美洲商陆再生及遗传转化体系的研究
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摘要
美洲商陆为商陆科多年生草本植物,我国具有丰富的野生资源。美洲商陆被广泛也应用于医药、病虫害防治、食品着色以及植物修复等方面。但由于野生型美洲商陆的生长周期较长,对美洲商陆的应用造成很大的困扰。因此建立一整套的美洲商陆快速繁殖体系和遗传转化体系十分必要,一方面有利于工业化生产多种重要次生代谢产物,并为从美洲商陆体内克隆药物基因提供植物材料；另一方面,为将来研究美洲商陆的富集机理和代谢途径以及研究美洲商陆重金属富集相关功能基因奠定基础,对今后美洲商陆的进一步开发应用具有重要的意义。
本实验通过组织培养技术培养无菌苗,建立了美洲商陆的高效再生体系,并结合农杆菌介导法建立了美洲商陆遗传转化体系。另外在组织培养条件下,研究了硫酸锰对美洲商陆无菌苗生长的影响及锰的富集特性。实验结果如下：
1.美洲商陆种子萌发及无菌苗培养的条件：种子在浓硫酸中处理12min,然后接种于MS+30g/L蔗糖+7g/L琼脂的培养基中,培养温度为26±1℃,先暗培养3-4天,然后转入光照强度为2000LUX、光暗周期为14h/10h条件下进行培养,2-3周后可获得商陆无菌苗,成苗率为100%。
2.美洲商陆高效再生体系的建立：以美洲商陆叶片、茎段、茎节为外植体,通过筛选其各个生长阶段最佳培养基,建立了其稳定高效的再生体系,结果表明：最适外植体为茎节,不定芽诱导分化最佳培养基为MS+6-BA3.0mg/L +NAA0.01mg/L+TDZ0.02 mg/L+蔗糖30mg/L+琼脂7g/L,增殖系数达12.7；不定芽伸长最佳培养基为MS+TDZ0.02 mg/L+6-BA3.Omg/L+NAAO.01mg/L +GA32.0mg/L+蔗糖30mg/L+琼脂7g/L,并具有较好的壮苗作用；生根最佳培养基1/2MS+NAA 0.4mg/L+蔗糖25mg/L+琼脂7g/L,生根率可达90%以上
3.美洲商陆遗传转化体系的建立：以美洲商陆无菌苗茎节为转化受体,预培养时间为5d,侵染时菌液的浓度为OD600=0.6,侵染时间为3～5mmin,共培养3d,在共培养时加入乙酰丁香酮可提高转化率。Cef在除菌过程中使用浓度为250mg/L,直至抗性芽的形成。
4. MnSO4处理对美洲商陆无菌苗的生长的影响：随MnSO4浓度的升高,无菌苗的株高、各器官的重量呈下降趋势；对美洲商陆无菌苗光合色素研究结果表明,MnSO4处理浓度从2mM上升至5mM,色素含量与对照相比没有显著性差异,说明低浓度Mn对美洲商陆无菌苗叶片色素未造成显著影响,而高浓度Mn处理下,美洲商陆无菌苗的色素含量下降的较为明显；MDA含量随MnSO4浓度的升高而增大。MnSO4处理下,美洲商陆无菌苗的根和叶片表现出了较为明显锰富集作用,但并没有达到野生美洲商陆的超富集植物的标准。因此,美洲商陆无菌苗可能不适合用来研究野生美洲商陆Mn超富集机制。
Phytolacca americana is a kind of perennial herb, belong to Phytolaccaceae. Its wild resources are rich in China. It has been widely used in medicine industry, pest controlling, food industry and phytoremediation and etc. However, the long growth cycle of P. americana obstructs its application for the industrial production of a variety of important secondary metabolites, the cloning the drug-related genes, the study on the Mn hyperaccumulating mechanism and Mn-enrichment related genes of P.americana, the establishment of rapid propagation system and genetic transformation system of P. americana are necessary. In this paper, we tried to establish an efficient regeneration system and a genetic transformation system of P. americana by using Agrobacterium tumefaciens. In addition, the effects of MnSO4 on the growth and manganese accumulation of P. americana aseptic seedling were studied in this paper. The major results were as follows:
1. The optimum culture condition for seed germination and aseptic seeding cultivation of P. americana are as follows:the seeds were first put into concentrated sulphuric acid for 12 minutes, then inoculated in MS basic medium supplemented with sucrose 20g/L and agar 7g/L and cultured in dark at 26±1℃for 3-4days; finally, they were cultured in 2000 Lux with a photoperiod of 14h/10h at 26±1℃for 2-3 weeks. Under such conditions, all seeds can grow into seeding.
