Hemant L. et al., In Vitro Cellular & Developmental Biology -Plant- 45:12–19 (2009)
Summary
Induction of high-frequency shoot regeneration using nodal segments containing axillary buds from a 1-yrold mother plants of Cannabis sativa was achieved on Murashige and Skoog (MS) medium containing 0.05–5.0 μM thidiazuron. The quality and quantity of regenerants were better with thidiazuron (0.5 μM thidiazuron) than with benzyladenine or kinetin. Adding 7.0 μM of gibberellic acid into a medium containing 0.5 μM thidiazuron slightly increased shoot growth. Elongated shoots when transferred to half-strength MS medium supplemented with 500 mg l−1 activated charcoal and 2.5 μM indole-3-butyric acid resulted in 95% rooting. The rooted plants were successfully acclimatized in soil. Following acclimatization, growth performance of 4-mo-old in vitro propagated plants was compared with ex vitro vegetatively grown plants of the same age. The photosynthesis and transpiration characteristics were studied under different light levels (0, 500, 1,000, 1,500, or 2,000 μmol m−2 s−1). An increase in photosynthesis was observed with increase in the light intensity up to 1,500 μmol m−2 s−1 and then decreased subsequently at higher light levels in both types of plants. However, the increase was more pronounced at lower light intensities below 500 μmol m−2 s−1. Stomatal conductance and transpiration increased with light intensity up to highest level (2000 μmol m−2 s−1) tested. Intercellular CO2 concentration (Ci) and the ratio of intercellular CO2 concentration to ambient CO2 (Ci/Ca) decreased with the increase in light intensity in both in vitro as well as ex vitro raised plants. The results show that in vitro propagated and hardened plants were functionally comparable to ex vitro plants of same age in terms of gas and water vapor exchange characteristics, within the limits of this study.
Objectives
large scale clonal production of screened and selected high yielding elite clones
Materials
screened and selected high-yielding C. sativa variety (MX-1) grown in a indoor cultivation facility housed at Coy-Waller laboratory, University of Mississippi.
Explant
nodal segments containing axillary buds (～1 cm in length)
Initial culture
Explants were surface-disinfected using 0.5% NaOCl and 0.1% Tween 20 for 20 min. The explants were washed in sterile distilled water three times for 5 min prior to inoculation on the culture medium.
Shoot multiplication
(Shoot proliferation and multiplication) Disinfected explants were inoculated on MS medium containing 3% (w/v) sucrose, 0.8% (w/v) type E agar (Sigma Chemical Co., St. Louis, MO) supplemented with 0.5μM TDZ in combination with 0.7μM GA3 adjusted to pH 5.7. Cultures were incubated at 25±2°C with 16-h photoperiod under fluorescent light with a photon flux of ≈52 μmol m−2 s−1.
Rooting
(Induction of rooting) The percentage of rooting from 2.5 cm length shoots was 80–95% on media 1/2-MS containing 2.5-5.0 μM IBA and 500 mg l−1 activated charcoal. Cultures were incubated at 25±2°C with 16-h photoperiod under fluorescent light with a photon flux of ≈52 μmol m−2 s−1.
Acclimation
(Acclimatization) Rooted plantlets were successfully transferred to thermocol cups containing potting mix. The plants attained 14-16 cm height exhibited 95% survival rate 8 wk after transfer.
Planting
Cultivation conditions
Traints of regenerants
The acclimatized plants exhibited normal development and no gross morphological variation was observed.