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Plant Science > Green Biotechnology > Flashcards

Green Biotechnology Flashcards

1
Q

Progress in plant biotechnology depends on:

A

1) Knowledge and understanding (of plants)
2) Development of new technologies
3) Commercialisation of plant biotechnologies

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2
Q

How can Plant Biotechnology deliver sustainable industry?

A

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

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3
Q

Future Directions of plant GMO through metabolic engineering and synthetic biology

A

1) Energy/ Biofuels
2) Pharmaceuticals and High Value Chemicals
3) High nutritional value food
4) Resistant Crops

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4
Q

Argued Benefits of Plant GMO

A

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

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5
Q

Plant Engineering Methods (Unit Coverage)

A

1) Nuclear Genome Engineering and Editing (e.g. over-expression, gene silencing, CRIPSR etc)
2) Chloroplast transformation and engineering

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6
Q

Applications of plant genetic engineering (Unit coverage)

A

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

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7
Q

Recent GM approval updates (there are others)

A

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.

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8
Q

Genetic Technology (Precision Breeding) Act UK

A

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).

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9
Q

Genetically Modified Organism (EU directive 2001)

A

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

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10
Q

DNA Insertion - altering genetic material in a way that does not occur naturally by mating and/or natural recombination

A
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11
Q

Transfection - definition

A

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

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12
Q

Transfection GM?

A

Transfection is not GM if no integration
If transfection leads to Transformation it is GM?

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13
Q

Transformation Definition:

A

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

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14
Q

How do you select for transformation?

A

Select for transformation using a chemical marker

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15
Q

Three ‘Modifiable’ Genomes in Eukaryotic Cells (e.g. Plants) - CHATGPT

A

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.

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16
Q
A

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.

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17
Q

Transformation Frequency (success rate)

A

<0.05% to 1%

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18
Q

What factors affect transformation frequency?

A
  • 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
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19
Q

Methods to transfect cell

A
  • Micro-injection using glass microcapillaries
  • electroporation of protoplasts
  • Biolistics: particle/gun/DNA gun/micropartivle bombardment
  • Agrobacterium tumefacians
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20
Q

Define cisgenic organism

A

An organism that has stably incorporated one or more genes from the same species, through genetic engineering

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21
Q

Define transgenic organism

A

an organism that has stably incorporated one or more genes (transgenes) through genetic engineering = GM

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22
Q

Breeding is not regulated by patents but by Plant Variety Rights

A
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23
Q

Patent v Breeding
Breeding

A

available to anyone to breed a better plant, must be able to show that plant created is different

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24
Q

Precision Breeding

A

Interface between breeding and GM

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25
Q

GM Labelling in UK

A

Any food product sold in UK that contains intentionally GM protein or DNA must be clearly labelled

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26
Q

UK Supermarkets and GM

A

Legal but too much trouble for them to sell GM products

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27
Q

GM Oil?

A

Oil from GM/Non Gm is basically identical, contains very little DNA (traces) but still noted as GM

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28
Q

Labelling is not required for

A

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).

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29
Q

Patents

A
  • DNA transfer protocols are patented
  • Regeneration protocols are patented
  • DNA sequences are patented
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30
Q

Technology

A
  • 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
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31
Q

GM in Plant Biotech

A

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

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32
Q

Mechanisms of gene silencing

A

Post-transcriptional silencing by small RNAs
RNA interference (RNAi) mediated by siRNAs, miRNAs, antisense

Directed DNA mutation
Site-specific nucleases (ZFNs, TALENs)
CRISPR/Cas

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33
Q

Define Gene Silencing

A

— 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)

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34
Q

Small RNA Gene Silencing - details

A
  • stable inherited in Mendelian Fashion
  • can be engineered to be very specific (target specific mRNA, single gene)
  • or multiple genes simultaneously
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35
Q

What are small RNAs?

