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Nikolai Lemeshev1, Lyudmila Rumyantseva2 and Robert C. Clarke3
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:
Table 1. Locations of the VIR/IHA Cannabis reproductions in 1994.
Station Latitude Longitude Pavlovsk, Russia
Ekaterinino, Russia
Ustimovka, Ukraine
Kuban, Russia
Trisaia, Italy59o 44'
52o 53'
49o 10'
44o 24'
40o 12'30o 24'
40o 29'
33o 30'
41o 32'
16o 40'
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.
References
Table 2. Results of 1994 VIR/IHA Cannabis germplasm reproduction. Pavlovsk Experiment Station
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) |
Table 3. Results of 1994 VIR/IHA Cannabis germplasm reproduction. Ekaterinino Experiment Station
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 |
Table 4. Results of 1994 VIR/IHA Cannabis germplasm reproduction. Kuban Experiment Station
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 |
Table 5. Results of 1994 VIR/IHA Cannabis germplasm reproduction. Ustimovka Experiment Station
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 |
Table 6. Results of 1994 VIR/IHA Cannabis germplasm reproduction. ENEA Trisaia, Italy
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