NDLI: Agrobacterium tumefaciens-mediated transformation of bush monkey-flower (Mimulus aurantiacus Curtis) with a new reporter gene ZsGreen

Content Provider	Springer Nature Link
Author	Susič, Nik Bohanec, Borut Murovec, Jana
Copyright Year	2013
Abstract	A successful in vitro Agrobacterium-mediated transformation protocol was developed for Mimulus aurantiacus, a model species for ecological and evolutionary genetics and a promising ornamental plant. Three binary vectors were tested, each containing the hptII selectable marker gene and one of the reporter genes: gusA, EGFP or ZsGreen, all of them under CaMV 35S promoter. Genetic transformation was achieved through 4 days of co-cultivation of leaf, petiole and hypocotyl explants with Agrobacterium tumefaciens strain LBA 4404. Explants produced transformed callus tissue on solid modified Murashige and Skoog medium supplemented with 1 mg L$^{−1}$ 6-benzylaminopurine, 0.5 mg L$^{−1}$ 1-naphthaleneacetic acid, 30 g L$^{−1}$ sucrose and 20 or 50 mg L$^{−1}$ hygromycin B. All three reporter genes were expressed in callus tissue but the intensity of expression gradually decreased during further plant development. The new reporter gene ZsGreen proved suitable for plant transformation experiments since very intense and bright fluorescence was detected. Out of 1,760 co-cultured explants, 110 plants were regenerated and all of them were found to be PCR positive for the selection and/or reporter genes. Chemiluminescent Southern blot analysis revealed that 91 % of the regenerated plants (100 T$_{0}$ plants) contained T-DNA integrated in their genome. Transformation efficiency varied from 1.4 to 23.3 % for hypocotyl and petiole explants, respectively. Integration of some backbone sequences in plant genomes was confirmed in 75.3 % of T$_{0}$ plants. Using this protocol, stable transformants expressing selectable marker gene hptII and one of the reporter genes (gusA, ZsGreen or EGFP) were obtained in 4–5 months.
Starting Page	243
Ending Page	251
Page Count	9
File Format	PDF
ISSN	01676857
Journal	Plant Cell, Tissue and Organ Culture (PCTOC)
Volume Number	116
Issue Number	2
e-ISSN	15735044
Language	English
Publisher	Springer Netherlands
Publisher Date	2013-11-09
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	EGFP GUS Fluorescent protein Model species Southern blot Vector backbone sequence Plant Sciences Plant Physiology Plant Genetics & Genomics Plant Pathology
Content Type	Text
Resource Type	Article
Subject	Horticulture

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