Plant Cell Rep (2012) 31:999–1007 1001 Fig. 1 Constructs, a the pUKN construct and b the pGEM-ubi-gfp construct Selective rooting medium: rooting medium supple- mented with 30 mg/L G418. Osmotic medium: MS medium supplemented with 0.2 M D-sorbitol and 0.2 M D-mannitol. Kill curve experimentation A kill curve experiment was carried out utilizing IEs at 2 weeks old after initiation on CIM. IEs were subcul- tured onto selective regeneration media containing G418 at 0, 10, 20, 30 and 50 mg/L (5 IEs per plate, three plates for each G418 treatment) and placed under lights. IEs were subcultured fortnightly. Four weeks later, the numbers of IEs generating shoots and surviving (the total number of IEs minus the number of necrotic IEs) were recorded. Microprojectile transformation IEs at 9–11 days old after initiation were used for micro- projectile transformation. Six IEs were placed at the center of a shallow petri dish 15 9 90 mm containing osmotic medium and stored for 2–3 h in the dark prior to bom- bardment (Fig. 6a). Operation of the particle inﬂow gun (PIG) was conducted as described by Vain et al. (1993) (Grootboom et al. 2010). For plasmids, DNA delivery occurred via 0.6 lm gold particles (0.42 mg per shot). The distance from the ﬁlter holder to the target cells was adjusted to 18.5 cm and the helium pressure was modiﬁed to 1,000 kPa. As much as 5 lg of each pUKN plasmid and pGEM-ubi-gfp plasmid were mixed and then equally loa- ded into the receptacle for six shots. A 500-lm nylon mesh screen was placed 8 cm above the target tissue and a vacuum of approximately -90 kPa was generated prior to shooting with a time duration of 0.05 s. Post-bombard- ment, IEs were kept on osmotic medium for 3–4 h before being transferred onto CIM. Selection of putative transgenics After IEs recovered on CIM for 3–4 days, they were transferred to selective regeneration medium and placed under lights in a tissue culture room. IEs were then sub- cultured fortnightly until putative shoots grew to 4–6 cm. Subsequently, shoots were then moved to selective rooting medium for 4 weeks without subculture. Once the plantlets had a well-developed root system ([10 roots of [1 cm length), the lids of petri dishes were opened for at least 3 days to acclimatize plantlets in tissue culture room. The plantlets were transferred into the containment glasshouse and planted in plastic pots (20 L) containing 15 L of pot- ting mix (Green Fingers). Plants were watered once per day. Reporter gene observation The gfp reporter gene produces a protein which ﬂuoresces in living cells when exposed to blue light at the wavelength of 395 nm (Ormo et al. 1996). The gfp gene expression in callus was monitored under the OLYMPUS BX60 ﬂuo- rescence microscope 3 and 10 days after bombardment. Each callus piece (from one IE) was then categorized (based on the number of GFP foci) into the following groups: 0–20, 21–50, 51–100 and [100. The gfp gene expression in root tips of putative transgenic plantlets and T1 seedlings was also monitored. PCR analysis Genomic DNA was extracted from young leaves of puta- tive transgenic and non-transgenic line Tx430. To conﬁrm the presence of nptII and gfp, fragments (722 bp of nptII and 732 bp of gfp) were ampliﬁed from genomic DNA using NPTII-speciﬁc primers: NPTII-1: 50-GCTAT- GACTGGGCACAAC-30; NPTII-2: 50-GTCAAGAAG 123