use sunlight as an energy source and soil, water, and
air as raw materials. Desired proteins are manufactured as the plant grows, and naturally
become concentrated within the rice-seed endosperm for easy harvest and purification.
Chloroplast Transgenic Plants
‘Transplastomic Plants’
Plastids
Family of plant organelles, different plastids are inter-convertible
All plastids contain DNA (identical genome in every plastid)
Plastid DNA
- Plastid DNA = Chloroplast DNA
- Has circular map
- many copies of plasmid DNA per plasmid
Overview of Plastid Genome
Higher plant chloroplasts xontain a small genome with -120 genes (sequenced in >1000 species)
50 photosynthetic protein genes
60 genetic system genes (rRNA, RNA polymerase, tRNA)
Plastid Genes
highly expressed in chloroplasts
large subunit of RuBisCO (30-50% tsp leaf)
tsp
total soluble proteins
rbcL
plastid gene encoding large subunit of RuBisCO
3 Steps to Transform Plastids
1) Vector Construction
2) Delivery of DNA into plastids
3) Selection of plastid transformants
2 components of plastid transformation vectors
1) Expression Cassette
2) Targeting DNA
Plastid Vector - Expression Casette
- plastid promoter
- ribosome binding site
- selectable marker gene (e.g. aadA)
- termination/ 3’ transcript processing
- function in plastids and bacteria but not in nucleus
- function best in leaves
aadA gene
resistance to streptomycin and spectinomycin
Plastid Vector - Targeting DNA
1) Left: Chloroplast DNA targeting arm
2) Marker
3) goi (gene of interest)
4) Right Chloroplast DNA targeting arm
Homologous recombination targets precise insertion of transgenes region between left and right arms exchanged between vector and chloroplast DAN
Homoplasmic Plant
a plant with a uniform population of plastid genomes
Heteroplasmic Plant
plant with a mixture of plastid genomes
Transplastomic Plant
a plant containing transformed (transgenic) plastids
Cytoplasmic Sorting
The process by which mixed plastids in a cell sort out to pure populations during growth and development
Top 4 Biotech Crops
Soybean
Oilseed Rape
Cotton
Maize
Chloroplast Expressed Proteins
- No glycosylation
- Compartmentation of toxic products
- Disulphide-bond formation & correct folding of human proteins
- Proteins cannot be exported
Nucleus/cytosol expressed proteins
- Glycosylation
- Toxic Proteins (pleiotropic effects)
- Disulphide-bonds requires targeting to the endoplasmic reticulum
- Proteins for all cellular compartments
Tobacco as a vehicle for making useful proteins
- non-food crop
- harvested before flowering
- no known wild relatives in North
- excellent biomass (100,000kg/ha)
- alternative used to smoking
- ICON Genetics and Chlorogen
Plastid Transformation Enables
- precise targeted insertion without marker genes
- enhanced gene containment (no pollen transmission)
- high levels of expression
- coordinated expression of multiple genes
Which components of the fruit and veg reduce non-communicable diseases
1) Macronutrients - contain energy (calories)
a) Carbs, fat, protein = fuel and building blocks
b) fibre - bowel health
2) Micronutrients - vitamins, trace minerals
3) Phytonutrients (protective functions)
Plant Secondary Metabolites
- organic chemicals that do not seem to play a direct role in growth and development
- some metabolites only made by a single family or species e.g. codeine, aspirin
- defence against pathogens, herbivores
- attract insects and animals
Phytonutrients = subclass of secondary metabolites
flavonoids, anthocyanins, related compounds
Isothiocyanates
- broccoli and other cruciferous vegetables
- may detoxify carcinogens
lycopene
- carotenoid abundant in tomatoes
- some studies show it protects against several forms of cancer
epigallocatechin gallate
green tea
may help elimnate immortality from some cancer cells
genistein
isoflavonoid from soybeans, may protect against cancer
Two science aims to improve dietary health
1) make fruit and vegetables cheaper
2) produce food enriched in phytonutrients
Glucoraphanin
glucosinolate that is cleaved by gut bacteria to produce sulforaphane, an isothiocyanate that has proposed anti-carcinogenic properties in humans
Super Broccoli = Beneforte
Broccoli crossed with wild relative containing high glucaraphanin (Bassica villosa)
Super Broccoli Method
Broccoli crossed with wild relative containing high glucaraphanin (Bassica villosa)
many back-crosses over 15 years to reach commercial broccoli with 3x glucoraphanin content
Genetic Basis of Super Broccoli
transcription factor Myb28 - previously determined methione-derived glucosinolate concentration = makes breading easier
Patenting of Beneforte
