Lecture 1 - Success stories

Question 1

Give an example of human selective breeding

Answer 1

The selection of Maize from the original plant, Teosinte.

Question 2

How was the generation of Maize an example of human selection?

Answer 2

It is not ‘natural’ selection

Plant would not exist without humans

Farmers in mexico found the maize mutant, selected for as Teosinte was small and not enough to feed lots of people

Question 3

Give examples of some of the traits selected for in crop improvement

Answer 3

1. Reduced branching: allows more resources available to a smaller number of seeds therefore bigger seeds
2. Reduced height: more resources available to seeds = bigger seeds
3. Reduced seed shattering: reduced loss before harvest
4. Pest resistance: lower input of conventional herbicides, lower costs, lower impact on human health
5. Herbicide tolerance: fewer treatments with a herbicide at the optimal time and optimal dose. Improved weed management, less ploughing, lower costs and lower energy input

Question 4

Give two examples of architechural modifications that have driven agricultural improvement

Answer 4

1. Modification of the TB1 gene 7,500 years ago during the domestication of teosinte to produce maize
2. Modification of the Reduced Height gene (RHT), 50 years ago, to permit the green revolution [less green parts more seeds]

An improved phenotype resulting from changes at the genetic level, although not involved GM in the popular sense

Question 5

What did Hubbard (2002) show about the Maize, Teosinte and Tb1 knockout mutant?

Answer 5

Maize: Has no branched shoot system and bigger ears

Teosinte: Branching and small ears

Tb1 mutant (Tb1 gene knocked out): Resulted in more branching than teosinte

Question 6

How does Tb1 act as a repressor?

Answer 6

When there is more Tb1 branching is more repressed, more of a maize phenotype.

When Tb1 is knocked out, branching is no longer repressed, more of a Teosinte phenotype.

Question 7

What is the action of Tb1?

Answer 7

Represses cell division in auxillary buds

Question 8

What is the story of the development of maize?

Answer 8

Mexican farmers found an unbranched plant in which the TB1 gene was over-expressed

Over-expression was due to a promoter mutation

And this promoter mutation is present in all modern maize varieties

Question 9

What is the phenotype of a loss of function maize TB1 allele?

Answer 9

A highly branched growth habit, even more extreme than teosinte

Question 10

How did Hubbard (2002) visualise the location of the expression of the TB1 gene?

Answer 10

Compared plant sections of WT to a tb1 mutant, identified that auxillary branches elongate in the tb1 mutant but not in the wild type. Used an insitu hybridisation to analyse where the gene was expressed.

Question 11

Outline the process of in situ hybridisation Hubbard (2002) used to visualise the location of the TB1 protein?

Answer 11

Aim: to analyse where in a tissue a specific gene is expressed

1. Generate probes in vitro to detect transcription by the use of anti-sense RNA
2. Visualise by a colour reaction as the anti-sense RNA binds to the specific DNA region

Control: tb1 Sense RNA as doesn’t bind but gives background staining

2nd Control: An antisense RNA probe of a gene that didn’t want to invesitgate but already knew about e.g. knotted1 antisense

Question 12

What was the result of the insitu hybridisation on the Tb1 (teosinte branched 1) gene in maize and teosinte by Hubbard? (2002)

Answer 12

For the Tb1 gene in Teosinte, there showed no staining, where Kn1 showed the location of the formation of the bud

For the Tb1 gene in maize there were slight stains at the location of the forming bud as shown by Kn1

Question 13

What would the knockout of Tb1 show in an in situ hybridisation for Tb1?

Answer 13

No staining whatsoever

Why was there then no staining in the Teosinte Tb1 localisation?

Question 14

What sensitive test could be used to determine if there was ANY expression of the Tb1 gene in teosinte?

Answer 14

PCR

Not northern blotting as it not as sensitive

Question 15

What is cited as the father of the Green revolution?

Answer 15

Norman Borlaug (1914-2009)

• Saved ~a billion lives when he died
• Achieved a almost doubling of wheat yields in Pakistan and India between 1964 and 1970
• Won Nobel Peace prize

Question 16

What is the Teosinte branched Tb1 gene?

Answer 16

A gene encoding for a transcription factor

A member of a transcription factor that represses cell cycle genes

Question 17

Where is the Tb1 gene expressed?

Answer 17

Expressed in buds (identified by in situ hybridisation)

Expressed in higher levels in maize than in teosinte

Question 18

In the in situ hybridisation (Hubbard, 2002) what probes and controls were used to identify the location of the Tb1 gene?

