typical of hairy root: three days post-infection, wounded hypocotyl areas became
swollen, 2–3 days later small calli started to growth from the wounds and thefirst
hairy root appears about 9 d post-infection and a cluster of hairy roots (up to
15 mm long) developed at infection site in 3–4 weeks (Fig.14.1a, b).
Aseptically-wounded plantlets, as control for callusing, produced no outgrowths.
Nicotiana tabacum, known to be highly susceptible toAgrobacterium, was used as
a positive control for root formation (Fig.14.1c). No substantial differences in hairy
root induction frequency between the different inocula assayed were observed.
Further,Agrobacteriumvirulence was little affected (12–20% over controls) by the
induction media used. Finally, the simplest procedure is used with satisfactory
results: 2-days-old bacteria grown on solid plates, pretreated with 20μM ace-
tosyringone, are washed off by sterile water until the bacterial suspension had a
milky appearance.
14.2.3 Wild-Type Transformed Tissues
In our laboratory, to study theAgrobacterium/hemp interaction, differentA. rhi-
zogenes (476, 477, 478, A4, A424, AR1601, AR10 and AR10GUS) and
A. tumefaciens(C58, IVIA251, LBA4404-rolA,LBA4404-rolB, LBA4404-rolC
and LBA4404-rolABC) strains and C. sativa(CAN0111, CAN0221, Futura77,
Delta-405 and Delta-llosa) varieties (Wahby et al. 2002 , 2004 , 2013 ) were avail-
able. TheA. rhizogenesstrains assayed were able to induce hairy roots on hemp
seedlings in short time, although with different frequency, and interestingly the
addition of the GUS-I plasmid to the strain AR10 does not appreciably alter the
virulence. On the other hand, also the hemp varieties considered were effectively
infected byA. rhizogenes, although quantitative variability in their response to the
infection is also observed. CAN0221 and Delta405 achieved higher frequency of
root induction, CAN0111 gave the highest root number and Futura77 attained
transformed roots with best growth. Similarly, an effective compatible interaction
between all the hemp varieties and theA. tumefaciensstrains used was visible as
tumor-like growth, at infection sites, in 5–8 days after inoculation. Tumors, only
one per infection site, were green in color, compact in texture and 3-10 mm in
diameter (Fig.14.1e), similar to galls formed onNicotiana tabacum(positive
control, Fig.14.1d). Differences between plant varieties or bacterial strains in tumor
induction frequencies were moderate. However, higher differences were apparent in
Fig. 14.1 Hemp response toAgrobacteriuminfection. Hairy root development from woundedc
hypocotyls of hemp (a,b) and tobacco (c) seedlings on solid B5½, four weeks after inoculation
withA. rhizogenesR1601 strain. Tumor development from wounded hypocotyls of tobacco
(d) and hemp (e) seedlings three weeks after inoculation withA. tumefaciensC58 strain on the
same medium (f) Histochemical staining of hemp hairy roots tissue transformed with the
A. rhizogenesstrain AR10GUS. Axenic transformed hemp root cultures on solid MS medium for
4 weeks exhibiting the thin (g) or thick (h) morphology (Wahby et al. 2013 )
304 I. Wahby et al.