Cannabis has been reported to contain over 560 different compounds, out of which 120 are cannabin... more Cannabis has been reported to contain over 560 different compounds, out of which 120 are cannabinoids. Among the cannabinoids, D 9-tetrahydrocannabinol and cannabidiol are the two major compounds with very different pharmacological profile and a tremendous therapeutic potential. However, there are many challenges in bringing cannabis from grow-farms to pharmaceuticals. Among many, one important challenge is to maintain the supply chain of biomass, which is consistent in its cannabinoids profile. To maintain this process, male plants are removed from growing fields as they appear. Even with that practice, still there are fair chances of cross fertilization. Therefore, controlled indoor cultivation for screening, selection of high yielding female plants based on their cannabinoids profile, and their conservation and multiplication using vegetative propagation and/or micropropagation is a suitable path to ensure consistency in biomass material. In this chapter, the botany and propagation of elite cannabis varieties will be discussed.
Terpenes are the major components of the essential oils present in various Cannabis sativa L. var... more Terpenes are the major components of the essential oils present in various Cannabis sativa L. varieties. These compounds are responsible for the distinctive aromas and flavors. Besides the quantification of the cannabinoids, determination of the terpenes in C. sativa strains could be of importance for the plant selection process. At the University of Mississippi, a GC-MS method has been developed and validated for the quantification of terpenes in cannabis plant material, viz., α-pinene, β-pinene, β-myrcene, limonene, terpinolene, linalool, α-terpineol, β-caryophyllene, α-humulene, and caryophyllene oxide. The method was optimized and fully validated according to AOAC (Association of Official Analytical Chemists) guidelines against reference standards of selected terpenes. Samples were prepared by extraction of the plant material with ethyl acetate containing n-tridecane solution (100 µg/mL) as the internal standard. The concentration-response relationship for all analyzed terpenes ...
The genetic stability of Stevia rebaudiana Bertoni plants, propagated through in vitro stored syn... more The genetic stability of Stevia rebaudiana Bertoni plants, propagated through in vitro stored synthetic seeds was assessed. In vitro grown micro shoots of Stevia rebaudiana were encapsulated in 5% sodium alginate and 50 mM CaCl2. The synthetic seeds were stored in vitro for 4 months and re-grown under the tissue culture conditions (16-h photoperiod, 25 oC) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ 0.2 mg L-1) [1]. Well rooted plantlets were successfully transferred to a climatic controlled indoor cultivation facility for further cultivation with mother plant. Leaf samples were taken from nine, randomly selected, fully developed, healthy in vitro propagated plants and a mother plant for the molecular analysis of genetic fidelity using eight inter simple sequence repeat (ISSR) markers. Each tested primer produced clear and scorable amplification products ranging in size from about 176 bp (UBC 817) to 1354 bp (UBC 826) with an average of 5.1 products per primer. A total of 41 bands were generated, giving rise to monomorphic patterns across all 10 plantlets analyzed. No ISSR polymorphism was observed in the micropropagated plants. Furthermore, leaf samples from the plants used for molecular analysis were also evaluated for rebaudioside A and stevioside content using high performance liquid chromatography (HPLC) [2]. Our data show no significant differences (p < 0.05) in rebaudioside A and stevioside content among re-grown plants and the mother plant following four months of in vitro storage, thus confirming the clonal fidelity of Stevia rebaudiana plants. Acknowledgement: This research was partially funded by a grant from the USDA, Agricultural Research Service Specific Cooperative Agreement No. 58 – 6408 – 1-603 – 07. References: [1] Hemant Lata, Suman Chandra et al. (2013) American Journal of Plant Sciences, 4: 117 – 128. [2] Yan-Hong Wang, Bharathi Avula et al. (2015) Food Additives & Contaminants: Part A, 32: 674 – 685.
Methods in molecular biology (Clifton, N.J.), 2016
Cannabis sativa L. (Marijuana; Cannabaceae), one of the oldest medicinal plants in the world, has... more Cannabis sativa L. (Marijuana; Cannabaceae), one of the oldest medicinal plants in the world, has been used throughout history for fiber, food, as well as for its psychoactive properties. The dioecious and allogamous nature of C. sativa is the major constraint to maintain the consistency in chemical profile and overall efficacy if grown from seed. Therefore, the present optimized in vitro propagation protocol of the selected elite germplasm via direct organogenesis and quality assurance protocols using genetic and chemical profiling provide an ideal pathway for ensuring the efficacy of micropropagated Cannabis sativa germplasm. A high frequency shoot organogenesis of C. sativa was obtained from nodal segments in 0.5 μM thidiazuron medium and 95 % in vitro rhizogenesis is obtained on half-strength MS medium supplemented with 500 mg/L activated charcoal and 2.5 μM indole-3-butyric acid. Inter Simple Sequence Repeats (ISSR) and Gas Chromatography-Flame Ionization Detection (GC-FID) are...
