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