[3-6] - Crop Improvement + GM

Question 1

Define Domestication in the context of crops

Answer 1

Domestication is the process of artificially selecting plants to improve their suitability for human use (e.g., taste, yield, storage and cultivation)

Question 2

Why did humans invent agriculture?

Answer 2

Evidence suggests that around 10,000 years ago, humans began to cultivate certain plant species - there are several proposed factors as to why:

OPPORTUNITY:
- Change to a dryer climate around 11000 years ago resulted in more Annual Plants
- Agriculture first appeared in areas which were ecologically rich
- There were an abundance of readily storable wild seeds, tubers and fibers
- RUBBISH HEAP HYPOTHESIS (see separate FC)

CRISIS:
- Food crisis due to cliamte or overpopulation may have pressured the change

Question 3

Explain the concept of the Rubbish Heap Hypothesis and evaluate how well supported it is

Answer 3

The theory is that gatherers may have brought back seeds and tubers, and some may have left on a dump heap and grown back the next year, which was eventually harnessed for intentional farming

However, the hypothesis does not explain why agriculture began when it did - there is evidence of association of weeds with human dwelling for thousands of years before the dawn of agriculture, so why didn’t this process begin then?

Additionally, the majority of wild ancestors of the main Near Eastern crops are not known as weeds, casting doubt on a “weedy origin”

Overall, the evidence points to long term interaction between humans and plants, driven by cultural forces and intentional domestication for nutritional features

Question 4

What are the main sources of evidence we can use to trace the origin of modern crops?

Answer 4

Centres of origin determined based on:

1. Areas where wild relatives occur
2. Areas where a particular crop shows the most natural variation and diversity (anywhere else it has been transported to will have less genetic diversity due to bottleneck)
3. Linguistic clues
4. Archaeology (with limits)
5. DNA clues/phylogenetics

Note: most were domesticated in a SINGLE REGION (apart from cotton, rice, yam)

Question 5

Explain what is notable about the TYPES of crops which were selected in each region

Answer 5

• Cereals (deficient in Lys)
• Pulses (deficient in Met)
• Fibers, fruits and tubers

Together, these crops provide all the essential amino acids

Although different crops were selected in different regions, there was similarity in the TYPES of crops selected

Question 6

Which species of cereal were first domesticated in the Fertile Crescent?

Answer 6

Three species:
- Einkorn wheat
- Emmer wheat
- Barley

Of these, only barley is still grown as a crop

Question 7

Describe the origin of BREAD wheat

Answer 7

Hexaploid wheat (Triticum aestivum) evolved after the origin of agriculture, from a hybridisation event between domesticated Emmer wheat and Wild Goat Grass

Hexaploid wheat eventually evolved into spelt wheat and bread wheat

Question 8

Describe the phenotypic changes seen in domesticated wheat and barley compared to their non-domesticated ancestors

Answer 8

THE NON-BRITTLE PHENOTYPE
- Seeds remain attached and must be threshed to release
- This is due to a single mutation

Can be distinguished by abscission scars:
- Smooth in wild spikelets due to natural dispersal
- Rough in domesticated spikelets due to threshing
This allows adoption of agriculture to be followed in the archaeological record

Question 9

Describe the (mentioned) phenotypic change seen in domesticated rice

Answer 9

In cultivated rice, domestication occurs suddenly, with the vast majority of seeds germinating at around the same time

In wild rice, germination happens more gradually, so that the entire generation will not be wiped out if a severe event such as frost wipes out germinated seeds

Question 10

List the changes in phenotype that make up “Domestication Syndrome”

Answer 10

1. Loss of Seed Dispersal (due to tough rachis mutation)
2. Loss of Seed Dispersal Aids (e.g., hairs and hooks)
3. Increase in seed size (large grains surviving deeper burial)
4. Loss of sensitivity to environmental cues for germination (mostly soon after planting)
5. Synchronous ripening of seeds/fruits (selected by cultivation in continuous annual cycle)
6. Compact growth habit

