(35% by weight, polyunsaturated fatty acid rich) is of nutritional and industrial
value (Callaway 2004 ; Li et al. 2010 ) and chemical compounds of pharmacological
implication are found throughout the plant (Turner et al. 1980 ; de Meijer et al.
2003 ).
Because of its high yielding with low chemical requirement, hemp culture is
viewed as a multipurpose crop with truly agricultural and environmental benefits
(Ranalli et al. 1999 ). Classical approaches for plant improvement have been applied
to hemp with some success (extensive work in France and Eastern Europe for
industrial hemp improvement). However, the progress of breeding programs is
more limited for the case of medicinalCannabis(de Meijer et al. 2003 ). Molecular
marker techniques are also coming to hemp research to help improvement in the so
called marker assisted selection (Pacifico et al. 2006 ). Other even more powerful
approaches i.e. in vitro plant regeneration and/or genetics transformation, however,
are less advanced. Among the different methods available to transfer foreign DNA
to plants, those mediated by the phytopathogenic soil bacteria Agrobacterium
tumefaciensandAgrobacterium rhizogeneshave been the most widely used to
generate transgenic plants from many species.
At the beginning of the last century, two plant diseases, crown-gall and hairy
root, caused significant losses in fruit tree nurseries. Phytopathogenic soil bacteria
of the genusAgrobacterium(Rhizobiaceaefamily) were identified, several years
after, as the etiological agent and the molecular mechanism underline these diseases
elucidated. It resulted from the ability ofAgrobacterium,a“natural genetic engi-
neer”, for transformation of plant cells. When susceptible plants are wounded at
about stem/root junction by nematodes, insect larvae, etc., release organic sub-
stances (some phenolics and sugars) that attract the bacteria and also induce the
expression of thevir(virulence) genes responsible for the transfer to plant cells of a
DNA fragment (the T-DNA) from the Ti (tumor induction) or Ri (root induction)
plasmids ofA. tumefaciensor A. rhizogenes, respectively (Hu and Du 2006 ;
Georgiev et al. 2007 , 2012 ).
The T-DNA in the Ti plasmids carries genes for plant hormone biosynthesis in a
way not controlled by the plant cell. Altered hormone balance induces cell
hypertrophy and hyperplasia leading to gall (tumor growth tissue) formation.
Tumor cells produce and secrete compounds (amino acid and sugar derivatives)
unique to these cells, called opines, that only can be used byAgrobacteriumas
energy source. T-DNA in Ri plasmid carries therolA,rolB,rolCandrolDgenes (in
the root locus) and theauxgenes for auxin biosynthesis. Plant cells at wound site
are induced byrolgenes to differentiate into roots (transformed or hairy roots that
also produce opines), whileauxgenes would have an accessory role in root dif-
ferentiation providing auxins when endogenous levels in the plant were insufficient
to initiate competent cell differentiation by the expression ofrolgenes. Ri plasmids
withoutauxgenes could induce normal hairy roots. Root loci are said to be essential
for induction of hairy root syndrome, however all common traits of these root were
observed when onlyrolABCgenes were transferred and expressed in plant cells,
thus these genes would really be the essential.Rolgenes alone could also induce
hairy roots though with quite different efficiency, beingrolBwhich evoke a stronger
300 I. Wahby et al.