Journal of Applied Research on Medicinal and Aromatic Plants, 2015
ABSTRACT The effect of different levels of photon flux densities (000, 400, 800, 1200, 1600 and 2... more ABSTRACT The effect of different levels of photon flux densities (000, 400, 800, 1200, 1600 and 2000 μmol m−2 s−1) on gas and water vapor characteristics of four high Δ9-tetrahydrocannabinol (Δ9-THC) yielding drug type varieties (HPM, K2, MX and W1) of Cannabis sativa was studied. Plants of each variety were grown from seeds. On flowering, male plants were removed and vegetatively propagated clones of selected female plants were used for gas and water vapor studies at different photosynthetic photon flux densities (PPFDs). Our data show an increasing trend in photosynthesis (PN), transpiration (E) and stomatal conductance (gCO2) with increase in PPFD up to 2000 μmol m−2 s−1 in all varieties at optimum growth temperature (25 ± 3 °C). However, the magnitude of increase and maximum rate of PN (PN max) varied considerably with the varieties. Highest PN was observed in W1 followed by MX, K2 and HPM. Water use efficiency (WUE) in W1, MX and HPM increased with PPFDs up to the highest level tested, whereas, in K2 the highest WUE was observed at 1600 μmol m−2 s−1. Our results suggest that this species is able to use high level of PPFDs for its PN and therefore, may be cultivated in sun exposed areas in the field or under high PPFDs using indoor grow lights for the optimum growth. Strict control of other environmental factors, however, needs to be maintained while growing the plants indoor.
Resumo: Mesmo sem ordenar uma rígida teoria de poesia, Manuel Bandeira reverbera como poucos na p... more Resumo: Mesmo sem ordenar uma rígida teoria de poesia, Manuel Bandeira reverbera como poucos na poesia brasileira. Desde a geração que fomentou o primeiro modernismo nos anos de 1920, passando pelos elegíacos de 1930 e 1940, por concretistas e neoconcretistas de 1950 e 1960, até a produção febril da atualidade, Bandeira ecoa em artífices moderados e radicais. Esparsas por crônicas, ensaios, memórias, cartas ou poemas, suas reflexões sobre o fazer poético desenham, no entanto, a busca por uma identidade artística orgânica, em diálogo constante com concepções decisivas para a lírica do século XX. Essas cogitações de poeta e estudioso de literatura que foi partem, em geral, de uma tensão de base entre arrebatamento e raciocínio, a qual, desde o período romântico, vem pautando as ações de um número grande de poetas e pensadores da poesia. Tal impasse, entre a poesia de inspiração e a tendência ao formalismo, assunto central do presente artigo, leva em conta, em Bandeira, certos postulados teóricos e poéticos de Stéphane Mallarmé, Paul Valéry e Mário de Andrade. Alguns intérpretes relevantes da obra do bardo serão, além disso, acionados ao longo da discussão, tais como Álvaro Lins, Davi Arrigucci Jr.
Cannabissativa L. (Cannabaceae) is an important medicinal plant that serves as a source of Cannab... more Cannabissativa L. (Cannabaceae) is an important medicinal plant that serves as a source of Cannabinoids, a unique class of terpenophenolic compounds which accumulates mainly in the glandular trichomes of the plant. Currently, about 110 phytocannabinoids have been isolated from C. sativa, the major biologically active compound being Δ9-tetrahydrocannabinol, commonly referred to as THC. Besides its psychoactivity, THC possesses analgesic, antiinflammatory, appetite stimulant, and antiemetic properties making it a very promising therapeutic agent especially for cancer and AIDS patients. Cannabidiol, Cannabichromene, Cannabigerol, Cannabinol, and Tetrahydrocannabivarin are other major bioactive phytocannabinoids present in the cannabis plant. Through a contract with the National Institute on Drug Abuse (NIDA), The University of Mississippi has been carrying out a variety of research activities dealing with cannabis, including growing, harvesting, and processing the cannabis biomass for research purposes making it available for licensed researchers across the country through NIDA. This chapter describes the role of biotechnology and our efforts to propagate C. sativa for the production of phytocannabinoids. This includes, screening of high yielding genotypes based on their chemical profile, propagation of these genotypes using biotechnological tools, comparison of micropropagated plants with the mother plants for consistency of chemical and genetic profiles and the utility of micropropagation in the conservation of elite clones for future use.
