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

2. Ways to Alter Plant Composition l Change how and/or where they are grown –Agronomics l Change genes –Traditional Breeding »Introduce new variability by crosses or induced mutations –Genetic Engineering »Introduce genes artificially (genetic transformation)

3. Advantages of Genetic Engineering Compared to Traditional Breeding Advantages of Genetic Engineering Compared to Traditional Breeding l Breeding –Genes from limited # of sources »sexually compatible relatives –Crosses change half the gene composition (genome) »Backcrosses to Adapted Varieties Needed l Genetic Engineering –Genes from any source »Natural genes modified for specific purposes »Chemically synthesized –Add one or a few known genes at a time

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

6. From Rosati et al., 2000 Changing Carotenoid Contents l Lycopene is an anti- oxidant l  - and  -carotenes are precursors of vitamin A l Tomato lycopene levels have been raised 2-3 fold u  -carotene synthesis has been engineered in tomatoes and rice

7. 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 High- Carotene Tomatoes

9. Engineering Vitamin A biosynthesis in rice seeds Engineering Vitamin A biosynthesis in rice seeds lCereal plants have carotenoids in their green tissues, but very little in their seeds lIn developing countries, about 250 million people don’t get enough Vitamin A in their diets lThis deficiency results in retarded growth and increased incidence of –Blindness –Infant and childhood mortality lThe Rockefeller Foundation funded a Swiss and a German group in a collaborative project to increase the  -carotene (pro-vitamin A) content of rice grains

10. From Hoa et al., 2003 “Golden Rice” lPeter Beyer and Ingo Potrykus groups added 2 genes in pathway to provitamin A –Daffodil phytoene synthase –Bacteria phytoene desaturase –Added seed-specific promoters l0.8-1.2  g per gram lAt typical rice consumptions levels in Asia, golden rice would supply about 1/3 RDA of  -carotene

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

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

13. From Pickering et al, 2003 Astragulus bisulcatus (locoweed) can accumulate as much as 2 mg selenium per gram

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

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

16. Experimental Plan lModify Astragalus SMT gene for expression in wheat seeds lTransform wheat with modified SMT gene lVerify transgene inheritance lMeasure amounts of SMT RNA and enzyme activity lMeasure accumulation of Se in seeds from transgenic wheat plants grown in selenate and selenite –How much Se? –In what chemical form?

17. The SMT Coding Region Was Inserted Between the Promoter and Transcription Terminator Regions of Wheat Glutenin Genes The SMT Coding Region Was Inserted Between the Promoter and Transcription Terminator Regions of Wheat Glutenin Genes Wheat Glutenin Promoter * 2945 bp2017 bp Wheat Glutenin Transcript Terminator Astragalus SMT Coding Region 1013 bp * Endosperm-Specific Expression

18. Biolistics (the “Gene Gun”) was used to introduce two DNAs into wheat embryos 1.Glutenin:SMT gene + 2. Herbicide (Bialaphos) resistance gene

19. Tissue Culture Steps for Wheat Transformation

20. Shoots and Roots are Regenerated Under Herbicide Selection

21. Inheritance of Glutenin:SMT Transgene 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

22. Transgene Messenger RNA Levels Transgene Messenger RNA Levels ActinSMTActinSMT 5 20 40 MM low expresserhigh expresser

23. Results I l 30 independent transgenic wheats containing the Glutenin:SMT gene l Expression ranged from 4x to 1/8x the levels of actin l Homozygous seeds from 2 medium- and 2 high-expressers were sent to Michael Grusak –USDA-ARS Children's Nutrition Research Center, Houston, TX

24. Results II Results II l Mike Grusak grew the wheats hydroponically with selenate added from spike emergence to harvest –10, 20, 30 and 40  M l Mike observed no differences between the the four transgenic and control plants – Plant and seed development – Seed set

25. Results from LeDuc et al., 2003 l Same Astragulus SMT gene l Engineered to be expressed in fast-growing mustard plants for phytoremediation l Transformed Arabidopsis and Brassica juncea l 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

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

27. What’s next for us? What’s next for us? l Michael Grusak will regrow the transgenic wheats with selenite supplementation l John Finley will measure SMT activity, Se amounts and forms in wheat flour l Feed rats?

28. Acknowledgements l Chika Udoh l Jeanie Lin

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

31. Dough Visco- Elasticity

32. 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.

33. Increases in native HMW-glutenin subunits increases dough strength Control (C) Transgenic (T) Dx5 Dy10 T C minutes 1030200 1.3x 1.9x

34. Dx5 1.5 2.7 Dy10 2.2 1.7 11.4% 11.7% Protein Content Mixing and Baking Results from Field-Grown Transgenic Wheats