Plant Bioengineering: plants as we want to see them....

Procedures

Current use

Future use

Concerns

How to genetically modify a plant

[Note the information below was taken from various sources including:

  • An excellent site: Biotechnology in the Food chain ( UK): How to genetically modify a plant : http://www.jic.bbsrc.ac.uk/exhibitions/bio-future/howto.htm -- from which most of this page was derived...I then added in materials from the following pages....
  • ENGINEERING GREEN MACHINES by Nevin Dale Young: http://www.plpa.agri.umn.edu/scag1500/handouts.html/greenmachines.html
  • Plant Genetics and Molecular Biology http://www.wam.umd.edu/~highway/WWW/lfsc/botn414/lecture_022795.html
  • http://www.csiro.au/enquiries/plbiotech.htm ]

I. Genetic modification of a plant using the techniques of biotechnology includes:

  • inserting isolated individual gene(s) into the genome of the plant, usually using plant tissue that has been prepared to take up the gene(s);
  • regenerating an intact plant from the genetically modified plant tissue;
  • make sure that the inserted genes work as expected;
  • if possible test that the gene inherited is in the offspring of the next generaton

Designer genes

Genes, either from the same or different species (transgenes), that are to be used to genetically modify a plant must be introduced into the plant linked to pieces of DNA that control how they work. Genes for proteins may need promotors for expression.

Controlling gene expression

Some promoters cause the genes to which they are linked to be expressed:

  • all the time,
  • expression only at certain stages of plant growth or
  • in certain plant tissues (e.g. roots or shoots), or
  • in response to external environmental signals (e.g. daylength).

Some promoters are weak and others strong, and this determines how much of the gene product is made.

There are obvious advantages in being able to control gene expression:

  • directing the plant's resources into growing or synthesizing desired molecules,
  • producing products for defense systems only when needed so resistance does not occur to readily .

An example is the culturing of plants that carry the BT insecticide gene from Bacillus thuringiensis. This soil bacterium produces a protein that disables the gut function of specific insects ; more than 200+ strains of the species exist prodcuing different forms of the BT specific to the insect they attack only. An altered form of the BT gene has been engineered and transferred into plants, making the transgenic plant resistant to the corresponding insect. Already resistance has ocuured in some insects and the widespread use of this engineering feat is of concern.In order to prevent this a promotor region that is turned on by chemical ( salicyic acid or ethylene) sprayed onto the plant when attacked is being developed up at Cornell. One of our students was part of this research effort last summer.

  • producing products in specific sites that are more harvestable.. etc.

Ripening genes are distinct from flavor genes, so plants that have delayed ripening can be stored and shipped over longer times without losing flavor, making them more attractive to consumers. The mechanism for turning off plant genes is antisense technology, in which the complementary version of the messenger RNA for a ripening gene is introduced into the plant cell. This complementary sequence interferes with the translation of the ripening gene product.

Marker genes

As part of the process of inserting transgenes into plants, 'marker' genes are usually linked to the transgene to facilitate its detection in plant tissue.

This enables only the plant tissue into which the transgene has been successfully introduced to be identified and regenerated into whole plants, thus saving considerable expense and effort.

Marker genes can confer a characteristic on the plant tissue that may be detected easily :

  • colour change in the laboratory which can be detected by a chemical test or viewed under specific conditons ( fluoresecence); The GUS reporter gene causes transgenic cells to turn blue in the presence of an indicator chemical while the Luciferase reporter gene, which comes from fireflies, causes transgenic cells to be luminescent when given the appropriate chemical. .
  • a marker gene can allow plant tissue into which it is inserted to survive experimental conditions (known as selection pressure) that would damage or kill tissue not containing the marker: for example, plant tissue containing a marker gene for antibiotic resistance would survive when antibiotics are added to the growing cells, while other plant tissue would die off. (There is some concern that this use of antibiotic-resistance genes will increase antibiotic resistance in humans and animals. Genes coding for resistance to non-medically important antibiotics are therefore preferred. In addition, alternative types of marker genes are also being developed).

