A Temporary Immersion System to Improve Cannabis sativa Micropropagation

Abstract

The aim of this study was to propagate axillary shoots of Cannabis sativa L. using liquid medium in temporary immersion bioreactors. The effect of immersion frequency (3 or 6 immersions per day), explant type (apical or basal sections), explant number (8, 10, and 16 explants), mineral medium (Murashige and Skoog half-strength nitrates, β-A and β-H, all supplemented with 2-μM metatopoline), sucrose supplementation (2, 0.5, and 0% sucrose), culture duration (4 and 6 weeks), and bioreactor type (RITA® and Plantform™) were investigated. As a result, we propose a protocol for the proliferation of cannabis apical segments in RITA® or Plantform™ bioreactors. The explants (8 per RITA® and 24 per Plantform™) are immersed for 1 min, 3 times per day in β-A medium supplemented with 2-μM metatopoline and 0.5% of sucrose and subcultured every 4 weeks. This is the first study using temporary immersion systems in C. sativa production, and our results provide new opportunities for the mass propagation of this species.

Keywords: Plantform™; RITA; bioreactors; liquid medium; mass propagation; sucrose.

Figure 1

Mati (A) and Beatriz (B) genotypes cultured in β-A semi-solid medium with 2% sucrose. Beatriz genotype cultured in RITA® with MS ½ N with 2% sucrose (C), β-A with 2% sucrose (D), MS–½N with 0.5% sucrose (E), and β-A with 0.5% sucrose (F). Mati (G) and Beatriz (H) genotypes cultured in β-A semi-solid medium with 0.5% sucrose, Beatriz genotype (I) cultured in Plantform™ with β-A medium and 0.5% sucrose. Bars, 10 mm.

Figure 2

Effect of immersion frequency (every 8 or 4 h) on proliferation rates of apical sections of Beatriz (A), Moniek (B), and Mati (C) genotypes cultured in RITA® vessels. Explants were grown in MS medium with half-strength nitrates and 2-μM metatopoline. Immersion duration was set at 1 min. Ten explants/RITA® were used for Beatriz and Moniek and 16 explants/RITA® for the Mati genotype. The data were recorded after 4 weeks of culture. The values represent the mean ± standard error. For each variable, different letters indicate significant differences at p < 0.05. NS, number of shoots per explant; MC, multiplication coefficient; SL, length of the longest shoot (mm); H, percentage of hyperhydric shoots.

Figure 3

Effect of initial explant number (8, 10, and 16 apical sections) on growth parameters of shoots of Beatriz (A) and Mati (B) genotypes cultured in RITA® vessels in MS medium with half-strength nitrates and 2-μM metatopoline. The explants were immersed for 1 min every 8 h. The data were recorded after 4 weeks of culture. The values represent the mean ± standard error. For each variable, different letters indicate significant differences at p < 0.05. NS, number of shoots per explant; MC, multiplication coefficient; SL, length of the longest shoot (mm); H, percentage of hyperhydric shoots.

Figure 4

Effect of the culture duration (4 or 6 weeks) on the growth of 8 Mati apical sections cultured in RITA® vessels with MS medium with half-strength nitrates and β-A medium. All media were supplemented with 2-μM metatopoline. The data recorded after 4 or 6 weeks of culture. The values represent the mean ± standard error. For each variable, different uppercase letters indicate significant differences in relation to the medium, and different lowercase letters indicate significant differences in relation to the duration of the culture (p < 0.05). NS, number of shoots per explant; MC, multiplication coefficient; SL, length of the longest shoot (mm); H, percentage of hyperhydric shoots.

Figure 5

Effect of sucrose and mineral medium on the growth of 8 apical sections of Beatriz genotype cultured in RITA® vessels. Explants were grown in MS medium with half-strength nitrates and in β-A medium, both supplemented with 2-μM metatopoline and 2 or 0.5% sucrose. The data were recorded after 4 weeks of culture. The values represent the mean ± standard error. For each variable, different uppercase letters indicate significant differences in relation to the medium, and different lowercase letters indicate significant differences in relation to sucrose supplementation (p < 0.05). NS, number of shoots per explant; MC, multiplication coefficient; SL, length of the longest shoot (mm); H, percentage of hyperhydric shoots.

Figure 6

Proliferation of apical vs. basal sections of Beatriz genotype cultured in RITA® vessels in β-A medium supplemented with 2-μM metatopoline. Explants (8 per vessel) were immersed for 1 min every 8 h. The data were recorded after 4 weeks of culture. The values represent the mean ± standard error. For each variable, different letters indicate significant differences at p < 0.05. NS, number of shoots per explant; MC, multiplication coefficient; SL, length of the longest shoot (mm); H, percentage of hyperhydric shoots.

Figure 7

Effect of the type of bioreactor (RITA® and Plantform^TM) on the proliferation of apical explants of cannabis genotype Beatriz cultured in β-A medium with 2 μM metatopoline and 0.5% sucrose. Explants were immersed for 1 min every 8 h. No additional aeration was provided to explants cultured in Plantform™ containers. The data were recorded after 4 weeks of culture. The values represent the mean ± standard error. For each variable, different letters indicate significant differences at p < 0.05. NS, number of shoots per explant; MC, multiplication coefficient; SL, length of the longest shoot (mm); H, percentage of hyperhydric shoots.