Science in Action: Part I. Engineering Plants Using Crown Gall Disease

Science in Action: Part I. Engineering Plants Using Crown Gall Disease
By Robert Ll. Morris

Ever since Mendel discovered that characteristics in pea plants could be inherited, scientists have been improving plants through hybridization; two related plants were crossed and the resulting offspring had characteristics of both parent plants. Breeders then selected and reproduced the offspring that had the desired traits. These conventional plant breeding techniques were relatively imprecise because they shuffled thousands of genes around and distributed them to the offspring just to get one important change in a plant that was economically worth pursuing.

One challenge encountered in Mendelian breeding is that generally only closely related species of plants could be crossed. If no closely related species with desirable traits existed, breeders had no way of passing on these traits to the other plant. Another problem was that some of the genes were linked to each other. This is seen today in tomatoes that have been bred so that they can be shipped long distances but with a substantial loss in flavor.

Since the early 1980’s scientists have been using the tools of modern biotechnology to insert a single gene, or just two or three genes, into a plant giving it new, advantageous characteristics. With this technology a single gene could be inserted into a plant giving it a desired characteristic instead of the mixing all the genes from two plants through traditional plant breeding and hoping for the best. This technique could develop a new plant with much more control and precision and at a rate much faster than ever before.

The bioengineering of plants emerged from discoveries by researchers in previous years on how bacteria caused plant tumors, how viruses protected plants from other viruses and what enabled some bacteria to kill insects. Some first major step toward biotechnology occurred early in the twentieth century with a plant disease called crown gall. Crown galls are tumor-like plant growths that occur on many woody plants including fruit trees, grape vines, and ornamentals. In 1907 researchers at the USDA discovered that the cause of crown galls was a soil bacterium, Agrobacterium tumefaciens. Other bacteria were known to cause plant diseases but A. tumefaciens had the unusual ability to cause the plant that was hosting it to grow a disfiguring tumor.

Forty years later in 1947 researchers at the Rockefeller Institute for Medical Research (now named Rockefeller University), curious about the crown gall bacterium and using it for insight into how tumors developed, grew crown gall tissue culturally free of any associated bacterium AND free of the plant host as well. They found that these uninfected crown galls could continue to grow, as crown gall tissue, independently of the crown gall bacterium and of the plant host for many years. It was concluded that normal plant growth in some unknown way had been permanently and irretrievably transformed by A. tumefaciens. Understanding how would have to wait nearly another thirty years.

During the 1950s and 1960s, scientists discovered DNA’s role in transmitting information from plant to plant and ultimately controlling plant growth. Armed with this new information, scientists began looking more closely at DNA’s role in the formation of crown gall. The crown gall mystery was attacked again when several investigators began, logically, looking for the tumor-inducing factor in the bacterium's DNA.

Bacterial DNA is relatively simple compared to other types of organisms since it can be normally found on a single chromosome. It wasn’t found there. Instead it was found by Flemish researchers on a smaller, mobile DNA unit called a plasmid that was not part of the bacterium's single chromosome. In a series of experiments at the University of Washington ending in 1977 researchers found that this bacterial plasmid was spliced into the chromosomes of plant cells when the bacteria infected the plants.

This was at the same time that researchers in other fields were just beginning to understand how to manipulate genetic information by a technique called, in lay terms, gene splicing – to cut and splice foreign DNA into the genetic code of an organism.

It became clear now that A. tumefaciens transmitted the genetic information needed to cause a plant to produce tumor-like growth through the transfer of a “packet” of information called a plasmid.  This plasmid altered the genetic makeup of the plant so that the infected cells of the plant were induced to divide continually, developing galls containing the genetic information from A. tumefaciens.

What if scientists could manipulate A. tumefaciens so that it no longer transferred the genetic information for creating crown gall but instead transferred genetic information into plants that produced desirable traits such as resistance to insects or disease?

To convert the A. tumefaciens plasmid into a beneficial plasmid (now called a vector) researchers first had to locate and then replace the tumor-inducing genes. By 1983, plant molecular biologists had developed the first plasmid vector for plants susceptible to crown gall from A. tumefaciens. The crown gall disease had changed plant breeding forever.

