Camelina regeneration medium (hereafter known as CR medium) was used for micropropagation of cultures and consisted of Murashige and Skoog (MS) salts and vitamins [21] , 30 g∙l⁻¹ sucrose, 6.6 µM BAP and 2.6 µM NAA. Medium pH was adjusted to 5.8 before adding 7.0 g∙l⁻¹ TC Agar (Phytotechnology Laboratories LLC, Shawnee Mission, KS, USA). The medium was autoclaved at 121˚C and 15 psi for 20 min, cooled down to 55˚C and 25 ml medium was dispensed in each 100 × 16 mm petri dish. Micropropagated cultures of C. sativa cultivars PI650159 and PI650161 were established from seeds, which were briefly immersed in 70% ethanol and transferred 25% commercial bleach solution containing one drop Tween 20. Seeds were surface-sterilized by constant agitation for 15 min in the solution, followed by two 5 min rinses with sterile distilled water, blotted dry and transferred to CR medium. Plates were incubated in the dark at 25˚C for 48 h and then placed in an Adaptis plant growth chamber (Conviron, Winnipeg, CA, USA) under white light (75 µmol∙m⁻²∙s⁻¹) at 25˚C ± 2˚C and 16 h light/8 h dark photoperiod. Following seed germination, shoot apical meristems from a single seedling were used to establish micropropagation cultures, which were increased in mass by transfer of proliferating shoot tips and nodes to fresh CR medium at 2 week-intervals.

In order to optimize kanamycin concentrations that inhibited growth of non-transformed cells while allowing for selection of transgenic cells, cultures were tested in CR medium containing kanamycin concentrations of 5, 10, 15, 20, 40, 80, and 100 mg∙l⁻¹. Filter-sterilized kanamycin at various concentrations was added to autoclaved CR medium cooled to 55˚C. Additionally, 50 mg∙l⁻¹ cefotaxime was added to all treatments to study potential inhibitory effects of the antibiotic on culture regeneration. Four shoot tips (10 mm) were transferred to each petri dish, and each treatment replicated thrice for all antibiotic concentrations. Cultures grown in the absence of kanamycin served as control. Cultures were grown in conditions mentioned above, and explant weight and appearance recorded after 4 weeks on CR medium. The experiment was repeated once. Data was analyzed using analysis of variance (ANOVA) and Tukey’s honest significant differences post hoc test to determine significance of mean separation between treatments. The single factor fixed effect ANOVA model, Y_ij = μ + αK_i + ε_ij, was used where Y is the explant weight when exposed to kanamycin concentration i and biological replicate j. The μ represents overall explant weight with adjustments from the effects of kanamycin concentration (αK), and ε_ij is the experimental error from concentration i and biological replicate j.

A binary vector containing the enhanced green fluorescent protein (egfp), β glucuronidase and neomycin phosphotransferase II (nptII) genes under the control of a CaMV 35S promoter was used to optimize transformation parameters [18] . The binary plasmid was transferred to Agrobacterium tumefaciens “EHA 105” by freezing in dry ice, thawing at 25˚C and used in transformation studies (Figure 1). Agrobacterium containing the binary vector was cultured overnight on an orbital shaker (180 rpm) at 26˚C in 30 ml liquid MG/L medium [22] containing 20 mg∙l⁻¹ rifampicin and 100 mg∙l⁻¹ kanamycin. The bacterial solution was centrifuged at 2200 ×g for 8 min and the supernatant discarded. The pellet was resuspended in liquid CR medium, optical density adjusted to 0.2 at 600 nm (OD₆₀₀), cultured for an additional 4 h, and used for co-cultivation. Shoot apical meristems from rapidly growing micropropagation cultures were excised and immersed in Agrobacterium culture for 7 min. Explants were then transferred to solid CR medium and co-cultivated for 2 days at 26˚C in dark.

