American chestnut tissue culture
Plant tissue culture (also known as micropropagation) is a method of propagating plants asexually in aseptic conditions. The four main steps of micropropagation are establishment, multiplication, rooting and acclimatization. An additional step, regeneration, is needed for embryo tissue culture. The following explains the process of tissue culture for American chestnut somatic embryos (non-zygotic embryos established from zygotic cells). All steps are performed in a laminar-flow hood that uses a HEPA filter to keep the air aseptic. The whole process can take between 18 to 24 months.
To establish American chestnut somatic embryos in tissue culture, the nut is removed from the spiny bur and then sterilized with bleach to rid it of microorganisms. The cleaned nut is cut open and the ovules are removed and placed on an embryo initiation medium (a semi-solid gel containing nutrients, vitamins, sugar and hormones) in a sterilize Petri dish. After a few weeks, some of the embryos will begin to grow, however, the majority of the embryos will either become contaminated or die. The embryos that do survive are now ready to be multiplied.
Once an embryo is established, it can be multiplied. By using a certain combination of hormones in the medium, new embryos will begin to grow out of the original one. As more embryos develop, the nutrients in the medium become exhausted. The new embryos are separated into smaller groups and transferred to fresh medium. Every two to three weeks, as more embryos grow, they are separated and transferred to fresh medium.
The next step is to regenerate the embryos into shoots. To do this, the concentration of nutrients, sugar and hormones in the medium is changed. There are a total of three different media used during this process. Once the shoots develop, they are multiplied on yet another medium. The shoots are cut into smaller segments and transferred to fresh medium once month.
Now the shoots are ready to be rooted. They are dipped into a liquid solution of a rooting hormone and then placed in a charcoal medium to absorb the excess hormone. Anywhere from 10 to 60 percent of the shoots going through this process will develop roots. These rooted plants are put in potting mix and then they go through an acclimatization process.
This has proven to be the most difficult part of American chestnut tissue culture. The plants are very sensitive to slight environmental changes and will die under the wrong circumstances. Lids are kept over the plants to reproduce the high humidity they were used to in tissue culture. The lids are removed in stages to let the plants get acclimated to ambient conditions slowly. They are watered and fertilized when needed. Only one to three percent of plants going through acclimatization survive to be planted in the field.
To add potential blight-resistance genes to the American chestnut genome, a method known as Agrobacterium-mediated transformation is being used. This method uses a bacterium called Agrobacterium tumefaciens, which is a natural genetic engineer. Wild-type Agrobacterium lives in the soil and colonizes small wounds near the root collar of many plant species. When the Agrobacterium encounters 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, and to produce food that only the Agrobacterium can use. Scientists have "tamed" Agrobacterium strains so that they insert genes of interest to the research project instead of the genes that are only adventageous to the bacterium.
To transform American chestnut somatic embryos, we use this Agrobacterium-mediated transformation method. This means we use a "dis-armed" strain of Agrobacterium containing our putative resistance enhancing gene and selectable and scorable marker genes. The Agrobacterium is mixed together with the embryos in a test tube. The bacterium is given enough time to inject the designer DNA into the plant cells and then the embryos are moved to a medium containing antibiotics that will kill the bacterium but not the plant cells. The plant cells that have not taken up the new DNA are eliminated with a tiny dose of an herbicide that won’t harm the transformed cells. The embryos that have been transformed are then regenerated into whole plants.
The pictures below follow the process from inoculating medium with the Agrobacterium (fig. 1), co-cultivating the Agrobacterium with chestnut embryos (fig. 2), desicating the Agro/embryo mix and then visualizing the transformed cells using the green fluorescent protein, GFP, marker (fig. 3), and the selection of transgenic embryo clumps again using the GFP marker (fig. 4). The embryos are then multiplied and regenerated into whole plants as described in process above. GFP was chosen as a marker because it have been show to be a safe and non-destructive marker system that can be used in transformation and subsequent environmental studies.
The first American chestnuts to be transformed this way contain an oxalate oxadase gene that originates from wheat (reference below). Several American chestnut plantlets with this gene have been planted outdoors and are doing well. Currently we are working on transforming more American chestnut somatic embryos using other blight-resistance genes.