title: In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation in asakura-sanshoo (Zanthoxylum piperitum (L.) DC. F. inerme Makino) an important medicinal plant.
abstract: BACKGROUND
  Asakura-sanshoo (Zanthoxylum piperitum [L.] DC. f. inerme Makino) is an important medicinal plant in East Asia. Transgenic technique could be applied to improve plant traits and analyze gene function. However, there is no report on regeneration and genetic transformation in Asakura-sanshoo.
  
  
  OBJECTIVE
  To establish a regeneration and Agrobacterium tumefaciens-mediated genetic transformation system in Asakura-sanshoo, which could be used for cultivar improvement and gene function analysis.
  
  
  METHODS
  The various combinations of indole-3-butyric acid (IBA), 6-benzylaminopurine (BA) and naphthalene acetic acid (NAA) were explored for the optimal plant regeneration from petiole and stem of Asakura-sanshoo. The half-strength woody plant medium (WPM) with different concentrations of NAA and IBA was used to induce root. For genetic transformation, A. tumefaciens strain EHA-105 harboring the plasmid pBin-Ex-H-ipt which carries the isopentenyl transferase (ipt) gene, β-glucuronidase (GUS) gene and kanamycin resistance gene neomycin phosphotransferase II (NPTII) were used. The transformation efficiency was detected by the kanamycin resistant frequency.
  
  
  METHODS
  Petioles and stems were obtained from the in vitro cultured Asakura-sanshoo. The petiole and stem segments were precultured for 3 days, and then inflected using the bacterium at the concentration of OD600 0.5-0.8 for 10 min, followed by 3 days co-cultivation. Selection of the transgenic plants was carried out after 7 days the regeneration using gradient kanamycin at 30 mg/L and 50 mg/L, respectively. Successful transformed plants were confirmed by GUS histochemical assays, polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR), and Southern blotting analysis.
  
  
  RESULTS
  The highest shoots regeneration was obtained on WPM supplement with 0.5 mg/L BA and 0.2 mg/L NAA. The optimal rooting medium was half strength macro-element WPM. The kanamycin resistant frequency of petiole and stem was 24.66% and 25.93%, respectively. Thirty-five shoots in thousands adventitious buds were confirmed through GUS histochemical assays, PCR, RT-PCR, and Southern blotting. The regeneration shoot per explants elevated 5.85 fold compared with the wild-type plants.
  
  
  CONCLUSIONS
  Individual transgenic Asakura-sanshoo lines were obtained. In this paper, it first revealed the expression of ipt gene significantly promoted the adventitious buds induction in Asakura-sanshoo as the same action as in other plants.
authors.affiliation: Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region, Ministry of Education, Institute of Agro-Bioengineering, Guiyang 550025, Guizhou Province, P.R. China ; The State Key Lab of Green Pesticide and Agricultural Biological Engineering, Center for Research and Development of Fine Chemicals, Guizhou University, Guiyang 550025, Guizhou Province, P.R. China.
authors.forename: Xiaofang
authors.initials: X
authors.lastname: Zeng
authors.affiliation: Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region, Ministry of Education, Institute of Agro-Bioengineering, Guiyang 550025, Guizhou Province, P.R. China ; The State Key Lab of Green Pesticide and Agricultural Biological Engineering, Center for Research and Development of Fine Chemicals, Guizhou University, Guiyang 550025, Guizhou Province, P.R. China.
authors.forename: Degang
authors.initials: D
authors.lastname: Zhao
keywords: Agrobacterium tumefaciens; asakura-sanshoo; isopentenyl transferase gene; regeneration; transformation
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