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  • Plant regeneration and genetic transformation of C. canadensis: a non-model plant appropriate for investigation of flower development in Cornus (Cornaceae).

Plant regeneration and genetic transformation of C. canadensis: a non-model plant appropriate for investigation of flower development in Cornus (Cornaceae).

Plant cell reports (2012-11-01)
Xiang Liu, Chun-Miao Feng, Robert Franks, Rongda Qu, De-Yu Xie, Qiu-Yun Jenny Xiang
ABSTRACT

KEY MESSAGE : Efficient Agrobacterium -mediated genetic transformation for investigation of genetic and molecular mechanisms involved in inflorescence architectures in Cornus species. Cornus canadensis is a subshrub species in Cornus, Cornaceae. It has recently become a favored non-model plant species to study genes involved in development and evolution of inflorescence architectures in Cornaceae. Here, we report an effective protocol of plant regeneration and genetic transformation of C. canadensis. We use young inflorescence buds as explants to efficiently induce calli and multiple adventitious shoots on an optimized induction medium consisting of basal MS medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.1 mg/l of 1-naphthaleneacetic acid. On the same medium, primary adventitious shoots can produce a large number of secondary adventitious shoots. Using leaves of 8-week-old secondary shoots as explants, GFP as a reporter gene controlled by 35S promoter and hygromycin B as the selection antibiotic, a standard procedure including pre-culture of explants, infection, co-cultivation, resting and selection has been developed to transform C. canadensis via Agrobacterium strain EHA105-mediated transformation. Under a strict selection condition using 14 mg/l hygromycin B, approximately 5 % explants infected by Agrobacterium produce resistant calli, from which clusters of adventitious shoots are induced. On an optimized rooting medium consisting of basal MS medium supplemented with 0.1 mg/l of indole-3-butyric acid and 7 mg/l hygromycin B, most of the resistant shoots develop adventitious roots to form complete transgenic plantlets, which can grow normally in soil. RT-PCR analysis demonstrates the expression of GFP transgene. Green fluorescence emitted by GFP is observed in transgenic calli, roots and cells of transgenic leaves under both stereo fluorescence microscope and confocal microscope. The success of genetic transformation provides an appropriate platform to investigate the molecular mechanisms by which the various inflorescence forms are developed in Cornus plants.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hygromycin B from Streptomyces hygroscopicus, powder, BioReagent, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
Hygromycin B from Streptomyces hygroscopicus, lyophilized powder
Sigma-Aldrich
Hygromycin B from Streptomyces hygroscopicus, suitable for plant cell culture, BioReagent, ≥60% (HPLC), lyophilized powder
Sigma-Aldrich
Hygromycin B solution from Streptomyces hygroscopicus, ≥60% (HPLC), 45-60 mg/mL in H2O, γ-irradiated
Sigma-Aldrich
Hygromycin B solution from Streptomyces hygroscopicus, ≥60% (HPLC), 45-60 mg/mL in H2O