2. Leaves, stems and nodes of P. americana aseptic seeding were used as explants to select the optimal explants, optimal culture medium and optimal culture condition. The results indicated that the optimal explant was node, the highest differentiation rate of adventitious buds reached 12.7 on the culture medium of MS+6-BA3.0mg/L +NAA0.01mg/L+TDZ0.02mg/L+sucrose30mg/L+agar7g/L; the optimized medium for adventitious buds elongation was MS+TDZ0.02mg/L+6-BA3.0mg/L +NAA0.01mg/L+GA32.0mg/L+sucrose30mg/L+agar7g/L; the optimized medium for rooting was 1/2MS+NAA0.4mg/L+sucrose25mg/L+agar7g/L, and the rooting rate could reach 90%.
3. The node of P. americana was used as transformation receptor. It was first pre-cultured for 5 days, then co-cultured with Agrobacterium culture fluid with an optimized OD value of 0.6 and supplemented with acetosyringone for 2-3days; Cef with a concentration of 250mg/L was used to inhibit the growth of Agrobacterium until plantlets formed.
4. With the increase of MnSO4 concentration, stem height, fresh weight and dry weight etc. of P. americana aseptic seedings were all decreased; pigment contents changed little when MnSO4 concentration was less than 5mM and decreased with MnSO4 concentration increased further; MDA contents increased with the increasing of MnS04 concentration. Under MnSO4 treatment, roots and leaves of P. americana aseptic seeding could accumulate Mn, but its degree did not reach the standard of hyperaccumulators. So, P. americana aseptic seedings are not suitable to be used as materials to study the Mn-hyperaccumulating mechanism of P. americana.wild plants.
本实验通过组织培养技术培养无菌苗,建立了美洲商陆的高效再生体系,并结合农杆菌介导法建立了美洲商陆遗传转化体系。另外在组织培养条件下,研究了硫酸锰对美洲商陆无菌苗生长的影响及锰的富集特性。实验结果如下：
1.美洲商陆种子萌发及无菌苗培养的条件：种子在浓硫酸中处理12min,然后接种于MS+30g/L蔗糖+7g/L琼脂的培养基中,培养温度为26±1℃,先暗培养3-4天,然后转入光照强度为2000LUX、光暗周期为14h/10h条件下进行培养,2-3周后可获得商陆无菌苗,成苗率为100%。
2.美洲商陆高效再生体系的建立：以美洲商陆叶片、茎段、茎节为外植体,通过筛选其各个生长阶段最佳培养基,建立了其稳定高效的再生体系,结果表明：最适外植体为茎节,不定芽诱导分化最佳培养基为MS+6-BA3.0mg/L +NAA0.01mg/L+TDZ0.02 mg/L+蔗糖30mg/L+琼脂7g/L,增殖系数达12.7；不定芽伸长最佳培养基为MS+TDZ0.02 mg/L+6-BA3.Omg/L+NAAO.01mg/L +GA32.0mg/L+蔗糖30mg/L+琼脂7g/L,并具有较好的壮苗作用；生根最佳培养基1/2MS+NAA 0.4mg/L+蔗糖25mg/L+琼脂7g/L,生根率可达90%以上
3.美洲商陆遗传转化体系的建立：以美洲商陆无菌苗茎节为转化受体,预培养时间为5d,侵染时菌液的浓度为OD600=0.6,侵染时间为3～5mmin,共培养3d,在共培养时加入乙酰丁香酮可提高转化率。Cef在除菌过程中使用浓度为250mg/L,直至抗性芽的形成。
4. MnSO4处理对美洲商陆无菌苗的生长的影响：随MnSO4浓度的升高,无菌苗的株高、各器官的重量呈下降趋势；对美洲商陆无菌苗光合色素研究结果表明,MnSO4处理浓度从2mM上升至5mM,色素含量与对照相比没有显著性差异,说明低浓度Mn对美洲商陆无菌苗叶片色素未造成显著影响,而高浓度Mn处理下,美洲商陆无菌苗的色素含量下降的较为明显；MDA含量随MnSO4浓度的升高而增大。MnSO4处理下,美洲商陆无菌苗的根和叶片表现出了较为明显锰富集作用,但并没有达到野生美洲商陆的超富集植物的标准。因此,美洲商陆无菌苗可能不适合用来研究野生美洲商陆Mn超富集机制。