II. How are foreign genes inserted into plants?

Gene transfer methods must get the transgene past the barriers of the cell wall, cell membrane and envelope that surrounds the nucleus, without affecting the cell's ability to survive.

Several methods are used to get genes into plant cells. If the introduced genes are functional, and the gene-product synthesized, the plant is said to be transformed.

a. Agrobacterium

 

Agrobacterium tumefaciens is a naturally occurring plant pathogenic bacteria. Agrobacterium contains a plasmid (the Ti plasmid) with the ability to enter plant cells and insert a portion of its genome into plant chromosomes. The Ti plasmid has been engineered to make it a vector for plant transformation by including sequences for replication in E coli and Agrobacterium, unique restriction sites for inserting foreign genes, and selectable markers.

+

1. Tissue from an herbicide susceptible plant is cultured to form undifferentiated mass of cells.....

2. These cells in cultture are now inoculated with agrobacterium carrying the altered Ti plasmid cells.....

3. Cells are dissociated and grown in liquid culture

 

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4. Herbicide is added to select cells that have incorporated the resistance DNA

5. The embryo grows into a plant which now contains the herbicide resistant gene

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This approach however did not work well with cereals, so alternative techniques were devised........

b. Protoplasts

Protoplasts are cells that have had their cell walls removed. This can be done mechanically, or by enzymic digestion. The 'naked' cells are surrounded only by a cell membrane and can be used in a variety of ways.

A mixture of carbohydrases can be used to degrade plant tissue, producing a protoplast suspension which can easily be seen under a microscope.

c. Gene-gun

The 'gene gun' or 'biolistics' method can be used with all plant species. This uses gold or tungsten microparticles, coated with transgene DNA, which are fired into the target tissue by an explosive discharge or pressurized helium. DNA that penetrates the nucleus of the plant cell may be incorporated among the plant's own genes. Unfortunately the gene may be expressed but often only temporallly... very often it is not truly incorporated into the genome and will not be expressed in the following generation.

Taken from CISCO educational page on bioengineering

Preparing samples of plant tissue for transformation using the 'gene gun'.

Finally, regeneration of whole plants

The unique ability of pieces of plant tissue and cells to regenerate into whole plants is used in most techniques of gene transfer. Plant cells or tissue into which genes have been introduced can be regenerated in the laboratory by the use of appropriate plant hormones, and careful culture, into whole plants. However, there is no universal method because tissues from different plants respond differently: culture and regeneration methods must be adapted depending on both plant and cell type.

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Rice embryos that have been successfully genetically modified using a 'gene gun'; the blue colour results from a laboratory test that identifies a 'marker' gene.

Tomato breeders find sweet spot ......HELEN PEARSON

Wild DNA is a sweet addition to new tomato breeds.

"If an experiment doesn't work you can always eat it," says Dani Zamir of his favourite fruit, the tomato. Unveiling a new super-sweet breed at the Human Genome Meeting in Edinburgh, he explained how his fieldwork could shed light on our own complex characteristics.

In search of a more ketchup-friendly fruit, Zamir and his colleagues from the Hebrew University of Jerusalem in Rehovot, Israel, crossed the familiar farmed red tomato with a small, green unappetising breed naturally found in the Peruvian Andes.

The group grew 100 strains, each containing a small chunk of 'wild' tomato DNA buried in the genome. On harvesting, one line proved particularly juicy, churning out more sugar than its fellow flora. "You can taste the difference," says Zamir.

By homing in on the 'wild' DNA, the group tracked down the juicy gene ; an 'invertase' enzyme that breaks down sugars.

"It's incredible," says Michel Georges at the University of Liege in Belgium, who maps sheep and cow genes in a similar attempt to raise farm productivity. Identifying the general area of a genome that affects a trait is hard enough ; zoning in on the gene itself can be fruitless. "No-one has ever been able to narrow it down to a few hundred base pairs before," says Georges.

Zamir claims "the sugar content of a tomato is very similar to human intelligence". 'Complex traits' like human IQ, weight and height vary from person to person because they are determined by many genes and their interactions with the environment. "We're trying to get an understanding of what these genes are," says Georges.