Answer 18

Hybridisation probes: tb1 antisense

Meristem control: knotted1 antisense

Question 19

What is referred to as the ‘Green Revolution’ gene?

Answer 19

Wheat Rht (Reduced height 1)

Ortholog of maize dwarf-8 gene and Arabidopsis GAI gene

Question 20

What is the function of the wheat Rht-1 gene?

Answer 20

Function in giberellin signalling

Question 21

How can the Rht-1 wheat gene be used to advantageous growth in other crops?

Answer 21

Semidominent mutant alleles confer the phenotype to other plaant species. Gene has been isolated from arabidopsis and introduced into rice (Peng et all 1999)

Question 22

How did Peng et al confer the reduced height phenotype into rice and consequently show that the green revolution genes encode mutant giberellin receptors?

Answer 22

Did a GA response assay on Basmati rice transformed with the arabidopsis GAI gene

GA involved in plant growth

• showed that w/o the transgene resulted in shoot elongation in response to GA
• with the transgene rice does not show elongation in response to GA

Transgenic plants smaller than wild type

Question 23

How is RHT involved in Giberellin signalling?

Answer 23

Bonetta and McCourt (2005) showed:

Reduced height is a DELLA domain protein acting as a repressor in the absence of GA. It binds ot the TF necessary to transcribe genes involved in the promotion of shoot elongation.

TF is sequestered by binding to the repressor therefore there is no transcript.

When GA present, binding of GA to its receptor and in conjunction with protein degradation machinery triggers the degradation of the repressor

DELLA domain required for degradation

Leads to the derepression of genes involved in shoot elongation

Question 24

What occurs if the DELLA domain is deleted from the RH1 protein?

Answer 24

If DELLA domain is deleted via genetic modification, this leads to a stabilisation of the repressor as it can no longer undergo degradation.

Therefore the repressor is always active meaning that there is no transcription of genes linked to shoot elongation

Question 25

What is the phenotype of the RHT1 knockout?

Answer 25

When the repressor is knocked out no repression of shoot elongation gene transcription and results in a very tall plant

Question 26

In general, how can traits other than height be identified?

Answer 26

Through the identification of natural genetic variation

e.g. for seed shattering. Often multiple genes are involved in the control of these traits (QTL). Carry out mapping and identify the location on the chromosome and what genes are involved in the control of the phenotype. This can be narrowed down and candiate genes investigated.

Question 27

What did Konishi et al (2006) discover about reducing seed shattering?

Answer 27

A single nucleotide polymorphism in the promoter of qSH1 leads to a loss of seed shattering. Discovered by QTL mapping.

• qSH1 codes for a transcription factor that (amongst other functions) is needed in the abscission layer of the grain. [where the seed is attached to the plant and is invovled in the shattering of seeds]
• a single base change eliminates the expression of SH1 in the abscission layer so the seed stayed on the plant and is not lost
• contributes to improved overall yield

Shows how a small mutation can have a large beneficial effect

Question 28

What is SPIKE and what are the phenotypic changes that result from its introgression from a rice landrace to indica cultivar? (Fujita et al 2013)

Answer 28

SPIKE: increased spiklet number due to a 3 amino acid change

Phenotypic changes of SPIKE: significant increase in yield, increase in spiklet number per panicle, thicker leaves, increased root dry weight, a reduction in poor quality seeds, increase in overall yield (20% improvement)

Question 29

What technique did Fujita et al (2003) do to observe the effect of SPIKE in an elite rice cultivar?

Answer 29

1. Developed a breeding programme to introgress SPIKE from a wild relative of rice into an elite cultivar
2. Through a series of backcrosses had the elite cultivar expressed phenotypic changes of SPIKE
3. Used lab and field analysis to narrow down the gene involved through genetic analysis

Question 30

What are the common themes noted in modern plant improvement?

Answer 30

• Many of the domestication genes encode transcriptional regulators
• Mutations leading to improved crop traits are not normally knockouts but subtle changes
  
  • often the function of a gene is still required, e.g. still need TF activity but just want subtle changes, whereas a knockout would have too many side effets
• Subtle changes have large effects

Question 31

Give an example of plants that have been engineered for increased insect resistancfe

Answer 31

The European corn borer

Question 32

why is the European corn borer problematic?

