fluorescent lamps). Thus, hemp seedlings of healthy appearance are obtained,
appropriate for in vivo (plantlets) or in vitro (explants) inoculation with
Agrobacterium. Moreover, plants could be maintained for up to 30 days, enough to
obtain good development of transformed tissues.
For the induction ofvirgenes inAgrobacterium, different treatments can be used
as described by Wahby ( 2007 ). One ml of the treatment solution is dispensed on
2-day-old bacterial plates and mixing the cells with a sterile loop, 5 h before
inoculation. For inocula preparation, different cultivation techniques (solid vs.
liquid medium; liquid carrier [water, LB, B5, YEM media]) and cell titres were
assayed. In our laboratory, for in vivo infection ofC. sativa,five-days-old axenic
plantlets are inoculated at four tissues by separate: hypocotyls, cotyledonary node,
cotyledons and primary leaves, with a syringe and 1-2 drops ofAgrobacterium
inoculum are applied on the wounds. After inoculation, the dishes are kept over-
night in the dark (20 °C) and then transferred to the growth chamber. For in vitro
infection of hemp material, explants (1 cm fragment of primary leaves and
hypocotyls and entire cotyledons) are excised from 7-days-old seedlings, placed in
Petri dishes (on wetfilter paper) and inoculated withAgrobacteriumon cut sur-
faces. After 2 d of coculture (solid medium, 3% sucrose and 60μEm−^2 s-1light
intensity), tissues are transferred to fresh medium supplemented with 500μgl−^1
cefotaxime for additional four weeks, under the same conditions.
14.2.2 Hemp Responses to Agrobacterium
Several transformed tissues (calli and hairy roots) ofC. sativacould be initiated
(Fig.14.1), with plant response depending on bacterial strain and, to some extent,
on plant variety. The transgenic nature of tissues was confirmed by PCR analysis
and histochemical localization of GUS activity (Fig.14.1f, Wahby et al. 2013 ).
Good responses toAgrobacteriuminfection are obtained by 5–7-days-old seedlings
with no substantial differences, respect to older ones, in the frequency of trans-
formed tissue induction. Younger plantlets, however, do not survive. Hypocotyls of
intact seedlings are the most susceptible material (Table14.1) responding in a way
Table 14.1 Response of hemp tissues to in vivo (intact seedlings) or in vitro (tissue explants)
infection withA. rhizogenes(Wahby et al. 2013 )
In vivo Response frequencya In vitro Response frequencyb
Hypocotyl 88.3±6.7 Hypocotyl 67.1±7.2
Cotyledonary node 58.7±5.8 Cotyledon 0
Cotyledon 0 Leaf 0
Leaf 0
Data were recorded three weeks after infection
aPercentage of infection sites with root induction
bPercentage of explants with root formation
14 Hairy Root Culture as a Biotechnological Tool inC. sativa 303