2 MATERIALS AND METHODS
2.1 Establishment of transgenic callus lines 2.1.1 Plant material
Embryogenic friable callus line CI07060 was established from integument-calli of clone PB260 Lardet et al. 2009. This callus line was sub-cultured every two weeks on a
maintenance culture medium MM containing macro-elements 20 mM NH
4
NO
3
, 20 mM KNO
3
, 3 mM MgSO
4
.7H
2
O, 2 mM NaH
2
PO
4
.H
2
O, 9 mM CaCl
2
, micro-elements 150.08
µM H
3
BO
3
, 100µM MoSO
4
.H
2
0, 5 µM KI, 1.01 µM CoCl
2
.6H
2
0, vitamins 300 µM inositol, 20 µM nicotinic acid, 3 µM pyridoxine-HCl, 2 µM thiamine-HCl, 0.2 µM biotine, 1 µM D-
calcium pantothenate, 1 µM ascorbic acid, 0.1 µM choline chloride, 60 µM L-cysteine-HCl, 5 µM glycine, 1 µM riboflavin, 1.35 µM benzylaminopurine BAP, 1.35 µM 3,4-
dichlorophenoxy-acetic acid 3,4-D, 100 µM FeSO
4
, 100 µM Na
2
EDTA, 30 µM AgNO
3
, 234 mM sucrose, 0.5 µM abscisic acid ABA and 2.3 g L
-1
Phytagel Carron et al. 2005; Lardet et al. 2007. The pH of all media was adjusted to 5.8 prior to autoclaving. Callus
cultures were grown in the dark condition at 27
o
C. Before Agrobacterium inoculation, the callus line was pre-cultivated for 15 days in glass tubes on two different pre-culture media
PM, namely a CaCl
2
-free MM medium supplemented either with 4.5 µM BAP and 3,4-D, or supplemented with 1,35 µM BAP and 3,4-D Montoro et al. 2003.
2.1.2 Binary vectors and Agrobacterium strain
Two binary vectors called pCamway binary destination vectors allow cloning of a candidate gene under the control of the 35S CaMV promoter or latex-specific HEV2.1
promoter Leclercq et al. 2015; Montoro et al. 2008. These vectors had a pCamway 2300 backbone with the NPTII gene conferring resistance to neomycin and a GFP reporter gene
under the control of the 35S CaMV promoter. Candidate genes HbERF-IXc4 and HbERF- IXc5 were cloned both under the control of 35S CaMV promoter and HEV2.1 promoter
Figure 29.
The binary vectors were introduced into Agrobacterium tumefaciens strain EHA105 by electroporation. For inoculation, bacteria were grown in liquid Lysogeny Broth medium
Duchefa, Haarlem, The Netherlands supplemented with 50 mg L
-1
kanamycin and 100 µM acetosyringone at 28
o
C until OD
600nm
= 0.6. After centrifugation at 1,000 g for 10 min, the pellet was dissolved to OD
600nm
=0.06 in liquid MM from which Fe-EDTA, CaCl
2
and growth regulators were eliminated and 100 µM acetosyringone were added Blanc et al.
2006.
Figure 29 Structure of T-DNA constructs using pCamway 2300 NPTII and GFP genes are
fused with the 35S CaMV promoter. Candidate genes HbERF-IXc4 and HbERF- IXc5 are under the control of the 35S CaMV promoter or the latex-specific
HEV2.1 promoter
2.1.3 Inoculation, coculture, and selection of transgenic callus lines
Inoculation was performed as described by Blanc and collaborators Blanc et al. 2006. Briefly, forty glass tubes containing precultured embryonic calli from clone PB260
were used Montoro et al. 2000b. Calli were immersed directly in the tube for 1 s in the Agrobacterium suspension prepared as described above. Two coculture durations 4 and 5
days were tested at 20
o
C Blanc et al. 2006. Six hundred small aggregates per treatment were then placed in 20 Petri dishes containing a decontamination medium DM, a MM
containing 500 mg L
-1
ticarcillin Sigma, Saint-Louis, USA, to prevent Agrobacterium growth.
GFP visualization was performed on callus at the end of each subculture under a fluorescence stereomicroscope and macroscope MZ FLIII, Leica Microsystems, Wetzlar,
Germany using the GFP2 filter 480 nm excitation filter510 nm barrier filter. To isolate transgenic callus lines, GFP-positive aggregates were successively sub-cultured every 3
weeks on DM and then several times on DM with increasing concentrations of paromomycin from 50 to 150 mg L
-1
Rattana et al. 2001. Finally, transgenic callus lines were established from sub-aggregates showing full GFP activity Leclercq et al. 2010. These calli were then
subjected to molecular characterization, plant regeneration andor cryopreservation according to the protocol described previously Lardet et al. 2007.
2.2 Cryopreservation of transgenic callus lines