Question 11

Describe and explain the two models proposed for how Neolithic populations domesticated crops in the Fertile Crescent

Answer 11

1. Monophyletic Origin and Fast Domestication
   - Genetic studies suggested that Einkorn and Emmer wheat were domesticated in southeast Turkey, while Barley was domesticated in southern Levant
   - Therefore, agriculture was thought to be invented by small, localised groups, then disseminated
   - Due to low frequency of relevant mutations in wild population, traits thought to be selected in a few generations by farmers who knew what they wanted
2. More Gradual, Complex Timeline
   - Examination of preserved seeds from archaeological sites suggests that humans were using a mix of wild and domesticated plants for around 4000 years before complete use of domesticated crops
   - Agriculture may have arisen gradually, at many places within the Fertile Crescent at the same time
   - Evidence: 19000 BP large quantities of wild barley at a site in Israel
   - Wild rye cultivated in Syria from 13000 BP
   - Fully domesticated barley and wheat present by 10000 BP

Question 12

What are the main reasons why it can be beneficial to study domestication?

Answer 12

1 - A Centre of Origin is a Centre of Diversity to be Preserved
-> Source of genes for new traits (e.g., resistance)
-> Improved understanding of control of plant architecture AND human evolution

2. Advantageous to release a GM crop OUTSIDE its area of origin (BR on why?)
3. Genetic knowledge of domestication processes may allow De Novo Domestication

Question 13

Define de novo domestication

Answer 13

De novo domestication is the introduction of domesticated traits into non-domesticated plants (e.g., wild plants, cultivars or landraces) via genetic engineering, such as CRISPR-Cas9

Question 14

Describe and explain the main phenotypical difference between domesticated barley and its wild ancestors, and the genetics underpinning this. Also, how does this relate to the two theories regarding the transition to agriculture?

Answer 14

Domesticated seed heads show a non-brittle phenotype (i.e., seeds remain attached after maturity, meaning they must be threshed to release seeds and are easier to harvest), whereas Wild seed heads show a brittle phenotype (i.e., seeds detach and fall to the ground upon maturity)

Genetics:
Gene cloning suggested that all modern barley cultivars are derived from recessive mutations in either BTR1 or BTR2 (which are very close together, meaning they had previously been mistaken for a single locus)

BTR2 is a ligand which binds to the PM receptor BTR1 to trigger the formation of the abscission zone (only occurs where expression of the two genes overlap, making it a narrow, highly regulated zone)

All modern cultivars are homozygous recessive for one of these two genes (most European/West Asian barleys have the mutant btr1 allele, most East Asian varieties have btr2) - the two deletions are monophyletic, and no double-homozygous line has been found

HOWEVER, a second BTR1 mutation also conferring the non-brittle phenotype was discovered in 2017 (a single Leu-Pro base substitution in the active part of the receptor, rather than in the hydrophobic TM domain) - showing that the origin of this trait is more complex than was previously thought, and supporting recent models suggesting the transition to agriculture in Southwest Asia was a protracted and multiregional process

Question 15

Describe the “routes” by which barley and agriculture spread into Europe

Answer 15

Around 8000-7000 years ago, barley spread from Turkey into Europe via two main routes:

1. A southern route into Spain (relatively quick, possibly because the climate is similar across the Mediterranean)
2. A northern route into Hungary and Germany (had several pauses of thousands of years at a time, possibly because barley needed time to adapt to different climates, or because local populations were resistant to take up agriculture)

Question 16

Explain how and why barley had to adapt to the climate of Northern Europe (including phenotype and genetics)

Answer 16

1. Flowering in most wild cereals is responsive to increasing day length (as this allows optimum seed production BEFORE the dry summers of the Fertile Crescent)
2. Flowering in some domesticated barleys (e.g., UK) is NON-responsive to day length (allowing flowering later in the year, controlled by time of planting - adapted to a temperate climate such as Northern Europe, with lower temperatures and moisture available for seed production during the summer)

In barley, flowering time is controlled by the Photoperiod Response Gene Ppd-H1 (ppd-H1 allele is NON-responsive to day length due to a single base mutation in exon 6 of the gene)

