The physical and chemical features of Cannabis plants grown in the United Kingdom of Great Britain and Northern Ireland from seeds of known origin
The physical and chemical features of Cannabis plants grown in the United Kingdom of Great Britain and Northern Ireland from seeds of known origin
Cannabis plants have been grown in the United Kingdom of Great Britain and Northern Ireland from seeds taken from seizures of cannabis of known geographical origin and chemistry. The gross physical appearance and cannabinoid patterns of many of the cannabis samples produced in the United Kingdom were closely related to those of the parents. However, some notable exceptions were recorded. There were wide variations in actual tetrahydrocannabinol content between plants grown from different seedstock and rather smaller variations within the groups grown from the same seedstock. Cannabis produced in the United Kingdom had higher tetra-hydrocannabinolic acid/tetrahyrocannabinol ratios than imported material.
In a study of the physical and chemical features of imported Cannabis 1products, Baker and others [ 1] found that the gross physical appearance and the cannabinoid pattern as revealed by thin-layer chromatography (TLC) correlated with the geographical origin for most of the samples examined. Consequently, if these features of a sample of unknown origin were compared with those of samples of known origin, an opinion as to the geographical origin of the unknown sample could be formed. Such information may be useful in criminal investigations relating to illicit Cannabisproduct importations and may aid the international control of the substance [ 2-5] . It may also be of considerable importance to know whether a seizure made within the United Kingdom was of foreign origin or whether the material was grown within the United Kingdom. It is thus necessary to study the physical and chemical features of Cannabis grown in the United Kingdom and in particular, to examine the cannabinoid patterns of Cannabis plants grown in the United Kingdom from seeds which are not only of known geographical origin but which are also derived from imported cannabis samples of known cannabinoid pattern and content.
Seeds from which Cannabis can be grown in the United Kingdom may be derived from three principal sources: ( a) from seed sold within the United Kingdom for fish bait or bird seed; ( b) from imported cannabis samples; and ( c) from fertile seeds produced from plants cultivated in the United Kingdom. Seeds sold legally within the United Kingdom have been shown to have poor germination rates [ 6] and are therefore unlikely to be used widely by those engaged in illicit Cannabis cultivation. The majority of seizures of cannabis examined at the Laboratory of the Government Chemist, London, contained fertile seeds. These were, however, unlikely to have been incorporated into cigarettes as they had negligible or zero cannabinoid content [ [ 6] , [ 7] ] and they thus provided a ready source of fertile seeds for cultivation. It was also necessary to study the physical and chemical features of plants grown from fertile seeds which themselves were produced in the United Kingdom.
The term Cannabis in this paper refers to Cannabis sativaL.; cannabis to marijuana; and cannabis resin to hashish.
A limited number of studies of Cannabis plants grown in the United Kingdom from seeds of known origin have been made. De Faubert Maunder [ 8] made a comparative study, using TLC, of the tetrahydrocannabinol (THC) content of an imported Nigerian cannabis seizure with plants grown in the United Kingdom from seeds contained therein. The original seizure and the siblings all showed the same basic cannabinoid pattern, namely absence of cannabidiol (CBD) and the presence of THC. Although TLC is essentially non-quantitative, de Faubert Maunder estimated that the siblings all contained less THC than the parent. A second study of plants grown from seeds derived from a seizure of South African cannabis gave similar results. Fairbairm and others [ 9] grew plants from seeds of known origin (although the cannabinoid pattern was not published) and examined the cannabinoid contents of the resulting plants. A Nepalese strain grown in different years showed different CBD and THC contents, but very similar CBD/THC ratios. A Turkish strain contained principally CBD with no THC content reported, and a Thai strain contained principally THC with a small amount of CBD. The authors recorded that the cannabinoids occurred in the plants principally as their acids. These same authors also observed [ 10] that conditions of growth affected the cannabinoid content by showing that plants grown in greenhouses had different THC content from plants grown from the same seed in the open. Care was taken in sampling as previous work had shown [ 9] that different parts of the same plant had differing canna-binoid contents.
