2.2.1 Cryopreservation procedure
Embryogenic callus was sampled after 12 days of culture on MM. Liquid cryoprotective medium, which is an MM modified with 1 M sucrose, was added to the callus
at a rate of 1 mL g
-1
of callus. Dimethylsulphoxide DMSO was added gradually over the first 30 min period to reach a final concentration of 10. The composition of DMSO solution
was 70 DMSO + 30 H
2
0. The callus suspension was gently shaken for 1 min and then callus suspension was pipetted and dispensed into cryovials 1 mL per cryovial Lardet et al.
2007.
2.2.2 Freezing
Each cryovial containing 120-160 mg callus fresh weight was placed in Nalgene Cryo 1C in the polystyrene box was placed in a -80 °C deep freezer and the temperature was
monitored by a thermocouple, which was placed in one of the cryovials. At -40 °C, the cryovials were rapidly immersed in liquid nitrogen for storage in a cryobiological storage
system LocatorJR Plus Thermolyne, Ohio, USA. The polystyrene box allowed a significant
decrease in the “Cryo 1C” cooling rate, with average cooling rates of 0.20 °C ± 0.06 min
-1
Lardet et al. 2007.
2.3 Plant regeneration
Production of somatic embryos and their conversion into plantlets were carried out as described in Lardet et al. 2007. Somatic embryogenesis was initiated for 4 weeks by sub-
culturing 1 g of callus showing full GFP activity in 250 mL flasks containing 50 mL of a semi-solid embryogenesis expression medium EXP, which was a modified MM medium
supplemented with 58.5 mM sucrose, 175.5 mM maltose, 0.44 µM BAP and 0.44 µM 3,4-D. Pro-embryo development was then carried out in a temporary immersion system RITA
®
, CIRAD, Montpellier, France for two subcultures of 4 weeks each with 1 min of immersion
per day in the liquid development medium DEV, which was a MM containing 234 mM sucrose and 3 mM CaCl
2
, without any growth regulator. Each RITA was considered as an experimental replication. Conversion of mature embryos was carried out according to Lardet
et al. 1999. Well-shaped mature embryos were collected and transferred to glass tubes on a semi-solid germination medium DEV3, which consisted of the MM medium supplemented
with 1.5 mM CaCl
2
solidified with 7 g L
-1
Agar Sigma, St. Louis, USA. Embryos were incubated under a light intensity of 60 µmol m
-2
s
-1
and a 12 h daydark photoperiod up to the full conversion of embryos into plants. Plantlets were then acclimatized in the greenhouse at
28
o
C with 60 relative humidity. To compare the regeneration ability of wild-type and transgenic callus lines, wild-type
callus line CI07060 was cultured over the duration of the transformation experiment and regenerated. Once enough calluses were produced, plant regeneration was initiated. For both
non-transformed and transgenic callus lines, the regeneration replication number, the number of total embryos g
-1
of callus T, the number of well-shaped embryos g
-1
of callus WS, the number of plantlets g
-1
of callus P and the conversion percentage PWS were recorded.
2.4 Genomic DNA extraction from leaves and Southern-blot hybridization
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