here - First Legume Society Conference (LSC1)

Book of Abstracts First Legume Society Conference 2013: A Legume Odyssey Novi Sad, Serbia, 9-11 May 2013
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Establishment of embryonic tip regeneration system and Transformation of iron uptake
genes in Azuki Bean (Vigna angularis Ohwi & Ohashi)
Ping Wan 1 ,Yu’e Tian 1 , Jinghong Cao 1 , Yisong Li 1 , Bo Zhao 1 , Kai Yang 1 , Huilan Wu 2 , Hongqing
Ling 2
1 College of Plant Science and Technology, Beijing University of Agriculture, Beijing, P. R. China
2 State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental
Biology, Chinese Academy of Sciences, Chaoyang District, Beijing, PR China
The embryonic tip regenerate system of azuki bean was firstly created. The best condition for
embryonic tip regeneration was MS medium with 0.2mg/L Indole butytic acid (IBA) and
0.2mg/L 6BA. The regeneration rate was nearly 109.7% under this condition. It needs 40 days to
be completed from inducing to obtain 3-5cm long shoots. The regeneration system of embryonic
tip has higher efficiency, time consuming and higher tolerance to agrobacterium tumefaciens than
hypocotyl’s and epicotyl’s. 100mg/ L Kanamycin and 5 mg/L Hygromycin B was suitable for
screen transformation plants in azuki bean. Embryonic tips of iron-deficiency sensitive Jingnong
5 variety were inoculated with Agrobacterium tumefaciens EHA105 harboring pCAMBIA1200-
35S::AtbHLH39GUS and pBI121-35S::FIT, respectively. AtbHLH39 and FIT are important iron
uptake regulating genes from Arabidopsis encoding bHLH protein.The frequency of GUS gene
transient expression was calculated to determine the suitable condition of agrobacterium tumefaciens
to inoculate embryonic tips. The infected method was standing and co-culture at 28ºC in dark.
The frequency of GUS gene transient expression was 48.78% under this condition. When the coculture
time was 72 hours, the frequency of GUS gene transient expression was 90.47%. Nine
positive FIT transgenic plants were obtained and identified by PCR and RT-PCR amplification
with specific primer pairs of FIT gene. The average transferred rate of FIT gene was 6.47%.
Acknowledgements
This research was sponsored by the Project of Beijing Municipal Education Committee Science and Technology
Research Fund (KM201010020004)
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