13

Selection for Non-Psychoactive Hemp Varieties
(Cannabis sativa L.) in the CIS (former USSR)

V. G. Virovets

Institute of Fiber Crops, Lenin Street 45, Glukhov, Sumy region, 245130 Ukraine

    Virovets, V. G. 1996.  Selection for non-psychoactive hemp varieties (Cannabis sativa L.) in the CIS (former USSR). Journal of the International Hemp Association 3(1): 13-15.  Results of research on the creation of new non-psychoactive hemp varieties are presented.  These hemp varieties are notable for their minimal cannabinoid content including tetrahydrocannabinol (THC), which excludes the possibility of the use of hemp as a drug.  Some varieties were produced that contain no cannabinoids and represent valuable initial material for further selection.

Introduction
   Hemp has been cultivated on a large scale as a bast crop because it allows relatively cheap fiber production.  Traditional use of Cannabis for psychoactive purposes in the southeastern countries of the former USSR is locally limited and it is only used by some ethnic groups at their places of residence.  As a rule, the industrial cultivation of hemp was excluded in these circumstances.  Due to a growing demand for articles made of natural fibers and as a raw material for paper making prospects for hemp use were increasing, but were curtailed by an outbreak of drug use.  In this connection, the Department of the Interior and the Department of Health and Agriculture of the former USSR in 1973 issued an ultimatum to the former All-Union Scientific and Research Institute of Bast Crops to either create non-psychoactive varieties or stop cultivating hemp.
    Under these circumstances, selection was used as a method to solve the problem of creating non-psychoactive varieties.  Because of the natural lack of varieties without cannabinoids, as well as a lack of a suitable selection method, we found ourselves in great difficulty.  The theoretical premise for selection was a law on hereditary variability formulated by N. I. Vavilov.  Successful selection to lower the levels of harmful substances had been successful in lupine, rape, tobacco, hops and other crops.
    The creation of hemp varieties was preceded by working out a selection method and by additional study of the psychoactive cannabinoids.  Three phenolic compounds prevail : cannabidiol (CBD), tetrahydrocannabinol (THC) and cannabinol (CBN), the first two existing predominantly as their carboxylic acid forms.
    Cannabinoids accumulate in different amounts in all parts of the plant.   Glandular hairs as well as cystolithic hairs are found on the upper leaves of the inflorescence.  The heads of the glandular hairs serve as reservoir for the resinous fluid.  The glandular hairs occur on the external surface of the bracts and up to 90% of their mass is cannabinoids (Clarke 1981, Hammond & Mahlberg 1973, Hammond & Mahlberg 1977).

Materials and methods
   During the selection process, we used varieties which had been created earlier at the Institute of Bast Crops and other establishments, which represent mainly Middle-Russian and Southern hemp types.  We used the middle-Russian hemp varieties Glukhovskaya 10, Starooskolskaya uluchshennays (SOU), Yermakovskaya local landrace and others.  Southern hemp was represented mainly by the following foreign varieties and samples : Szegedi 9 and Kompolti (Hungary), Fibrimon 56 (France), Napoletana and CS (Italy) and Belobzhevskaya (Poland).  The initial material was obtained mainly by the classical methods of selection and hybridization.  The apical part of an inflorescence was taken from each plant to determine cannabinoid content by thin-layer chromatography (TLC).  Controlled cross-pollination and field analyses of the plants for cannabinoids were made in separate plant nurseries.

Results and discussions
   At the beginning of the work, the maximum threshold for tetrahydrocannabinol (THC) content as the main cannabinoid component was 0.3 % and was then lowered to 0.2 %.  Recently, this index has been lowered twice again to 0.1 %.  The initial selection step was to study all of the known hemp varieties as well as samples from the world hemp collection.  The results of our past researches into cannabinoid contents were rather broad (Zakharova 1973).  There were no varieties that completely lacked cannabinoids.  A rather high content of these substances was found in some varieties and hybrids was determined and they were excluded from cultivation (Table 1).  The results obtained have shown that hemp cultivated in more northerly areas is naturally rich in cannabinoids, and that the only way of lowering their content is through selection.

    We chose the method of family-group selection among highly productive varieties as a method of lowering cannabinoid content.  Preliminary research was made with hybrids from the crossing of separate low yield hemp strains (collection of the All-Union Institute of Agriculture) with low cannabinoid content.   However, the work with low productivity hybrids was discontinued because of the necessity to produce industrial varieties with high fiber and seed yields.  As a result of this intensive selection work the first three highly productive hemp varieties, (USO-14, USO-16 and Dnepro-petrovskaya monoecious-6) containing less than 0.2 % THC (Table 2), were created and subsequently distributed (beginning in 1980) (Virovets et al. 1980).  The monoecious hemp variety USO-16 was also characterized by high fiber and seed yield.  Advanced hemp farms in the Cherkassy region harvested 1.7-1.8 t/ha of fiber and 0.7-0.8 t/ha of seeds.  Expansion of the selection work was carried out simultaneously with the elaboration of a selection technique and a method for the determination of cannabinoid content.

