Optimization of Induction and Culture Conditions for Hairy Roots of <i>Anisodus luridus</i>

Kai-hui ZHAO; Yu-fang FAN; Yuan-jiang XU; Fang CAO; Xiao-zhong LAN; Zhi-hua LIAO

doi:10.13718/j.cnki.xdzk.2017.04.006

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Anisodus luridus, belonging to the Solanaceae family, is a Tibetan endemic plant species producing pharmaceutical tropane alkaloids. In order to provide a theoretical basis for the scale production of tropane alkaloids with A. luridus hairy roots as the raw material, a study was conducted in which Agrobacterium C58C1 (pRiA4) was used to infect the cotyledon, leaf or stem of A. luridus, and its hairy root induction was recorded. High frequencies of hairy root induction were obtained from stem (60.00%) and leaf (53.33%); while cotyledon produced no hairy roots. Then, the typical hairy root line was used to screen the optimal medium for root culture growth and alkaloid production. Of the seven types of liquid media used in this study (1/2MS, MS, 1/2B5, White, WPM, B5 and N6), B5 was the best one for both biomass accumulation and TA production. The fresh weight of hairy root cultures in B5 was 6.9±0.94 g/flask (or 0.44±0.06 g/flask dry weight). Further, HPLC analysis results showed that hairy root cultures in B5 produced hyoscyamine (3.49±0.13 mg/g) at a higher level than that in other media. The highest scopolamine contents were detected in hairy root cultures in B5 and 1/2B5, which were respectively 1.37±0.06 mg/g and 1.43±0.22 mg/g. However, there was no significant difference in scopolamine contents between hairy root cultures in B5 and that in 1/2B5. Finally, it was found that hairy root cultures in B5 produced total TAs at the highest level (2.1±0.29 mg/flask).

Corresponding author: Zhi-hua LIAO

Received Date: 10/12/2015

Available Online: 20/04/2017

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Abstract: Anisodus luridus, belonging to the Solanaceae family, is a Tibetan endemic plant species producing pharmaceutical tropane alkaloids. In order to provide a theoretical basis for the scale production of tropane alkaloids with A. luridus hairy roots as the raw material, a study was conducted in which Agrobacterium C58C1 (pRiA4) was used to infect the cotyledon, leaf or stem of A. luridus, and its hairy root induction was recorded. High frequencies of hairy root induction were obtained from stem (60.00%) and leaf (53.33%); while cotyledon produced no hairy roots. Then, the typical hairy root line was used to screen the optimal medium for root culture growth and alkaloid production. Of the seven types of liquid media used in this study (1/2MS, MS, 1/2B5, White, WPM, B5 and N6), B5 was the best one for both biomass accumulation and TA production. The fresh weight of hairy root cultures in B5 was 6.9±0.94 g/flask (or 0.44±0.06 g/flask dry weight). Further, HPLC analysis results showed that hairy root cultures in B5 produced hyoscyamine (3.49±0.13 mg/g) at a higher level than that in other media. The highest scopolamine contents were detected in hairy root cultures in B5 and 1/2B5, which were respectively 1.37±0.06 mg/g and 1.43±0.22 mg/g. However, there was no significant difference in scopolamine contents between hairy root cultures in B5 and that in 1/2B5. Finally, it was found that hairy root cultures in B5 produced total TAs at the highest level (2.1±0.29 mg/flask).

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铃铛子(Anisodus luridus)为茄科多年生宿根草本植物，藏语名为“唐冲那薄”，主要分布在西藏等海拔在2 500~4 000 m的山坡草地、林缘及田地宅旁.铃铛子根和种子可供药用，有抗痉挛和止痛作用，医药上常用其根提取莨菪碱类生物碱^[1].

托品烷生物碱(tropane alkaloids，TAs)是一类来自茄科植物的生物碱，是副交感神经系统的有效抑制剂，主要药用成分为莨菪碱和东莨菪碱^[2].由于托品烷生物碱的化学合成成本较高，目前其供应仍然依赖于从植物中提取.托品烷生物碱在野生茄科植物中质量分数很低，故而寻求新途径来提高托品烷生物碱的产量尤为重要.铃铛子作为托品烷生物碱的药源植物之一，其野生资源正在逐渐枯竭.目前发根培养成为研究药用植物次生代谢产物的有效手段，而关于铃铛子发根培养条件的优化尚未有报道.本研究建立了铃铛子发根的转化体系，并对最适于铃铛子发根生长和生物碱积累的培养基进行优化筛选，为获得托品烷生物碱提供了新的途径，具有重要的理论意义和应用价值.