A tool for introducing genes into plants is useful only if scientists have found genes that they want to transfer. Enter Monsanto. In the late 1960’s researchers at Monsanto wanted to know what made the nonselective herbicide glyphosate (Roundup^TM) a potent killer of so many different kinds of plants; weeds and crop plants as well. It seemed reasonable that if you could alter crop plants so that they were resistant to glyphosate, then spraying an herbicide like glyphosate “over the top” of a mixture of emerging combination of resistant crops and weeds would kill the weeds but not the crop.

Through combined research starting early in the 1970’s glyphosate’s genetic “mode of action’, destruction of an enzyme vital to all plants, was specifically identified by research performed at Monsanto and by German researchers.

In 1983 researchers at Calgene and Monsanto identified the gene, and Monsanto modified the gene, so that the enzyme it produced was no longer sensitive to glyphosate. The A. tumefaciens plasmid vector was used to introduce the modified gene into crop plants. The new tomatoes, petunias and other modified plants were resistant to damage from glyphosate which was reported in the research in1985.



Gene gun. More on this subject at

http://users.comcen.com.au/~biodynamic/pages/what_is_it.html

The USDA requires field testing of genetically modified plants for several seasons before its release and that testing includes potential changes in food safety, nutrient levels, development of potentially toxic substances and safety to the environment. In 1996, the first glyphosate-resistant soybean, cotton, canola, and corn seeds were made available to farmers.

During the1980s and 1990s other ways were developed to introduce beneficial genes into plants. One is called a "gene gun," which literally shoots DNA-covered particles attached to metal “bullets” through plant cell walls and membranes to the cell nucleus. Inside the nucleus the foreign DNA combines with the plant's own DNA and transforms the plant. Other techniques involve electrical or chemical treatments assisting DNA molecules to pass through plant cell walls and membranes barriers and combine with a plant’s genetic information, a high-tech form of plant breeding. Wouldn’t Mendel be impressed?

Previously published in Southwest Trees and Turf

My Beans Emerged From the Soil and Died

A good indicator of collar rot is when the plant shows less vigor than it
should or less vigor than its neighbors

Q. I recently planted bean seeds in pots on my porch here in Las Vegas.  However, after the bean stalks were half a foot tall, they bent over, turned dark and died.

A. This is probably crown rot. If you plant beans when the soil is too cold they have a huge chance of developing crown rot at the point where the bean stem enters the soil. This causes the stem rot, the plant dies or we say “collapses”. If you plant them when the soil is warmer, they have less chance of this happening.

Once the plant is lifted from the soil you can inspect the stem at the soil
level. Where the brown part of the stem and the green part starts was
the soil level. This is also the place where the bean stem was attacked
by collar rot fungi. Avoid planting in soils that are too cold.

 

What to do When Your Indoor Palm Has Sticky Leaves

Q. My indoor palm plant has developed a sticky-looking, shiny appearance on the leaves.  Also, small, 1/16" brown spots/bumps on the leaves and stems.  What is this?

Scale on palm leaf but unfortunately the plant also has spider mites
judging from the yellow speckled appearance of the leaf

A. This is probably one of the scale insects. It is living under this brown bump and sucking plant juices while totally protected by this bump or shell they build on top of themselves.

            This brown bump is what keeps them alive when pesticides are sprayed on the plant. BUT they are susceptible to poisons or insecticides that are taken up into the juices INSIDE the plant.

            So, systemic insecticides, those that are applied to the soil or sometimes sprayed on the leaves and taken up through the leaves, can be quite effective on these protected insects. Since it is a palm and you are not eating anything from it you can use these types of poisons on these plants.

            You will have to try to find one at a nursery or garden center that carries a systemic insecticide that has a label specifically for houseplants and controlling scale insects. The shiny stuff on the leaves is sticky since this is the sugary excrement of these scale insects falling on the leaves. Frequently it attracts ants.

Is Now a Good Time to Fertilize Shade Trees?

Apply fertilizer to the irrigated areas and just
outside the irrigated areas, just below the
soil surface, no deeper than this

Q. There are six trees on our property; a Raywood ash which is 10 years old, Arizona ash also 10 years old, California pepper (taller than our 2 story house and 8 years old), Bradford pear also 10 years old, a young African sumac and a young palo verde. Is now (October - November) a good time of year to deep feed these trees? Also, we've got a three foot long tree root from the pepper that's growing along the top of our lawn. Can I cut that part out this time of year?