Cannabis has been reported to contain over 560 different compounds, out of which 120 are cannabin... more Cannabis has been reported to contain over 560 different compounds, out of which 120 are cannabinoids. Among the cannabinoids, D 9-tetrahydrocannabinol and cannabidiol are the two major compounds with very different pharmacological profile and a tremendous therapeutic potential. However, there are many challenges in bringing cannabis from grow-farms to pharmaceuticals. Among many, one important challenge is to maintain the supply chain of biomass, which is consistent in its cannabinoids profile. To maintain this process, male plants are removed from growing fields as they appear. Even with that practice, still there are fair chances of cross fertilization. Therefore, controlled indoor cultivation for screening, selection of high yielding female plants based on their cannabinoids profile, and their conservation and multiplication using vegetative propagation and/or micropropagation is a suitable path to ensure consistency in biomass material. In this chapter, the botany and propagation of elite cannabis varieties will be discussed.
Terpenes are the major components of the essential oils present in various Cannabis sativa L. var... more Terpenes are the major components of the essential oils present in various Cannabis sativa L. varieties. These compounds are responsible for the distinctive aromas and flavors. Besides the quantification of the cannabinoids, determination of the terpenes in C. sativa strains could be of importance for the plant selection process. At the University of Mississippi, a GC-MS method has been developed and validated for the quantification of terpenes in cannabis plant material, viz., α-pinene, β-pinene, β-myrcene, limonene, terpinolene, linalool, α-terpineol, β-caryophyllene, α-humulene, and caryophyllene oxide. The method was optimized and fully validated according to AOAC (Association of Official Analytical Chemists) guidelines against reference standards of selected terpenes. Samples were prepared by extraction of the plant material with ethyl acetate containing n-tridecane solution (100 µg/mL) as the internal standard. The concentration-response relationship for all analyzed terpenes ...
The genetic stability of Stevia rebaudiana Bertoni plants, propagated through in vitro stored syn... more The genetic stability of Stevia rebaudiana Bertoni plants, propagated through in vitro stored synthetic seeds was assessed. In vitro grown micro shoots of Stevia rebaudiana were encapsulated in 5% sodium alginate and 50 mM CaCl2. The synthetic seeds were stored in vitro for 4 months and re-grown under the tissue culture conditions (16-h photoperiod, 25 oC) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ 0.2 mg L-1) [1]. Well rooted plantlets were successfully transferred to a climatic controlled indoor cultivation facility for further cultivation with mother plant. Leaf samples were taken from nine, randomly selected, fully developed, healthy in vitro propagated plants and a mother plant for the molecular analysis of genetic fidelity using eight inter simple sequence repeat (ISSR) markers. Each tested primer produced clear and scorable amplification products ranging in size from about 176 bp (UBC 817) to 1354 bp (UBC 826) with an average of 5.1 products per primer. A total of 41 bands were generated, giving rise to monomorphic patterns across all 10 plantlets analyzed. No ISSR polymorphism was observed in the micropropagated plants. Furthermore, leaf samples from the plants used for molecular analysis were also evaluated for rebaudioside A and stevioside content using high performance liquid chromatography (HPLC) [2]. Our data show no significant differences (p < 0.05) in rebaudioside A and stevioside content among re-grown plants and the mother plant following four months of in vitro storage, thus confirming the clonal fidelity of Stevia rebaudiana plants. Acknowledgement: This research was partially funded by a grant from the USDA, Agricultural Research Service Specific Cooperative Agreement No. 58 – 6408 – 1-603 – 07. References: [1] Hemant Lata, Suman Chandra et al. (2013) American Journal of Plant Sciences, 4: 117 – 128. [2] Yan-Hong Wang, Bharathi Avula et al. (2015) Food Additives & Contaminants: Part A, 32: 674 – 685.
Methods in molecular biology (Clifton, N.J.), 2016
Cannabis sativa L. (Marijuana; Cannabaceae), one of the oldest medicinal plants in the world, has... more Cannabis sativa L. (Marijuana; Cannabaceae), one of the oldest medicinal plants in the world, has been used throughout history for fiber, food, as well as for its psychoactive properties. The dioecious and allogamous nature of C. sativa is the major constraint to maintain the consistency in chemical profile and overall efficacy if grown from seed. Therefore, the present optimized in vitro propagation protocol of the selected elite germplasm via direct organogenesis and quality assurance protocols using genetic and chemical profiling provide an ideal pathway for ensuring the efficacy of micropropagated Cannabis sativa germplasm. A high frequency shoot organogenesis of C. sativa was obtained from nodal segments in 0.5 μM thidiazuron medium and 95 % in vitro rhizogenesis is obtained on half-strength MS medium supplemented with 500 mg/L activated charcoal and 2.5 μM indole-3-butyric acid. Inter Simple Sequence Repeats (ISSR) and Gas Chromatography-Flame Ionization Detection (GC-FID) are...