Genotyping of 170 landraces from the 20th century reveals the increasing prevalence of ppdH1 (non-responsive allele) further north in Europe

Note: wild plants with mutant ppd-H1 do exist - mainly in mountainous areas of Iran where summers are less hot and more moisture is available (in these environments, flowering later would allow more time for the mother to grow, without the summer being too dry for the seeds to germinate

Relate to big picture: importance of flowering time and climate for yield (new climate, same flowering time = less yield)

Question 17

What is the negative side effect of crop improvement by inbreeding which was highlighted by the case study of the Tomato? What key concept did we learn about that aims to correct this

Answer 17

It often results in a loss of genetic diversity which can, in turn, cause a loss of certain beneficial traits (e.g., salt tolerance in tomatoes) - which may have been unnecessary in the environment where it was first domesticated

DE NOVO domestication can be used to induce beneficial traits of domesticated plants (e.g., day-length neutrality, increased size and number of fruit, ripening synchrony) but WITHOUT losing other traits (e.g., salt tolerance!)

Question 18

Which (mentioned) monogenic traits have been identified as possible targets of de novo domestication in tomatoes? [Bonus points for naming the relevant genes]

Answer 18

Compact plant architecture and ripening synchrony -> SP

Day-length neutrality -> SP5G

Enlarged fruit size -> SICLV3 and SIWUS

Increased VitC level -> SIGGP1

Question 19

How successful was de novo domestication of tomatoes?

Answer 19

It was considered successful (i.e., beneficial domestication traits were induced while salt tolerance and resistance to bacterial spot disease were maintained)

HOWEVER, fruit size was limited compared to existing domesticated tomatoes - possibly because fruit size is a more complex phenotype than previously thought

Question 20

Describe and explain the key case study demonstrating domestication of an orphan crop

Answer 20

The Groundcherry - grown in South America for its sweet berries, but limited due to sprawling growth habit and small fruits (1g) that drop to the ground

By sequencing the groundcherry genome, and using the well-studied tomato genome as a guide for the genes underlying these undesirable traits, researchers were able to target these traits/genes with CRISPR-Cas9 to improve fruit size, determinate growth and flower number [couldn’t identify genes to induce loss of husk, optimal flowering time or loss of fruit abscission]

Mechanistic explanation for determinate growth:
- Identified the SELF-PRUNING (PR) gene in tomato, and a mutation in this gene that provided compact ‘determinate’ growth, with a burst out flowers and fruits at a single point, enabling large-scale field production
- Using the tomato gene as a guide, they transferred this trait into groundcherry

Question 21

What are the caveats mentioned in “The Taming of the Shrub” with regard to the groundcherry case study?

Answer 21

Domesticating an orphan crop involves many challenges:
- It requires multiple tools, e.g., well sequenced genome, understanding of paralogue structure and expression, and a reliable delivery system for editing the genome
- Must be able to predict which modification will yield phenotype of interest (can infer from history of related crops, but still complex and varied)
- Knockout may be inadequate, necessitating an alternative, more subtle allele
- Some orphan crops may have resisted domestication precisely because their key traits are under more complex control than in other plants

Question 22

State the major types of genetic improvement of agriculture that have happened throughout history

Answer 22

Distant past:
- Crop plant domestication

Recent past:
- Hybrid seed
- Advances in breeding tech
- Genetic manipulation

Now and the future:
- Plants for improved human health
- Plants for environmental stress tolerance
- Precise genome editing
- Synthetic biology

Question 23

Why might it be worth using genetic manipulation instead of simply breeding?

Answer 23

For traditional marker-assisted breeding:
- the desired trait must already be present in the population of the plant species
- some genetic information (i.e., molecular markers) MUST be available for the plant in question

With genetic manipulation:
- the gene can come from any source
- no genetic map or genome sequence is required (although still helpful)

Overall, GM is simply an alternative method to alter plant characteristics, which CAN be faster and more efficient than conventional breeding

Question 24

State the two broad approaches to Plant GE (extremely vague!)

