Lecture 5 - Genetic modifications and biosafety

Question 1

What are the various uses of transgenic plants?

Answer 1

• herbicide resistance
• resistance towards pests and pathogens
• tolerance of extreme environments
• increased yields
• modification of development
• nutritional modification of food
• growth on marginal soils
• bioremediation
• moelcular farming of proteins
• production of useful metabolites

Question 2

What biosafety issues are there with the use of transgenic plants?

Answer 2

• transgene spread
• environmental pollution
• contamination of human food
• product safety

Question 3

How might transgenes be spread into the environment?

Answer 3

• horizontal gene transfer to microorganisms (bacteria can pick up a gene transfer and problematic if use antibiotics that are used in making people better)
• outcrossing (transgenic pollen) gene flow into wild relatives (e.g. bad if herbicide resistance genes spread into weeds)
• colonisation of natural ecosystems by transgenic plants (percieved as a risk however if working with crop varieties it is unlikely that will become an agressive weed species

Question 4

How can transgene spread by horizontal gene transfer be avoided?

Answer 4

• removing markers form transgenic plants
• rare and inefficient as long as the recipient has no selective advantage
• use alternatives to antibiotic resistance markers

Question 5

How can transgene spread by outcrossing be prevented?

Answer 5

• physical containment
• removal of flowers
• harvest crops before flowering
• border rows to trap pollen
• apomixis (reproduction without fertilisation)
• physical or genetic male sterility
• excision of transgenes from pollen
• chloroplast tranformation

Question 6

How can transgene spread by colonisation of natural ecosystems be prevented?

Answer 6

• physical containment
• harvest crops before seed setting (leafy crops only)
• suicide genes (failure of embryo development)
• control of seed dormancy or shattering

Question 7

What unwanted foreign DNA is there in transformed crops that may need to be removed? What does this acheive?

Answer 7

• removing antibiotic resistance genes
• removing vector DNA
• removing surplus copies of the transgene (increases stability and expression - too many increases liklihood of silencing)

addresses the risk of horizontal gene transfer, respoding to public concerns

Question 8

What tool is used to excise antibiotic resistance markers?

Answer 8

Marker free transgenic plants by autoexcision - Using site specific recombinase system

1. Used construct introduced into plants to produce transgenic lines by a resistance marker flanked by sites that are recognised by site specific recombinase. e.g. cre recombinase
2. Recombinase gene also in the region flanked by the recombination site under the control of a germline specific promoter
3. In the second generation recombinase acts on the recombination site and excises all the internal genetic material
4. Construct introduced into plants which are transformed and regenerated after selfing of the primary transformant

Question 9

What is the result of generating marker free transgenic plants by autoexcision?

Answer 9

• observe a high frequency of excision

Question 10

What is the structure of the autoexcision construct?

Answer 10

• construct v complicated within the region flanked by recognition sites (lox sites) recognised by the cre recombinase
• contains antibiotic resistance gene under control of monopoline synthase promoter and terminator
• intron containing recombinase gene under germline specific promoter
• counter selectible marker - makes system more efficient as can select against plants that contain the region

Question 11

how can you test that marker free transgenic plants have been generated by autoexcision?

Answer 11

• isolate DNA of offspring
• PCR analysis (design efficient primers)
• in case the region was still present, used primers
• the absence of a band demonstrate that the plant is marker free and also the presece of a band at 724bp demonstrated that excision had occured

Question 12

What are the advantages of chloroplast formation?>

Answer 12

• multiple transgene copies (100 chloroplast/cell x 100 copies/chloroplasts)
• integration of the transgene at the target site by HR
• no transgene silencing, high yields
• usually no chloroplasts in pollen, no outcrossing
• operon gene organisation, several genes under the control of one promoter

Question 13

What are the cons to chloroplast transformation?

Answer 13

• cannot use agrobacterium (have to use particle bombardment)
• still difficult in many plant species
• yet to be demonstrated in monocots
• no glycoslyation of proteins
• risk of horizontal gene transfer (control elements similar to prokaryotes, extreme copy number, decaying plant material DNA can be picked up by microbes)

Question 14

How can you obtain transplastomic plants?

Answer 14

1. Introduce vector into plant tissue via particle bombardment
2. Some reach chloroplasts and and as the region of interest is flanked by homologous region on plasmid DNA get integration by HR at specific sites on the plasmid chromosome
3. Grow callus on selective media and test by PCR for homoplasmic calli that are homogeneous and don’t carry the untransformed genetic material (otherwise result in a dilution of effect)
4. Regenerate shoots and roots of plants that have had the region of interest integrated into the plasmid chromosome
5. Can be made marker free by adding a restriction site in the construct to allow the excision of the marker by HR, as long as the marker is flanked by a region homolgous to itself to permit a deletion event by HR or use site specific recombination system with lox sites and plant expressing cre recombinase or marker could be cotransformed on a different construct (although still have to identify events with successful transformation but could then segregate the antibiotics resistance marker

Question 15

How can markers be removed from transgenic plants?

