4.5. GUS Histochemical and Fluorometric Assays

The progenies resulting from the crosses with transgenic pear plants were screened for GUS expression by a qualitative histochemical GUS assay [71]. Young leaves from seedlings were tested using X-Gluc as substrate. Three (2007, 2009, 2010) of four (2008) plants were randomly selected from GUS-positive progeny of each transgenic line for quantitative assessment of GUS activity. Three leaves from the middle of the shoot were used in one-year-old greenhouse-grown plants (early June) and a mixture of several leaves from the three top lateral shoots in field-grown plants (early July). Fluorometric assay was conducted as described by Scott et al. [71] using 4-methyl-umbelliferyl-β-D-glucuronide (MUG) as substrate. Fluorescence of released 4-methylumbelliferone (4-MU) was measured using the Infinite 200 multifunctional microplate reader (Tecan Group Ltd., Switzerland) with excitation at 360 nm and emission at 465 nm (Pushchino Center for Collective Use of Science Equipment). The amount of total soluble protein was determined according to the Bradford method with bovine serum albumin as standard [72]. GUS activity was expressed as picomoles of 4-MU produced per minute per milligram of protein. Pear crosses from 2006 were evaluated during four years in the greenhouse (2007) and in the field (2008–2010). Other seed progenies were evaluated once as greenhouse-grown one-year plants (2010) or three-year plants in the field (2010 and 2011). The grafted plants were tested for GUS activity in 2009 and rooted cuttings in 2010–2011 in three randomly selected individuals under field conditions.