After 2 days of co-cultivation, explants were transferred to petri dishes containing solid CRck40 (CR medium containing 50 mg∙l⁻¹ cefotaxime and 40 mg∙l⁻¹ kanamycin) and maintained in conditions mentioned above for 2 weeks. Transgenic cultures were identified on the basis of EGFP fluorescence, GUS histochemical staining assay and kanamycin resistance, and separated from non-transformed cultures. Resulting putative transgenic cultures were transferred to fresh CRck medium every 10 days for 8 weeks. Transient transgenic status of shoot cultures was screened for EGFP and GUS expression.

Transient EGFP expression in transformed cultures was detected using a Zeiss Stemi SV11 microscope (Carl Zeiss AG., Oberkochen, Germany) with an X-Cite 120 fluorescence illumination system (X-Cite, Quebec, Canada). Characteristic green fluorescent emission observed in co-cultivated explants was scored as transient expression. Transient GUS activity in co-cultivated explants was determined by an enzymatic reaction with X-Gluc (5-bromo-4-chloro-3-indolyl glucuronide) substrate. Co-cultivated explants were incubated in 1.5 ml microcentrifuge tubes containing 500 µl GUS staining solution (10 mM sodium phosphate pH 7.0, 10 mM EDTA, 10% Triton X-100, 1 mM potassium ferricyanide, and 2 mM X-Gluc) at 37˚C in dark for 24 h and transferred into 95% ethanol for

24 h to remove chlorophyll. Non-transformed shoot tips were used as a negative control. Transient GUS expression was recorded as the percentage of explants that exhibited a blue stain versus the total number of explants detected by a Zeiss Stemi SV11 microscope (Carl Zeiss AG., Oberkochen, Germany). After 4 weeks of culture on regeneration medium, leaves from transgenic shoots were used to analyze EGFP and GUS expression as described above.

Shoots 3.0 cm or longer were transferred to CR medium containing 0.4 mg∙l⁻¹ NAA and 16 mg∙l⁻¹ kanamycin for rooting (CRT). Plants with a well-developed shoot and root system were transferred to sterile Promix BX (A. H. Hummert Seed Co., St Louis, MO, USA) and acclimatized in a clear plastic dome under conditions of high humidity for 3 weeks. Plants were enriched with liquid 2:2:2 (NPK) fertilizer at weekly intervals and ultimately transferred to a greenhouse. Transgenic seeds were collected and EGFP fluorescence detected as mentioned above.

Genomic DNA was extracted from six transgenic plant lines (three from each cultivar) using the Qiagen DNeasy plant extraction kit (Qiagen, Valencia, CA, USA). PCR and quantitative real-time PCR was performed to confirm transgene presence and copy number in transgenic plants. A forward primer EG-51 (5’-ATGGTGAGCAAGGGCGAGGAGCTGT-3’) and a reverse primer EG-32 (5’-CTTGTACAGCTCGTCCATGCCGAGA-3’) were used to amplify a 717 bp DNA fragment from the egfp/nptII fusion gene. Conditions for PCR reactions were: 95˚C for 4 min, 40 cycles at 94˚C for 1 min, 58˚C for 1 min, 72˚C for 1 min and a final cycle at 72˚C for 4 min [23] . DNA from a non-transformed plant was used as a negative control. PCR products were run on a 0.6% agarose gel and DNA bands were observed with a UV transilluminator.

