Lecture 10 -Transgenic plants and animals and how to make them

Question 1

What is genetic modification?>

Answer 1

the selective and purposeful alteration of genes by humans including methods of incorperating new genes or traits into anorganism through genetic engineering

Question 2

What is genetic engineering?

Answer 2

introduction of ‘foreign’ DNA by transformation

Question 3

Outline the traditional breeding strategy

Answer 3

1) Cross an elite line with another line of an organism showing the desired trait
2) Resulting F1 hybrid contains organism heterozygous for the desired trait
3) Identify phenotypic expression of desired trait in F2
4) Backcross F2 desired trait to elite line

Question 4

Outline transgenic strategy

Answer 4

1) use gene transfer methods to incorperate a desired trait from foreign agents
2) results in only desired trait being incorperated

Question 5

What factors significantly contribute to the yield gap in crop improvement?

Answer 5

Abiotic - drought, cold, heavy metals, salt
Biotic - plant pathogens, insects, weeds
Political - conflict, infgrastructure diseases education

Question 6

What are the aims of crop improvement?

Answer 6

• increase productivity
• increase nutritional benefit
• use ‘marginal’ land
• avoid post harvest stresses
• reduce chemical lost
• increase economic gain
• increase food security
• avoid environmental harm

Question 7

What are the requirements of making a transgenic plant?

Answer 7

• DNA construct to express gene of interest
• method of DNA delivery into plant tissue e.g. agrobacterium mediated, particle bombardment
• method to regenerate whole plants carrying gene of interest

Question 8

What must the promotor in front of a gene of interest contain?

Answer 8

regulatory elements to allow transcription in plants

• consituative expression
• tissue specific
• inducible

Question 9

What must the gene of interest contain for creation transgenic plant?

Answer 9

-an open reading frame (codons may have to be optimised for expression in plants)

Question 10

What must the terminator contain for creation of a transgenic plant?

Answer 10

transcription termination

poluadenylation

Question 11

How can gene expression be supressed in plants?

Answer 11

using RNA interference (RNAi)

Question 12

What are the features of RNA interference?

Answer 12

• target squence must be inverted repeat
• transcribed RNA is self complimentary and can form dsDNA
• dsRNA is trigger of the RNA interference pathway
• RNA interference can be used for targeted supression of gene expression

Question 13

What is the mechanism of RNA interference?

Answer 13

1) dsRNA or shRNA cut by a dicer to expose P groups on 3’ ends of DNA forming a siRNA duplex
2) Argo enzyme leads to single stranded RNA and formation of the RISC complex
3) RISC binds to mRNA forming a siRNA/mRNA complex which splices mRNA, silencing it

Question 14

What is Agrobacterium tumefaciens?

Answer 14

• bacteria induces crown gall disease in plants

- bacteria live within the gall, which is a tumour like proliferating mass of undifferentiated plant cells

Question 15

What do plant cells produce that are used by Agrobacterium tumefaciens and how?>

Answer 15

1) opines used by A.bacterium as carbon and nitrogen source

2) Hormones to enable cell proliferation

Question 16

What is the Ti plasmid?

Answer 16

A tumour inducing plasmid found in A.bacterium

Question 17

What is T-DNA?

Answer 17

Segment of the Ti plasmid integrated into host plants genome

Question 18

What are some features of the Ti plasmid?

Answer 18

• 200kb long
• contains T-DNA (tumour production and Nopaline synthesis) vir genes, nopaline utilisation and an ORI for replication in agrobacterium

Question 19

What do wounded plants release and what does this lead to?

Answer 19

release phenolic compounds e.g. acetosyringone

• attract Agrobacterium to wound site
• involved in the induction of Ti vir genes

Question 20

What are vir genes responsible for?

Answer 20

act upon T-DNA to transfer of genomic DNA within cell nucleus

Question 21

What is the structure of the TDNA?

Answer 21

• has left boarder and right boarder with genomic DNA between (only right boarder necessary)
• DNA contains a selectable marker e.g. for antibiotic or herbicide resistance
• Gene of interest

Question 22

What is the purpose of the selectable marker?

Answer 22

allow for selection of plant cells that have recieved TDNA

Question 23

What is needed for binary vector system for Agrobacterium mediated plant transformation?

Answer 23

Need:
Disarmed Ti plasmid - contains vir genes A.tumefaciens ORI, TDNA deleted
Binary vector - ORI for E.coli and A.tumefacien, within TDNA left boarder and right boarder repeats have selectable marker gene and site for introducing gene of interest

Question 24

What is binary vector system for Agrobacterium mediated plant transformation?

Answer 24

• binary vector engineered in E.coli and transferred to strain of A tumefaciens harboring a helper Ti plasmid
• vir genes act in trans and TDNA is transferred to plant cell where integrates into plant genome
• cells are selected for T-DNA uptake in planta as they will express the selectable marker

Question 25

What is the process of regeneration of transformed plants using A bacterium?

Answer 25

1) remove discs from tobacco leaf
2) leaf disks are incubated with genetically engineered Agrobacterium for 24 hours
3) selection medium only allows plants that have acquired DNA from bacteria to proliferate
4) disks grown on a shoot inducing medium
5) Shoot transferred to a root inducing medium
6) Grow rooted seedling leading to an adult plant that carrys transgene originally present in bacteria

Question 26

What is the process of regeneration using tranformation by particle bombardment?

Answer 26

1) delivery of gold or tungsten microparticle projectiles carrying DNA into cells (DNA has been precipitated onto microparticles)
2) load projectile microparticles into particle gun that contains a firing pin, blank charge, plastic bullet (macroprojectile) microparticles
3) microparticles are accelerated towards plant tissue by explosive force and DNA delivered to plant nucleus through gaps in cell wall