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Robert C. Clarke

International Hemp Association, Postbus 75007, 1070 AA Amsterdam, The Netherlands Tel/Fax: +31 20 618-8758, e-mail: iha@euronet.nl

        Clarke, Robert C. 1998. Maintenance of Cannabis germplasm in the Vavilov Research Institute Gene Bank - Five year report. Journal of the International Hemp Association 5(1): 75-79. The Vavilov Research Institute / International Hemp Association (VIR/IHA) Cannabis Germplasm Preservation Project (CGPP) spanned five years and involved cooperative participation from several research institutes in Russia as well as in Italy, the Ukraine and Yugoslavia. During the project 379 of the 496 Cannabis sativa L. seed accessions now contained in the Industrial Crops section of the VIR Gene Bank in St. Petersburg, Russia were reproduced.

Introduction
        The most important project of the International Hemp Association (IHA) is a grant of humanitarian aid to the N. I. Vavilov Institute of Plant Industry (VIR) in St. Petersburg, Russia. The institute is the longest existing seedbank in the world and houses a priceless collection of over 400,000 seed accessions. Their Department of Industrial Crops maintains the largest Cannabis germplasm collection in the world. The initial collection represented wild populations and traditional cultivated varieties as well as products of plant improvement programs from three basic geographical groups; Northern-, Middle-, and Southern-ripening, collected from 26 regions. The vast majority of the accessions are classified as low-THC chemotypes of Cannabis sativa L., and many of them are found in no other gene banks. Under normal storage conditions, hemp seeds can be kept for about five years before losing viability. Maintaining a Cannabis collection, therefore, involves the provision of long-term storage facilities, or reproducing the accessions at least once every five years. Since the dissolution of the Soviet Union, funding for the VIR has dramatically decreased, threatening the maintenance of its collections.
        In 1993, the VIR, with the sole support of the IHA, began a four year program to preserve and evaluate its Cannabis germplasm. At that time, the collection consisted of 397 accessions. The collection now numbers 496 accessions (Table 1). The original plan was to reproduce all of the accessions during the first four years, and in 1997, samples of the entire reproduced collection were to be grown in replicate gardens, each accession characterized, and the results published. We also hoped to install freezers for the long term storage of a portion of each accession. Due to slow progress with reproductions, and the inclusion of additional accessions, the CGPP was extended to a fifth year and at this time remains incomplete.

Materials and methods
        Accessions of Cannabis contained in the germplasm collection of the Department of Industrial Crops at the VIR were sown at various locations (Table 2) with the purpose of germplasm maintenance, replenishment of seed stocks for distribution, and installation of seed into long-term storage.
        Information concerning materials and methods, climatic conditions at individual reproduction sites and other details can be found in past June issues of this journal.

Results
        The project has proceeded more slowly than expected for several reasons. Low amounts of initial seed, poor germination, abnormal weather conditions and thefts have lowered the yields of reproduced seed. This has caused us to reproduce some accessions more than once to ensure that sufficient seed is placed in long-term storage. Refrigerated long-term storage units have been installed at the VIR and part of their capacity is reserved for the freshly reproduced Cannabis accessions. One more year of reproductions is required to complete the preservation portion of the project.
        Eighty-five accessions of hemp contained in the germplasm collection of the VIR were sown in 1993. The accessions were planted at four VIR Experiment Stations in Russia; Pavlovsk near St. Petersburg, Yekaterinino, Kuban near Krasnodar, and Volgograd and 60 were reproduced. In 1994, the VIR had its second successful year of reproductions by growing out 94 endangered seed accessions and reproducing 72. Additional grow-out locations were arranged in 1995 in Italy. The 1995 reproductions were also successful. The sowing of 106 accessions resulted in 84 reproductions. In 1996, 112 accessions were grown only at Russian research institutes, and 90 were reproduced. In 1997, the project was expanded to include research institutes in Yugoslavia and the Ukraine, resulting in a total of 105 successful reproductions from 140 sowings. One hundred and seventeen accessions remain to be reproduced successfully for the first time, while another approximately 100 must be reproduced in sufficient quantity for long-term storage and/or distribution to researchers. A block of 77 accessions from Germany appear to be individual selections from (possibly selfed) monoecious individuals. All of the seed was last reproduced in 1987 and 1988 and some accessions have very little seed. We suggest that these accessions be grouped in 10 to 15 batches and each batch be reproduced in isolation as a separate accession. This will ensure the multiplication of these small and old accessions and reduce the total number of isolations required by 10 to 15 fold.
        Seeds are preserved at the VIR in a "working collection" of various amounts stored at room temperature (used for reproductions or distribution to researchers), a "cool storage" of either 50 g or 75-100 g stored at 4-6º C., and a "long-term storage" of 75 g amounts at minus 18º C. The VIR also maintains seeds in "temporary storage" at nearby Pushkin at minus 10º C. All of the accessions are represented in the working collection and cool storage, 28 recently reproduced accessions are stored in the newly established long-term collection, and 42 accessions are stored in the temporary storage. Varying amounts of seed of 214 accessions are also stored at room temperature in the National Seed Storage facility at Kuban, Russia.
        Efficiency and progress of the CGPP are assessed by measuring (a) the percentages of successful reproductions that assure genetic preservation (>1000 plants) and (b) the number of reproductions of sufficient size (>120 g) that allow distribution to researchers (Table 4).

