An Alternative In Vitro Propagation Protocol of Cannabis sativa L. (Cannabaceae) Presenting Efficient Rooting, for Commercial Production

Abstract

An alternative in vitro propagation protocol for medical Cannabis sativa L. cultivars for pharmaceutical industrial use was established. The aim of the protocol was to reduce the culture time, offering healthy and aseptic propagating material, while making the whole process more economic for industrial use. The propagation procedure was performed using plastic autoclavable vented and non-vented vessels, containing porous rooting fine-milled sphagnum peat moss-based sponges, impregnated in ½ Murashige and Skoog liquid growth medium, supplemented with indole-3-butyric acid (IBA) at various concentrations (0, 2.46, 4.92, and 9.84 µM) or by dipping nodal cuttings into 15 mM IBA aqueous solution. The highest average root numbers per cutting, 9.47 and 7.79 for high cannabidiol (H_CBD) and high cannabigerol (H_CBG) varieties, respectively, were achieved by dipping the cuttings into IBA aqueous solution for 4 min and then placing them in non-vented vessels. The maximum average root length in H_CBD (1.54 cm) and H_CBG (0.88 cm) was ascertained using 2.46 μM filter sterilized IBA in non-vented vessels. Filter-sterilized IBA at concentrations of 2.46 μM in vented and 4.92 μM in non-vented vessels displayed the maximum average rooting percentages in H_CBD (100%) and H_CBG (95.83%), respectively. In both varieties, maximum growth was obtained in non-vented vessels, when the medium was supplemented with 4.92 μM filter-sterilized IBA. Significant interactions between variety and vessel type and variety and IBA treatments were observed in relation to rooting traits. Approximately 95% of plantlets were successfully established and acclimatized in field. This culture system can be used not only for propagating plant material at an industrial scale but also to enhance the preservation and conservation of Cannabis genetic material.

Challenges and potentials of new breeding techniques in Cannabis sativa

“…Rooting is very important for successful micropropagation and has been a topic in several papers (e.g. : Zarei et al., 2021 ; Ioannidis et al., 2022a ; Kurtz et al., 2022 ; Stephen et al., 2023 ).…”

Investigation of the Effect of the Auxin Antagonist PEO-IAA on Cannabinoid Gene Expression and Content in Cannabis sativa L. Plants under In Vitro Conditions

“…However, multiplication and rooting still represent, to some extent, a bottleneck for trouble-free in vitro cultivation of cannabis. Although the rate of new scientific publications on the in vitro cultivation of cannabis is accelerating, adapting protocols for the micropropagation of different genotypes remains challenging [18][19][20][21]. Generally, the successful cultivation of plants under in vitro conditions highly depends on the genotype, the explant type, the composition of the medium, light intensity and quality, carbohydrate source, and, above all, the presence of phytohormones in the medium [22][23][24].…”

“…Generally, the successful cultivation of plants under in vitro conditions highly depends on the genotype, the explant type, the composition of the medium, light intensity and quality, carbohydrate source, and, above all, the presence of phytohormones in the medium [22][23][24]. Various phytohormones have been tested to improve cannabis micropropagation protocols, such as thidiazuron (TDZ) [17,25], 1-naphthalene acetic acid (NAA), and 6-benzylaminopurine (BAP) [26] for shoot proliferation and regeneration, and indole-3-butyric acid (IBA) [21] and NAA [27] for rooting induction, while Lata et al (2016) tested meta-topolin (mT) for a shoot and root induction from nodal segments [18]. Large-scale micropropagation, genetic conservation, and the development of plant biotechnologies for advanced new plant breeding technologies (NPBTs) might be promising tools for future research and commercial production of cannabis, although the plant is still understudied [10,[28][29][30].…”

Genetic Evaluation of In Vitro Micropropagated and Regenerated Plants of Cannabis sativa L. Using SSR Molecular Markers

“…Aiming at increasing bioactive substances with medicinal properties in Cannabis, agricultural geneticists and breeders have detected and selected several cannabis varieties or strains that produce high amounts of cannabinoids such as cannabidiol (CBD) [ 13 , 14 , 15 , 16 ], cannabigerol (CBG) [ 15 , 16 , 17 , 18 ], and Δ 9 -tetrahydrocannabinol (Δ 9 -THC) [ 19 , 20 , 21 ]. This phenotypic selection would lead to the formation of varieties rich in specific phytocannabinoids [ 22 ], which should present phytochemical profile stability.…”

“…Previous studies performed by this scientific team [ 15 , 16 ] have successfully accomplished the development of efficient in vitro micropropagation protocols for mass production. Moreover, we have developed an in vitro plant regeneration process through callus formation, i.e., indirect organogenesis, which is presented in the present study.…”