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

Two applications of biotechnology for crop plants… 1.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

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

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

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)

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)

3. Genes or sequences can be tracked through DNA fingerprinting techniques. Marker-Assisted Selection

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

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

from Liu, et al., MPMI (2004), 17:1146 # of defense response genes in a plant line Amount of disease 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 An example of the utility of molecular applications:

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

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

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

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 of surviving cells are growing and look healthy--dead and dying cells look brown. Selection for transformed cells is generally used.

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. Callus can be regenerated to form a whole plant (photos of rice tissues from Yinong Yang)

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

Vitamin A (trans-retinol) deficiency: In Southeast Asia, approximately 5 million children develop xeropthalmia (impaired vision) each year 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

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

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

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

(Nature Biotechnology, March 2005)

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…)