Zeng Xiaofang, Zhao Degang
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.
Pharmacogn Mag. 2015 Apr-Jun;11(42):374-80. doi: 10.4103/0973-1296.153092.
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.
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.
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.
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.
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.
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.
糙叶花椒（Zanthoxylum piperitum [L.] DC. f. inerme Makino）是东亚一种重要的药用植物。转基因技术可用于改善植物性状和分析基因功能。然而，目前尚无关于糙叶花椒再生和遗传转化的报道。
建立糙叶花椒的再生及根癌农杆菌介导的遗传转化体系，用于品种改良和基因功能分析。
探索吲哚 - 3 - 丁酸（IBA）、6 - 苄氨基嘌呤（BA）和萘乙酸（NAA）的不同组合，以实现糙叶花椒叶柄和茎段的最佳植株再生。使用含有不同浓度NAA和IBA的半强度木本植物培养基（WPM）诱导生根。对于遗传转化，使用携带质粒pBin - Ex - H - ipt的根癌农杆菌菌株EHA - 105，该质粒携带异戊烯基转移酶（ipt）基因、β - 葡萄糖醛酸酶（GUS）基因和卡那霉素抗性基因新霉素磷酸转移酶II（NPTII）。通过卡那霉素抗性频率检测转化效率。
从体外培养的糙叶花椒中获取叶柄和茎段。将叶柄和茎段预培养3天，然后用浓度为OD600 0.5 - 0.8的菌液侵染10分钟，随后共培养3天。在再生7天后，分别使用30 mg/L和50 mg/L的梯度卡那霉素进行转基因植株的筛选。通过GUS组织化学分析、聚合酶链反应（PCR）、逆转录 - PCR（RT - PCR）和Southern杂交分析确认成功转化的植株。
在添加0.5 mg/L BA和0.2 mg/L NAA的WPM培养基上获得了最高的芽再生率。最佳生根培养基是半强度大量元素WPM。叶柄和茎段的卡那霉素抗性频率分别为24.66%和25.93%。通过GUS组织化学分析、PCR、RT - PCR和Southern杂交确认了数千个不定芽中有35个芽。与野生型植株相比，每个外植体再生芽数提高了5.85倍。
获得了单个转基因糙叶花椒株系。本文首次揭示ipt基因的表达与在其他植物中一样，显著促进了糙叶花椒不定芽的诱导。