Conclusions
        In reviewing the progress of the CGPP, it is instructive to understand the original aims of the project. Our optimistic prediction in 1993 was that:
        "All of the accessions will be reproduced during the first three years, and in 1996 the entire collection will be grown in replicate gardens and each accession will be characterized."
        After slow progress the first year, we realized that a fourth year would be required to complete the reproductions. Following the third year, due to continued slow progress and the addition of nearly 100 accessions, it became apparent that a fifth year must be added. At this point a sixth year will be required to complete the reproductions. The six years required to complete the project certainly exceed the time allowances predicted at the outset.
        When we began, we realized that two key requirements must be fulfilled before an accession could be considered adequately reproduced. The primary goal of a germplasm preservation project is the conservation of the entire gene pool of each accession. It is very important that the population size of each individual reproduction is large enough to ensure that nearly all of the genes within each accession’s gene pool are reproduced in the resultant seed. We attempted to set a minimum limit of 1000 plants in each population. This should ensure 99% preservation of the gene pool.
        The second goal was to reproduce the accession in sufficient amounts to distribute seed to researchers worldwide. Initially, we set the minimum amount of seed produced at 200 g per accession. Since the installation of long-term storage facilities in St. Petersburg, this threshold has been lowered to 120 g, which still allows for sufficient seed for long-term storage of each accession. Although our success rate for reproductions increased steadily from 71% to 81%, the percentage of reproductions for distribution fluctuated between 45% and 64%.The greatest hindrance in producing enough seed for distribution has been the size of the reproductions. Due to limitations in the size of growing space, and the amount of viable seed, it was often not possible to grow 1000 individuals or 120 g of seed. Very few of the accessions have been lost, but several have very few seeds, and now must be multiplied from a possibly more limited genetic base. Seeds produced by the VIR/IHA Cannabis Germplasm Preservation Project are made available to Cannabis breeders worldwide. Since 1993, 301 seed accessions have been reproduced in sufficient quantity to allow distribution to researchers. The VIR freely distributes approximately 7-9 g amounts of seed to researchers who complete a Material Transfer Agreement (MTA) stating that the recipient of the seeds agrees to use the seeds for evaluation trials only, not use the offspring for breeding, not transfer the seeds to a third party, and share any data from the trials with the VIR. The use of VIR germplasm in plant improvement programs must be arranged in an agreement of scientific cooperation. There are no restrictions on the use of VIR germplasm, as long as agreements are in place. A complete list of the VIR Cannabis seed accessions, including experimental agronomic data such as growth period, oil percentage and fiber and seed yields, as well as cannabinoid and essential fatty acid contents, is available to IHA members for US$50.00.
        Operating with a very small budget, much of our success has resulted from direct IHA participation in administering the CGPP, without the expense and hindrance of administrative overhead. This was made possible because the contribution made by the IHA is handled as humanitarian aid rather than as a research grant and is not administered by the VIR budget office or the Russian government. The complete budget for the five years of the CGPP is found in Table 5. The funds have been dispersed directly by the director of the VIR’s Industrial Crops section, presently Dr. Sofia Kutuzova. Dr. Sergey Grigoryev currently manages the Cannabis germplasm collection.
        The most immediate obstacle the CGPP faces is locating funding for the projected 1999 reproductions. Evaluation of the collection in common gardens should take place in 2000, requiring cooperative agreements with foreign institutions and a new research budget.
        There is a pressing need to preserve the germplasm in the VIR collection until agronomic research can realize its potential. For example, the VIR Cannabis accessions k-313 and k-315 were the basic building blocks of ‘FIN-314’, a new seed variety developed for northern latitudes and currently the leading variety for use in hempseed oil production. We feel that the project should certainly be continued. Persistence and considerable additional financing will be required to complete the project within the near future. The present requirements to reach our goals for the re-establishment of Cannabis as a major industrial crop are the same as when the project began:
        "Only one thing is certain, we will never succeed if we do not focus our most concerted efforts on Cannabis germplasm conservation."

Doubovskaja, A. A. Sonnino, U. De Corato, L. T. Rumyantseva and R. C. Clarke 1996. Report of Italian VIR/IHA Cannabis Germplasm Preservation Project - 1995. Journal of the International Hemp Association 3(1): 7-9.

Kutuzova, S. N., S. V. Grigoryev, L. T. Rumyantseva, J. Berenji and R. C. Clarke 1998. Maintenance of Cannabis germplasm in the Vavilov Research Institute Gene Bank - 1997. Journal of the International Hemp Association 5(1): 28-31.

Kutuzova, S. N., L. T. Rumyantseva and R. C. Clarke 1996. Maintenance of Cannabis germplasm in the Vavilov Research Institute Gene Bank - 1995. Journal of the International Hemp Association 3(1): 10-12.

Kutuzova, S. N., L. T. Rumyantseva, S. V. Grigoryev and R. C. Clarke 1997. Maintenance of Cannabis germplasm in the Vavilov Research Institute Gene Bank - 1996. Journal of the International Hemp Association 4(1): 17-21.

Lemeshev, N., L. T. Rumyantseva and R. C. Clarke 1994. Maintenance of Cannabis germplasm in the Vavilov Research Institute Gene Bank - 1993. Journal of the International Hemp Association 1(1): 1, 3-5.

Lemeshev, N., L. T. Rumyantseva and R. C. Clarke 1995. Report on the maintenance of hemp (Cannabis sativa L.) germplasm accesioned in the Vavilov Research Institute Gene Bank - 1994. Journal of the International Hemp Association 2(1): 10-13.