    Since hemp populations are characterized by panmixia, there was a tendency for spontaneous maintenance of heterozygosity of a given trait, in spite of repeated strict selection.  However, on the whole the proportion of plants with a complete absence or minimal quantity of THC increased (Table 3).

    Artificially excluding plants with high cannabinoid content from the population (before flowering) convinced us of the efficiency of controlled pollination.  In 1980, only the population of individuals without THC were allowed to flower.  The next year the number of cannabinoid-free plants in the population doubled from 39.9 % to 76.3 %.
    As the demand for increased plant analysis throughput accelerated, the rather labor-intensive thin layer chromatography (TLC) technique was replaced by a simplified method that made it possible to determine cannabinoid content rapidly and easily.  This provided an opportunity to divide plants on qualitative characters : varieties containing some THC and varieties without any THC (Sazhko et al. 1985).   The rapidity of analysis of one plant (11 sec.) with simultaneous removal of undesirable individuals has made it possible to extend investigations and develop a method for effective selection of new initial material for breeding.
    Simultaneous selection for both cannabinoids and productivity has made it possible for the new varieties to occupy great industrial areas in the Middle-Russian hemp sowing zone and in the Northern Caucasus.  The variety USO-14 has been the best among the southern-maturing hemp varieties, with stem fiber content of 30 % or higher.   USO-16 and Dnepropetrovskaya monoecious-6 are also examples of varieties with reduced cannabinoid content.  According to data from the Central Laboratory of the State Commission on Agricultural Testing of the former USSR for 1978-79, the THC content of the variety USO-14 has been lowered to 0.08 % while in the initial variety USO-1 this index was equal to 0.44-0.49 %.
    The accumulated experience of putting this selection method into practice, combined with a good choice of diverse initial breeding stocks, has made it possible to obtain a number of new varieties, among them the variety Zolotonoshskaya USO-11.  In 1984, it was distributed in the Cherkassy and Poltava regions of the Ukraine to replace the variety USO-16.  In 1987, in the Sumy region of the Ukraine, and in the Oryol, Penza and other regions of Russia, the new variety USO-31 has been distributed and is no less productive than the variety USO-14, which contains twice the cannabinoids.  In the regions of the Northern Caucasus were distributed the new southern hemp varieties Zolotonoshskaya-13 (1986) and Zenitsa (1991).  These are also characterized by high productivity and lowered THC content (Goloborod’ko et al. 1993).  In 1993, a decision was made to put the southern hemp Dneprovskaya monoecious-14 into production in the Ukraine.
    The main achievement of the intensive selection work has been the creation of the two hemp varieties USO-42 and USO-45 with not only a minimal quantity of THC, or even its complete absence, but a considerably lowered content of the other cannabinoid components as well (Goloborod’ko et al. 1993).  These new standard varieties also possess resistance to infections and pests.

Conclusion
   We have studied the peculiarities of accumulation and localization of cannabinoids in plant organs, the inheritance of cannabinoid production and the modification of cannabinoid production depending on different factors.  This has made it possible to elaborate a selection method and to obtain considerable lowering of cannabinoid quantity.
    With further selection work, we foresee creation of new varieties with high yield indices, a stable minimum THC content (or complete absence of THC) and other cannabinoids, and resistance to pests and infections, with the aim of excluding hemp as a potential drug crop.

References

• Clarke, R. C. 1981. Marijuana Botany Ronin Press, Berkeley, CA USA: 197 pp.
• Goloborod’ko, P. et al. 1993.  Sozdaniye visokoproduktivnykh sortov konopli, ne obladayushckikh narkoticheskoi aktivnostyu (Creation of highly pro-ductive non-psychoactive hemp varieties) Mezhdunarodniy sel. - khoz. zhurnal 4: 50-54.
• Hammond, Ch. T. and Mahlberg, P. G. 1973.  Morphology of glandular hairs of Cannabis sativa from scanning electron microscopy Amer. J. Botany 60(6): 524-528.
• Hammond Ch. T. and Mahlberg, P. G., 1977.  Morphogenesis of capitate glandular hairs of Cannabis sativa (Cannabaceae) Amer. J. Botany 64(8): 1023-1031.
• Sazhko, M. M. et al. 1985.  Sposob otsenki rasteniy konopli na nalichiye kannabinoidov.  Avtorskoye svidetelstvo USSR, N 1188926 ot 01.07.1985.   (The method of determination of cannabinoid content in hemp plants.   Author’s certificate of the USSR, N 1188926 dated 01.07.1985).
• Virovets, V. G. et al. 1980.  Novje sorta odnodomnoi konopli.   (New vari-eties of monoecious hemp). Len i konoplya I: 28-29.
• Zakharova, N. S. 1973.  Soderzhaniye kannabinoidov v kollektsion-nykh obraztsakh konopli.  (Cannabinoid content in collected hemp samples.   Synopsis of thesis for a candidate’s degree in agriculture: 24).