Establishing Homozygous Transgenic Petunia Lines

Transformants were transferred into soil (Metro-Mix 200, Sun Gro, Bellevue, WA, USA) and grown under artificial lighting (∼40 μM m⁻² s⁻¹, 16 h photoperiod, 25°C). The stigma of each flower was artificially pollinated and the flower without petals was enveloped with tape to avoid cross-pollination. T1 seeds were collected and sown on MS (Sigma, USA) medium with 20 mg ml⁻¹ hygromycin (Sigma, USA) for the further selection of homozygous lines. The following procedure was performed: T1 seeds were first surface-sterilised in 15% bleach (Clorox, USA) with 0.01% Tween-20 (Sigma, USA) for 20 min and then 70% ethanol for 45sec, followed by three washes with sterile distilled water. The plates (20 seeds/plate) were kept at room temperature under continuous low fluorescence light (∼40 μM m⁻² s⁻¹) for 21 days. T1 seeds which displayed a 3∶1 ratio of survival on hygromycin medium were retained and moved to pots for harvesting T2 seeds. One hundred seeds from each line were germinated again on MS medium containing 20mg ml⁻¹ hygromycin, and lines having 100% survival were identified as homozygous and used for further experiments.
PCR amplification was performed for further identifying transgenic lines. Genomic DNA of the petunia transformed Arabidopsis etr1-1 gene was extracted using a hexadecyltrimethylammonium bromide (CTAB) method, as described previously [19] (link). PCRs were carried out in a total reaction volume of 20 µl, according to the procedure as the following: 5 min at 95°C followed by 40 cycles of 30 s at 94°C, 30 s at 56°C, and 1min at 72°C. Primer pairs: 5′-CCATCACACTAAATCTTGCACCA-3′ and reverse 5′- TTCGGTATGCCCGACTGTTTAG- 3′ were used. 1.0% agarose gel electrophoresis was performed using standard protocols.