The Canadian Certification Program Application of Tissue Culture at the Nursery

By: Judit Monis, Ph.D.  

Last March I was invited to present for the Canadian Grapevine Certification Network (CGCN RCCV). Ethan Churchill, the CGCN-RCCV program manager did an introduction to their certification program standards.  Rob Haynes described the application of tissue culture and other practices at his Upper Canada Growers Nursery.  Robin Ross presented his research on tissue culture techniques. Tanja Voegel and myself did presentations on crown gall disease caused by Agrobacterium vitis.  While I have written before and plan to do an update on crown gall in the future, this article will focus on the Canadian Grapevine Certification Network program with emphasis on the application of tissue culture of the Upper Canada Growers Nursery in their production practices.

  In June of 2019 over two million dollars in funding through the Canadian Agricultural Partnership’s AgriAssurance program became available to start a network of virus-free grapevines in Canada.   The program would provide clean planting material to Canadian growers to assure the viability of their wine industry.  The funding is being allocated to both the interim verification standard and the long-term Canadian certification programs.  An interim verification standard consists of visual inspection and testing existent nursery blocks for the presence of Grapevine leafroll associated viruses (GLRaV) -1 and -3; Grapevine red blotch virus (GRBV), and Grapevine Pinot Gris virus (GPGV).  The sample collection and testing are performed in the laboratory of the Cool Climate Oenology and Viticulture at Brock University at a 50/50 cost share between nurseries and the CGCN-RCCV (until March 2023).  The network works with partner nurseries for general propagation and with vineyard growers and wineries for custom propagation of their own vineyard planting material.  The testing protocol is as follows: an initial random sampling of 10% of the vineyard that includes visual inspection and RT- PCR testing for the presence of the viruses of concern (GLRaV-1 and -3, GRBV, GPGV).  If 15% or more of the vineyard block is found to be infected it is dropped from the program.  If the vineyard block is found to be less than 15% infected, it is moved to the second phase of testing which consists on testing the entire vineyard. Samples are composites of leaves from five vines or canes from two vines.  The threshold for acceptance to the program is 0.1% (i.e., less than one infected vine in 1000 vines).  If the block is found to be infected more than 0.1%, the nursery has the option of testing each vine in the positive composite sample individually and remove the infected vines from the block.  Once the vineyard is tested and confirmed to be under the 0.1% threshold, plants propagated from those vines are deemed verified through CGCN-RCCV, Of course, similar to other certification programs there is no guarantee that the vines produced by the nurseries are virus free.  The program includes yearly audits with visual inspection and virus testing (10% random sampling) of the nursery blocks to ensure the lack of infection or spread to planting material.  Participating nurseries as well as available certified varieties are listed in the CGCN-RCCV website (see: https://www.cgcn-rccv.ca/site/about-cgcn).  Custom propagation of vineyard or winery planting material is subjected to a protocol similar to the one described for nursery blocks.

  Rob Haynes presented information on the practices of his family nursery operation (see: https://www.uppercanadagrowers.ca). The family purchased Mori Nursery in 2016 and started the Upper Canada Growers Nursery.  The use and application of tissue culture technology is what sets this nursery apart from others. It all stated ten years ago when the nursery had problems with the propagation of apples due to the presence of viruses and the fire blight bacterial pathogen.  At the time researchers at Cornell University had developed apple rootstocks that were resistant to these pathogens (Geneva rootstocks).  However, these rootstocks were difficult to propagate.  The company turned into using tissue culture (a collaboration with the University of Guelph) to develop their tissue culture program.  After a long and slow learning process the nursery is now ready to release two million tissue cultured propagated apple, hazelnuts, and plum plants (apples being the larger part of the production).  The company also has grapevines in the pipeline.  Although there is no timeline for the release of grapevines, so far, the process appears to be easier than the other crops produced at the nursery.  To summarize, the propagation material starts in the laboratory (as tissue culture plants), once the plants are rooted and grown to a certain size, the plants are transferred to the greenhouse in misting tents (the humidity must be kept very high.  The plants continue to grow in the greenhouse up to a certain point and are transferred to the field.  However, the plants are not planted directly in the field but inside “smart pots”. The plants (in pots) are constantly being screened from insects and the environment to avoid infection.  Therefore, from start to end, plants are being grown in a clean environment to ensure that pathogens are not present. In the fall the plants senesce and go dormant, being finally moved into a cold storage unit   The final planting product described is different from the dormant bare root plants that the orchard farmers are used to planting.  Instead, the plants produced are kept inside containers in peat /perlite mix media.  The roots appear to be further developed compared to plants grown in a standard production.  The technology is not new to the fruit (or grape) growing industry, but the nursery has developed specialized media that has made the process very successful. 

  The same process developed for fruit and nut crops at the nursery is being transferred to grapevines. The nursery approached CGCN-RCCV to work on the commercial release of certified grapevine planting stock.  The material being propagated was initially sent to the Canadian Food Inspection Agency for virus indexing.  This allowed the nursery to determine that they were starting with clean propagation material.  Indexed grapevine cuttings were sent to the University of Guelph who set up all the protocols to initiate the material in tissue culture (media, growth conditions, etc.).  Once the plants were initiated in tissue culture, the material was transferred to the nursery’s laboratory for further propagation and to develop a commercial product.  There are many advantages to the propagation system:  the nursery is starting with a clean product (virus tested), grown in a sterile/aseptic media (tissue culture) and subsequently grown in hermetic greenhouses (air showers and other mitigation practices to avoid the entrance of insect vectors/pathogens), to finally being moved to a screened area in the field.

  Rob Haynes mentioned that initially there were concerns from the industry that the apple tissue culture plants would be juvenile (which they were during the first two years).  However, the apple plants appear to start production much faster in the first five years, than apple trees propagated normally.  By the 10th year the production is expected to even out, but the quality of tissue culture propagated trees seem to be superior to the standard propagated trees.  Presently, Canadian grape growers are not familiar with tissue culture produced plants but at some points when these plants become available, the nursery expects these plants will be superior to standard propagated grapevine plants. 

  The Nursery has an active research and development program.  One current program is applied to understand the microbial population (microbiome) around the roots and within the plants.  It is known that tissue cultured plants are grown aseptically, consequently many of the microbes that were present in the original plants have been removed.   Some of the microbes (bacteria, fungi, and/or viruses) may be harmful to the plant growth.  However, some can be beneficial and help the plants absorb nutrients or may have other important functions. Learning about the plant’s microbiome will allow the isolation of the beneficial microbes while eliminating the harmful ones.  The ultimate goal, is to replenish (at a later stage of production) the beneficial microbes that were removed during the tissue culture process to develop of stronger and healthier plants.    Judit Monis, Ph.D. provides specialized services to help growers, vineyard managers, and nursery personnel avoid the propagation and transmission of disease caused by bacteria, fungi, and viruses in their vineyard blocks.   Judit (based in California) is fluent in Spanish and is available to consult in all wine grape growing regions of the word.  Please visit juditmonis.com for information or contact juditmonis@yahoo.juditmonis@yahoo.comcom to request a consulting session at your vineyard.

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