Fig. 4
Production and characterization of transgenic plants of T. timopheevii (Zhuk.). a. Transient GFP gene expression in cells of explant subjected to bombardment, 16 h after the plasmid delivery. b. Formation of GFP-positive morphogenic callus, 40 days of culture. c. Fluorescent embryoids, 65 days of culture. d. The putative transgenic plant of T. timopheevii before transfer to soil. e. Tiller set of primary transgenic plants. f. GFP expression in anther and pollen grain of primary transgenic wheat plant Tw1. g. Inheritance of GFP expression in T1 seeds of primary transgenic plant Tw2. h. Primary plants were analyzed for the presence of BAR gene by PCR amplification (left panel) and GFP expression by RT-PCR (right panel); lane M, DNA ladder as molecular weight marker; lane P, psGFP-BAR; lane WT, untransformed T. timopheevii plant; lanes Tw1 and Tw2 represent putative transgenic plants; +, a sample with addition of reverse transcriptase; −, the same sample without addition of reverse transcriptase. i. Southern blot analyses of genomic DNAs from PCR-positive primary transgenic plants Tw1 and Tw2 (T0) and three progeny plants (T1) of the transgenic line Tw2; genomic as well as plasmid control DNA was digested with HindIII and the membrane was probed using the 510-bp Ubi1-BAR probe; lane P, psGFP-BAR (1.0 ng); lane WT, negative control representing DNA from untransformed T. timopheevii plant. Tissues (a,b,c,f,g) were photographed under white light or UV light using the GFP filter set (EX BP 470/40, BS FT 495, EM LP 550)