葡萄（Vitis vinifera L.）细胞悬浮培养基和培养条件的优化
摘要
为了探索工业化生产植物次生代谢产物白藜芦醇的新途径,本文以巨峰葡萄(Vitis vinifera L.)幼苗细胞为研究对象,以建立并优化葡萄幼苗细胞悬浮培养体系为研究目标。通过单因子试验和正交试验等,分别对葡萄愈伤组织诱导、悬浮细胞系的初步建立、细胞悬浮培养基及其培养条件的优化等方面进行了研究。
本实验以葡萄的叶、叶柄和茎分别作为外植体,在不同激素组合的培养基上进行愈伤组织的诱导和继代培养；并利用获得的松散的愈伤组织在不同条件下建立细胞悬浮培养体系；在整个过程中利用高效液相色谱法(HPLC)测定白藜芦醇的含量。
以葡萄叶为外植体时,诱导的愈伤组织的出愈率很低,均在10%以下,以叶柄和茎为外植体时,愈伤组织的出愈率均大于65%,且叶柄的愈伤组织最高诱导率为96.7%,茎的愈伤组织最高诱导率为94.2%。利用叶柄和茎获得松散愈伤组织时其诱导培养基组成分别是：B5培养基+6-BA0.5 mg/L+IBA0.1 mg/L和B5培养基+6-BA 1.0mg/L+IBA 0.2 mg/L,其继代培养基均为B5培养基+6-BA 1.0 mg/L+2,4-D 0.1 mg/L。
通过单因子及正交试验结果表明,葡萄细胞悬浮培养最佳的培养体系为：以叶柄在基本培养基B5+0.1mg/L NAA+1.Omg/L 2,4-D上诱导的愈伤组织为起始细胞；接种量为40%(20mL液体接种8mL)；25℃、振荡速度为110r/min、遮光培养；并在培养的第3d添加2mL的1mol/L的蛋白胨溶液、第8d添加4mL的1mol/L蔗糖溶液。此时的总白藜芦醇达到203.7μg/20mL,细胞干重为62.5mg/mL。
本文通过葡萄形成愈伤组织外植体的筛选、松散型愈伤组织的获得和细胞悬浮培养基和培养条件的优化,为大规模工业化生产白藜芦醇积累了初步的实验参数。
To study on a new industrialization pathway that some plant cells produce resveratrol (a kind of secondary metabolin), the grape cells were used as a research object in this paper. The foundation and optimization of a suspension culture system were acted as the aims. Based on some single factor tests and the orthogonal trials of four main factors, grape callus were induced, suspension cell system were preliminary erected and suspension culture medium and condition were optimized.
The leaves, leafstalk, and stems from grapes were used as explants to induce calli and subculture in medium with different plant hormone combination; Te relaxed calli was used to erect suspension cell system, and then the resveratol content was determinated by using HPLC.
The results were indicated that, the calli development rate from leaves inducement was lower(under 10%) than that from leafstalk(maximum 96.7%), and young stems(maximum 94.2%),。The inducement medium were as follow, respectively:for the leafstalk, B5 medium with 6-BA 0.5mg/L+IBA0.1 mg/L, and for stems, B5 medium with 6-BA 1.0 mg/L+IBA0.2 mg/L, and subculture medium both were B5 medium with 6-BA1.0 mg/L +2,4-D0.1 mg/L.
Through single factor and orthogonal experiment, this study ascertains the best condition of grape cell suspension culture was that during cell suspension culture,2 mL of peptone (1M) at the 3rd day and 4 mL of sucrose (1M) at the 8th day were added into the medium, respectively; at subculture,40% inocula were maculated to fresh medium; in all suspension cultures, the temperature was at 25℃, the shaking spreed was 110 r/min under darkness. At that time, the content of total resveratrol was 203.7μg/20mL, and the dry cell weight was 62.5mg/mL.
In this research, screening the explants of grape, getting the relaxed callus and optimizing cell suspension culture medium and condition, it will provide industrialization production of resveratrol to accumulate preliminary paramete
本实验以葡萄的叶、叶柄和茎分别作为外植体,在不同激素组合的培养基上进行愈伤组织的诱导和继代培养；并利用获得的松散的愈伤组织在不同条件下建立细胞悬浮培养体系；在整个过程中利用高效液相色谱法(HPLC)测定白藜芦醇的含量。
以葡萄叶为外植体时,诱导的愈伤组织的出愈率很低,均在10%以下,以叶柄和茎为外植体时,愈伤组织的出愈率均大于65%,且叶柄的愈伤组织最高诱导率为96.7%,茎的愈伤组织最高诱导率为94.2%。利用叶柄和茎获得松散愈伤组织时其诱导培养基组成分别是：B5培养基+6-BA0.5 mg/L+IBA0.1 mg/L和B5培养基+6-BA 1.0mg/L+IBA 0.2 mg/L,其继代培养基均为B5培养基+6-BA 1.0 mg/L+2,4-D 0.1 mg/L。
通过单因子及正交试验结果表明,葡萄细胞悬浮培养最佳的培养体系为：以叶柄在基本培养基B5+0.1mg/L NAA+1.Omg/L 2,4-D上诱导的愈伤组织为起始细胞；接种量为40%(20mL液体接种8mL)；25℃、振荡速度为110r/min、遮光培养；并在培养的第3d添加2mL的1mol/L的蛋白胨溶液、第8d添加4mL的1mol/L蔗糖溶液。此时的总白藜芦醇达到203.7μg/20mL,细胞干重为62.5mg/mL。
本文通过葡萄形成愈伤组织外植体的筛选、松散型愈伤组织的获得和细胞悬浮培养基和培养条件的优化,为大规模工业化生产白藜芦醇积累了初步的实验参数。
To study on a new industrialization pathway that some plant cells produce resveratrol (a kind of secondary metabolin), the grape cells were used as a research object in this paper. The foundation and optimization of a suspension culture system were acted as the aims. Based on some single factor tests and the orthogonal trials of four main factors, grape callus were induced, suspension cell system were preliminary erected and suspension culture medium and condition were optimized.
The leaves, leafstalk, and stems from grapes were used as explants to induce calli and subculture in medium with different plant hormone combination; Te relaxed calli was used to erect suspension cell system, and then the resveratol content was determinated by using HPLC.
The results were indicated that, the calli development rate from leaves inducement was lower(under 10%) than that from leafstalk(maximum 96.7%), and young stems(maximum 94.2%),。The inducement medium were as follow, respectively:for the leafstalk, B5 medium with 6-BA 0.5mg/L+IBA0.1 mg/L, and for stems, B5 medium with 6-BA 1.0 mg/L+IBA0.2 mg/L, and subculture medium both were B5 medium with 6-BA1.0 mg/L +2,4-D0.1 mg/L.
Through single factor and orthogonal experiment, this study ascertains the best condition of grape cell suspension culture was that during cell suspension culture,2 mL of peptone (1M) at the 3rd day and 4 mL of sucrose (1M) at the 8th day were added into the medium, respectively; at subculture,40% inocula were maculated to fresh medium; in all suspension cultures, the temperature was at 25℃, the shaking spreed was 110 r/min under darkness. At that time, the content of total resveratrol was 203.7μg/20mL, and the dry cell weight was 62.5mg/mL.
In this research, screening the explants of grape, getting the relaxed callus and optimizing cell suspension culture medium and condition, it will provide industrialization production of resveratrol to accumulate preliminary paramete
引文
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