Monitoring the expression of green fluorescent protein in carrot

Abstract

Green fluorescent protein (GFP) was successfully used as a visual reporter at various stages of carrot (Daucus carota L.) transformation. GFPfluorescence was non-invasively observed in protoplasts, callus and plants after the delivery of mgfp5-er gene using two transformation methods: direct DNA transfer into polyethylene glycol (PEG) -treated protoplasts and inoculation of root discs with Agrobacterium rhizogenes. Transient GFP-expression was detected in the treated protoplasts and monitored during the first week of the cell culture until the stable level of expression was observed. It was useful for the comparison of protoplast susceptibility to DNA uptake and the transgene expression as the fluorescence declined with various rates depending on the used carrot genotype and PEG-concentration. GFP-monitoring in callus enabled the selection of stably expressing lines. It also allowed verification of the homogeneous tissue composition with regard to the expression of the transgene. In plants, GFP-performance depended on the assayed tissue and organ despite of the constitutive 35S promoter. The expression was visually detected in both vegetative and generative parts, but particularly strong fluorescence was observed in leaf marginal meristems, petioles, stems, and styles. Those tissues can be convenient for examination of the transgenic plants during their growth. The results encourage that GFP is a valuable reporter and can be routinely used for optimization of transformation protocol, selection of transformants and monitoring transgenic carrot.

Stable transformation of Mesembryanthemum crystallinum (L.) with Agrobacterium rhizogenes harboring the green fluorescent protein targeted to the endoplasmic reticulum

“…Some previous reports have suggested that GFP can replace antibiotic selection (e.g. Yancheva et al, 2006;Baranski et al, 2006Baranski et al, , 2007. In our study, single green fluorescent roots could produce GFP-positive calli when maintained on media supplemented with kanamycin.…”

“…As observed in other studies, the production of chimeric calli with green fluorescence in some, but not all, cells would be a possible outcome as a result of transgene silencing in continued culture (e.g. Baranski et al, 2007;Rakosy-Tican et al, 2007). However, as shown here, the intensity of green fluorescence of calli produced from transformed roots of the common ice plant did not change, and individual callus cells exhibited similarly high, uniform levels of GFP fluorescence even after 2 years of culture, indicating high transgene stability.…”

“…The intensity of GFP fluorescence depends on several factors, such as the GFP gene variant and the type of promoter as well as the target tissue and the species used (Hraška et al, 2006). The CaMV 35S promoter that we used in our work does not ensure uniform expression of the transgene and is suggested to favor actively dividing cells like those in meristems (Nagata et al, 1987;Baranski et al, 2007). This may partly account for the strongest GFP signal being found at the apical region of ice plant roots and along cambial zone within vascular cylinder.…”

Chitinase CHIT36 from Trichoderma harzianum Enhances Resistance of Transgenic Carrot to Fungal Pathogens

“…sativus L.) cultivar ‘Koral’ were maintained in ZSMI, a modified B5 Gamborg et al. (1968) medium [B5 salts, 500 mg/l KNO 3 , 250 mg/l casein hydrolysate, 0.5 mg/l nicotinic acid, 0.1 mg/l thiamin, 0.1 mg/l pyridoxin, 20 g/l sucrose and 0.4 mg/l 2,4‐dichlorophenoxyacetic acid (2,4‐D), pH 5.7] in dark at 25°C and the protoplasts were released from well‐established cell suspension as described previously (Baranski et al., 2007b). The pGJ2350 vector was delivered into protoplasts essentially according to Radchuk et al.…”

Overexpression and characterization of cyprosin B in transformed suspension cells of Cynara cardunculus

“…GFP has proved to be a well-suited and very useful visual marker for transgenic plant screening (Baranski et al 2006(Baranski et al , 2007Borisjuk et al 1999;Elliott et al 1999;Niedz et al 1995;Tian et al 1999;Vain et al 1998;Yancheva et al 2006).…”