A
  • 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
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36
Q

RNAi via SiRNAs

A

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

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37
Q

RNAi for containment of leafy trees

A

Silencing gene called LEAFY need for flower development

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38
Q

miRNA = type of siRNA

A
  • 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
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39
Q

Generation of inverted repeat RNA silencing plasmids

A

Invert repeat expression vectors can be designed for the artificial construction of double-stranded hairpin RNA (hpRNA) to induce targeted RNA silencing

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40
Q

miRNA mechanism

A

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

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41
Q

miRNAs and plant biotechnology

A
  • 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
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42
Q

Biotechnological potential miRNA examples

A
  • miRNA
    o e.g. miRN156 increased biomass
    o reduced pathogen infection on mir393 mutants
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43
Q

Use of artificial miRNAs

A

engineering via LKR/SDH

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44
Q

RNAi to delay tomato ripening

A
  • 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
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45
Q

Pros and Cons of RNA silencing

A
  • 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’
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46
Q

Random DNA Mutagenesis

A

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

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47
Q

Directing ‘random’ DNA mutagenesis

A

Targeted mutagen = site specific nuclease e.g. ZFN and TALEN

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48
Q

Zinc Finger Nucleases

A
  • 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
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49
Q

TALENs (TALE Nucleases)

A
  • 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
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50
Q

Pros and Cons of ZFN and TALEN gene silencing

A
  • 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
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51
Q

Gene mutation by CRISPR/Cas

A
  • 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)
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52
Q

Potential of CRISPR mutations

A
  • 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.
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53
Q

Pros of Using Transgenic Plants as Production Platforms

A
  • 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
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54
Q

Cons of Using Transgenic Plants as Production Platforms

A
  • regulation and containment
  • time to isolate stable transgenic plants (>12 months) = transient expression = solution
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55
Q

Gene Transfer Methods

A
  • Agrobacterium tumefaciens = gene transfer to the nucleus
  • Particle Bombardment
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56
Q
  • Agrobacterium tumefaciens
A

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

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57
Q

Particle Bombardment

A
  • 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
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58
Q

Particle Bombardment and Tissue Culture in Wheat

A

First report in 1991, now routine in few labs

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59
Q

Marker Genes and Selection

A

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

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60
Q

Plant Expression Hosts

A
  • tobacco
  • tomato
  • seeds
  • cell culture
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61
Q

Tobacco as Plant Expression Host

A
  • high biomass
  • 100,000kg per hectare
  • non-food
  • rapid scale up through seeds
  • harvested before flowering
  • leaves dried or frozen for transport
62
Q

Tomato as Plant Expression Host

A
  • high biomass
  • 70,000kg per hectare
  • edible
  • growth in greenhouses (containment)
63
Q

Maize (corn) as plant expression hosts

A

High biomass (12,000kg per hectare)

Long term stability in seeds

64
Q

Hydroponic culture

A

Well established in commercial horticulture = reduced risk of escape

65
Q

Prodigene proteins from transgenic plants

A

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

66
Q

Importance of Regulating GM Crops

A

Enhance containment of transgenic crops

Various requirements exist for growing pharma plants in USA

67
Q

Plant Expressed Vaccine Epitopes

A

Hep B (1992)
Rabies and HIV (1997)

Many many more

68
Q

Cell Culture Expression Hosts

A
  • plant cell cultures required sucrose
  • no plant pathogens
69
Q

Tissue Expressed Vaccines

A
  • Newcastle Disease for use in poultry
70
Q

Plant based insulin - SemBioSys

A
  • 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
71
Q

Stratosome Biologic System

A

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

72
Q

Transgenic Plants v Transient Expression

A
  • for crops can take >12 months to obtain transgenic seeds
  • transient expression = alternative faster method, often one week
  • transient = stable plant lines not made
73
Q

Transient Expression Method

A

Viral Vectors and Agroinfection
a) grow plants and bacteria
b) agroinfilitration
c) plant incubation
d) biomass harvest
e) extraction
f) protein purification

74
Q

ICON GENETICS GMBH

A

World leader in developing and using expression systems for production of recombinant proteins in green plants.

High & Rapid Yields of Recombinant Proteins

75
Q

Magnifection

A
  • transient amplification of viral vectors delivered to entire plant using Agrobacterium
  • marker genes are not required
  • containment is enhanced
76
Q

Ebola Virus Treatment example

A

Zmapp

77
Q

Zmapp method

A
  • 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
78
Q

Transient Expression

A

Ebola antibodies
Flu vaccine
Covid Vaccine - Medicago
Covid Antibody - Medicago

79
Q

cGMP

A

current good manufacturing practice

80
Q

cGMP for recombinant protein production in plants

A

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

81
Q

Molecular Pharming - key steps for commercialisation

A
  • product yield (>1% total plant protein)
  • protein stability
  • protein quality
  • extraction and downstream processing
  • quality assurance and quality control
  • regulatory approval
  • clinical trials
82
Q

Commercialisation of plant-dereived pharmaecutical proteins example

A

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.

