银杏愈伤组织生长、褐化与黄酮积累研究
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摘要
旨在建立稳定的银杏愈伤组织继代体系,筛选细胞活性强且黄酮产量高的细胞培养条件,为工业化生产提供一定参考。以MS培养基为基本培养基,在40 d的继代周期内设计正交实验研究外植体月份、外源激素与抗褐化剂交互作用下的银杏愈伤组织生长、褐化与黄酮积累,并对比了4、5、6月中旬银杏叶片黄酮含量。结果表明,银杏愈伤组织继代中的最佳生长组合为:4月叶片诱导的愈伤组织+3.0 mg/L NAA+0.5 mg/L KT+5 mg/L VC;最佳抗褐化组合为:4月叶片诱导的愈伤组织+1.0 mg/L NAA+1.0mg/L KT+10 mg/L VC;银杏叶黄酮含量从4-6月呈上升趋势,且不同月份叶片诱导的愈伤组织黄酮含量与之呈正相关,以6月叶片诱导的愈伤组织+2.0 mg/L NAA+1.0 mg/L KT+5 mg/L VC为最佳黄酮积累条件;结合愈伤组织干重,总黄酮实际产量最高组合为:5月叶片诱导的愈伤组织+2.0 mg/L NAA+0.5 mg/L KT+5 mg/L VC。银杏愈伤组织的适宜继代周期为24-32 d,生长量较大且褐化率低。
The aim of this study is to establish a stable callus succession system of Ginkgo biloba,to screen the condition of culturing cells with high activity and flavonoids,and thus to provide references for industrial production. Using MS medium as the basic medium,we investigated the G. biloba callus growth,browning and flavonoid accumulation under the interactions among explant month,exogenous hormone and anti-browning agent by an orthogonal experiment of 40 d succession period,and compared with the contents of G. biloba leaves' flavonoids in April,May,and June. The results showed that the condition for optimal growth of G. biloba callus was callus induced by April leaf + 3.0 mg/L NAA + 0.5 mg/L KT + 5 mg/L VC. The optimal anti-browning condition was callus induced by April leaf +1.0 mg/L NAA+1.0 mg/L KT+10 mg/L VC. The flavonoid content of G. biloba leaf tended to increase from April to June,and the flavonoid content of callus at different month was positively correlated with its explants,the condition of callus induced by June leaf + 2.0 mg/L NAA + 1.0 mg/L KT + 5 mg/L VC was the optimal for flavonoids accumulation. Combined with the dry weight of callus,the maximum flavonoid yield was from the combination of callus induced by May leaf + 2.0 mg/L NAA + 0.5 mg/L KT + 5 mg/L VC. The appropriate succession period for G. biloba callus was 24-32 d,with large growth and low browning rate.
The aim of this study is to establish a stable callus succession system of Ginkgo biloba,to screen the condition of culturing cells with high activity and flavonoids,and thus to provide references for industrial production. Using MS medium as the basic medium,we investigated the G. biloba callus growth,browning and flavonoid accumulation under the interactions among explant month,exogenous hormone and anti-browning agent by an orthogonal experiment of 40 d succession period,and compared with the contents of G. biloba leaves' flavonoids in April,May,and June. The results showed that the condition for optimal growth of G. biloba callus was callus induced by April leaf + 3.0 mg/L NAA + 0.5 mg/L KT + 5 mg/L VC. The optimal anti-browning condition was callus induced by April leaf +1.0 mg/L NAA+1.0 mg/L KT+10 mg/L VC. The flavonoid content of G. biloba leaf tended to increase from April to June,and the flavonoid content of callus at different month was positively correlated with its explants,the condition of callus induced by June leaf + 2.0 mg/L NAA + 1.0 mg/L KT + 5 mg/L VC was the optimal for flavonoids accumulation. Combined with the dry weight of callus,the maximum flavonoid yield was from the combination of callus induced by May leaf + 2.0 mg/L NAA + 0.5 mg/L KT + 5 mg/L VC. The appropriate succession period for G. biloba callus was 24-32 d,with large growth and low browning rate.
引文
[1]朱红威,邵菊芳,李庆,等.不同培养条件对银杏愈伤组织生长及黄酮含量的影响[J].食品科学, 2007, 28(11):102-105.
[2]张辉,于荣敏,姚新生.银杏愈伤组织和悬浮细胞培养及黄酮类化合物的产生[J].沈阳药科大学学报, 1999(2):54-58.
[3]Bilia AR. Ginkgo biloba L.[J]. Fitoterapia, 2002, 3:276-279.
[4]曹福亮.中国银杏志[M].北京:中国林业出版社, 2007.
[5]Laurain D, Trémouillaux-Guiller J, Chénieux JC, et al. Production of ginkgolide and bilobalide in transformed and gametophyte derived cell cultures of Ginkgo biloba[J]. Phytochemistry, 1997, 46(1):127-130.
[6]陈西娟,王成章,叶建中.银杏叶化学成分及其应用研究进展[J].生物质化学工程, 2008, 42(4):57-62.
