with 3 ppm 2,4,5-T. More biosynthesis studies in 1987 were conducted by Braemer
and Paris ( 1987 ) for investigating the conversion offlavonoids to glucosides using
suspension cultures ofC. sativa. The cells were grown in B5 medium supplemented
with 0.5 mg/l KIN and 1 mg/l 2,4-D. After a gap of almost a decade,
Flores-Sanchez et al. ( 2009 ) employed elicitation using biotic and abiotic elicitors
on cannabinoid production inC. sativacultures. The cell cultures initiated from leaf
explants were maintained in MS medium supplemented with B5 vitamins, 1 mg/l 2,
4-D and 1 mg/l KIN. However, no cannabinoids were found in elicited or con-
trolled cultures. Lata et al. ( 2010 ) used young leaf tissues as explant for obtaining
callus on MS medium supplemented with different concentrations (0.5, 1.0, 1.5, and
2.0μM) of IAA, IBA, NAA, and 2,4-dichlorophenoxyacetic acid (2,4-D) in
combination with 1.0μM TDZ for the production of callus. The optimum callus
growth and maintenance was in 0.5μM NAA plus 1.0μM TDZ. On the other
hand, Jiang et al. ( 2015 ) have used internodes of the new cultivar Long-ma ofC.
sativaas explants for tissue culture. Best combination for callus induction has been
reported on MS medium supplemented with 1 mg/l BAP and 0.5 mg/l NAA. In
another recent study conducted by Movahedi et al. ( 2015 ), the best callus were
obtained using cotyledon explant treated with 2 mg/1 TDZ and 0.5 mg/1 IBA.
13.2.4 Agrobacterium Mediated Transformation
The use of hairy root cultures technology has revolutionized the role of plant cell
culture technology infine chemical synthesis (Toivonen 1993 ). In addition, the
hairy root technology offers an alternative and a promising in vitro source for the
production of valuable secondary metabolites as compared to plant suspension
cultures due to more biochemical and genetic stability (Liu et al. 1998 ; Farag and
Kayser 2015 ).
Thefirst reports ofCannabistransformation was reported by Feeney and Punja
( 2003 ). The agrobacterium transformation approach resulted in well developed calli
on MS medium with B5 vitamins supplemented with 5lM 2, 4 D and 1lM KIN.
However, the cultures were unresponsive to plant regeneration. One of the early
attempts of working withCannabisroot infection usingA. rhizogenesin 2006,
Wahby et al. identified secondary metabolites (choline and atropine) inCannabis
roots. Wahby et al. later extended the work in 2013, on transformation ofCannabis
roots withA. rhizogenesand transformingCannabiscalli withA. tumefaciens.
Hypocotyl of intact seedlings was reported as the most responsive material for the
establishment ofC. sativahairy root cultures, however, no regenerated shoots were
observed. Most recently, Farag and Kayser ( 2015 ) have reported hairy root cultures
ofC. sativafrom callus induced using B5 medium supplemented with 4 mg/l NAA
under dark conditions, for the production of cannabinoids. However, very low
amount of cannabinoids have been detected.
13 Micropropagation ofCannabis sativaL.—An Update 291