Hairy roots can also serve as starting material to regenerate transgenic plants.
Transformed roots from a number of plant species have been shown to be able to
easily regenerate transformed plants both spontaneously (Lee et al. 2004 ) or after
treatments with growth regulators (Crane et al. 2006 ). Transformed plants show
several morphological and developmental abnormalities, i.e., wrinkled leaves,
reduced apical dominance, reduced internode length and leaf size, etc., collectively
known as the hairy root syndrome. However,A. rhizogenes-mediated transforma-
tion could be useful for recalcitrant species. On the other hand, it has also been
reported thatrolgenes can segregate out, of other introduced genes, in the progeny
of these plants, being possible to obtain transgenic plants of normal appearance.
14.2 AgrobacteriumTransformation of Hemp:
Establishment and Applications of Transformed
Root CulturesAgrobacterium-mediated transformation of plants is commonly carried out on small
pieces of plant organ/tissues, called explants, by infection/cocultivation of these
materials with cell suspensions of the appropriate bacterial strains. Alternatively, it
could also be performed by direct inoculation of axenically-grown seedlings fol-
lowed by isolation and culture of transformed tissues. This last approach is referred
as in vivo inoculation. To date, the list of plant species really transformed via
Agrobacteriumis quite long and a great body of information has been generated.
Nonetheless, plant species of interest, for a variety of purposes, still resist such
genetic engineering and for this reason they are said to be recalcitrant.
The case of hemp (C. sativa), an important medicinal and/or industrial crop is
illustrative of that situation and although some progress has really been made, it still
continues to be a difficult to transform plant. In our laboratory, a number of factors
considered to be determinant to get a compatible plant-Agrobacteriuminteraction
have been studied forC. sativa(Wahby 2007 ; Wahby et al. 2013 ).
14.2.1 Axenic Growth of Hemp Seedlings
and Infection Protocol
Surface-sterilized seeds of hemp varieties were germinated in the dark at 25 °C for
2 – 4 days (seed viability and endogenous contamination were important draw-
backs). Seedlings (3–4 mm radicle) were transferred to Petri dishes on the top of
agar slopes of selected medium (½B5) with root tips dipped into the medium.
The dishes, partially sealed with Parafilm™are incubated at vertical position in a
growth chamber (16 h photoperiod, 25/20 °C day/night and a 400–700 nm photon
flux of 350μEm−^2 s−^1 supplied by Philips Cool White and Sylvania Gro-lux
302 I. Wahby et al.