Report on the maintenance of hemp (Cannabis sativa L.) germplasm accessioned in the Vavilov Research Institute Gene Bank- 1994

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

On the isolated plots of the Pavlovsk, Ekaterinino, Ustimovka, and Kuban Experimental Stations of the VIR, and the Trisaia facility of ENEA in Italy were maintained 94 samples of the VIR Cannabis collection. Reproductions were received from 72 accessions, two yielded no seeds, 4 were cut prematurely, 4 perished from drought, and 12 were insufficiently isolated to ensure genetic purity.

Introduction
        The current situation is that the hemp industry in Russia is plagued by declining production levels and decreasing quality. Hemp production declined from 202,000 ha. in 1970 to less than 100,000 ha. by 1988 (FAO Yearbook) and has continued to fall during the early 1990s. As a result of this situation, hemp production satisfies only about one half of the demands of industry. Hemp is receiving renewed interest from the agricultural community, so that the present situation will change. The VIR Cannabis collection must be supported and maintained in living condition as the initial breeding material for future breeding programs. It is the main task of this project to maintain Cannabis germplasm in viable condition.
        Genetic seed longevity, which retains full genetic seed value requires 80-100% germination. Storage conditions at the VIR can preserve Cannabis accessions for only 3-4 years before germination drops below 80% (Khoroshailov and Zhukova 1978). Therefore each accession must be reproduced every 3-4 years. Two requirements must be fulfilled before an accession can be considered adequately reproduced. The primary goal of a germplasm preservation project is the conservation of the entire gene pool. It is very important that the population size be large enough to ensure that nearly all of the genes within the gene pool are reproduced in the resultant seed. We have set a minimum limit of 1,000 plants in each population. This should ensure 99% preservation of the gene pool of monoecious varieties, and 95% of the gene pool of dioecious varieties. A population size of 2,000 for dioecious accessions is required to ensure that 99% of the gene pool will be preserved (Crossa 1993).
        The second goal is to reproduce the accession in sufficient amounts to distribute seed to researchers worldwide. We have set a minimum limit of 200 grams of seed for storage and later reproductions. Two hundred grams is approximately 10,000 seeds. This will allow 5,000 seeds to be stored at an ambient temperature ~15o C and moisture content around 10% in an active collection at the VIR in St. Petersburg for distribution and reproduction, and 5,000 seeds to be kept at 4-6o C and 7% moisture content in medium-term storage (up to 10 years) at the Kuban Research Station. A third long-term (more than 10 years) reserve collection stored at -20o C and 6% moisture content will be established at the VIR in St. Petersburg. Frozen storage will mean that viability of accessions can be maintained for 10 to 20 years or more and that accessions can be reproduced much less frequently, based on demand for seed from the scientific community.
        Seeds can only be released to the research community if more than 200 grams of seed from each accession is held by the VIR. Two hundred grams of seed must be reproduced with proper population size and sufficient isolation for an accession to be considered successfully reproduced. If the accession's population size was too small, but over 200 grams of its seed were produced, then distribution will be made with the understanding that some of the compliment of genes may have been lost during multiplication.

Results
Ninety-four accessions were planted in 1994 on isolated plots in different climatic conditions at 5 experimental stations:

