Discovery of auxin: Went 1928

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Discovery of auxin Went (1928)
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Chemical structure of auxin
                             
Auxins                                   
Indole-3-acetic acid (IAA)
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Properties of auxin

• Mainly produced in actively growing regions,
  particularly shoot
• (or coleoptile) tips

• Transport polar, down the plant

• Unstable photo-oxidised, or oxidised by
  IAA-oxidase

• Two fractions Diffusable (undergoing
  transport) and bound, functioning through
  interaction with a receptor

• Effective at very low concentrations

• Synthetic compounds have auxin activity

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Main functions of auxin

• Cell expansion

• Cell differentiation

• Bud dormancy (apical dominance)

• Leaf and fruit fall

• Root initiation and development

• Flower development

• Fruit development

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Discovery of cytokinins Skoog (1950s)
Induction of continuous cell division in tobacco
callus
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Effect of auxincytokinin ratio on development of
tobacco callus (Skoog and Miller 1957)
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Properties of cytokinins

• Mainly produced in root tips, but also developing
  seeds

• Transport in the xylem, from the roots. Therefore
  primarily up the plant.

• Cytokinins are mostly isoprenoid derivatives of
  purine bases

• As well as free base they also exist in
  conjugated forms, eg. ribosides or ribotides

• Synthetic alternatives to the most abundant
  naturally occurring cytokinins (zeatin and
  isopentenyl adenine) include kinetin and
  benzylaminopurine

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Main functions of cytokinin

• Cell division

• Morphogenesis (bud initiation)

• Lateral bud growth

• Leaf growth

• Delaying leaf senescence

• Chloroplast development (conversion of etioplasts
  to chloroplasts)

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Potatoes
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Sterile potato shoot cultures maintained by
axillary shoot culture
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Potato shoot cultures maintained under short days
develop microtubers
Microtubers
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Microtubers packaged for delivery
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Genetic Transformation
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Crown gall disease caused by Agrobacterium
tumefaciens
Infection by the bacterium causes cell
proliferation to form a tumour. The tumour can be
cut off and grown as a callus in tissue culture,
without auxin or cytokinin
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Infection of plant by A. tumefaciens
Infection depends on a large tumour inducing (Ti)
plasmid, part of which (T-DNA) is transferred to
the plant cell
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T-DNA transfer into infected plant cell
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Ti Plasmid
X
X
X
X
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Discs cut from sterile leaves
Transformation using disarmed Ti plasmid
of A. tumefaciens
Discs floated on Agrobacterium suspension
Discs placed on medium containing cytokinin.
Shoots regenerate from transformed cells
Shoots rooted on medium without cytokinin
Transformed plant in the soil
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Requirements for transformation
1. Selectable marker
nptII encodes enzyme neomycin phosphotransferase
which confers resistance to kanamycin
hpt encodes enzyme hygromycin phosphotransferase
which confers resistance to hygromycin
aadA resistance to spectinomycin
Herbicide resistance genes
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Requirements for transformation
2. Suitable expression signals
a) Constitutive promoters
Cauliflower Mosaic virus (CaMV) 35S
promoter Agrobacterium tumefaciens noplaline
synthase (nos) promoter
b) Tissue specific promoters Eg. leaf specific
chlorophyll a/b binding protein, Rubisco small
subunit
c) Inducible promoters Eg. Heat shock promoters
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Binary vectors, contain two plasmids
ii. Vector with left and right borders,
selectable marker gene, multiple cloning site. In
lacZ gene, plus additional antibiotic resistance
genes. Can be manipulated in E. coli
i. Ti plasmid without T-DNA. Vir genes ensure
virulence
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Use of gene gun for biolistic delivery of DNA
to plant cells
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Comparison of Agrobacterium and biolistic DNA
delivery
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Genetic Transformation as a basic research tool
Case history The role of cytokinins in delaying
senescence

• Cytokinins synthesised in the roots are believed
  to delay the senescence of leaves

• Genes for cytokinin synthesis are available.
• Eg. The ipt gene from the T-DNA of Agrobacterium
  tumefaciens. This is one of the oncogenes. It
  encodes the enzyme isopentenyl transferase which
  makes the cytokinin Isopentenyl adenine

• Therefore will this gene introduced into plants
  keep the leaves green?

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The problem
Introducing ipt under control of a constitutive
promoter, produces to much cytokinin, so a very
abnormal plant (small and bushy, with tiny leaves
results.
The solution
Use the promoter of a gene associated with
senescence
These are well known in Arabidopsis thaliana. Eg.
the gene SAG12 (encoding a protease) is only
turned on at the onset of senescence
Therefore can transform plant with chimeric
construct pSAG12-ipt
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What happens (shown in tobacco by Gan and
Amasino, 1995)

• Young leaves are healthy because of cytokinin
  from the roots

• Older leaves start to senesce due to reduced
  supply of cytokinin

• Senescence activates pSAG12 promoter, and
  therefore ipt gene

• This makes enough cytokinin to reverse senescence

• Senescence reversed pSAG12 no longer active.
  Therefore ipt no longer transcribed. No build up
  of cytokinin

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Delayed senescence in cauliflower leaves
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GM crops

• Flavr-Savr tomato
• First GM crop to get FDA approval in US.
  Anti-sense for polygalacturonase gene, which
  causes softening of fruit.
• Result
• Fruit has longer shelf-life and better solids
  content

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2. Insect resistance

• cry genes encoding crystal proteins, toxic for
  specific insect groups.
• Engineered into several crops
• Cotton resistant to bollworm
• Maize resistant to corn borer

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Insecticidal protein in tobacco leaves
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3. Herbicide resistance
Resistance to the herbicide glyphosate. Engineere
d into soybean, maize, sugarbeet, among
others Roundup Ready varieties
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Other areas under development

• Vitamin A producing rice (golden rice)

• Virus and blight resistant potato

• Improved oil quality (eg oilssed rape)

• Improved wood quality in forestry species
  (modified lignin

• Vaccine and antibody production