Dr. Ken Korth Department of Plant Pathology University of Arkansas, Fayetteville

1. Dr. Ken Korth Department of Plant Pathology University of Arkansas, Fayetteville biotechnology - Using an organism to make a product, …or using advanced methods to study an organism GMO - Genetically Modified Organism Transgenic - describing material where a gene from one organism has been transferred into another

2. Two applications of biotechnology for • crop plants… • Using techniques to identify desired plants, for example through Marker-Assisted Selection. • -- to speed up the selection process in breeding programs • -- to identify and track genes that might have minor effects (quantitative trait loci, QTL) • 2. Through the introduction of novel genes in crop plants (GMOs). • -- as a research tool to identify gene function • -- to develop novel plants for release

3. It starts with a gene... What is a gene? What is it made of?

4. Double helical structure of DNA G pairs with C A pairs with T Each cell in a rice plant has about 400 million base pairs of DNA.

5. DNA provides the “code” that determines which proteins are made DNA Nucleus Transcription RNA Enzymes, structural proteins, signaling factors, hormones, energy storage, metabolism Processed RNA Protein RNA Translation

6. Marker-Assisted Selection 1. A first step is to identify DNA sequences associated with some trait – these can originate is any variety of interest. (Image from Goff and Salmeron, Sci Amer. 2004)

7. Marker-Assisted Selection 2. After crossing with an elite cultivar, progeny that carry the desired marker can be identified – at a very young age. (Image from Goff and Salmeron, Sci Amer. 2004)

8. Marker-Assisted Selection 3. Genes or sequences can be tracked through DNA fingerprinting techniques. (Image from Goff and Salmeron, Sci Amer. 2004)

9. Marker-Assisted Selection 4. Genes or sequences of interest can be combined into new cultivars using traditional crossing techniques. (Image from Goff and Salmeron, Sci Amer. 2004)

10. An example of the utility of molecular applications: Five genes that respond to blast infection were first identified through basic molecular studies… Amount of disease # of defense response genes in a plant line from Liu, et al., MPMI (2004), 17:1146

11. An example of the utility of molecular applications: Each gene by itself has a minor effect, but when combined lead to high levels of resistance – presence of the genes is determined via molecular analysis Amount of disease # of defense response genes in a plant line from Liu, et al., MPMI (2004), 17:1146

12. Some potential uses for transgenic plants……. • enhanced insect and herbicide resistance • a research tool to identify gene function • enhanced pathogen resistance • foods with enhanced nutritional value • enhanced salt or drought tolerance • “farmaceuticals” production

14. Agrobacterium is a plant pathogen—it can transfer DNA into plant cells A crown gall tumor on tomato. The stem was wounded with a needle carrying Agrobacterium tumefaciens--this photo was taken one month after inoculation

15. A whole plant can be grown from a single cell… Callus Stages of plant regeneration in wheat

16. Selection for transformed cells is generally used. Callus tissue (undifferentiated cells) is placed on selective media--only cells with the introduced DNA should survive Closeup of a clump of callus on selective media--sectors ofsurviving cellsare growing and look healthy--dead anddying cellslook brown.

17. Callus can be regenerated to form a whole plant (photos of rice tissues from Yinong Yang) By changing levels of plant hormones in the media, callus is induced to form plant shoots, then shoots are transferred to new media and induced to form roots.

18. Agrobacterium plant transformation… Pick single colony and grow cells Introduce DNA of interest into bacteria Inoculate plant tissue and regenerate whole plant on selective media

19. GOLDEN RICE Vitamin A (trans-retinol) deficiency: In Southeast Asia, approximately 5 million children develop xeropthalmia (impaired vision) each year. 0.25 mil eventually go blind correlated with diarrhea, respiratory diseases, measles According to UNICEF, Vitamin A supplements could prevent 1- to 2-million childhood deaths per year

20. The biochemistry of Golden Rice… The protein in this step adds the double bonds The protein that performs this step modifies the ends…

21. A recent improvement has led to Golden Rice that produces levels of b-carotene over 20X higher than the original Golden Rice Normal rice Original Improved (Paine, et al., (2005) Nature Biotech., 4:482)

22. New techniques in plant biology are leading to… • Improved tools for plant breeders and • biologists • The ability for wide transfer of genes • Complex issues

23. (Nature Biotechnology, March 2005)

25. Current Biotech/Genomics applications in rice • Although transgenic (“GMO”) rice is being grown in other parts of the world (China, Iran, ?), it is not currently being commercially grown in the U.S. • The complete sequence of the rice genome is known. This information can make it easier to identify and characterize specific genes of interest, not only in rice but also in related species (e.g., corn, wheat, barley, etc…)