Answer 24

1. Manipulate genes which ALREADY exist in the plant
2. Introduce foreign (NOVEL) genes into the plant

Question 25

Explain the principle of GE via manipulating existing genes in plants (and give two examples)

Answer 25

Can either increase activity of specific genes (e.g., overexpression of BBX17 gene in tomatoes to increase heat tolerance;

OR

Can switch off/silence specific genes (e.g., silencing Arah2 and Arah6 genes in peanuts to reduce allergic reactions)

Question 26

Explain the principle of genetic manipulation via novel genes, and give two examples

Answer 26

Can either improve existing characteristics (e.g., improved insect resistance via expression of insecticidal toxins encoded by cry genes from Bacillus thuringiensis)

OR

Can introduce completely novel characteristics (e.g., TNT and RDX tolerance via introduction of NR and XplA bacterial genes, which convert TNT and RDX into non-toxic chemicals) -> this allows plants to grow on contaminated soil, e.g., former war zones, firing ranges, etc., and decontaminate these zones

Question 27

What is the main tool used for genetic engineering of plants, and how (briefly) does it function?

Answer 27

CRISPR (clustered regularly interspaced short palindromic repeats) - Cas9

This system uses sgRNAs to target nucleases to specific sites, thereby inducing desired mutations, insertions or deletions

Question 28

What are the most important factors to consider when planning the genetic modification of plants

Answer 28

1. The gene to be transferred (i.e., the transgene) -> which gene is most suitable for the desired characteristic?
2. The host cells/tissues in which the gene should be expressed -> not all plants are equally amenable to manipulation
3. Mechanism of transfer
4. Method of regeneration and selection

Question 29

What are the main transformation methods for plant GM and what are some pros and cons of each?

Answer 29

1. Agrobacterium-mediated gene transfer
   - Highly effective
   - BUT lower copy numbers and doesn’t work as well on monocots
2. Microprojectile bombardment
   - Easy and effective, high copy numbers, wide range of plants
   - Random integration
3. Viral vectors
   - Not very effective, limited to hosts
4. Protoplasts
   - Regeneration problems
5. Microinjection
   - Tedious and slow
6. Electroporation
   - High efficiency
   - BUT large foreign DNA, regeneration problems

The first two are the only methods which have consistently proven effective

Question 30

What is Agrobacterium and why is it useful?

Answer 30

Agrobacterium tumefacins is described as a “natural” genetic engineer

• Gram-negative, rod shaped bacterium
• Found in rhizosphere
• Causal agent of crown gall tumours in plants
• Can be used for precise, effective genetic modification of plants

Question 31

How does Agrobacterium form crown galls (and, briefly, how can we exploit this for genetic modification)?

Answer 31

Via a process of horizontal gene transfer - the transfer of DNA from one species to another (in this case, from a bacterium to a plant)

Agrobacterium inserts a Ti plasmid [containing genes for auxin, cytokinin and opine synthesis between the left and right borders (LB and RB), as well as genes for virulence and opine catabolism] into the host plant cell, where the region defined by LB and RB becomes integrated into the plant DNA

The host cell is co-opted to produce auxin and cytokinins, which form a gall via cell proliferation, and opines, which act as an energy source for Agrobacterium

Since only the T-DNA region (defined by LB and RB) becomes integrated into the plant DNA, and these genes are not involved in the transformation process itself, we can replace them with genes of interest which we want to introduce, without disrupting the transformation

Question 32

Describe the basic features of a vector for plant transformation

Answer 32

A T-DNA plasmid (around 20 kb) is constructed, with the T-DNA region between the LB and RB sequences containing a marker gene (e.g., Kan resistance) and a gene of interest behind a promoter (e.g., CaMV 35S)

Outside the border sequences, there may be an E. coli origin or replication (ori), an Agrobacterium origin of replication (ori) and a selectable marker gene for both E. coli and Agrobacterium (needed for identification of the plasmid in bacteria and plants)

Question 33

What are the key required features of a transgene in order to function in a plant?