Fairbairn and Liebmann [ 11] grew 12 strains of Cannabis in the open in southern England and found considerable variations between plants of the same strain grown together and harvested at the same time. Considerable variations within plants were once again noted. However, the basic cannabinoid ratios remained the same in plants of the same strain despite these quantitative variations. Results from other groups of workers who had grown plants from the same batch of seeds in different parts of the world indicated that the same qualitative picture of cannabinoid pattern was always exhibited [ [ 7] , [ 12] , [ 13] ]. On the basis of these observations, Fairbairn and Liebmann [ 11] suggested that there may be two chemical races of Cannabis plants, one being THC-rich and the other CBD-rich. They considered that this ratio was independent of environmental conditions, but qualified their opinion by suggesting that several generations of plants should be grown to confirm this.
Rowan and Fairbairn [ [ 6] , [ 14] ] presented further evidence for the existence of two chemical races of Cannabis in a study of the cannabinoid patterns of Cannabis seedlings. THC-rich and CBD-rich patterns were shown to be established when plants were small. In the THC-rich strain, cannabichromene (CBC) was present, whereas in the CBD-rich strain, CBC was absent.
There have been a number of studies of Cannabis plants grown from seeds of known origin in other parts of the world. Fetterman and others [ 7] in a study of Mexican and Turkish seeds grown in Mississippi, United States of America, proposed that Cannabisplants fell into two groups which were termed phenotypes, corresponding to fibre-producing and to drug-producing plants. These authors suggested that the phenotypes should be classified as phenotype I (drug-type) if the ratio (% THC + % CBN)/(% CBD) was greater than 1.0 and phenotype II (fibre-type) if this ratio was less than 1.0. All plants of known origin and history could be classified using this system; only plants of unknown history could not be so classified and the authors suggested that these samples could have contained mixtures of different types of cannabis. From their data, the authors concluded, the phenotype of one variety of cannabis remained the same regardless of the year of planting or place of growth of the sample analysed. In a related study, Doorenbos and others [ 15] found most samples to have a phenotype ratio greater than 5 or less than 0.2. They concluded, as a result of their studies of cannabinoid content of plants of the same variant grown in different countries and plants grown through three generations, that environmental factors were not as important as heredity in determining cannabinoid patterns of Cannabis. The authors noted the different cannabinoid contents in plants of the same variant grown under different conditions and that most of the cannabinoids were present as their acids. Ohlsson and others [ 16] , in a study of Cannabis plants grown in Sweden, concluded that the type of cannabinoid produced by the plant depended on the seed and that the influence of climate was limited. Turner and Hadley [ 17] grew three generations of plants from seeds originating in South Africa and noted some fluctuation in cannabinoid content, particularly an increase in cannabigerol monomethyl ether (CBGM) percentages. The authors point out that no major attempt was made to avoid cross-pollination and so the significance of the increase in CBGM content (a cannabinoid in plants from north-east Asia [ 18] ) cannot be properly assessed. Small and Beckstead [ 12] grew plants from 350 different seeds at Ottawa, Canada, and increased the number of phenotypes by the addition of a third group where CBD/THC = 1.0 and a fourth group from north-east Asia containing CBGM. These authors correlated phenotype with the latitude of the seed source as follows: phenotype I from south of 30°N had THC > 0.3% and CBD < 0.5% and female plants similar in cannabinoid content to male plants; phenotype II had THC > 0.3% (in female plants) and CBD > 0.5% and originated from north of 30°N with female plants of much higher cannabinoid content than males; phenotype III had THC < 0.3% and CBD > 0.5%, but was otherwise similar to phenotype II, and phenotype IV contained CBGM. The authors considered that climate might affect the actual amount of THC in a plant, with a more southerly location meaning a longer growing season resulting in most high-THC (drug-type) plants being produced from seeds originating from south of 30°N. Krejci and others [ 19] demonstrated that seeds from Turkish fibre-producing plants, even when grown under favourable conditions did not produce appreciable amounts of THC and that seeds derived from South Africa and Thailand (drug-type), even when grown under unfavourable conditions, still produced relatively large amounts of THC. Boucher and others [ 20] grew South African Cannabis in France and observed that there were two types, either tetrahydrocannabinolic acid (THCA)-rich or tetrahydrocannabinvarinic acid (THVA)-rich, an observation also recorded by Field and Arndt [ 21] and Baker and others [ 1] . After three generations, however, all the THVA-rich plants had become THCA-rich, indicating that THCA/THVA ratios might be under environmental control. Both Novotny and others [ 22] and Turner and others [ 23] suggested that minor cannabinoids might be liable to variations when plants were grown in different environments.