Answer 32

ECB multiples in the shoots and eats the shoot material leading to plant toppling. Mould grows in these cavities which is toxic, and the plant cannot be used in the harvest, leading to yield loss

Question 33

What organic farming technique is used to deal with the european corn borer?

Answer 33

Bt produce a toxin which is deposited in a crystalline form in the bacteria, when the spores are sprayed on plants, the toxin is taken up by insects and this is bound by receptors in the gut of the insects where some parts of the toxin are integrated in to the intesticular membrane causing it to be leaky and ending in death.

Question 34

Why is it important that many dfferent Bt toxin (Cry) genes have been isolated?

Answer 34

Different forms have different actions against other bacteria or could be used in combination for better results or an avoidance of growing resistance

Question 35

What strategies are used to prevent insects from becoming resistant to the Bt toxin?

Answer 35

• Use of refuges to allow any resistance alleles to remain heterologous
  
  • These are areas of untransgenic plants. Insects may gain a mutation in one chromosome which allows resistnace however need it to be homozygous for the allele to become fixed in the population. Refuges are resevoirs of susceptibility, meaning that it will take a very long time for population to become fully homozygous as there is an area without selection pressure.
• Using plants expressing different Bt toxins at a time.
  
  • If different mechanisms are operating the insect would need to have multiple resistance mechanisms
• Supression of resistance by releasing sterile insects
  
  • mutants cannot pass on resistant allele

Question 36

Why are multiple strategies required to prevent insect resistence to transgenic Bt plants?

Answer 36

Becuase experience an ‘arms race’ with the ECB adapting to the control methods, multiple methods make this adaptation far slower and harder to acheive

Question 37

Who demonstrated the benefit of multiple expression of Bt toxins on growing insect resistance?

Answer 37

Zhao (2003) showed that the # of larvae and pupar per Bt plant resumed to normal levels after time when only one was used, however when two were used in combination the numbers did not appear to recover, the treatment was more stable and sdifficult for resistance to develop

Question 38

Aside from using multiple Cry genes, what else could be used?

Answer 38

Cry genes could be used in combination with other toxin genes with different targets, meaning it would be very difficult for insects to overcome the pressure.

Yes there is a high selection pressure each particular method consequently has its own selection pressure reduced.

Question 39

How was the eradication of pink bollworm in Arizona acheived and why is it a success story?

Answer 39

• Many years had a problem getting rid of PB using common insecticides.
• Using a combination og Bt cotton with the release of sterile insects
• Results in an eradication of PB and a dramatic decline of # of insecticide sprays

Question 40

What two herbicides have been used in engineering herbicide resistance?

Answer 40

Glufosinate, phosphinothricin

And

Glyphosate

Question 41

What pathway is the herbicide glufosinate/phosphinothricin involved in? And how was herbicide resistance introduced?

Answer 41

Involved in the repression of the activity of glutamine synthase which is necessary for the production of glutamine. Results in the accumulation of ammonia which is toxic. To introduce herbicide resistance, bar gene was taken from streptomyces hygroscopicus transcribing an acetly transferase. The acelyated version of the herbicide is inactivated.

Question 42

What are the benefits of indroducing herbicide resitance into crop plants?

Answer 42

• less herbicide treatments
• reduces costs and energy inputs

Question 43

How does the herbicide glyfosate act on susceptible plants?

Answer 43

Acts in the pathway of chorismate synthesis, which is neccessary for the synthesis of the amino acids Typtophan, Tyrosine and phenylalanine.

Acts to inhibit the EPSP synthase meaning that PEP + Shkimate-5-P is not converted to EPSP (3-Enolpyrovylshikimate-5-phosphate).

Lack of amino acid synthesis results in plant growth inhibition

Question 44

How have glyphosate tolerent plants been developed?

Answer 44

Mutant aroA gene identified from S.typhimurium resistance EPSP synthase. Change in the structure means that the enzyme is no longer inhibited by the herbicide and the biosynthetic pathway remains intact.

Question 45

What is the trend for GM crop use worldwide?

Answer 45

Between 1994 and 2014 there has been over an 100 fold increase in the global area of biotech crops (million hectares). 28 countries worldwide using biotech crops. Many not in europe however.

The total #hectares that biotech crops are grown on is an area more than 7* the UK.

4 Plants make up the bulk of transgenic plants, Soybean, Maize, Cotton, Canola. 82% of soybean worldwide is biotech.

GM traits are mostly for herbicide resistance, but # of plants with stacked traits (both insect and herbicide tolerence) have been increasing.