patented because crossings eventually had to dissociate wild myb28 gene from a c-segregating ELONG gene coming from wild species. ELONG was detrimental to glucaraphanin concentration
Health benefits of Glucoraphanin
- molecule is taken up from diet
- protective benefits against colorectal cancer
- may reduce risk of prostate cancer
- diet-human gene interactions
Diet-human gene interactions
complicate picture of biofortification
‘Green Revolution’
1960s-1970s increased high yielding cereal production in developing countries at expense of nutrient-rich pulse/legumes
Iron Deficiency
60% world population suffer from Fe deficiency
Weakens immune function and generally impairs growth and development
Zinc (Zn)
Over 30% of the world population suffer
Impairs development and immune system
Iodine (I)
30% world population are I deficient
Impaired thyroid function (Goitre disease), impaired infant development
Vitamin A = retinol
250m children affected by vitamin A deficiency
Deficiency can cause blindness, xerophthalmia, reduced immune function
500,000 children become blind every year, half die within 12 months of losing sight
severe defeciency = dryness and ulceration of cornea
modest deficiency = increase morbidity and mortality from infectious diseases by impairing immune function and leads to night blindess
Folate (vitamin B9)
numbers unclear but many people in developing countries known to be at risk
foetal neural tube effects, spina bifida, risk of cardiovascular disease
Biofortification
increase plant nutrient content pre-harvest
a) Agronomic (fertiliser use)
b) Genetic (selective breeding)
c) Transgenic (genetic engineering)
Potential of Transgenic Biofortification
↑ mineral accumulation
↑ mineral transfer into leaves/grains/tubers
↑ mineral storage
↑ mineral bioavailability
↑ vitamin biosynthesis in edible parts of plant
Sources of Vitamin A in Diet
- only animal products contain preformed vitamin A (fish oil, liver, dairy products)
- plants which are good sources of provitamin A carotenoids (a and b carotenes) can be converted by animals into retinal and retinoic acid (active forms of Vitamin A)
- b carotene = antioxidant = evidence role for cancer risk reduction
Golden Rice
Rice line engineered to produce B carotenene in its endosperm
Multigene Transfer Methods
1) Stacking - successive rounds of crosses between different transgenic lines
2) Sequential transformation - retransform a previously generated transgenic line
3) Co-transformation - simultaneous introduction of multiple genes either unliked (each gene on separate plasmid) or linked (multiple genes on same plasmid)
Evaluation of Multigene Transfer Methods
Stacking and sequential transformation are labour intensive, take consuming, require multiple selection markers, and transgenes can segregated in later generations
Unlinked co-transformation is inefficient and can generate complex loci
Linked co-transformation by Agrobacterium or direct transfer methods for small numbers of transgenes >5-10 inefficient for large genes and large no. genes
Barriers and Lessons
- intellectual property (freedom to operate, licensing, volume)
- economic value (viable business proposition v social value)
- regulatory approval (cost, time)
- consumer and societal acceptance
Golden Rice Trials
Philippines 2013 - destroyed anti-GM activists
GR2(R1) - poor in field compared to non GM lines
GR2-R1 molecular characterisation (transgene cassette integrated into 1st exon on OSAux1 disrupting auxin transport
Philippines Rice Research Institute - 2017 - application biosafety permit and field trial Philippines/Bangladesh
2021 - approved in Philippines as world’s first commercialised GM biofortified crop
Opposition to Golden Rice
- consistent political resistance to GMOs
- GR as ‘trojan horse’ for other GM crops
- excessive regulation takes a long time to overcome, preventing use by farmers
- “unjustifiable loss of millions lives”
- GE has unprecedented safety record, more precise and predictable than unregulated breeding technology
Economic Opposition to Golden Rice
- 10year delay = $700m (1.4m life years)
- high perceived costs sway governments
Uganda - Vit A Deficiency
- 20% children (6mths - 5yrs), 19% women (15-49yrs)
- Banana staple diet (75% farmers, 7% global production)
- estimated consumption 220-250kg /year
- Low levels essential micronutrients (Fe,Zn), provitamin A
Uganda - Biofortification
- conventional breeding (low fertility, lack of pro-vitamin A varieties in gene pool)
- gene modification (Banana21 project)
Banana21 Project - Method
- GR2 Strategy
- Suitable transgenes - banana dervied cisgene(s) - Asupina, MtPsy2a
- suitable promoters
- local banana varieties - Nakitembe, M9, Sukali Ndiizi (popular with small children)
- Training Program
