After a further 3 months of culture on selective medium, the proliferating EM were considered to be putatively transgenic clones and were selected for molecular and histochemical selleck chem inhibitor analysis.3.3. Maturation and Conversion into PlantsFor maturation, kanamycin and cefotaxime were removed. No Agrobacterium regrowth was observed. Mature embryos were obtained from 6 out of 10 transgenic lines (Figure 2). The number of mature embryos showed a high variability among lines, on average with 13.8 �� 3.4 mature embryos per gram of fresh mass. This value was significantly lower than 231.9 �� 7.1 mature embryos per gram of fresh mass in the untransformed line L01 at the same age. No correlations were observed among maturation capacity, number of T-DNA insertions, and GUS activity (Figure 2).

Transgenic lines that showed mature embryos were cryopreserved and recovered. No reduction in maturation capacity was observed after cryopreservation.Figure 2Fluorometric assay of 10 kanamycin-resistant lines. Different bar fills indicate transgene copy number: 1 copy (solid black), 2 copies (striped grey), and 3 or more copies (solid grey). The presence (+) or absence (?) of maturation is depicted …After 1 month on germination medium, 68.2% of embryos showed radicle elongation and bud-break. These were transferred to peat-vermiculite substrate and plant conversion was 71.4 %. No significant differences on germination or acclimatization percentages were observed when the transgenic embryos were treated with BA before transferring to germination medium compared with nontreated embryos.

In addition, various axillary shoots per plantlet (5-6) were obtained after 3 months of culture on germination medium. The lateral shoots were isolated and rooted (85%). No plagiotropic growth was observed and the plants showed a well-developed root system capable of sustaining further shoot outgrowth. 3.4. Molecular AnalysisTen putative transgenic lines resistant to kanamycin were tested by PCR to detect the nptII and uidA genes (included in the T-DNA) and the virG gene (to detect bacterial contamination). All of the lines were PCR-positive for both nptII and uidA genes, so no escapes were detected. The virG gene was only amplified in the positive control (AGL1 pBINUbiGUSint). No amplification was detected in the negative control (nontransformed L01 line) (Online Resource 5).

The five transgenic plants (one-year-old) from germinated embryos and the five rooted shoots tested for the nptII and uidA genes were PCR-positive. No amplification Brefeldin_A was detected for the virG gene (Online Resource 6). Copy number estimation by the comparative Ct method showed one copy in five lines, two copies in two lines and three or more copies in three lines (Figure 2). 3.5. ��-Glucuronidase Assay during Embryo DevelopmentThe presence of GUS activity in the EM and embryos harboring the uidA gene was investigated by histochemical assay.