Answer 33

Some foreign gene promoters and 5’ and 3’ UTRs may not be recognised in plants

Therefore, chimeric transgenes must have Plant-gene Regulatory Regions flanking the ORF (i.e., the coding region) to allow expression

The ORF sequence itself may also have to be modified

Essentially, need to make plant transgenes “as plant-like as possible, without affecting the desired function of the gene”

Question 34

Describe how transfection of plant cells can be achieved via Biolistics

Answer 34

The basic principle is bombarding plant cells with DNA using a particle gun

Small gold particles are coated with DNA, placed on a small metallic macroprojectile, then shot out of a barrel towards the target tissue (e.g., a leaf)

Gold particles will pass through, leaving the DNA in the leaf and allowing it to be taken up (transfection)

Question 35

Describe the major steps in the process of plant transformation and regeneration (in plant GM)

Answer 35

1. Identify a suitable Explant (e.g., leaf piece)
2. Either co-cultivate with Agro OR bombard with DNA
3. Kill Agro with a suitable antibiotic that does not harm the plant
4. Select for transformed cells using a selectable marker
5. Regenerate whole plants
   (Regeneration requires hormones - cytokinin to induce shoot formation, and auxin for root formation)

In the regeneration step, balanced cytokinin/auxin induces cell proliferation (callus growth), while high cytokinin induces shoot formation and high auxin induces root formation

Overall, plant regeneration can be very slow (months) and is very difficult to achieve in some species

Question 36

Describe the Alternative method of plant transformation that avoids the need for tissue culture

Answer 36

For some plants (e.g., Arabidopsis) we can use a “Floral Dip” method - directly infect and transform ovules of developing flowers

1. Dip young flowers into Agrobacterium + surfactant (a detergent)
2. Allow plants to set seed
3. Select for transformed plant cells using selectable marker

Only around 0.1-1% of seeds are transgenic, but if large enough numbers of seeds are produced, the overall transformation efficiency is acceptable

Question 37

Describe how “Alternative Fast-track Engineering” can be carried out in plants

Answer 37

In addition to the transgene, developmental genes can be transformed into a whole plant, to induce formation of transgenic roots

Question 38

Describe and explain the alternative type of plant GM besides nuclear genes

Answer 38

Chloroplast Engineering:

Each cell has both a nuclear genome and a plastid (chloroplast) genome (roughly 10,000 circular genomes per cell)

Engineering of plastids can offer:
- Precise insertion of genes
- Very high levels of gene expression
- Co-ordinated expression of multiple genes
- Enhanced gene containment
- No risk of GM-gene “escape” to a non-GM plant, as chloroplasts are maternally inherited, and not transmitted through pollen

Question 39

What is the Anti-GM Lobby, and how justified are their concerns?

Answer 39

There is a strong anti-GM lobby which argues that there may be detrimental health impacts of GMOs

No detrimental health effects have yet been proven, and GM-plant-derived food has been consumed by over 1 billion people

However, long-term studies on the safety of GM plants are certainly needed

Also, many studies on the health impacts of GM foods are carried out by biotech companies involved in its commercialisation, which could be a conflict of interest.

Question 40

How widespread is GM crop commercialisation now?

Answer 40

After 20 years of commercialisation, GM plants are now grown in nearly 30 countries by 17 million farmers (mostly soybean, maize, cotton and canola)

Most GMOs available are for pest control (e.g., weed control via glyphosate resistance, insect resistance via production of Bt toxin, or other disease resistance, e.g., virus/bacteria)

Question 41

Explain the significance and benefits of currently used GM crops

Answer 41

By providing resistance to weeds and pests, GM plants reduce reliance on chemicals - a cost AND environmental benefit

This provides continuous protection, when and where needed

Question 42

Define plant stress, name the two types, and give some key examples

Answer 42

Any kind of non-optimal conditions that impact plant growth can be considered stress

Abiotic stresses include intense light, herbicides, ozone, flooding, heavy metals water-deficit stresses (which includes drought and salinity, as well as temperature stresses such as heat, chilling and freezing)

Biotic stresses include pathogens, pest damage, wounding and oxidative stresses

Question 43

How might GM be used to generate “healthy” plant food?

