Altering Plants to Increase Nutritional Value

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Altering Plants to Increase Nutritional Value

• Ann E. Blechl
• USDA Agricultural Research Service
• Albany, CA

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Ways to Alter Plant Composition

• Change how and/or where they are grown
• Agronomics
• Change genes
• Traditional Breeding
• Introduce new variability by crosses or induced
  mutations
• Genetic Engineering
• Introduce genes artificially (genetic
  transformation)

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Advantages of Genetic Engineering Compared to
Traditional Breeding

• Breeding
• Genes from limited of sources
• sexually compatible relatives
• Crosses change half the gene composition (genome)
• Backcrosses to Adapted Varieties Needed

• Genetic Engineering
• Genes from any source
• Natural genes modified for specific purposes
• Chemically synthesized
• Add one or a few known genes at a time

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Disadvantages of Genetic Engineering

• Unintended side effects of tissue culture or gene
  insertion
• Also an issue for induced mutations in
  traditional breeding
• Currently limited to varieties that regenerate
  from tissue culture
• Public Acceptance
• Costly to clear regulatory and intellectual
  property hurdles

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Some Targets for Increased Nutritional Value

• Increased essential amino acids to make seeds
  complete protein sources
• Increased lysine in cereal grains
• Increased methionine in beans
• Low-Phytate Grains
• Increased bio-available iron and zinc up to 50
• Decreased phosphate waste
• Changes in fatty acid composition of oil seeds to
  less saturated types
• Changes in soybean anti-oxidant composition
• Vitamin E, shift tocopherol profiles to mainly
  ?-form

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Changing Carotenoid Contents

• Lycopene is an anti-oxidant
• ?- and ?-carotenes are precursors of vitamin A
• Tomato lycopene levels have been raised 2-3 fold
• ?-carotene synthesis has been engineered in
  tomatoes and rice

From Rosati et al., 2000
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High- Carotene Tomatoes
Fig. 2. Phenotypic analysis of high -carotene
transgenic and control Red Setter tomato plants.
Transgenic (right) and Red Setter (left). All
parts of the transgenic fruits (columella,
pericarp and placenta) are intensely orange
coloured.
From DAmbrosio et al., 2004
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Engineering Vitamin A biosynthesis in rice seeds

• Cereal plants have carotenoids in their green
  tissues, but very little in their seeds
• In developing countries, about 250 million people
  dont get enough Vitamin A in their diets
• This deficiency results in retarded growth and
  increased incidence of
• Blindness
• Infant and childhood mortality
• The Rockefeller Foundation funded a Swiss and a
  German group in a collaborative project to
  increase the ?-carotene (pro-vitamin A) content
  of rice grains

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Golden Rice

• Peter Beyer and Ingo Potrykus groups added 2
  genes in pathway to provitamin A
• Daffodil phytoene synthase
• Bacteria phytoene desaturase
• Added seed-specific promoters
• 0.8-1.2 ?g per gram
• At typical rice consumptions levels in Asia,
  golden rice would supply about 1/3 RDA of
  ?-carotene

From Hoa et al., 2003
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High-Selenium Beef, Wheat and Broccoli a
Marketable Asset?

• USDA IFAFS grant
• One goal Engineer wheat to accumulate increased
  levels of selenium in flour

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Metabolism of Selenate and Selenite in Most Plant
Cells
Glutathione
wheat

• Generally, plants accumulate Se in proportion to
  its concentration in soil
• 10 - 100 ?g per gram dry weight

Adapted from LeDuc et al., 2004
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Astragulus bisulcatus (locoweed) can accumulate
as much as 2 mg seleniumper gram
From Pickering et al, 2003
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Metabolism of Selenate and Selenite in Plant
Hyper-accumulators
Glutathione
Adapted from LeDuc et al., 2004
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Sequence of the Astragalus gene encoding
selenocysteine methyltransferase (SMT)
From Neuhier et al, 1999
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Experimental Plan

• Modify Astragalus SMT gene for expression in
  wheat seeds
• Transform wheat with modified SMT gene
• Verify transgene inheritance
• Measure amounts of SMT RNA and enzyme activity
• Measure accumulation of Se in seeds from
  transgenic wheat plants grown in selenate and
  selenite
• How much Se?
• In what chemical form?

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The SMT Coding Region Was Inserted Between the
Promoter and Transcription Terminator Regions of
Wheat Glutenin Genes
2945 bp
2017 bp
1013 bp
Wheat Glutenin Promoter
Wheat Glutenin Transcript Terminator
Astragalus SMT Coding Region
Endosperm-Specific Expression
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Biolistics (the Gene Gun) was used to introduce
two DNAs into wheat embryos

• GluteninSMT gene
• 2. Herbicide (Bialaphos) resistance gene

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Tissue Culture Steps for Wheat Transformation
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Shoots and Roots are Regenerated Under Herbicide
Selection
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Inheritance of GluteninSMT Transgene
M - 1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8 M
656 bp
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Transgene Messenger RNA Levels
Actin
SMT
Actin
SMT
M
M
5 20 40
5 20 40
5 20 40
5 20 40
low expresser
high expresser
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Results I

• 30 independent transgenic wheats containing the
  GluteninSMT gene
• Expression ranged from 4x to 1/8x the levels of
  actin
• Homozygous seeds from 2 medium- and 2
  high-expressers were sent to Michael Grusak
• USDA-ARS Children's Nutrition Research Center,
  Houston, TX

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Results II

• Mike Grusak grew the wheats hydroponically with
  selenate added from spike emergence to harvest
• 10, 20, 30 and 40 ?M
• Mike observed no differences between the the four
  transgenic and control plants
• Plant and seed development
• Seed set

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Results from LeDuc et al., 2003

• Same Astragulus SMT gene
• Engineered to be expressed in fast-growing
  mustard plants for phytoremediation
• Transformed Arabidopsis and Brassica juncea
• Transgenics
• Accumulated SMT enzyme
• Tolerated higher concentrations of selenate and
  selenite than their non-transformed parents
• Accumulated more Se (2-4x)
• Accumulated more MethylSelenoCysteine (1.5-10x)
• Produced up to 2.5x more volatile Se

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Proposed Fates for Selenate and Selenite in
Mustard Plants Expressing Astragalus SMT
Limiting in mustards
Glutathione
Enzyme?
Adapted from LeDuc et al., 2004
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Whats next for us?

• Michael Grusak will regrow the transgenic wheats
  with selenite supplementation
• John Finley will measure SMT activity, Se amounts
  and forms in wheat flour
• Feed rats?

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Acknowledgements

• Chika Udoh
• Jeanie Lin

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Acknowledgement of Support

• USDA IFAFS grant
• High-Selenium Beef, Wheat and Broccoli a
  Marketable Asset?
• Agricultural Research Service

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Dough Visco-Elasticity
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The biotechnology approach use genetic
transformation to add HMW-glutenin genes

• Dough strength depends on flour proteins.
• Especially important are the larger type of
  glutenin proteins, HMW-Glutenins.
• We have added glutenin genes to change the
  proportion of these proteins in wheat flour.
• Flours from these wheats have differing mixing
  and baking properties.

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Increases in native HMW-glutenin subunits
increases dough strength
T C
Dx5
1.9x
Transgenic (T)
Dy10
1.3x
Control (C)
10
30
20
0
minutes
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Mixing and Baking Results from Field-Grown
Transgenic Wheats
Protein Content
11.4
11.7
Dx5 1.5 2.7 Dy10
2.2 1.7