Phytolacca americana is a kind of perennial herb, belong to Phytolaccaceae. Its wild resources are rich in China. It has been widely used in medicine industry, pest controlling, food industry and phytoremediation and etc. However, the long growth cycle of P. americana obstructs its application for the industrial production of a variety of important secondary metabolites, the cloning the drug-related genes, the study on the Mn hyperaccumulating mechanism and Mn-enrichment related genes of P.americana, the establishment of rapid propagation system and genetic transformation system of P. americana are necessary. In this paper, we tried to establish an efficient regeneration system and a genetic transformation system of P. americana by using Agrobacterium tumefaciens. In addition, the effects of MnSO4 on the growth and manganese accumulation of P. americana aseptic seedling were studied in this paper. The major results were as follows:
1. The optimum culture condition for seed germination and aseptic seeding cultivation of P. americana are as follows:the seeds were first put into concentrated sulphuric acid for 12 minutes, then inoculated in MS basic medium supplemented with sucrose 20g/L and agar 7g/L and cultured in dark at 26±1℃for 3-4days; finally, they were cultured in 2000 Lux with a photoperiod of 14h/10h at 26±1℃for 2-3 weeks. Under such conditions, all seeds can grow into seeding.
2. Leaves, stems and nodes of P. americana aseptic seeding were used as explants to select the optimal explants, optimal culture medium and optimal culture condition. The results indicated that the optimal explant was node, the highest differentiation rate of adventitious buds reached 12.7 on the culture medium of MS+6-BA3.0mg/L +NAA0.01mg/L+TDZ0.02mg/L+sucrose30mg/L+agar7g/L; the optimized medium for adventitious buds elongation was MS+TDZ0.02mg/L+6-BA3.0mg/L +NAA0.01mg/L+GA32.0mg/L+sucrose30mg/L+agar7g/L; the optimized medium for rooting was 1/2MS+NAA0.4mg/L+sucrose25mg/L+agar7g/L, and the rooting rate could reach 90%.
3. The node of P. americana was used as transformation receptor. It was first pre-cultured for 5 days, then co-cultured with Agrobacterium culture fluid with an optimized OD value of 0.6 and supplemented with acetosyringone for 2-3days; Cef with a concentration of 250mg/L was used to inhibit the growth of Agrobacterium until plantlets formed.
4. With the increase of MnSO4 concentration, stem height, fresh weight and dry weight etc. of P. americana aseptic seedings were all decreased; pigment contents changed little when MnSO4 concentration was less than 5mM and decreased with MnSO4 concentration increased further; MDA contents increased with the increasing of MnS04 concentration. Under MnSO4 treatment, roots and leaves of P. americana aseptic seeding could accumulate Mn, but its degree did not reach the standard of hyperaccumulators. So, P. americana aseptic seedings are not suitable to be used as materials to study the Mn-hyperaccumulating mechanism of P. americana.wild plants.
引文
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