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Altering Plants to Increase Nutritional Value

Altering Plants to Increase Nutritional Value. Ann E. Blechl USDA Agricultural Research Service Albany, CA. 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

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Altering Plants to Increase Nutritional Value

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  1. Altering Plants to Increase Nutritional Value Ann E. Blechl USDA Agricultural Research Service Albany, CA

  2. 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)

  3. 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 Advantages of Genetic Engineering Compared to Traditional Breeding

  4. 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

  5. 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

  6. 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

  7. 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 D’Ambrosio et al., 2004

  8. 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 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

  9. “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

  10. “High-Selenium Beef, Wheat and Broccoli: a Marketable Asset?” • USDA IFAFS grant • One goal: Engineer wheat to accumulate increased levels of selenium in flour

  11. Metabolism of Selenate and Selenite in Most Plant Cells Glutathione • Generally, plants accumulate Se in proportion to its concentration in soil • 10 - 100 g per gram dry weight wheat Adapted from LeDuc et al., 2004

  12. Astragulus bisulcatus (locoweed) can accumulate as much as 2 mg seleniumper gram From Pickering et al, 2003

  13. Metabolism of Selenate and Selenite in Plant Hyper-accumulators Glutathione Adapted from LeDuc et al., 2004

  14. Sequence of the Astragalus gene encoding selenocysteine methyltransferase (SMT) From Neuhier et al, 1999

  15. 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?

  16. The SMT Coding Region Was Inserted Between the Promoter and Transcription Terminator Regions of Wheat Glutenin Genes 2945 bp 1013 bp 2017 bp Wheat Glutenin Promoter * Astragalus SMT Coding Region Wheat Glutenin Transcript Terminator *Endosperm-Specific Expression

  17. Biolistics (the “Gene Gun”) was used to introduce two DNAs into wheat embryos • Glutenin:SMT gene • + • 2. Herbicide (Bialaphos) resistance gene

  18. Tissue Culture Steps for Wheat Transformation

  19. Shoots and Roots are Regenerated Under Herbicide Selection

  20. Inheritance of Glutenin:SMT Transgene M + - 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 M 656 bp

  21. Transgene Messenger RNA Levels M SMT Actin SMT Actin M 5 20 40 5 20 40 5 20 40 5 20 40 low expresser high expresser

  22. Results I • 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

  23. 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

  24. 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

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

  26. What’s 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?

  27. Acknowledgements • Chika Udoh • Jeanie Lin

  28. Acknowledgement of Support • USDA IFAFS grant “High-Selenium Beef, Wheat and Broccoli: a Marketable Asset?” • Agricultural Research Service

  29. Dough Visco-Elasticity

  30. 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.

  31. Increases in native HMW-glutenin subunits increases dough strength T C Dx5 1.9x Transgenic (T) Dy10 1.3x Control (C) 0 10 20 30 minutes

  32. 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

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