Answer 15

• Can be made marker free by adding a restriction site in the construct to allow the excision of the marker by HR, as long as the marker is flanked by a region homolgous to itself to permit a deletion event by HR
• or use site specific recombination system with lox sites and plant expressing cre recombinase
• or marker could be cotransformed on a different construct, still have to identify events with successful transformation but could then segregate the antibiotics resistance marker to remove

Question 16

How was chloroplast transformation demonstrated in tomato plants?

Answer 16

Ruf et al 2001

1. Used particle bombarment on tomato leaf explants
2. Callus grown
3. any resistant tissue would continue gorwing on selective media and plants were regenerated from growing callus
4. Before regeneration plants checked using PCR analysis, for a homoplasmid situation, want band that demonstrates a successful integration
5. Tested protein levels in leaves and used a second selectable marker for indication of successful integration
6. resulted in extreme levels of protein synthesis
7. useful if want to produce lots of pharmaceutical compounds

Question 17

Give an example of chloroplast transformation with the Bt toxin

Answer 17

1. overexpressed Bt toxin gene with the help of additional proteins encoded naturally in bacteria where CRY genes were identified. Additional genes act as chaperones in protein folding - useful to take the whole operon (Bt cry2Aa2 operon) and express this in order to acheive a high level of protein formation
2. Showed that when added whole operon as opposed to just the CRY gene alone produced far more protein. production levels were so extreme that it lead to a formation of insecticidal crystals

Question 18

What was acheived through the use of plastid transformation for metabolite engineering”?

Answer 18

1. Used plastid transformation for metabolite engineering of various compounds.
2. Achieved high levels of molecules that are used as scavengers of ROS (tocopherol’s act against ROS)
3. Compared WT plants with transformed plants under stress conditions they functioned better. Wt exhibited bleaching.

Question 19

How can seed germination be prevented in transgenic plants?

Answer 19

GURT (genetic use restriction technology)

1. Need three constructs. Introduce toxic gene under the control of an embryo active promoter. This is not active whilst there is a blockr active between the promoter and coding region. Blocker needs to be excised and is flanked by restriction sites. (Embryo active promoter)
2. Second contruct is a repressible promoter that drives the expression of a recombinase which acts on the recombination sites, but is inactive when the repressor is bound. (Repressible pormoter)
3. Repressor is produced by the third construct containing an active promoter in front of the repressor coding sequence. (Plant active promoter)

All of these together in the plant means there is no activity.

4. Before sale the seeds are treated with an inducer that removes the repressor (binding to the repressor initiating a conformational change that reduces its affinity to the promoter). The recombinase is not active and the blocker is excised.
5. When the plants set flowers the seeds become sterile

Question 20

What are the advantages and disadvantages of GURT?

Answer 20

No germination of trangenic or hybrid seed

Pro: Useful for companies to develop seed stock that could not be propagated by farmers

Cons: Common practice in poorer countries that farmers reuse seeds but these do not germinate

Question 21

What compounds are of interest to biosafety considerations?

Answer 21

• In plants: toxins, secondary metabolites
• On plants: field chemicals (herbicides, pesticides)
• Allergens

Question 22

What are the percieved risks of GM crops?

Answer 22

• Foreign genes may have unpredictable effects
• Theoretically this could have unpredictable effects*
• Enhanced fitness -> super weeds
• Unlikely as cultivars are not fit enough to compete with wild plants*
• Toxic compounds might be produced
• toxic xompounds cannot come out of nowhere, and products will be extensively tested*
• It is not natural
• Invalid*

Question 23

Give some examples of some “unnatural crops”

Answer 23

Golden rice

• could not have been acheived naturally
• Developed to have high beta-carotene levels as rice is not high in vitamins, lacks of vitamin in many poorer countries which rely on rice
• Ignoring that we can prevent vitamin deficiencys by not using the tools that are available

Multivitamin maize

• cannot be acheived other than introducing specific genes that encodes biosynthetic pathway allowing a synthesis of the vitamins
• 170 fold more carotene
• 6 fold ascorbate
• 2 fold folate

Question 24

How have advances in molecular techniques helped to support conventional breeding?

Answer 24

• Improved yield, stress tolerance can be achieved by plant breeding and can be assisted by molecular plant breeding techniques
• very successful to introgress traits from wild relatives into plants - can make use of already present gene pool - otherwise tedious process can be made better by genotyping chromosomes
  
  • if introgress trait after many rounds of backcrossing just have the locus of the chromosome from the wild relative and rest of the genome is cleaned up
  • molecular tools can help by identifying these individuals by genotyping, early on to speed up
• accepted by the public
• no enhanced risk beyond the normal

Question 25

How is reverse breeding achieved by suppression of CO?

Answer 25

• Reverse breeding - related to hybrid breeding
• companies able to produce seed stocks that are homozygous and if these are crossed produce hybrids that are better than the original
• Need to maintain beneficial hybrid genes in the next generation through a segregation of all combined beneficial loci - segregation hard to control in the next generation
• Use chemicals on the starting hybrid that supress CO
• If meiotic CO is supressed there is no mix up of chromosomes and every chromosome stays as it is
• However, CO is essential for correct chromosomal segregation
• By genotyping and microscopy select gametes with full chromosome set in combination
• regenerate plants from gametes and cross to reconstitute starting hybrid
• generates population of double homozygous plants that can be then combined in a cross to get the combination of genes in the starting hybrid

Question 26

How can you achieve the silencing of a gene?