To help their search, geneticists should take tips from the tomato breeders, Zamir suggests. Those working on mice could source strains from Siberia or the Sahara desert. "Shouldn't we go to exotic species with natural variation and try and learn from them?" he asks.

"It's a fair point," Georges agrees. More genetic diversity already exists in nature than can be created by artificially knocking out or altering gene function. But the Israeli group analysed 14,000 plants to zone in on the sweetness gene. "It's harder to generate that many mice," Georges points out.

Make sure your read this.... relates to above topic and defense.

Nature's gems Text by Melissa Rake

Photography by Dan Dry

Scrawny trees grow out of the arid desert that sweeps across Sudan in northern Africa. Covered with thorns and cracked limbs, they look like unruly weeds compared to this continent's diverse offerings of exotic flora. But in the thick of the heat, the skinny acacia senegal oozes a sticky, iridescent substance that's proved useful to humans for thousands of years. Ancient Egyptians used this special "sap" to make flaxen wrappings for embalming mummies. Today, it's an indispensable ingredient in soft drinks, foods, and other products.

Ohio University biochemist Marcia Kieliszewski admires the biology behind this natural syrup, called gum arabic, that protects the acacia senegal when it's been injured.

"As with any of my research, I simply want to understand how plants grow," says Kieliszewski, an assistant professor of chemistry and biochemistry.

But Kieliszewski's project has transcended basic research. She wants to take the prized feature of this unattractive tree and transfer it into domestic crops, including tomato and tobacco plants, through genetic manipulation. "Gum arabic possesses certain properties coveted by the food and pharmaceutical industries," she says. "Why not try to impart these properties in other plants?" 

The United States imports several thousand tons of gum arabic from Africa each year. Because of its unique emulsification and thickening properties, the substance mostly is used as an additive in foods. Its applications are countless: preventing sugar crystallization in confections, replacing fat in foods, securing flavors, improving the texture of ice cream, adhering seasonings to snack foods, and stabilizing foam in beer. In fruit-based soft drinks, such as Mountain Dew, it keeps fruit oils from settling to the bottom.

Outside the food industry, gum arabic is used as a gelling agent in cosmetics, an adhesive for postage stamps, and is an ingredient in textiles, pharmaceuticals, inks, and paints. It's a valuable product, considering there is no substitute.

"So far, there's no known substitute for gum arabic. You can't produce it anywhere else in the world, and it's so important to our businesses," says Sean McBride, director of communications for the National Soft Drink Association.

And that worries American industries. Most of Africa's gum arabic is produced in Sudan, a country south of Egypt notorious for its civil struggles, political instability, and alleged support of terrorism. In 1997, President Bill Clinton issued an order prohibiting U.S. trade with Sudan. Although the U.S. government is allowing the temporary importation of gum arabic because it can't be found elsewhere, American industries worry that the product someday will become completely inaccessible.

The acacia senegal plant grows only in Sudan - and in a greenhouse at Ohio University, thanks to the nurturing of scientist Marcia Kieliszewki.  

A natural defense

The acacia senegal, although short and skinny, survives in conditions that would devastate most plants. In fact, gum arabic is produced best in unhealthy trees exposed to dry, hot weather and poor soil. When the bark of the tree is cut or damaged, it exudes gum at the wound site to stop dehydration and ward off pathogens.

"Think about it," Kieliszewski muses. "Plants can't go get stitches when they're wounded, so they have their own type of defense mechanism. It's an evolutionary strategy for survival, and obviously it's worked well because plants are more abundant than we are."

In the plant world, this kind of defense reaction isn't uncommon. Other trees excrete sap - cherry, peach, even pine trees. But the sap from these trees is contaminated with mild poisons and foul-tasting substances. Gum arabic and three other African and Indian gums - gum ghatti, gum karaya, and gum tragacanth - are the only ones clear of contaminants.

In Sudan, gum industry workers speed the production of gum arabic by cutting and stripping the bark of acacia senegals. A few weeks later, they pick off nodules of the hardened gum that have formed on the trees' wounds. Once the pieces are sorted, cleaned, and graded, they resemble amber-colored crystals.

