tumor growth, suggesting some differential response to Agrobacteriumwithin
C. sativagermplasm. Finally, tumors could be cultured in vitro and vigorous tumor
lines were obtained in six weeks (data not shown).
Transformed roots from allA. rhizogenes-plant variety combinations could be
cultured in vitro and a number of actively growing root clones were stabilized. They
all displayed the typical hairy root phenotype of plagiotropic growth, high inci-
dence of lateral branching and abundance of root hairs (Fig.14.1g, h). Two mor-
phological phenotypes, on the basis of gross morphology, were distinguished in
these root lines: the thin morphology (Fig.14.1g) and the thick morphology
(Fig.14.1h). Thefirst one was much more frequent but clones with thick mor-
phology grow faster (Table14.2) and both remain stable over more than two years.
These differences, however, apparently neither relate with bacterial strains or plant
variety nor with root capacity to synthesize ethylene (Table14.2).
14.2.4 RolABC,rolA,rolBandrolCTransgenic Roots
To study the ability of individualrolgenes, by separate, or combinations of them,
to induce transformed roots on a particular plant species, those genes could be
cloned in plant binary vectors, introduced in the appropriateA. tumefaciensstrain
and then inoculated in the desired plant. In our laboratory, theA. tumefaciens
LBA4400 harboringrolABC, rolA, rolBand rolCcloned in the binary vector
pBin19 was used to infect hemp seedlings as above. The strains harboringrolA,
rolBandrolCalone induced weak plant responses, with roots that were few in
number and did not survive and thus were not further considered. The construction
including the threerol A,BandCgenes, however, induced transgenic roots with a
frequency and other characteristics similar to those observed with the complete
T-DNA. Interestingly, these roots also were of thin morphology (Fig.14.1g). These
rolABCtransgenic roots also offer, relative to wild-type transformed roots, a further
advantage of not accumulating opines.
Overall results show thatC. sativa, a difficult to transform plant, is susceptible to
a number ofAgrobacteriumwild-type strains, which induced transformed tissues in
a short time, in high number and with high vigor and survival rates (Fig.14.1a, b),
but different frequency. Moreover, hairy roots induced by AR10GUS strain, har-
boring the binary p35SGUS intron plasmid, showed normal pattern of GUS positive
Table 14.2 Growth (fresh weight), growth index (harvest FW per inoculum FW) and ethylene
evolution of hemp hairy roots lines after four weeks of culture on MS medium (Wahby et al. 2013 )
Root
morphologyFW (mg
plate−^1 )Growth
indexEthylene evolution (pmol
C 2 H 4 gDW−^1 h−^1 )
Thick 403 ± 53 100 178.3±46.0
Thin 214 ± 37 53 172.3±39.7
Inoculum FW was 4 mg (ca. 15 mm length)
306 I. Wahby et al.