[7]程勤贤,陈鹏,何爱华.银杏种质资源分类研究综述[J].江苏林业科技, 2007, 34(4):44-47.
[8]St?ckigt J, Obitz P, Falkenhagen H, et al. Natural products and enzymes from plant cell cultures[J]. Plant Cell Tissue&Organ Culture, 1995, 43(2):97-109.
[9]孙楠楠,许锋,黄小花,等.银杏组织培养及次生代谢分子生物学的研究进展[J].贵州农业科学, 2011, 39(10):27-31.
[10]胡选萍,张晓娟,任鹏斌,等.不同激素对银杏愈伤组织黄酮积累的影响[J].中成药, 2015, 37(12):2787-2790.
[11]Cheng SY, Zhang WW, Sun N, et al. Production of flavonoids and terpene lactones from optimized Ginkgo biloba tissue culture[J].Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 2014, 42(1):88-93.
[12]程水源,王燕,李俊凯,等.银杏叶黄酮含量变化及分布规律的研究[J].园艺学报, 2001, 28(4):353-355.
[13]戴莹,杨世海,赵鸿峥,等.药用植物组织培养中褐化现象的研究进展[J].中草药, 2016, 47(2):344-351.
[14]罗晓芳,田砚亭,姚洪军.组织培养过程中PPO活性和总酚含量的研究[J].北京林业大学学报, 1999, 21(1):98-101.
[15]陈学森,邓秀新,章文才.培养基及培养条件对银杏愈伤组织黄酮产量的影响[J].园艺学报, 1997, 24(4):62-66.
[16]房慧勇,朱虹,丁海麦,等.影响愈伤组织中次生代谢产物含量的因素研究进展[J].中国中药杂志, 2014, 39(15):2846-2850.
[17]崔刚,唐蕾,王武.培养基和外源激素对银杏愈伤组织诱导、生长及叶绿素含量的影响[J].食品与生物技术学报, 2008,27(2):117-122.
[18]高红兵,杜凤国,王欢.抗褐化剂对天女木兰芽外植体褐化与酚酸氧化的影响[J].林业科学研究, 2017, 30(3):525-532.
[2]张辉,于荣敏,姚新生.银杏愈伤组织和悬浮细胞培养及黄酮类化合物的产生[J].沈阳药科大学学报, 1999(2):54-58.
[3]Bilia AR. Ginkgo biloba L.[J]. Fitoterapia, 2002, 3:276-279.
[4]曹福亮.中国银杏志[M].北京:中国林业出版社, 2007.
[5]Laurain D, Trémouillaux-Guiller J, Chénieux JC, et al. Production of ginkgolide and bilobalide in transformed and gametophyte derived cell cultures of Ginkgo biloba[J]. Phytochemistry, 1997, 46(1):127-130.
[6]陈西娟,王成章,叶建中.银杏叶化学成分及其应用研究进展[J].生物质化学工程, 2008, 42(4):57-62.
[7]程勤贤,陈鹏,何爱华.银杏种质资源分类研究综述[J].江苏林业科技, 2007, 34(4):44-47.
[8]St?ckigt J, Obitz P, Falkenhagen H, et al. Natural products and enzymes from plant cell cultures[J]. Plant Cell Tissue&Organ Culture, 1995, 43(2):97-109.
[9]孙楠楠,许锋,黄小花,等.银杏组织培养及次生代谢分子生物学的研究进展[J].贵州农业科学, 2011, 39(10):27-31.
[10]胡选萍,张晓娟,任鹏斌,等.不同激素对银杏愈伤组织黄酮积累的影响[J].中成药, 2015, 37(12):2787-2790.
[11]Cheng SY, Zhang WW, Sun N, et al. Production of flavonoids and terpene lactones from optimized Ginkgo biloba tissue culture[J].Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 2014, 42(1):88-93.
[12]程水源,王燕,李俊凯,等.银杏叶黄酮含量变化及分布规律的研究[J].园艺学报, 2001, 28(4):353-355.
[13]戴莹,杨世海,赵鸿峥,等.药用植物组织培养中褐化现象的研究进展[J].中草药, 2016, 47(2):344-351.
[14]罗晓芳,田砚亭,姚洪军.组织培养过程中PPO活性和总酚含量的研究[J].北京林业大学学报, 1999, 21(1):98-101.
[15]陈学森,邓秀新,章文才.培养基及培养条件对银杏愈伤组织黄酮产量的影响[J].园艺学报, 1997, 24(4):62-66.
[16]房慧勇,朱虹,丁海麦,等.影响愈伤组织中次生代谢产物含量的因素研究进展[J].中国中药杂志, 2014, 39(15):2846-2850.
[17]崔刚,唐蕾,王武.培养基和外源激素对银杏愈伤组织诱导、生长及叶绿素含量的影响[J].食品与生物技术学报, 2008,27(2):117-122.
[18]高红兵,杜凤国,王欢.抗褐化剂对天女木兰芽外植体褐化与酚酸氧化的影响[J].林业科学研究, 2017, 30(3):525-532.