        On isolated plots at the Pavlovsk Experiment Station 17 accessions of hemp belonging to.the Middle Russian group were planted. The distance between plots was about 3-4 km. In each plot one hemp accession was planted. The accessions were sown according to their ripening period, with early-ripening varieties planted nearer to late-ripening varieties.
    Agrometeorological conditions during 1994 were unfavorable for the growth and development of hemp. In May and June cold and humid weather conditions were frequent and the temperature did not exceed 13-14° C while rains in June were more frequent than usual. The plots were planted after the soil had warmed on June 14-17. The seed was sown in densely planted rows spaced 20 cm apart. The short growing season at Pavlovsk does not allow plants to develop many branches and seeds are produced in a single apical inflorescence. The size of the plot depended on the quantity of seeds and varied from 2-10 m² depending on the number of seeds available. One accession was sown on each plot.
        The soils of Pavlovsk Experimental Station are loamy. Frequent rains, and strong cold winds produced a thick crust (3-5 cm) which resulted in great difficulty for seedlings which started to appear 10-12 days after sowing. The seedlings were also damaged by hail which fell for two hours and covered the seedlings to a depth of 5-6 cm.
        Agricultural maintenance of the plants in the isolation plots were performed according to methods developed by the Department of Industrial Crops: thinning where excessive plants remained, weeding, fertilization, removal of ripened staminate hemp; covering of pistillate plants with gauze during the period of seed ripening to provide protection against birds.
        Ripening of the seeds took place this year in the end of September and the beginning of October. During ripening, individual harvesting took place, biometrical analyses were made, the sheaves were dried, and the seeds were threshed.
        During the vegetative cycle, morphological characters were analyzed. It was observed that some accessions were not typical when compared with the original forms. The morphological and agronomic description of each accession, including its typical traits, were established in 1975 under common garden conditions, and the data are recorded in the accession notes of the Vavilov Institute. For example, in such accessions as Toguchinskaya (Cat. No. 78) and Chuvashskaya (Cat. Nos. 352 and 354), plants were found with non-typical habit and long inflorescence. It is necessary to make a special planting of these accessions in 1995 in order to clean up the gene pools and establish a typical forms. Thus out of the 17 collection accessions planted at the Pavlovsk Station, 13 accessions yielded from 60 to 500 grams of seed, 2 accessions did not ripen, and 2 accessions were stolen (Table 2).
        The remaining 70 collection accessions of the Central and Southern eco-geographical groups were sown in isolated plots at the Ekaterinino, Kuban, and Ustimovka Experiment Stations. Twenty two accessions were planted at the Ekaterinino Station and yielded from 4 to 954 grams of seed. On three isolated plots the seed yield was very low because the majority of plants were stolen and in one accession few seeds were harvested because they matured very late (Table 3).
        Twenty-two accessions were planted at the Kuban Station and 16 accessions yielded from 1-670 grams of seed and 4 accessions perished due to drought (Table 4). Sowing lasted from April 28 through May 24, mainly because of poor organization in preparing the land for the isolated plots. The accessions sown on time developed normally and yielded a sufficient amount of seeds. Those planted late lagged behind in their development and were so damaged by high temperatures that they did not produce any seeds. Had they been sown earlier in the spring, the summer heat and drought conditions would have caused less damage.
        Twenty-one accessions were planted at the Ustimovka Station and yielded from 45-1,000 grams of seed (Table 5). Sowing was performed in due time on isolated plots protected by forests. Seedlings appeared 8 days after sowing. The weather in early summer was favorable for growth and development of hemp, but dry and cold weather in mid-summer negatively affected the formation and the final yield of seed. Some accessions were not morphologically uniform and did not correspond to the original forms.
        Twelve accessions were planted at the Ente per le Nuove Tecnologie, L'Energia, e L'Ambiente (ENEA) at Trisaia in southern Italy. The soil is of the red alumina type and the climate is semi-arid Mediterranean allowing reproduction of later maturing accessions. The soil was fertilized prior to sowing with the equivalent of 100 kg of nitrogen (N), 100 kg of phosphorous (P), and 100 kg of potassium (K) per hectare. The accessions were sown in June in a single plot. Late sowing caused the crop to be short at maturity. The equivalent of 20,000 m³/ha of irrigation water was required during crop growth. Population sizes were too small to assure successful reproduction of the entire gene pool. Isolation was inadequate. Paper envelopes were used to individually isolate branches and the tops of the plants in an effort to reproduce accessions in a common garden rather than by spatial isolation. July and August temperatures often reached 45° C. The bags heated up in the sun and condensed water, so that much of the pollen was destroyed. The weather throughout the growing season was favorable for growth and development of southern ripening hemp, but failure of the isolation method negatively affected the formation, the genetic purity, and the final yield of seed. The attempted reproductions yielded no seed suitable for distribution (Table 6). Geographical and temporal isolation will be used to effectively reproduce only 4-6 accessions at Trisaia in 1995.

