High levels of BA completely inhibited HRM response and also promote best calli
growth (Fig.14.3b), being mean callus size between 1.5 and 2.0 cm in diameter
after six weeks of culture. Further 2,4-D also abolished HRM (Fig.14.3e) but callus
growth was significantly lower (0.5–1.0 cm) in the same period. All the calli,
however, were brown in color and never developed green areas, indicative of
ageing and degeneration. In MS medium supplemented with 1.5 and 0.05μgml−^1
of BA and ANA, respectively, root explants developed mostly into non-rhizogenic
calli (75% of total) and these small friable calli, white in color, developed intense
green areas on the surface (Fig.14.3f). Consecutive subculturing cycles have been
reported to improve organogenesis response in different species (Hamza and
Chupeau 1993 ; Stiller et al. 1997 ; Gurriarán et al. 1999 ) includingC. sativa
(Chandra et al. 2013 ). Thus actively growing green nodules of calli were succes-
sively subcultured in the same medium or with a ratio BA/NAA of 0.5/0.025μg
ml−^1 for up to 10 weeks. The new calli grew vigorously, reaching 2 cm in size in
four weeks, with intense green nodular areas on surface (Fig.14.3c), which was
stable over the entire culture period. Unfortunately, these calli were unable of bud
or shoot regeneration. Green calli fragments were also subcultured on media
including high levels (3.0 and 5.0μgml−^1 ) of 2,4-D, with which some occasional
shoot regeneration events, from untransformedC. sativaexplants, were reported by
Mandolino and Ranalli ( 1999 ). In these media, however, the callus fragments lost
the green color, stopped growth and died after two weeks. Explant source, i.e.
transformed vs. untransformed material, tissue/organ considered and plant genotype
among others might be important factors conditioning such differential behaviors.
Overall results show that some media inhibited HRM response and promoted good
callus growth and appearance, but these calli continued to be unable of bud or shoot
regeneration.
Rolgenes and probably other oncogenes strongly influence hormone balances
and perception in plant cells (Tarkowski and Vereecke 2014 ; Matveeva et al. 2015 ).
Particularly,rolBappears to alter auxin signalling in the plant cell where it is
expressed. All this might, at least in part, explain the responses ofC. sativahairy
root-derived calli to the hormone combination and concentration in culture media
used in our laboratory, and their inability for shoot regeneration. Finally, root
explants are inoculated in MS medium supplemented with different concentrations
(2.1, 4.3, 8.6, 15 and 21.1μgml−^1 )ofp-chlorophenoxyisobutyric acid (PCIB) as
auxin action inhibitor (Oono et al. 2003 ) in combination with 1.0μgml−^1 BA for
the production of calli.
PCIB decreased callusing response by roughly 36%, on average, but also
affected callus growth and appearance (Fig.14.3d). After four weeks of culture,
calli were small (0.5–1.0 cm), quite friable and to some extent they resembled the
so called callus-like root morphology described for otherA. rihizogenestrans-
formed roots (Mallol et al. 2001 ; Bandyopadhyay et al. 2007 ; Tiwari et al. 2007 ).
On the surface of some calli, especially in the presence of 8.6μgml−^1 PCIB,
started to grow (at fourth week) secondary calli of compact texture and green color
(similar to those in Fig.14.3f). These structures ( 3 – 4 mm) were isolated and
subcultured on the same medium for an additional four week period. Calli grew
312 I. Wahby et al.