Altering Plants to Altering Plants to Increase Nutritional Increase Nutritional
ValueValue
Ann E. BlechlUSDA Agricultural Research Service
Albany, CA
Ways to Alter Plant Ways to Alter Plant CompositionComposition
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)
Advantages of Genetic Advantages of Genetic Engineering Compared to Engineering Compared to
Traditional BreedingTraditional 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
Disadvantages of Genetic Disadvantages of Genetic EngineeringEngineering
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
Some Targets for Increased Some Targets for Increased Nutritional Value 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
From Rosati et al., 2000
Changing Carotenoid Changing Carotenoid ContentsContents 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
Fig. 2. Phenotypic analysis of high -carotene transgenic and controlRed Setter tomato plants. Transgenic (right) and Red Setter (left).All parts of the transgenic fruits (columella, pericarp and placenta) are intensely orange coloured.
From D’Ambrosio et al., 2004
High- High- CarotenCaroten
e e TomatoTomato
eses
Engineering Vitamin A Engineering Vitamin A biosynthesis in rice seedsbiosynthesis in rice seeds Cereal plants have carotenoids in their green tissues,
but very little in their seeds In developing countries, about 250 million people
don’t 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
From Hoa et al., 2003
““Golden Golden Rice”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
““High-Selenium Beef, High-Selenium Beef, WheatWheat and Broccoli: a and Broccoli: a
Marketable Asset?”Marketable Asset?”
USDA IFAFS grant
One goal: Engineer wheat to accumulate increased levels of selenium in flour
Adapted from LeDuc et al., 2004
MetabolisMetabolism of m of
Selenate Selenate and and
Selenite in Selenite in Most Plant Most Plant
CellsCells Generally, plants
accumulate Se in proportion to its concentration in soil
10 - 100 g per gram dry weight
Glutathione
whe
at
From Pickering et al, 2003
Astragulus Astragulus bisulcatus bisulcatus (locoweed)(locoweed)
can can accumulate accumulate
as much as 2 as much as 2 mg seleniummg selenium
per gramper gram
Adapted from LeDuc et al., 2004
Metabolism Metabolism of Selenate of Selenate and Selenite and Selenite
in Plant in Plant Hyper-Hyper-
accumulatoraccumulatorss
Glutathione
From Neuhier et al, 1999
Sequence of Sequence of the Astragalus the Astragalus gene encoding gene encoding selenocysteinselenocystein
e e methyltransfermethyltransfer
ase (SMT)ase (SMT)
Experimental PlanExperimental 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?
The SMT Coding Region Was Inserted The SMT Coding Region Was Inserted Between the Promoter and Between the Promoter and
Transcription Terminator Regions of Transcription Terminator Regions of Wheat Glutenin Genes Wheat Glutenin Genes
Wheat Glutenin Promoter *
2945 bp 2017 bp
Wheat Glutenin Transcript Terminator
Astragalus SMT Coding
Region
1013 bp
*Endosperm-Specific Expression
Biolistics (the “Gene Gun”) was Biolistics (the “Gene Gun”) was used to introduce two DNAs into used to introduce two DNAs into
wheat embryoswheat embryos1. Glutenin:SMT gene
+2. Herbicide (Bialaphos)
resistance gene
Tissue Culture Tissue Culture Steps for Wheat Steps for Wheat TransformationTransformation
Shoots and Shoots and Roots are Roots are
Regenerated Regenerated Under Herbicide Under Herbicide
SelectionSelection
Inheritance of Inheritance of Glutenin:SMT TransgeneGlutenin:SMT Transgene
M + - 1 2 3 4 5 6 7 8M + - 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 M1 2 3 4 5 6 7 8 M
656 bp
Transgene Messenger RNA Transgene Messenger RNA LevelsLevels
ActinActin SMTSMT ActinActinSMTSMT5 20 405 20 40 5 20 40 5 20 40
M M
low expresser high expresser
Results Results II 30 independent transgenic wheats
containing the Glutenin:SMT 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
Results Results IIII 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
Results from LeDuc et al., 2003Results 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
Adapted from LeDuc et al., 2004
Proposed Fates Proposed Fates for Selenate for Selenate
and Selenite in and Selenite in Mustard Plants Mustard Plants
Expressing Expressing Astragalus SMTAstragalus SMT
Limiting in mustards
Enzyme?
Glutathione
What’s next for What’s next for us?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?
AcknowledgementsAcknowledgements
Chika Udoh
Jeanie Lin
Acknowledgement of Acknowledgement of SupportSupport USDA IFAFS grant
“High-Selenium Beef, Wheat and Broccoli:
a Marketable Asset?”
Agricultural Research Service
DoughDough Visco- Visco-
ElasticityElasticity
The biotechnology The biotechnology approach: use genetic approach: use genetic transformation to add transformation to add HMW-glutenin genesHMW-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.
Increases in native HMW-Increases in native HMW-glutenin subunits increases glutenin subunits increases
dough strength dough strength
Control (C)
Transgenic (T) Dx5
Dy10
T C
minutes10 30200
1.3x
1.9x
Dx5 1.5 2.7Dy10 2.2 1.7
11.4% 11.7%Protein Content
Mixing and Baking Results Mixing and Baking Results from Field-Grown from Field-Grown
Transgenic WheatsTransgenic Wheats