1. 材料

1.1. 植物

铃铛子种子由西藏农牧学院兰小中教授鉴定并提供.

1.2. 菌株

农杆菌C58C1 (pRiA4) 由西南大学天然产物与代谢工程实验室保存.

2. 方法

2.1. 铃铛子发根的获得

分别剪取约1 cm见方的叶片，1.5 cm长的茎段，0.5 cm长的子叶放入已活化好的菌液中，振荡浸泡约10 min后，用无菌滤纸吸干多余菌液.将叶片、茎段和子叶分别平铺在MS(含100 μmol/L的乙酰丁香酮)固体培养基上，于25±1.0 ℃恒温暗培养48 h后转入含抗生素400 mg/L头孢噻肟钠的MS固体培养基上除菌培养，待发根生长到2 cm左右时剪下，转移到新的含400 mg/L头孢噻肟钠的MS固体培养基中培养，每20 d继代1次，直至除菌完全.

2.2. 发根rolB基因和rolC基因的PCR检测

取200 mg除菌彻底的发根应用CTAB法^[3]提取基因组DNA，同时提取铃铛子无菌苗的根DNA作为阴性对照.另取200 μL活化的农杆菌C58C1菌液，12 000 r/min离心2 min，去上清，20 μL ddH₂O悬浮，沸水煮5 min，12 000 r/min离心2 min，上清作为阳性模板.所用rolB基因和rolC基因引物参照Zhang等^[4].

2.3. 发根生长最优培养基的筛选

选取长势较好、分支较多的真叶上诱导出的发根T3株系，作为铃铛子发根生长研究的实验对象.为找出铃铛子发根生长的最适培养基，本文选取了7种不同培养基作为测试培养基：N6，1/2B5，B5，WPM，MS，1/2MS和White，蔗糖作为碳源，质量浓度为30 g/L.将各培养基pH调至5.8，分装于250 mL的广口三角瓶中，每瓶100 mL，121℃灭菌20 min.每瓶发根的接种量为10根2 cm左右的发根，鲜质量大约为100 mg，每种培养基3瓶重复，于110 r/min，25℃摇床暗培养.

2.4. 托品烷生物碱的提取和高效液相色谱HPLC分析

托品烷生物碱的提取及HPLC分析参照Wang等^[2]的方法.

4. 讨论

农杆菌种类、被感染部位细胞类型以及生理状况等因素的不同，使T-DNA插入植物基因组的位置、长度和拷贝数千差万别^[5].本实验分别选取了真叶、茎段和子叶作为外植体，其受到农杆菌C58C1 (pRiA4) 侵染后的反应有很大的差异，子叶对于侵染完全没有反应，茎段上诱导出的发根生长状态较弱，从而选取无菌苗的真叶作为最佳的外植体.

各种培养基由于所含的成分和比例不同，因此对发根的生物量、生长状态和次生代谢产物的形成有不同的影响^[6-10].将真叶上诱导的发根转接到N6，1/2B5，B5，WPM，MS，1/2MS和White 7种不同的液体培养基中，以B5液体培养基中培养的发根生物量积累速度最快，莨菪碱碱质量分数最高，总生物碱产量最高，最适宜培养铃铛子发根.这是由于B5液体培养基中含有较低浓度的铵态氮和较高浓度的硝态氮，而培养基中铵态氮和硝态氮之间的比例会影响发根生长和次生代谢产物的合成^[11]，硝态氮比例高，铵态氮的浓度低，有利于铃铛子发根生物量的积累和生物碱的合成，具体的原因还需要进一步研究.

本实验成功建立了铃铛子发根诱导和培养体系，同时对铃铛子发根的培养条件及其次生代谢产物累积进行了初步研究.结果表明铃铛子无菌苗的真叶为农杆菌诱导发根的最佳外植体，铃铛子发根在B5液体培养基培养时生物量积累最多，托品烷生物碱合成最多，对进一步规模化生产莨菪碱和东莨菪碱奠定了基础.

Figure (3) Table (3) Reference (11)

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Optimization of Induction and Culture Conditions for Hairy Roots of Anisodus luridus

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Optimization of Induction and Culture Conditions for Hairy Roots of Anisodus luridus

Corresponding author: Zhi-hua LIAO