Answer 43

It is proposed that GM could be used to enrich plants in Vitamin A (since 250 million children suffer from VitA deficiency, which causes blindness and reduced immune function), and Iron (as 60% of the world population is deficient, which causes weakened immune system and growth)

Question 44

What are some non-food applications of GM in plants?

Answer 44

Plant secondary metabolites (i.e., useful products produced by a plant which are not necessary for its survival, such as defence, colour and scent compounds -> alkaloids, anthocyanins, flavenoids, steroids, terpenoids, etc.) can be a source of drugs

GM can be used to increase content of these useful secondary metabolites (e.g., overexpression of SalAT enzyme alters metabolism of morphinan alkaloids in opium poppies, thus increasing morphine, codeine and thebaine content)

GM can also be used to generate plants that produce therapeutic proteins (which were traditionally synthesised using recombinant microbes or mammalian cells) - essentially, plants can now be used as vectors for these.
-> Plant-Derived Pharmaceutical Products (PDPs) include antibodies, vaccines (e.g., ZMAPP for Ebola and a Covid-19 vaccine), enzymes used in diagnostic kits, etc.

This is known as biopharming.

Question 45

What are the main advantages and disadvantages of Plant-Derived Drugs?

Answer 45

ADVANTAGES:
- Production scale and economy (no need for large-scale fermentation or bioreactor systems - plants are cheaper, even when hi-tech greenhouses are factored in)
- Product safety (less potential for contamination of protein product with human/animal toxin)
- Ease of storage and distribution (can stored/distributed as seed)
- Opportunities for low-cost drugs and vaccines (purification might not be needed if engineered in edible plants)

DISADVANTAGES:
- Unclear whether consistent quality can be delivered (natural variation and inconsistencies in growth, soil and weather conditions)
- Differences in post-translational modifications of plant proteins compared to animals and microbes (particularly a problem with antibodies)
- Risk of entry into food chain (especially if edible plants are used)

Question 46

Explain how GM of terrestrial plants can be applied in the energy sector

Answer 46

Photosynthetic organisms can be used as a source of biofuel -> LIQUID fuels will be needed in short/medium term, as solar/batteries unfeasible for shipping and aviation

Currently, bioethanol is made from maize and sugarcane, and can be used as a fuel additive

HOWEVER, it is imporant that biofuels are made sustainably and independently from food crops

GM of plants may be able to overcome the problem of lignocellulose recalcitrance in biomass - cellulose is a very abundant polymer of glucose in plant cell walls, but is often in the form of lignocellulose (difficult and expensive to break down); GM could:
-> increase hemicellulose and cellulose content to increase ethanol yield
-> reduce lignin content or change lignin composition (to improve access to cell walls for degrading enzymes)
-> express cell wall degrading enzymes in plants themselves, to improve cell wall digestion

Question 47

Explain how GM of non-terrestrial organisms could be used in the energy sector

Answer 47

Microalgae (single-celled algae - as opposed to macroalgae such as seaweed) can be used as a source of biofuel, as they accumulate high quantities of starch or oil for conversion to bioethanol or biodiesel

A system of algal growth, harvesting + dewatering, lipid extraction, then conversion to fuel is not yet economically viable, but it is possible that GM could improve the economic viability without the need for controversial subsidisation

Algal biofuels rely on biosynthesis of storage lipids in cells - non-membrane glycerolipids (e.g., Triacylglycerol) form in the cytoplasm under nitrogen deficiency [downstream of Acetyl Co-A from Calvin Cycle]

It might be possible to engineer algae to produce more lipid, or to reduce BREAKDOWN of lipids:
-> Knockout of the LIP4 lipase gene via CRISPR resulted in increased TAG content under nitrogen deficiency, AND reduced TAG breakdown following nitrogen resupply!

HUGE potential for BR when it comes to the essay hehe