Journal of Applied Research on Medicinal and Aromatic Plants, 2015
ABSTRACT The effect of different levels of photon flux densities (000, 400, 800, 1200, 1600 and 2... more ABSTRACT The effect of different levels of photon flux densities (000, 400, 800, 1200, 1600 and 2000 μmol m−2 s−1) on gas and water vapor characteristics of four high Δ9-tetrahydrocannabinol (Δ9-THC) yielding drug type varieties (HPM, K2, MX and W1) of Cannabis sativa was studied. Plants of each variety were grown from seeds. On flowering, male plants were removed and vegetatively propagated clones of selected female plants were used for gas and water vapor studies at different photosynthetic photon flux densities (PPFDs). Our data show an increasing trend in photosynthesis (PN), transpiration (E) and stomatal conductance (gCO2) with increase in PPFD up to 2000 μmol m−2 s−1 in all varieties at optimum growth temperature (25 ± 3 °C). However, the magnitude of increase and maximum rate of PN (PN max) varied considerably with the varieties. Highest PN was observed in W1 followed by MX, K2 and HPM. Water use efficiency (WUE) in W1, MX and HPM increased with PPFDs up to the highest level tested, whereas, in K2 the highest WUE was observed at 1600 μmol m−2 s−1. Our results suggest that this species is able to use high level of PPFDs for its PN and therefore, may be cultivated in sun exposed areas in the field or under high PPFDs using indoor grow lights for the optimum growth. Strict control of other environmental factors, however, needs to be maintained while growing the plants indoor.
Resumo: Mesmo sem ordenar uma rígida teoria de poesia, Manuel Bandeira reverbera como poucos na p... more Resumo: Mesmo sem ordenar uma rígida teoria de poesia, Manuel Bandeira reverbera como poucos na poesia brasileira. Desde a geração que fomentou o primeiro modernismo nos anos de 1920, passando pelos elegíacos de 1930 e 1940, por concretistas e neoconcretistas de 1950 e 1960, até a produção febril da atualidade, Bandeira ecoa em artífices moderados e radicais. Esparsas por crônicas, ensaios, memórias, cartas ou poemas, suas reflexões sobre o fazer poético desenham, no entanto, a busca por uma identidade artística orgânica, em diálogo constante com concepções decisivas para a lírica do século XX. Essas cogitações de poeta e estudioso de literatura que foi partem, em geral, de uma tensão de base entre arrebatamento e raciocínio, a qual, desde o período romântico, vem pautando as ações de um número grande de poetas e pensadores da poesia. Tal impasse, entre a poesia de inspiração e a tendência ao formalismo, assunto central do presente artigo, leva em conta, em Bandeira, certos postulados teóricos e poéticos de Stéphane Mallarmé, Paul Valéry e Mário de Andrade. Alguns intérpretes relevantes da obra do bardo serão, além disso, acionados ao longo da discussão, tais como Álvaro Lins, Davi Arrigucci Jr.
Cannabissativa L. (Cannabaceae) is an important medicinal plant that serves as a source of Cannab... more Cannabissativa L. (Cannabaceae) is an important medicinal plant that serves as a source of Cannabinoids, a unique class of terpenophenolic compounds which accumulates mainly in the glandular trichomes of the plant. Currently, about 110 phytocannabinoids have been isolated from C. sativa, the major biologically active compound being Δ9-tetrahydrocannabinol, commonly referred to as THC. Besides its psychoactivity, THC possesses analgesic, antiinflammatory, appetite stimulant, and antiemetic properties making it a very promising therapeutic agent especially for cancer and AIDS patients. Cannabidiol, Cannabichromene, Cannabigerol, Cannabinol, and Tetrahydrocannabivarin are other major bioactive phytocannabinoids present in the cannabis plant. Through a contract with the National Institute on Drug Abuse (NIDA), The University of Mississippi has been carrying out a variety of research activities dealing with cannabis, including growing, harvesting, and processing the cannabis biomass for research purposes making it available for licensed researchers across the country through NIDA. This chapter describes the role of biotechnology and our efforts to propagate C. sativa for the production of phytocannabinoids. This includes, screening of high yielding genotypes based on their chemical profile, propagation of these genotypes using biotechnological tools, comparison of micropropagated plants with the mother plants for consistency of chemical and genetic profiles and the utility of micropropagation in the conservation of elite clones for future use.
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