Transgene copy number was determined using quantitative real-time PCR. Assays were carried out in an ABI QuantStudio 12K instrument equipped with a 96-well plate and CopyCaller^TM Software (Life Technologies, Carlsbad, CA, USA). Oligonucleotide primers for amplification of a 340-bp target fragment from the egfp gene included a forward primer ERT-51 (5'-CCATCCTGGTCGAGCTGGAC-3'), a reverse primer ERT-32 (5'-TTCAGCTCGATGCGGTTCAC-3'), and a probe (5’-FAM-GCAAGCTGACCCTGAAGTTC-MGB-3’). All reactions were carried out in a 20 μl final volume containing 4 μl sample DNA (total of 20 ng), 10 μl of 2× Taqman genotyping master mix, 1 μl of 20× Taqman assay mix (containing primers and probe), 1 μl of 20× Taqman copy number reference assay and 4 μl sterile water. Real-time PCR reaction samples were replicated 4 times for each line tested. For accurate gene expression, the C. sativa actin gene primer-probe set (fp: 5’-ACAATTTCCCGCTCTGCTGTTGTG-3’, rp: 5’-AGGGTTTCTCTCTTCCACATGCCA-3’, probe: 5’-VIC-TGTTTCAAACGCTCTATCCCTCGCTC?MGB-3’) was used as reference. Quantitative real-time PCR conditions were as follows: 95˚C for 10 min followed by 40 thermal cycles of 95˚C for 15 s and 60˚C for 1 min. Fluorescence signals were analyzed by the method utilized by CopyCaller^TM software (Life Technologies, Carlsbad, CA, USA). This analysis method utilized cycle threshold (Ct) values to extrapolate initial concentration of target DNA in each sample.

The effect of increasing kanamycin levels on growth and proliferation of transformed and non-transformed shoots was determined by measuring fresh weight after 2 weeks of cultivation. At 5 mg∙l⁻¹ and 10 mg∙l⁻¹ kanamycin, we observed an initial inhibitory effect, however, shoots eventually outgrew this effect after 2 weeks (data not shown). By contrast, kanamycin over a concentration of 40 mg∙l⁻¹ caused complete necrosis and inhibited growth of explants within 3 weeks, resulting in a significant decrease in average weight of explants in both the cultivars. While the weight of explants grown in the absence of kanamycin was 0.20 ± 0.029 g in cv. PI650159, explants grown in 40 mg∙l⁻¹ kanamycin measured 0.06 ± 0.005. Similar results were observed in cv. Pl650161 where control explants measured 0.17 ± 0.019 g compared to 0.05 ± 0.005 g in those exposed to 40 mg∙l⁻¹ kanamycin (Table 1).

Shoot tips that expressed EGFP produced a bright green fluorescence when observed under a microscope equipped with epi-fluorescence illumination (Figure 2(a) and Figure 2(b)). We also observed distinct EGFP expression in all putatively transgenic seeds and pods (Figure 2(c) and Figure 2(d)). In addition, GUS staining of 60 transformed explants from each cultivar revealed transient transformation efficiencies of 53.33% and 98.33% for Pl650159 and Pl650161

^aMean weights for three independent biological replicates were calculated. Identical letters followed by values denote no significant difference between treatments within the same column (P > 0.05).

respectively (Table 2), represented by blue stain on explants (Figures 2(e)-(h)). Complete leaf areas including veins were substantially and uniformly stained in the transformed tissues. In addition, we detected significant GUS expression in the stems as well. On the contrary, no GUS activity was observed in non-transformed explants. Additionally, transgenic cells carrying these marker genes selectively grew on culture medium containing kanamycin while inhibiting the growth of non-transformed cells.

Genomic DNA extracted from the transgenic and control plant lines were amplified and tested using gene specific primers. PCR revealed the expected plasmid-encoding fragment of 717 bp in all EGFP positive transgenic plants, while DNA from the negative control did not. These results indicated that all lines expressing EGFP contained the gene (Figure 3). Molecular analysis using quantitative real-time PCR demonstrated variable copies of the egfp transgene. A single copy integration of the gene was confirmed in a cv.Pl650161 line. In cv. Pl650159 the lowest copy number detected was two, while another line of this cultivar had six copies and the remaining line contained a high copy number of the transgene (>10 copies) (Table 3).

Explants of both cultivars in CRT medium produced roots in 3 - 4 weeks. All regenerated plantlets that were transferred to PROMIX survived. The in vitro propagated shoots started forming new leaves within 2 - 3 weeks after transferring them to pots containing PROMIX.

^aCSActin was used as the endogenous reference gene; ^bAverage of three technical replicates.