Conclusions
        Ninety-four accessions were planted in 1994 at three locations in Russia, one location in Ukraine, and one location in Italy. Seeds were received from 72 accessions, two did not ripen, 4 were stolen, 4 perished from drought, and 12 were insufficiently isolated to ensure genetic purity.
        The results of the first two years of the VIR/IHA Cannabis Germplasm Preservation Project have been somewhat disappointing. We attempted to reproduce a total of 172 accessions during 1993 and 1994. Only 11 accessions were reproduced with both sufficient population - size (1,000 plants) and sufficient seed yield (200 grams) and 71 accessions were reproduced in sufficient quantity to allow distribution to researchers. The low rate of successful reproductions results from two factors. Most unfortunately, 77 of the accessions do not contain enough viable seed to produce populations of 1,000 plants and therefore can't be reproduced to the project standard. Also, the project is faced with the vagaries of reproducing Cannabis accessions outdoors. This is necessary because it is the least expensive way to grow large population sizes with sufficient isolation. Isolation is established spatially by distances of at least two kilometers, and isolation is provided temporally by sowing early-maturing and late-maturing varieties alternately, thereby effectively doubling the isolation distance.
        The system works well when the weather is favorable. However, frost, hail, drought, and unseasonably heavy rains have all delayed or damaged the reproductions periodically, especially at the more northern locations. The most difficult problem to control is theft of the plants. Local people occasionally steal the tops of the plants, either for the edible seeds, or because they mistake them for marijuana.
        Glasshouse reproductions in screened positive airflow pollen exclusion tents are technologically possible and may be more effective, but the equipment is very expensive, especially when compared to the cost of outdoor reproductions. The necessity of reproducing nearly 400 accessions in four years, with populations of 1,000 plants, would require a large sophisticated facility capable of providing 100 isolation opportunities each year. The cost of building such a facility would be prohibitive given the small project budget.
        The most immediate and cost eftective way to increase the number of reproductions is to locate additional gardens. The Ukraine and Italy were added to the project in 1994 and this increased the capacity for reproductions. Additional southern locations are still required. 'The majority of the VIR Cannabis collection consists of southern ripening types. At the end of 1995, reproductions of the northern and middle ripening accessions appropriate for the climate and latitude at Pavlovsk Research Station near St. Petersburg will be completed. Therefore the Pavlovsk Research Station will no longer be used and our total capacity for reproductions will be reduced unless new locations are found. The VIR is exploring the possibility of working with the former VIR research stations in Turkmenistan near Iran and Afghanistan.
        Once the VIR Cannabis collection has been reproduced in its entirety, all of the remaining accessions must be grown in common gardens at varying latitudes and characterized for their agronomic traits. Molecular data must also be collected in order to determine evolutionary relationships and areas of genetic overlap between the accessions. Only then can the collection be used to its full potential by modern plant breeders.

Acknowledgments
The VIR/IHA Cannabis Germplasm Preservation Project was made possible by a grant of humanitarian aid from the International Hemp Association to the Vavilov Research Institute. This paper was presented by Robert C. Clarke at the Bioresource Hemp symposium in Frankfurt, Germany March 2-5, 1995.

Catalog No.	Name and/or origin	TotalPlants	SeedYield(g)
41 78 91 141 414 422 347 348 349 352 354 361 158 308 99 48 85	Trubchevskaya Toguchinskaya Altaiskaya Altaiskaya Kazakhstan Kazakhstan Maryiskaya Maryiskaya Maryiskaya Chuvashskaya Chuvashskaya Udmurtskaya Udmurtskaya Glukhovskaya Keshtovskaya Orlovskaya Tyumenskaya	3,400 560 240 360 540 200 150 100 120 240 180 321 15 1,200 1,000 960 1,050	500 300 120 200 150 80 60 270 100 150 160 110 190 ⁽²⁾ ⁽³⁾ ⁽³⁾ ⁽²⁾

