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The character of inheritance of differences in cannabinoid content in hemp (Cannabis sativa L.)

V. P. Sytnik1 and A. F. Stelmah2

1 The Institute of Bast Crops, Glukhov, Sumy region, Ukraine and 2 New York Academy of Sciences

        Sytnik, V. P. and A. F. Stelmah 1998. The character of inheritance of differences in cannabinoid content in hemp (Cannabis sativa L.). Journal of the International Hemp Association 6(1): 8-9. Analyses of F2 hybrid and backcross populations from crosses between inbred monoecious and dioecious populations reveal that inheritance of cannabinoid production is controlled by closely linked, but independent genes for the biosynthesis of CBD and THC. Monohybrid segregation of cannabinoid production should simplify selection for Cannabis with particular cannabinoid phenotypes.

Introduction
        In the selection process to develop hemp (Cannabis sativa L.) varieties with less psychoactivity an important area is to study is the inherited differences of the cannabinoid content of hybrid crosses. The main terpenophenolic substances in hemp are cannabidiol (CBD), tetrahydrocannabinol (THC) and the THC degradation product, cannabinol (CBN). THC is responsible for most psychoactivity. Antibiotic qualities are present in CBD. Other cannabinoids are not considered to be significant drugs. (Machbert and Lukowicz 1971; Lazurevsky and Nikolaeva 1972).
        At the Ukrainian Institute of Bast Crops, new lines of non-psychoactive varieties have been created by means of linear selection directed at lowering the cannabinoid content. Many of the lines were non-psychoactive. The results of the selections confirm the inheritance of cannabinoid content from the mother line. Data show that inheritance of cannabinoid production may be by blending, polygenic and sex-linked.
        The separate lines of hemp, practically devoid of THC and CBD, obtained by several generations of inbreeding, enabled research into the inheritance of cannabinoid content in hybrid generations.

Materials and methods
        The research on inheritance of differences in cannabinoid content of hemp was conducted in 1991-1995 at the Institute of Bast Crops of the Ukrainian Academy of Agrarian Sciences under their leadership. Double crossings of lines of monoecious hemp of the variety ‘USO-45’, with almost complete absence of THC and other cannabinoids, with families of dioecious hemp, variety ‘US-9’, containing large amounts of cannabinoids, were completed. F1 and F2 hybrids and back crosses were tested for cannabinoid content.
        Every plant of hybrid and parental forms was analyzed for cannabinoid content at biological maturity by the semi-quantitative method of thin-layer chromatography. Plants were scored for cannabinoid content.
        Genetic analysis was used to compare the modification of cannabinoid content in hybrids and parental forms. F2 hybrids and back crosses were divided into classes, based on similarity to one of the parents (P1 or P2) or the F1 hybrids (P1 X P2 or P2 X P1). A correspondence to theoretical segregation was found in the F2 on P1 or P2 : (F1+P2 or P1) and in back crosses on P1 or P2 : F2.

Table 1. Cannabinoid content of F2 hybrid and backcross combinations.
Cross Number of
plants		 Cannabinoid
content
high: low		 Segregation
Actual		 Segregation
Theoretical		 X2 p
   

Cannabidiol (CBD) content

    
F2
(P1 X P2)		 39 (F1 + P2): P1 27 : 12 29 : 10 0.68 0.25-0.50
F2
(P2 X P1)		 24 (F1 + P2): P1 18 : 6 18 : 6 0.00 1.00
BC1
(P1 X P2) X P1		 30 F1: P1 15 : 15 15 : 15 0.00 1.00
BC2
(P2 X P1) X P2		 25 F1: P1 14 : 11 12.5 : 12.5 0.36 0.50-0.75
   

Tetrahydrocannabinol (THC) content

    
F2
(P1 X P2)		 39 (F1 + P2): P1 29 : 10 29 : 10 0.00 1.00
F2
(P2 X P1)		 24 (F1 + P2): P1 17 : 7 18 : 6 0.23 0.50-0.75
BC1
(P1 X P2) X P1		 30 F1: P1 15 : 15 15 : 15 0.00 1.00
BC2
(P2 X P1) X P2		 25 F1: P1 19 : 9 12.5 : 12.5 1.69 0.10-0.25

P1 is an inbred line I6 of monoecious 'USO-45', P2 is a family of 'US-9'.

 

Results and discussion
        Individual analyses of cannabinoid contents of the offspring allowed us to divide the populations of F2 hybrids and back crosses into groups by the level of cannabinoids (high or low) shown by one of the parents and to determine the cannabinoid phenotype ratios. Two pairs of reciprocal crossings were taken as examples of segregation and the data is given in Table 1.
        In the F2 generation, a segregation of about a quarter of the plants with low CBD and THC content equal to ‘USO-45’, can be seen (Table 1). The remainder of the F2 offspring resemble either the F1 or the high cannabinoid parent ‘US-9’. In the first back cross with the parental form ‘USO-45’ (BC1), low cannabinoid content plants constitute about half of the off-spring. In the second back cross with the parental form ‘US-9’ (BC2), half of the offspring have a high cannabinoid content equal to that of ‘US-9’. The balance of plants in the back crosses resemble the F1.

Conclusions
         Monohybrid segregation indicates the monogenic character of cannabinoid levels in the crossed forms of ‘USO-45’ and ‘US-9’. This could mean, that if the control of biosynthesis of the different cannabinoid components is performed by the same gene, then the real segregation of CBD and THC, would be exactly the same in all combinations of crossings. But since, as is seen from Table 1, this is not true, then it is more logical to conclude that the content of each of the cannabinoids is controlled by closely linked independent genes.
        The presence of the above mentioned source of heredity of differences in cannabinoid components greatly simplifies the selection of hemp. It is now possible to quickly create varieties and hybrids, that are non-psychoactive, based on easily realized methods of selection.

References