Genomic DNA extraction from leaves and Southern-blot hybridization

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

DNA from leaves from wild-type and transgenic lines were isolated as described in Leclercq et al. 2010. One gram of tissue was ground in liquid nitrogen and then mixed with 6 mL of MATAB buffer 100 mM Tris-HCl pH 7.5, 2 MTAB, 0.4 wv sodium sulphite, 1 PEG 6000, 1.4 M NaCl, 20 mM EDTA. Extracts were maintained at 74 o C for 20 min, and proteins removed using an equal volume of 24:1 chloroform-isoamyl alcohol CIAA followed by centrifugation at 6,220 g for 10 min. Supernatants were transferred to clean tubes and DNA precipated with 5 mL of isopropanol followed by centrifugation at 13,000 g for 15 min. DNA pellets were re-suspended in 300 L µM of TE buffer. Ten micrograms of genomic DNA were fragmented with EcoRI restriction enzyme and fractionated by electrophoresis in a 0.8 agarose gel in TAE 1x buffer. After transfer onto a Hybond N + nylon membrane Amersham TM Megaprime DNA Labelling System, Buckinghamshire, UK, hybridization was performed as described in Sambrook et al. 1989, using random primed 32 P radio-labelled probes corresponding NPTII genes amplified with the following primers: NPTII- F: 5’-CCGGCTACCTGCCCATTCGA-3’ NPTII-R: 5’-GCGATAGAAGGCGATGCG-3’ The numbers of bands reflected the number of T-DNA insertions. 2.5 RNA extraction from leaf and bark Twelve-month-old plants per line were used for gene expression analysis. Leaf and bark samples were collected from wild-type CI07060 and transgenic lines TS18A09, TS18A13, TS18A37, TS 19A46, TS19A59, TS19A90, TS20A69, TS20A75, and TS20A82. The RNA extraction procedure used has been describing in Duan et al. 2010. Briefly, 1 g of leaves from fresh matter was ground in liquid nitrogen and 30 mL of extraction buffer 4 M guanidium isothiocyanate, 1 sarcosine, 1 polyvinylpyrrolidone PVP, and 1 β- mercapto-ethanol was added to the powder. After homogenization, tube was kept on ice and then centrifugated at 13,000 g at 4 o C for 30 minutes, the supernatant was loaded on 8 mL of 5.7 M CsCl. Ultracentrifugation was carried out at 89,705 g, at 20 o C for 20 hours in a swinging bucket. After discarding the supernatant and the cesium cushion, the RNA pellet was washed with 70 ethanol, air dried and dissolved in 200 µM of sterile water. Total RNAs were quantified with Nanoquant Tecan, Männedorf, Switzerland and conserved at - 80 o C.

2.6 Complementary DNA cDNA synthesis