As a result of the studies described above, the majority of workers have concluded that although actual THC content, and possibly minor cannabinoid content, may be under environmental control, the ratios of the principal cannabinoids CBD and THC and the absence of the former in some Cannabissamples [ 1] were controlled by the genetic material in the seed. However, there was a need for multigeneration growing trials before this opinion could be confirmed [ 11] .
Although the geographical origin of the seeds was known in many of the studies described, in no case had the parent material (as produced before export) been adequately analysed in order that changes (if any) produced by growing seeds in a totally alien environment could be observed. It was therefore considered to be both necessary and useful to grow plants from seeds whose origin was known and whose parent chemistry could be studied in detail. The Laboratory of the Government Chemist was fortunate in possessing a large number of cannabis samples of known origin containing fertile seeds which were used as a basis for a study of Cannabis grown in the United Kingdom.
Seeds were taken from illicitly imported samples of cannabis of known origin. The country of origin was assigned by taking into account information from the carrier, from Officers of Her Majesty’s Customs and Excise and from the route of importation. The cannabis samples were chosen for having particularly characteristic cannabinoid patterns or content which might be easily followed through subsequent generations. Although homogeneity of seeds within a seizure could not be completely guaranteed, the homogeneity of seizures as regards cannabinoid pattern made it a reasonable assumption that seeds would also be homogeneous.
The first generation of United Kingdom grown plants of Indian and Thai origin were cultivated indoors in a poorly lighted and unheated room in 1979. The second generation of the above plants, together with the first generation of the others listed in table 1 were
Hermaphroditism in Marijuana (Cannabis sativa L.) Inflorescences – Impact on Floral Morphology, Seed Formation, Progeny Sex Ratios, and Genetic Variation
Punja ZK and Holmes JE (2020) Hermaphroditism in Marijuana (Cannabis sativa L.) Inflorescences – Impact on Floral Morphology, Seed Formation, Progeny Sex Ratios, and Genetic Variation. Front. Plant Sci. 11:718. https://doi.org/10.3389/fpls.2020.00718.
Cannabis sativa L. (hemp, marijuana) produces male and female inflorescences on different plants (dioecious) and therefore the plants are obligatory out-crossers. In commercial production, marijuana plants are all genetically female; male plants are destroyed as seed formation reduces flower quality. Spontaneously occurring hermaphroditic inflorescences, in which pistillate flowers are accompanied by formation of anthers, leads to undesired seed formation; the mechanism for this is poorly understood. We studied hermaphroditism in several marijuana strains with three objectives: (i) to compare the morphological features of this unique phenotype with normal male flowers; (ii) to assess pollen and seed viability from hermaphroditic flowers; and (iii) to assess the effect of hermaphroditism on progeny male:female (sex) ratios and on genetic variation using molecular methods. The morphological features of anthers, pollen production and germination in hermaphroditic flowers and in staminate inflorescences on male plants were compared using light and scanning electron microscopy. Seeds produced on hermaphroditic plants and seeds derived from cross-fertilization were germinated and seedlings were compared for gender ratios using a PCR-based assay as well as for the extent of genetic variation using six ISSR primers. Nei’s index of gene diversity and Shannon’s Information index were compared for these two populations. The morphology of anthers and pollen formation in hermaphroditic inflorescences was similar to that in staminate flowers. Seedlings from hermaphroditic seeds, and anther tissues, showed a female genetic composition while seedlings derived from cross-fertilized seeds showed a 1:1 male:female sex expression ratio. Uniquely, hermaphroditic inflorescences produced seeds which gave rise only to genetically female plants. In PCR assays, a 540 bp size fragment was present in male and female plants, while a 390 bp band was uniquely associated with male plants. Sequence analysis of these fragments revealed the presence of Copia-like retrotransposons within the C. sativa genome which may be associated with the expression of male or female phenotype. In ISSR analysis, the percentage of polymorphic loci ranged from 44 to 72% in hermaphroditic and cross-fertilized populations. Nei’s index of gene diversity and Shannon’s Information index were not statistically different for both populations. The extent of genetic variation after one generation of selfing in the progeny from hermaphroditic seed is similar to that in progeny from cross-fertilized seeds.