A. You can deep root feed these trees (put the fertilizer in the root and watered area at about half a foot, no more) now that the trees are preparing for winter and stopped growing but they should still have green leaves on the tree.

            If there is no grass there then apply it to the surface of the soil and water it in. Water the fertilizer in the root zone three times over the period of one week and then continue to cut back on your irrigation for these trees as you normally would for the winter.



            The living green leaves on the tree will help move the water laced with fertilizer into the trees and into storage for next spring's growth. It also places fertilizer in the area of the roots for next season provided you don’t water too much. You can then skip a spring application of fertilizer.

            If the leaves have turned yellow or are starting to drop, you missed it for this year. Wait until next spring. There is no advantage to applying it in the fall vs. spring except possibly convenience.

Not All Cactus Fruit Are Desirable for Eating

Bird damage to nopal fruit called tunas in Mexican Spanish.
Bird damage to cactus fruits is a good indicator tunas will
have good quality for human consumption. Tunas are high
in vitamin C.

Q. I have a large cactus garden in my front yard and much fruit.  My cactus are the"bunny ears" type.  Is the fruit of this type good to eat and does it have health benefits?  I am a retired teacher and just read your article on cactus fruit.  I have made jelly from them in the past.  I suffer with Crohn's disease and wonder if this fruit is comparable to the Nepolia (sp?) health drink that costs so much. Since Crohn's is an auto immune illness, I wonder if this fruit has some good anti-inflammatory properties? Thank you for your time and any help you can give me.

A. Sorry to hear about your health concerns. The fruit would definitely be edible BUT not all Opuntia type cacti are as desirable as others for eating. The nopal cactus is also a “bunny ears” type but the fruit may not be as edible.

            In some nopal cactus the sugar content may be as high as 30% which would rival fully mature wine grapes in sugar content. I normally will get about 16 to 18% which would rival a really good apple or peach.

            A good indicator about whether it is going to be a good one is how the birds use the fruit. If birds are devouring the fruit then it will have some really good characteristics for human consumption. If the birds leave it alone, well… it probably has low desirability.

            So look for bird damage to ripe fruit. Nopal or bunny ears cactus is selected for edibility so not all of these cacti are as desirable as others for food.

            As far as health benefits goes, I have heard that it is of course high in fiber content, helps to lower cholesterol, helps reduce high blood pressure, is normally high in Vit C (reds are the best) but I have not heard anything about auto immune system benefits unless you want to count the Vit C content as part of that. But I am no health expert and you would probably have to do some digging in the literature online for that information. There are some good papers out of the University of California with the University of Sonora on this subject in the past.

            Also if you want to start growing cactus for food you will have to alter your irrigations to push new succulent growth starting in about March. A deep irrigation every two weeks is all that is needed along with a fertilizer application high in nitrogen in the spring. Compost applications at the time of planting and on the surface each year will also help push new growth.

How Far to Cut Back Ash Without Killing Them?



Example of a thinning cut made to peach. The cut is made
directly above a side branch going in a preferred direction
or a smaller one that will help to reduce the size of the plant.

Q. We have two fantex ash trees, 15 years old. They are spreading out too far. How far can we cut them back without killing them?

A. The problem with ash is that it does not have much ability to come back from cut limbs if you cut back too far. You can begin structuring the tree if you do it fairly early and stay on top of it but if you let it go too long and then cut it back you may have some problems.
 

            You can cut it back to side branches that are growing in a desirable direction but you cannot prune it back by what we call heading cuts (stubbing it back) and hoping these dead end cuts will resprout. You can cut back into about second or maybe three year old wood (there are still side buds remaining that can grow) but if you cut into a limb with no buds present it will probably die back to a major limb.

            So cut back to a branch at a crotch going in the direction you want it to grow. When limbs are growing the wrong direction, eliminate them back to a crotch or another limb. Do not leave any stubs (dead end cuts).

The Modesto ash on the right was "topped" and because of the nature of
ash it never came out of it but had to be removed. The one on the left was
cut back early enough so that it could resprout from young wood.
Consequently the one on the left developed swellings just under the cuts
that are full of tissue that can generate new growth. The one on the right
could not.

            I hope this makes sense. I attached a picture of a thinning cut made removing a larger limb going up…to a smaller limb going out and toward the camera. The direction of growth of the limb was changed without leaving a stub.