transformed root cultures together with elicitation approaches (Mehrotra et al. 2015 )
as systems for biotechnological production of these active compounds should not
be discarded. Finally, quantification of atropine and muscarine inCannabismate-
rials would also be of biological significance as, to our knowledge, quantitative data
for these alkaloids have not previously been reported out of Solanaceae plants or
poisonous mushrooms, respectively.
14.3 Callusing Responses and Regeneration
Ability of Hemp Hairy Roots
Our group was aimed to genetically transformC. sativaand to identify experi-
mental conditions for regeneration of transgenic plants from hemp transformed
tissues. As stated before, hairy roots are also useful as starting material to regenerate
transgenic plants. Transformed roots from a number of plant species easily
regenerate complete plants both spontaneously (Lee et al. 2004 ;
Trémouillaux-Guiller 2013 ; Mehrotra et al. 2015 ) or after treatment with plant
growth regulators (Crane et al. 2006 ;Lütken et al. 2012 ) and despite several growth
and developmental abnormalities (the hairy root syndrome) of these plants, theA.
rhizogenestransformation approach is considered valuable for recalcitrant species.
In our laboratory, root clones derived from different plant varieties (Futura77,
CAN0221)/bacterial strains (A4, AR10, R1601, LBA-rolABC) combinations
(Wahby et al. 2004 ; Wahby 2007 ) were used to assay their ability for callusing and
plant regeneration. Root explants (1 cm segments of apical regions) were cultured
in B5 solid medium (pH 5.5, 2% sucrose and 0.6% agar) supplemented with
0.5μgml−^1 of benzyladenine (BA) and 0.05μgml−^1 ofa-naphtalenacetic acid
(NAA), a combination of growth regulators successfully used to regenerate plants
fromLotus corniculatus (Stiller et al. 1997 ),Populus (Han et al. 1997 ) and
Catharanthus roseus(Choi et al. 2004 ) hairy roots. In this medium all root lines
showed a high callusing response, being the frequency of callus induction (per-
centage of explants that forms calli) near 100% after three weeks in culture. These
calli (0.5–1 cm in diameter) were white in color, friable in texture and developed
abundant roots on the surface (Fig.14.3a). Isolated and cultured on the same
regeneration medium, such roots behave in a similar way. However, when cultured
on hormone-free MS medium, they grew very fast showing the characteristic hairy
root phenotype (Wahby et al. 2013 ). Thus this root neoformation on calli surface
was called hairy root memory (HRM) response. Rhizogenic calli also developed
from untransformed hemp tissues in the presence of NAA as an auxin (Fisse et al.
1981 ; Feeney and Punja 2003 ). At this point it might be highlighted that both HRM
and other rhizogenic responses in calli would be indicative of no good competence
for regeneration inC. sativaas well as in other species.
In the absence of bud or shoot organogenesis responses, additional conditions
were explored. Media were B5 (as above) or MB (MS salts with B5 vitamins plus
310 I. Wahby et al.