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Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons

Content Provider	Springer Nature Link
Author	Cheng, Lin Li, He Ping Qu, Bo Huang, Tao Tu, Jin Xing Fu, Ting Dong Liao, Yu Cai
Copyright Year	2010
Abstract	A protocol for chloroplast transformation of an elite rapeseed cultivar (Brassica napus L.) was developed based on optimized conditions for callus induction and regeneration from cotyledonary tissues. Comparison of six different media with three elite cultivars showed that B5 medium plus 3 mg/l AgNO3 supplemented with 0.6 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-furfurylaminopurine was optimal for callus formation and maintenance without differentiation, while the medium suitable for regeneration was B5 medium supplemented with 1 mg/l 6-benzylaminopurine, 1 mg/l 6-furfurylaminopurine and 0.5 mg/l α-naphthaleneacetic acid. A rapeseed-specific chloroplast transformation vector was constructed with the trnI and trnA sequences amplified from the rapeseed chloroplast genome using two primers designed according to Arabidopsis homologs. The aadA gene was used as a selection marker regulated by the ribosome-binding site from the bacteriophage T7 gene 10L, the tobacco 16S rRNA promoter and the psbA terminator. After bombardment, cotyledonary segments were cultured for callus formation on media containing 10 mg/l spectinomycin and regeneration was carried out on medium with 20 mg/l spectinomycin. Heteroplasmic plastid transformants were isolated. An overall efficiency for the chloroplast transformation was one transplastomic plant per four bombarded plates. Southern blot analyses demonstrated proper integration of the target sequence into the rapeseed chloroplast genome via homologous recombination. The expression of the aadA gene was confirmed by Northern blot analysis. Analysis of T1 transplastomic plants revealed that the transgenes integrated into the chloroplast were inheritable with a ratio of about 8%. These results suggest that rapeseed may be a suitable crop for chloroplast transformation with cotyledons as explants under appropriate conditions.
Starting Page	371
Ending Page	381
Page Count	11
File Format	PDF
ISSN	07217714
Journal	Plant Cell Reports
Volume Number	29
Issue Number	4
e-ISSN	1432203X
Language	English
Publisher	Springer-Verlag
Publisher Date	2010-02-24
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Bombardment Brassica napus Callus induction Chloroplast transformation Cotyledonary tissue Shoot regeneration Plant Biochemistry Biotechnology Plant Sciences Cell Biology
Content Type	Text
Resource Type	Article
Subject	Plant Science Medicine Agronomy and Crop Science

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