Two articles in the September/October issue of the Forest Owner, the first by Herbert Darling and the second by Elizabeth Densmore, discussed the importance of the American chestnut (Castanea dentata), the disastrous introduction of the chestnut blight fungus, and the role that the American Chestnut Foundation is playing to save this once-magnificent species from extinction. I would like to discuss our chestnut research at the College of Environmental Science and Forestry. The project, partially funded by grants from the New York Chapter of the American Chestnut Foundation, is a joint effort between Dr. William Powell in the Faculty of Environmental and Forest Biology and myself in the Faculty of Forestry and a team of dedicated graduate students. We are attempting to use genetic engineering to design an entirely new gene for blight resistance. There are two parts to the project. The first part is to identify a naturally-produced compound capable of stopping the growth of the blight-causing fungus. The second part is to develop a method of delivering the gene that codes for that compound into individual cells of chestnut and regrowing those cells into a whole tree. Within each of these two areas of research, there are dozens of steps, any one of which may take weeks to months to accomplish. It will be a long project.
The first step in identifying a potential resistance gene is to examine ways in which other organisms combat fungal attacks. One defense mechanism to combat fungi directly is with enzymes designed to break down their cell walls. Many plant species, including chestnut, use this defense mechanism. Unfortunately, the blight fungus evades the chestnutís defense enzymes by producing enough acid in the immediate area of a developing canker to inactivate the enzymes.
Another defense mechanism is for an organism to release very short strands of specialized defense proteins just long enough to span the cell membranes of attacking fungi. Dozens of these small proteins clump together, forming small holes in the membrane. Essential minerals leak out and the fungus dies.
Although not as direct an attack as an enzyme that simply rips apart the fungal cell wall, these tiny proteins can be just as effective. A number of different organisms including some moths, frogs, bees, pigs, and probably many others, produce mini proteins with these properties but to our knowledge, American chestnut does not yet.
Dr. Powell and his graduate students are testing several of these small proteins and plan to test more to see which are most effective against the blight pathogen. The ideal mini protein would be one that is deadly to the fungus at a very low concentration, but causes the plant no harm even at a much higher concentration. In addition, it should be completely non-toxic to humans or wildlife who will someday eat the chestnuts. After identifying a mini protein that comes closest to this ideal, the next phase of the project will be to build a gene to make these mini proteins inside the plant cell.
The second part of the project involves actually transferring the gene into cells of a chestnut and regenerating those cells back into a whole tree. Since the mini proteins are still under development, we are working with other engineered genes to develop the gene-transfer and plant-regeneration techniques. In this way both parts of the project can move forward simultaneously.
The gene transfer process will actually be carried out by a bacterium called Agrobacterium tumefaciens, a natural genetic engineer. Wild-type Agrobacterium lives in the soil and invades small wounds near the root collar of many plant species, including chestnut. When Agrobacterium invades a wound, it attaches itself to plant cells, pokes a microscopic hole into the cell, and injects small pieces of DNA. The DNA travels to the nucleus and is incorporated into the chromosomes of the plant. Wild-type Agrobacterium injects genes that cause the plant's cells to divide rapidly, producing a warty gall, called a "crown gall." The bacteria then live and multiply happily inside the gall.
Bacteria are much easier to manipulate than plant cells. It is a routine procedure to remove the small segments of DNA that Agrobacterium injects into the plant, cut out the gall-inducing genes, replace them with designer genes,Ó and reinsert the segment of DNA back into Agrobacterium. These "tamed" laboratory strains can no longer cause gall formation but they can transfer the desired DNA to plant cells.
The final step is to regenerate whole plants from the transformed cells. At the tip of every growing shoot is a region of a few cells called the apical meristem. These cells are unique in that they grow and divide very rapidly and are capable of producing all the above-ground portions of a plant. We are now attempting to cut thin slices through the tips of tiny chestnut shoots and transform them with Agrobacterium. We know that we can regenerate new shoots from these shoot tip slices, but we do not know yet if the Agrobacterium can successfully transform them. This work is just beginning, and we won't know if it is successful for several months.
What I have outlined above is a research plan and a description of work in progress, not a finished project. We have enough results to be excited about continuing, but we are many years away from having blight-resistant trees. However, the basic philosophy and procedures for genetic engineering of plants outlined above have been used successfully in transferring useful genes to other valuable crop species such as tomato, corn, and potato. Closest to commercial release is the Flavr SavrÆ tomato. This new variety produces fruit that turns bright red and sweet just like the standard varieties, but the flesh stays firm much longer. Typical "store-bought" tomatoes must be picked green so that the fruit can withstand shipping but Flavr Savrs can be vine ripened and should arrive in the grocery store with much more of their "just-picked" flavor. Thanks to biotechnology, Christmas dinner may include salads topped with sweet juicy tomatoes and American chestnuts roasted once again over the open fire (or perhaps in the microwave.)