biotechnology - Using an organism to make a product,

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Dr. Ken Korth Department of Plant Pathology
University of Arkansas, Fayetteville
biotechnology - Using an organism to make a
product, or using advanced methods to study an
organism GMO - Genetically Modified
Organism Transgenic - describing material where
a gene from one organism has been transferred
into another
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• Two applications of biotechnology for
• crop plants
• Using techniques to identify desired plants, for
  example through Marker-Assisted Selection.
• -- to speed up the selection process in breeding
  programs
• -- to identify and track genes that might have
  minor effects (quantitative trait loci, QTL)

2. Through the introduction of novel genes in
crop plants (GMOs). -- as a research tool to
identify gene function -- to develop novel
plants for release
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It starts with a gene...
What is a gene?
What is it made of?
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Double helical structure of DNA G pairs with C A
pairs with T
Each cell in a rice plant has about 400 million
base pairs of DNA.
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DNA provides the code that determines which
proteins are made
DNA
Nucleus
Transcription
RNA
Enzymes, structural proteins, signaling factors,
hormones, energy storage, metabolism
Processed RNA
Protein
RNA
Translation
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Marker-Assisted Selection
1. A first step is to identify DNA sequences
associated with some trait these can originate
is any variety of interest.
(Image from Goff and Salmeron, Sci Amer. 2004)
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Marker-Assisted Selection
2. After crossing with an elite cultivar,
progeny that carry the desired marker can be
identified at a very young age.
(Image from Goff and Salmeron, Sci Amer. 2004)
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Marker-Assisted Selection
3. Genes or sequences can be tracked through DNA
fingerprinting techniques.
(Image from Goff and Salmeron, Sci Amer. 2004)
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Marker-Assisted Selection
4. Genes or sequences of interest can be
combined into new cultivars using traditional
crossing techniques.
(Image from Goff and Salmeron, Sci Amer. 2004)
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An example of the utility of molecular
applications
Five genes that respond to blast infection were
first identified through basic molecular studies
Amount of disease
of defense response genes in a plant line
from Liu, et al., MPMI (2004), 171146
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An example of the utility of molecular
applications
Each gene by itself has a minor effect, but when
combined lead to high levels of resistance
presence of the genes is determined via molecular
analysis
Amount of disease
of defense response genes in a plant line
from Liu, et al., MPMI (2004), 171146
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Some potential uses for transgenic plants.

• enhanced insect and herbicide resistance
• a research tool to identify gene function
• enhanced pathogen resistance
• foods with enhanced nutritional value
• enhanced salt or drought tolerance
• farmaceuticals production

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Agrobacterium is a plant pathogenit can transfer
DNA into plant cells
A crown gall tumor on tomato. The stem was
wounded with a needle carrying Agrobacterium
tumefaciens--this photo was taken one month after
inoculation
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A whole plant can be grown from a single cell
Callus
Stages of plant regeneration in wheat
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Selection for transformed cells is generally
used.
Callus tissue (undifferentiated cells) is placed
on selective media--only cells with the
introduced DNA should survive
Closeup of a clump of callus on selective
media--sectors of surviving cells are growing
and look healthy--dead and dying cells look brown.
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Callus can be regenerated to form a whole plant
(photos of rice tissues from Yinong Yang)
By changing levels of plant hormones in the
media, callus is induced to form plant shoots,
then shoots are transferred to new media and
induced to form roots.
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Agrobacterium plant transformation
Pick single colony and grow cells
Introduce DNA of interest into bacteria
Inoculate plant tissue and regenerate whole plant
on selective media
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GOLDEN RICE
Vitamin A (trans-retinol) deficiency In
Southeast Asia, approximately 5 million children
develop xeropthalmia (impaired vision) each
year. 0.25 mil eventually go blind correlated
with diarrhea, respiratory diseases,
measles According to UNICEF, Vitamin A
supplements could prevent 1- to 2-million
childhood deaths per year
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The biochemistry of Golden Rice
The protein in this step adds the double bonds
The protein that performs this step modifies the
ends
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A recent improvement has led to Golden Rice that
produces levels of b-carotene over 20X higher
than the original
Golden Rice
Normal rice Original Improved
(Paine, et al., (2005) Nature Biotech., 4482)
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• New techniques in plant biology are leading to
• Improved tools for plant breeders and
• biologists
• The ability for wide transfer of genes
• Complex issues

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(Nature Biotechnology, March 2005)
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• Current Biotech/Genomics applications in rice
• Although transgenic (GMO) rice is being grown
  in other parts of the world (China, Iran, ?), it
  is not currently being commercially grown in the
  U.S.
• The complete sequence of the rice genome is
  known. This information can make it easier to
  identify and characterize specific genes of
  interest, not only in rice but also in related
  species (e.g., corn, wheat, barley, etc)