83
Q

Strengths of nuclear transgenic planta

A

variety of crops, accumulation in different parts, different subcellular components or secreted into medium (rhizosecretion), highly scalable from seeds

84
Q

Negatives of Nuclear Transgenic Plants

A

recombinant protein accumulation concentration can be low (<1% total protein)
slow

85
Q

Plant Cell Vaccine System

A

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

86
Q

Transient expression

A

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

87
Q

Negative of Transient Expression

A
  • Leaves contain agrobacterium and associated endotoxins (lipopolysaccharides) - immune response -> septic shock
  • each cycle requires re-infection with Agrobacterium (adds costs)
88
Q

Chloroplast (plastid) transformation main method

A

main = particle bombardment

89
Q

Pros of stable chloroplast transformation

A

Very high accumulation of recombinant proteins, highest in leaves, subcellular compartmentalisation to chloroplasts, highly scalable, gene flow through pollen is restricted

90
Q

Cons of chloroplast (plastid) transformation

A
  • slow to get stable transgenic plants 6-12 months
  • not suitable for glycosylated proteins
91
Q

Delivering DNA into Plasmids

A

Bio-Rad PDS 1000/He particle delivery system

Fires microprojectiles coated with a plastid transformation vector at cells

92
Q

PlyGBS (phage lytic protein)

A

Protein accumulation does not decline with leaf age, but plastid protein biosynthetic capacity does, indicating the PlyGBS is extremely stable inside chloroplast

93
Q

Molecular Pharming: key steps for commercialisation

A

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

94
Q

Diamante

A

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

95
Q

ExpressTec

A

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.

96
Q

Chloroplast Transgenic Plants

A

‘Transplastomic Plants’

97
Q

Plastids

A

Family of plant organelles, different plastids are inter-convertible

All plastids contain DNA (identical genome in every plastid)

98
Q

Plastid DNA

A
  • Plastid DNA = Chloroplast DNA
  • Has circular map
  • many copies of plasmid DNA per plasmid
99
Q

Overview of Plastid Genome

A

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)

100
Q

Plastid Genes

A

highly expressed in chloroplasts
large subunit of RuBisCO (30-50% tsp leaf)

101
Q

tsp

A

total soluble proteins

102
Q

rbcL

A

plastid gene encoding large subunit of RuBisCO

103
Q

3 Steps to Transform Plastids

A

1) Vector Construction
2) Delivery of DNA into plastids
3) Selection of plastid transformants

104
Q

2 components of plastid transformation vectors

A

1) Expression Cassette
2) Targeting DNA

105
Q

Plastid Vector - Expression Casette

A
  • 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
106
Q

aadA gene

A

resistance to streptomycin and spectinomycin

107
Q

Plastid Vector - Targeting DNA

A

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

108
Q

Homoplasmic Plant

A

a plant with a uniform population of plastid genomes

109
Q

Heteroplasmic Plant

A

plant with a mixture of plastid genomes

110
Q

Transplastomic Plant

A

a plant containing transformed (transgenic) plastids

111
Q

Cytoplasmic Sorting

A

The process by which mixed plastids in a cell sort out to pure populations during growth and development

112
Q

Top 4 Biotech Crops

A

Soybean
Oilseed Rape
Cotton
Maize

113
Q

Chloroplast Expressed Proteins

A
  • No glycosylation
  • Compartmentation of toxic products
  • Disulphide-bond formation & correct folding of human proteins
  • Proteins cannot be exported
114
Q

Nucleus/cytosol expressed proteins

A
  • Glycosylation
  • Toxic Proteins (pleiotropic effects)
  • Disulphide-bonds requires targeting to the endoplasmic reticulum
  • Proteins for all cellular compartments
115
Q