What creates this candy-like appearance in the hardened nodule is sugar, which constitutes 90 percent of the gum. Glycoproteins, the focus of Kieliszewski's research, make up the rest. Glycoproteins are part of a plant's extracellular matrix, the "skin" of the plant that gives it form and structure. They are involved in all aspects of plant growth and development, including fertilization and invoking disease and stress responses.

And for tomato and tobacco plants to produce gum arabic, Kieliszewski must make them manufacture glycoproteins.

For a detailed graphical explanation of Kieliszewski's experiment,    

Improving on nature

Kieliszewski began her long and complex genetic manipulation project a few years ago in her lab at Ohio University. First, she purified some glycoproteins by grinding up pieces of gum arabic. She stripped the sugar from the glycoproteins, exposing each protein's chain of amino acids, called the polypeptide backbone.

The polypeptide backbone then was sent to a lab off campus to be sequenced, revealing the protein's intricate order of amino acids. Using this information, Kieliszewski designed an artificial gene that would encode instructions for the production of glycoproteins.

Kieliszewski now is introducing the synthetic gene into tomato cells by inserting the gene into a plasmid - a piece of DNA - and then injecting the plasmid into bacteria that infects tomato cells. The bacteria adds the new DNA to the plant's genome.

"Once it's in the plant, the glycoprotein should be expressed," she says.

The process is working, a discovery Kieliszewski's made with the lights off. When tomato cells begin creating glycoproteins, the cell culture radiates a fluorescent green in the dark. That's because Kieliszewski attaches a jellyfish protein gene to the synthetic gene that makes glycoproteins glow when they are expressed.

"It looks pretty good so far," she says. "The plant cells aren't seeing it as a weird gene. It means that tomatoes have the right enzymes to do this kind of stuff. The real test is when we get enough of the gum arabic produced to see if it has the same physical properties as the real gum. From everything I've seen so far, I don't see why it won't."

The next step is to test gum arabic production in adult tomato and tobacco plants, Kieliszewski says. It could be several years before her research is fully tested and patented. Meanwhile, commercial prospects look promising.

"If the product or the ingredient would be approved for use by the U.S. Food and Drug Administration and it would live up to the standards that our companies are used to, we'd be interested in taking a look at it," says McBride of the National Soft Drink Association.

But even that doesn't excite Kieliszewski as much as the basic research she's doing on glycoproteins and their diverse functions in plants. She recently received a five-year, $500,000 Career Grant from the National Science Foundation to study them.

"Determining the function of these types of glycoproteins in plants really is what interests me," she says. 

The addition of a jellyfish protein gene causes tomato cells to glow a fluorescent green when they express the gum arabic DNA introduced in Kieliszewski's experiments.   Plant ethics

As she delves deeper into plant bioengineering, Kieliszewski's research edges closer to other types of progressive projects in the field, such as the production of human antibodies in plants and engineering plants to yield more vitamins. Like human genetic engineering, plant bioengineering is controversial because it involves changing nature's design.

"Some people argue that this is tampering with natural selection and evolution, and they say that's always dangerous," says Arthur Zucker, associate professor of philosophy and director of the Institute for Applied and Professional Ethics at Ohio University. "But at the same time, we wouldn't have things like antibiotics and medicine if it weren't for our tinkering."

Debate has erupted surrounding questionable cases of plant bioengineering. For example, a process patented in March 1998 by the United States Department of Agriculture and the Delta and Pine Land Company in Mississippi programs a plant's DNA to kill its own embryos, rendering the seed sterile. Some believe this "terminator gene," as it's called, interferes with a plant's natural biology and prevents farmers from saving seeds for the next year's crops, forcing them to buy new seed annually.

Kieliszewski believes her brand of bioengineering won't have any negative impact on the nature and quality of the tomatoes and tobacco plants that carry the artificial gene. Still, she says, scientists must be mindful of the risks that come with rearranging the building blocks of life.

"We're messing with genes that Mother Nature has taken billions of years to evolve. We're plowing ahead, but we all have to be careful." 

For more information on this project, e-mail Marcia Kieliszewski at kielisze@oak.cats.ohiou.edu.