13.2.5 Regeneration
Efficient plant regeneration protocol is essential for mass production of pharma-
ceutically superior elite clones ofC. sativa. The induction of direct shoot regen-
eration depends on the nature of the plant organ from which the explants were
derived and the interaction between endogenous growth substances and the syn-
thetic growth regulators added to the media (George and Eapen 1994 ; Jones et al.
2007 ). There are only few reports on induction of organogenesis ofCannabis
sativa.Early reports dates back to 1970s, Hemphill et al. ( 1978 ), obtained root
development but no shoot formation from callus. Similar results were reported by
Fisse et al ( 1981 ). In 1985, Fisse and Andres used different explants (stem, leaf,
cotyledon, root and callus cultures) forCannabismicropropagation. NAA stimu-
lated rhizogenesis and gibberellic acid was reported to promote stem elongation.
Richez-Dumanois et al. ( 1986 ) induced direct shoot multiplication of explants from
apical and axillary buds using BAP. Whereas, Mandolino and Ranalli in ( 1999 ),
demonstrated occasional shoot regeneration of hemp (C. sativaL.) from leaf callus.
Mackinnon et al. ( 2000 ) obtained root development but no shoot formation from
callus. Slusarkiewicz-Jarzina et al. ( 2005 ), too reported shoot regeneration ofC.
sativa, from calli regenerated from different explants (internodes, axillary buds and
petioles) on MS media supplemented with various combinations of KN, NAA,
2,4-D and dicamba. However, only 2% of calli were able to regenerate into whole
plants of which highest regeneration frequency was obtained from petiole explants
on medium supplemented with 2.0 and 3.0 mg/l dicamba. Plawuszewski et al.
( 2006 ), worked on three different polish cultivars of hemp to regenerate in vitro
growth from explants, roots, leaves and stem grown on DARIA medium. But only
were able to obtain partial regeneration. Shoot regeneration from calli was extended
to 14% by Weiglus et al. ( 2008 ) using different explants from cotyledons, stem and
root on DARIA medium. It was observed that interaction between tested explant
and cultivar (cv.) had significant effect on the efficiency of plant regeneration, with
highest regeneration observed for cotyledon explants (cv. Beniko) and the lowest
for stem explants (cv. Silesia). Casano and Grassi ( 2009 ), reported a higher
micropropagation rate of meristem of selected clones ofCannabisin Formulab
based medium as compared to the MS based medium.
Further studies on organogenesis inCannabisshow the predominance of use of
TDZ in inducing shoot morphogenesis. Thidiazuron, is a substituted phenylurea
(N-phenyl-1,2,3-thidiazol-5-ylurea) with intrinsic cytokinin like activity (Huetteman
and Preece 1993 ). Compared to most other compounds, with cytokinins activity, TDZ
can stimulate better shoot proliferation and regeneration (Lata et al.2009a; Parveen and
Shahzad 2010 ). The use of lateral buds obtained from germinating seeds were inves-
tigated by Bing et al. ( 2007 ), using a combination of TDZ and NAA for shoot
regeneration and IBA for rooting on MS medium. Lata et al. (2009a) have successfully
established a direct organogenesis protocol using nodal segments containing axillary
buds as explants. The quality and quantity of regenerants were better with thidiazuron
(0.5lM thidiazuron) than with benzyladenine or kinetin. Adding 7.0lM of gibberellic
292 H. Lata et al.