Tobacco as a vehicle for making useful proteins

A
  • 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
116
Q

Plastid Transformation Enables

A
  • precise targeted insertion without marker genes
  • enhanced gene containment (no pollen transmission)
  • high levels of expression
  • coordinated expression of multiple genes
117
Q

Which components of the fruit and veg reduce non-communicable diseases

A

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)

118
Q

Plant Secondary Metabolites

A
  • 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
119
Q

Phytonutrients = subclass of secondary metabolites

A

flavonoids, anthocyanins, related compounds

120
Q

Isothiocyanates

A
  • broccoli and other cruciferous vegetables
  • may detoxify carcinogens
121
Q

lycopene

A
  • carotenoid abundant in tomatoes
  • some studies show it protects against several forms of cancer
122
Q

epigallocatechin gallate

A

green tea
may help elimnate immortality from some cancer cells

123
Q

genistein

A

isoflavonoid from soybeans, may protect against cancer

124
Q

Two science aims to improve dietary health

A

1) make fruit and vegetables cheaper
2) produce food enriched in phytonutrients

125
Q

Glucoraphanin

A

glucosinolate that is cleaved by gut bacteria to produce sulforaphane, an isothiocyanate that has proposed anti-carcinogenic properties in humans

126
Q

Super Broccoli = Beneforte

A

Broccoli crossed with wild relative containing high glucaraphanin (Bassica villosa)

127
Q

Super Broccoli Method

A

Broccoli crossed with wild relative containing high glucaraphanin (Bassica villosa)

many back-crosses over 15 years to reach commercial broccoli with 3x glucoraphanin content

128
Q

Genetic Basis of Super Broccoli

A

transcription factor Myb28 - previously determined methione-derived glucosinolate concentration = makes breading easier

129
Q

Patenting of Beneforte

A

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

130
Q

Health benefits of Glucoraphanin

A
  • molecule is taken up from diet
  • protective benefits against colorectal cancer
  • may reduce risk of prostate cancer
  • diet-human gene interactions
131
Q

Diet-human gene interactions

A

complicate picture of biofortification

132
Q

‘Green Revolution’

A

1960s-1970s increased high yielding cereal production in developing countries at expense of nutrient-rich pulse/legumes

133
Q

Iron Deficiency

A

60% world population suffer from Fe deficiency

Weakens immune function and generally impairs growth and development

134
Q

Zinc (Zn)

A

Over 30% of the world population suffer
Impairs development and immune system

135
Q

Iodine (I)

A

30% world population are I deficient

Impaired thyroid function (Goitre disease), impaired infant development

136
Q

Vitamin A = retinol

A

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

137
Q

Folate (vitamin B9)

A

numbers unclear but many people in developing countries known to be at risk

foetal neural tube effects, spina bifida, risk of cardiovascular disease

138
Q

Biofortification

A

increase plant nutrient content pre-harvest

a) Agronomic (fertiliser use)
b) Genetic (selective breeding)
c) Transgenic (genetic engineering)

139
Q

Potential of Transgenic Biofortification

A

↑ mineral accumulation
↑ mineral transfer into leaves/grains/tubers
↑ mineral storage
↑ mineral bioavailability
↑ vitamin biosynthesis in edible parts of plant

140
Q

Sources of Vitamin A in Diet

A
  • 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
141
Q

Golden Rice

A

Rice line engineered to produce B carotenene in its endosperm

142
Q

Multigene Transfer Methods

A

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)

143
Q

Evaluation of Multigene Transfer Methods

A

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

144
Q

Barriers and Lessons

A
  • intellectual property (freedom to operate, licensing, volume)
  • economic value (viable business proposition v social value)
  • regulatory approval (cost, time)
  • consumer and societal acceptance
145
Q

Golden Rice Trials

A

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

146
Q

Opposition to Golden Rice

A
  • 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
147
Q

Economic Opposition to Golden Rice

A
  • 10year delay = $700m (1.4m life years)
  • high perceived costs sway governments
148
Q

Uganda - Vit A Deficiency

A
  • 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
149
Q

Uganda - Biofortification

A
  • conventional breeding (low fertility, lack of pro-vitamin A varieties in gene pool)
  • gene modification (Banana21 project)
150
Q

Banana21 Project - Method

A
  • 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
151
Q
A

Plant Science (5 decks)

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