The USDA’s National Clean Plant Network is a program that began with the 2008 Farm Bill and administered by the United Stated Department of Agriculture. It provides resources to develop, maintain and distribute virus and other pathogen-indexed plant material (plant material clean of known viruses) to propagators of important clonally propagated cultivars of key plant commodities valuable to U.S. agriculture. Here, David C. Zlesak, Ph.D., explains the testing protocols for roses conducted at the Foundation Plant Services facility.

Foundation Plant Services (FPS), located on the University of California-Davis campus, has supported an extensive clean stock rose program since the 1960s through the pioneering efforts of Dr. George Nyland. FPS likely has the largest clean plant collection for roses in the world with eight rootstock varieties and more than 500 cultivars. Over the years the FPS research team has been conducting valuable rose research characterizing new rose viruses, optimizing tissue culture methods to clean infected roses of virus, and documenting the impact virus infection (a single virus or multiple viruses in combination) has on rose propagation rates for industry and plant performance.

Before a new rose can be entered into the clean stock block, the rose needs to pass three kinds of testing. First, roses undergo molecular tests (PCR/ELISA) to determine if they are positive for the rose viruses that have been well characterized and detection protocols are readily available. If they pass that test, then there are two biological tests they will also need to pass. One is being bud-grafted onto a virus sensitive rootstock called R. multiflora ‘Burr’s Multiflora’ that shows strong symptoms when infected. The rootstock is checked for symptoms over two years. The other biological test is bud-grafting the rose in question onto ‘Shirofugen’ cherry, another virus sensitive indicator plant.

The biological tests are very helpful, but do not tell us which virus(es) are present, just that there appears to be a virus or virus-like pathogen of some kind. In addition, there may be a virus present that the indicator plant may not show symptoms for. That is partly why two different indicator plants are used. Passing all three tests offers strong peace of mind that the rose is practically clean and FPS can feel comfortable including it in the clean stock collection.

How roses are tested

Typically five plants are provided when industry members submit a new cultivar to FPS for inclusion into the clean plant collection. Each of the plants is independently screened using PCR and/or ELISA methods for the customary rose viruses (for example, PNRV: Prunus Necrotic Ringspot Virus; ApMV: Apple Mosaic Virus; Arabis Mosaic Virus; and so on). Up to three plants that test clean for all key viruses are retained to move forward for biological indexing. If only one or two plants test clean, only those plants move forward. If all test positive for one or more viruses, the cultivar will need to undergo meristem culture to try to generate a plant of the cultivar that is clean of those virus(es). When the plants that are regenerated from meristem culture are large enough, they are retested using PCR and/or ELISA and the process continues again.

Biological tests

For biological testing, both the bud grafting of ‘Burr’s Multiflora’ and ‘Shirofugen’ cherry can be performed the same summer. Judy Lee coordinates the biological assays. Clean plants of ‘Burr’s Multiflora’ are planted out in the field, typically as hardwood cuttings the fall before bud grafting. There are at least two rootstock plants budded from each of the three putatively clean plants retained of each cultivar after the molecular tests. Rootstock shoot growth is not removed after grafts take, because the goal is to observe the leaves and stems of the rootstock for virus symptoms. If the grafted cultivar is clean, the rootstock looks normal and vigorously overshadows the budded growth. For virused plants, the rootstock is often severely stunted, and one can easily see the growth of the rose cultivar originating from the graft. Throughout the screening field, rootstock plants are bud grafted with cultivars known to be positive for particular viruses for use as controls/comparisons.

The plants are rated for virus symptoms on the rootstock during  the first and second spring after grafting. Signs of stunting, mottling, streaking and watermarks on the new growth are noted. By the spring after grafting, virus has typically replicated and transferred to the rootstock in abundance. Spring is the best time to notice symptoms for most viruses. As the season heats up, virus concentration typically declines. Newer growth in the heat of summer may still show some stunting, but yellow patterns in the foliage are often less obvious.

The ‘Shirofugen’ cherry biological assay is a much faster test than ‘Burr’s Multiflora’ and is rated between 30 to 35 days after bud grafting. Long, current-season stems of the cherry are bud grafted with often several buds from various roses in tandem. At least two buds originating back to each starting cultivar plant are budded to ensure replication and greater confidence in the results; this is similar to the ‘Burr’s Multiflora’ biological assay.

After 30 to 35 days following bud grafting, the stems are harvested and each bud is assessed. Virused grafts show a gummy, sticky, discolored reaction around the bud. When grafted stems are removed within about a month, virus should not have moved into the rest of the cherry tree. After removal of the grafted stems, the cherry tree should be clean and can be used for grafting again in subsequent years. As they age, these trees eventually have stout bases and in some ways are similar in appearance to bonsai. An established cherry tree can support many long and sturdy stems for serial bud grafting.

The identity of the stock plant from which the buds used for bud grafting both the ‘Burr’s Multiflora’ and ‘Shirofugen’ cherry came is retained. The original stock plants are maintained in large pots in the greenhouse or shadehouse throughout the biological indexing. At the end of the study, one can go back to the stock plants that passed the molecular and biological indexing and propagate plants to go to the clean stock field. If there are no plants of a cultivar that passed all three molecular and biological tests, the cultivar is meristem-cultured, and the process starts again.

Read more: Keeping track of rose rosette disease

FPS Meristem Culturing

Tissue culture is a highly controlled environment where nutrition, temperature, light and moisture can be optimized and microbes excluded. Most viruses move from cell to cell using channels called plasmodesmata. The very youngest cells in the growing points have not developed plasmodesmata yet and, therefore, are typically not yet infected. These small groups of dividing cells can be carefully excised and placed onto nutrient rich medium with the hope that they continue to divide and develop stem and leaf tissue. Months later, whole plants with roots are regenerated and can be taken out of culture and grown on in the greenhouse.

It takes significant skill and dexterity to effectively perform meristem culture and ultimately clean plants of virus. Dissecting microscopes are used to better see and isolate the desired cells from growing points, and one needs to work relatively fast so the cells do not dry out between isolation and being put on moist culture medium. The larger the clump of cells the greater the chance of survival, but also the greater the chance of still being infected.

Rose cultivars respond differently to plant tissue culture. Some are forgiving and have a wider tolerance range of nutrition and other conditions, while others struggle and are difficult to have survive and grow to a suitable size to take out of culture and transition to the greenhouse. One can work to optimize conditions for problematic roses, but that becomes time consuming.

Susan Sim coordinates activities in the FPS Plant Tissue Culture Lab and Waclawa Pudlo is the primary researcher who conducts the meristem culturing. It was a special treat when Waclawa took the time to walk me through her techniques and kindly invited me to spend some time practicing in the lab. I learned a lot and recently was able to secure a teaching grant to purchase a higher quality dissecting microscope so students and I can do a better job at virus clean up in our plant tissue culture teaching laboratory I manage at UW-River Falls.

David C. Zlesak, Ph.D., is Associate Professor of Horticulture in the Department of Plant and Earth Science at the University of Wisconsin-River Falls; he can be reached at david.zlesak@uwrf.edu.

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