Answer 26

• Old method: introduce a construct (silencing construct) to suppress the activity of a gene
• mutagenesis and identification of plants by reverse genetics (TILLING or sequencing)

Question 27

How can genetic enginnering in plants be done without the introduction of foreign DNA?

Answer 27

• Identify regions of the plant genome that could be used as tools within plants for GE.
• Rommens et al study attempted to identify sequence within plants related to T-DNA left and right border sequences
• Found many sequences in different plants that were highly related
• These were used to replace T-DNA border sequences
• In arabidopsis there was a single region that was related and resulted in a transformation efficiency comparative to that of TDNA borders.

Plant derived transfer DNAs (P-DNAs)

Question 28

How was potato improved by an all-native DNA transformation?

Answer 28

• Rommens et al follow up study
• Formed an all native contruct and this was compared to a stardard construct where parts were foreign e.g. kanamycin resistant and promoters
• Transformed potato targetting a gene so that the glucose released from starch in potato would be reduced
• Achieved in many individuals. (french fries didnt get browning due to over glucose production) Acylamide levels also reduced (french fries contain substantial amounts of acrylamide)
• Got substantial restults using all native construct from plants
• 3 genes silenced that conferred unwanted properties

Question 29

How can genetic modifications be achieved? Should regulatory procedures be based on the process or the outcome?

Answer 29

1. Conventional breeding including mutagenesis
   
   • New combinations of genes within the same or related species
2. Intragenic/cisgenic genetic modification
3. Transgenic GM: plants carry foreign DNA that they could not have recieved by conventional breeding

Conventional breeding including mutagenesis and intragenic/cisgenic modification: uses different processes bu with the same results (phenotypes)

Intragenic/cisgenic and transgenic GM: same process, possibly different results

Public care more about the process than the result (in EU)

Question 30

What are the process and product differences in the main breeding techniques?

Answer 30

1. Introgression breeding
   
   • No GM techniques used in the process
   • Product not GMO​​
   • still have regions didn’t want to introduce
2. Transgenesis (inserting new gene from unrelated genome using agrobacterium)
   
   • ​​GM techniques used in process
   • Product GMO
3. Cisgenesis (using agrobacterium to insert new gene from wild relative)
   
   • ​​GM techniques used in process
   • Product GMO (has agrobacterium border sequences)
4. Precision breeding from wild relative into crop
   
   • ​​GM techniques used in process (no new gene material inserted in product)
   • product GMO? (no foreign border sequences)
   • uses site specific nucleases to precisely introduce genes using CRISPR/cas9 system, tools can be crossed out
   • no regulatory industry would be able to tell whether GE or classical

Question 31

What are the two main types of regulatory procedures?

Answer 31

Comparative approach: If the product is substantially equivilent it is okay (USA)

Precautionary principle: (EU) One cannot excluded risks, therefore extra measures are required to anticipate and minimize potential risks

Question 32

How might sweet potatoes be considered GM?

Answer 32

The genome of cultivated sweet potato contains Agrobacterium TDNAs with expressed genes - a naturally transgenic food crop

These genes have been selected for when breeding. Identified two T-DNAs - one present in closely related wild relative, therefore during conventional breeding and selection T-DNA was kept in the crop, therefore transgenic plants have been used for thousands of years with no problem

291 accessions of cultivated sweet potato all contained one or more T-DNA sequences

Question 33

What argument is there that organic breeding could benefit from transgenic techniques?

Answer 33

Organic farming based on concept of working with nature

Could make use of the gene pool of wild relatives

Question 34

What is the mutagenic chain reaction (MCR)?

Answer 34

A technique that converts heterozygous into homozygous mutations

A mutation can spead from a single modified individual thoughout a whole population (gene drive system)

Question 35

How does MCR work?

Answer 35

1. Introduce construct with Cas9 nuclease and guide RNA
2. Guide RNA guides Cas9 ends to the target and then the sequences left and right of the cut site of cas9 result in a cut in the target site. Produces a dsbreak then construct can be integrated by HR at the cut site
3. Result in a chromosome that has been modified by insertion of a whole casette then a gene can be introduced
4. Cas9 RNA region can act on a second allele in a diplod organism -> homology directed repair result in both chromosomes carrying the construct
5. Take insect that has been modified by an embryo injection
6. results in a single chromosome modification which will then spread to the next
7. if mutant crossed to the WT get a recombination resulting in heterozygotes which will also be homogenised by the activity of the construct
8. End up with the introduction of a single mutant to spread the genetic modification throughout the whole population
9. 100% observed efficiency
10. Safety considerations: would have to have a tool to reverse if it went wrong

Question 36

What is the current status of crop yeild increase?

Answer 36

In 60s rice, maize and wheat were increasing in yeild linearly. If continue with linear increase would not reach reported yeild increase required for the expanding population by 2050