Catalog No.	Name and/or origin	TotalPlants	SeedYield(g)
17 11 404 375 376 384 119 130 135 121 131 124 125 199 205 206 278 60 a 366 312 115 70	Local, Yugoslavia Domaca, Yugoslavia Boloniska, Yugoslavia Navosadska, Yugoslavia Bolognese, Yugoslavia Local, China Transcarpathia Transcarpathia Transcarpathia Transcarpathia Transcarpathia Transcarpathia Transcarpathia Local, Ukraine Local, Ukraine Local, Ukraine Napoletana, Italy Local, China Lovrin, Rumania Chernoviskaya Krasnodarskaya Gorkovskaya	124 230 110 200 48 85 340 365 540 460 138 250 200 740 136 50 58 44 91 36 120 250	22 30 200 360 50 152 630 802 470 210 10 ⁽³⁾ 300 200 ⁽³⁾ 954 70 34 ⁽³⁾ 4 ⁽²⁾ 46 316 80 290 460

Catalog No.	Name and/or origin	TotalPlants	SeedYield(g)
476 441 18 22 57 403 117 396 171 195 279 377 378 179 180 182 184 301 326 336 327 329	Local, Poland Carmagnola, Yugoslavia Local, Yugoslavia Local, Yugoslavia Local, Spain N555, Bulgaria Fleischman, Hungary Hybrid, Hungary Linia 13/167, Rumania Local, Italy Bolognese, Italy Leskovaskea, Yugoslavia Carmagnola, Yugoslavia Chui-din, China Da-van, China Chain-chgo, China Tin-yan, China No. 9, China Lun-dzin-da, China Dun-ma, China Huan-da-ma, China Shui-ma, China	170 258 102 490 302 21 117 40 120 40 49 47 5 10 17 124	410 20 ⁽⁶⁾ 15 655 550 ⁽⁶⁾ 71 ⁽¹⁾ 670 3 ⁽⁶⁾ 3 ⁽³⁾ 3 ⁽³⁾ 560 13 1 16 3 ⁽³⁾ ⁽⁶⁾ ⁽³⁾ 100

Catalog No.	Name and/or origin	TotalPlants	SeedYield(g)
5 7 177 338 462 442 402 106 507 463 203 201 335 443 435 128 475 369 401 469 169	Bolonska, Yugoslavia Fleischnian, Yugoslavia Lai-sui, China Cu-ma, China Fibranova, Italy ? Novosadska, Yugoslavia No. 556, Bulgaria Yuzhnaya Krasnodarskaya Hybrid Glukhovskiy C. S., Italy Local, Ukraine Local, Ukraine Local, Bulgaria C. Sl. gigantea, Poland Fibrimon, Italy Transcarpathia Local, Poland Local, Bulgaria G-s (J3 ?), Bulgaria Fibrimulta, Rumania Rumania	560 640 300 200 94 456 940 126 48 164 1,110 840 260 148 56 250 340 570 210 360 420	220 1,000 55 60 130 45 110 140 65 70 90 210 120 205 80 115 175 150 180 155 150

Catalog No.	Name and/or origin	TotalPlants	SeedYield(g)
109 173 321 398 499 500 541 557 562 563 564 565	Proskurovskaya, Russia Kompolti F, Hungary Local, China Fatza (Turkey), France Yuznaya Odnovremenne Sozrevayushchaya 14 (USO-14), Russia USO-16, Russia USO-31, Russia Zoltonoshskaya 13, Russia Dnepropetr. 84, Russia USO-43, Russia USO-40, Russia USO-34, Russia	112 female 54 female 31 female 46 female 39 83 106 55 female 39 female 134 142 103	0 0 0 0 0 0 0 0 0 0 0 0

1 Late Head of the Department of Industrial Crops, Vavilov Research Institute (VIR), St. Petersburg, Russia
2 Chief Researcher, VIR Cannabis germplasm collection and Manager IHA/VIR Cannabis Germplasm Preservation Project
3 Projects Manager, International Hemp Association (IHA), Amsterdam, The Netherlands

Station	Latitude	Longitude
Pavlovsk, Russia Ekaterinino, Russia Ustimovka, Ukraine Kuban, Russia Trisaia, Italy	59o 44' 52o 53' 49o 10' 44o 24' 40o 12'	30o 24' 40o 29' 33o 30' 41o 32' 16o 40'