石榴愈伤组织的诱导和细胞悬浮培养体系的建立
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摘要
本文以石榴(Punica granatum L)植株为材料,利用植物组织和细胞培养技术,以诱导愈伤组织和建立石榴愈伤组织悬浮培养体系为目的,系统地对石榴愈伤组织的诱导和状态调控、分化了的疏松愈伤组织颗粒悬浮培养体系的特点和生长规律等几个方面进行研究。使用分光光度法测量石榴不同来源组织的黄酮类化合物含量,为工厂化生产黄酮类化合物提供实验和理论依据。
愈伤组织的诱导阶段,研究了不同外植体类型、不同灭菌条件、不同激素种类和水平对石榴愈伤组织诱导率、疏松程度和褐化率的影响。结果表明:靠近顶端的成熟叶片更容易诱导为疏松愈伤组织,体积分数为75%酒精消毒5s和质量分数为0.1%的升汞消毒4min组合的消毒效果较好,石榴愈伤的最佳诱导培养基激素配方为:MS + 1.0 mg/L 2,4-D + 1.0mg/L 6-BA+1.0mg/LNAA。
石榴愈伤组织的继代过程中,出现愈伤组织生长过于致密,不利于实现悬浮培养,因此获得疏松、生长快速的愈伤组织是成功实现石榴细胞悬浮培养的前提。研究不同激素种类和水平对疏松愈伤组织的影响。结果表明石榴最佳继代培养配方为:MS + 1.0mg/L2,4-D +0.1mg/LKT。同时确定了石榴愈伤组织在36天的动态生长曲线,伤组织在这个培养阶段内,呈现出“S”型生长周期,第12d进入对数生长期,在培养的第30d,达到细胞生物量的最大值,为6.48g,增长了5.34倍。
建立石榴细胞悬浮培养体系,研究接种量、摇床转速、蔗糖浓度、装液量、激素配方、培养过程中pH的变化等因素对细胞悬浮培养体系的影响。同时还对悬浮培养中细胞动态生长曲线的测定和黄酮类化合物的积累做了部分研究。
本文得出石榴细胞悬浮培养的最佳条件如下:以MS基本培养基的成分和含量为标准,配制成液体培养基,调节蔗糖浓度为3%,即30g/L,培养瓶的装液量为25mL/150mL,每瓶接种3g愈伤组织,以110rpm的摇床转速,最佳激素配方为:MS+1.0mg/L2,4-D+0.1 mg/L KT。在以上条件下进行培养,悬浮细胞在6d后进入对数生长期,在21d细胞生物量达到最大值,干重达到21.68g/L。21d后细胞生物量开始下降,进入生长静止期,次级生物代谢产物细胞总黄酮含量在此阶段出现高峰值,含量为69.05mg/g。在培养过程中pH值有变化,先降低,后有升高趋势。
用分光光度法,对定期采收的石榴皮、石榴叶和固体培养中的石榴愈伤组织、液体培养中的细胞,测量其总黄酮含量,并分析在不同时间的变化趋势。结果表明:石榴皮的总黄酮含量随时间变化而呈逐步下降趋势,在六月份底石榴皮黄酮含量达到高峰值,为124.7mg/g,到九月底十月初果实成熟时,石榴皮总黄酮含量基本稳定,为64.8mg/g。石榴叶的总黄酮含量变化不大,在九月下旬有一个高峰期,浓度达到30.40mg/g。石榴叶片通过组织培养诱导的愈伤组织和悬浮培养的细胞组织,分别为73.13mg/g和69.05mg/g,它们的总黄酮含量和石榴皮的平均水平相当,高于石榴叶的总黄酮含量。
In this paper,the different organs tissues of pomegranate(Punica granatum L) plant are selected as the material, plant tissue and cell culture technology were used to induce callus and establish cell suspension culture system. The callus inducement and regulation , and the characteristics and growth regularity of differentiated loose callus suspension particles in cultural system, were studied systematically. The amount of flavonoids from different sources of pomegranate tissues was measured by spectrophotometric method, to provide the experimental and theoretical basis for the industrial production of flavonoids.
In the Callus inducement period, the effects of different explants types,different sterilization conditions, different hormones and hormones levels on pomegranate callus induction rate, the raritas rate and the browning rate were studied.The results showed as follow: raritas callus were more easily induced from the mature leaves near the top, the combination of alcohol with a volume fraction of 75% 4 s and mercuric chloride with a mass fraction of 0.1% 4 min has a better effect for disinfection. The best callus inducement medium hormone prescription of pomegranate callus was: MS + 1.0 mg / L 2,4-D + 1.0mg / L 6-BA +1.0 mg / LNAA.
In the continuing generation process of Pomegranate callus, as it appeared that the callus grew excessively dense and it was not conducive to achieve suspension culture, so the premise of successfully accomplishing pomegranate cell suspension culture was to obtain loose, rapidly growing callus. The effects of raritas callus by different types and different levels of hormone were studied. The results showed that The best secondary culture prescription for Pomegranate was:MS + 1.0mg/L2 ,4-D +0.1 mg / LKT. At the same time, developmental growth curve in 36 days of the pomegranate callus was determined. In this cultivating stage, injured tissues showed a "S"-type growth cycle, entered to logarithmic phase in the 12th day, and in the 30th day the cell biomass reached its maximum of 6.48g, which has increased 5.34 times.
The cell suspension culture system of pomegranate was established, the effects of cell suspension culture systems by the inoculation capacity, shaker speed, sucrose concentration, liquid volume, hormone prescriptionss, such factors as pH change in the culture process were studied. At the same time the determination of developmental growth curve in the cell suspension culture and the accumulation of flavonoids were partly researched.
In this paper, the optimum conditions of pomegranate derived cell suspension culture are as follows: with the composition and content of standards of MS basic medium, liquid medium was prepared , the sucrose concentration was regulated to 3%, that is, 30g / L, the liquid volume of the culture bottles is 25mL/150mL, 3g calli were inoculated in every bottle. The shaker speed was 110rpm, the best prescription for hormones was:MS +1.0 mg/L2 ,4-D +0.1 mg / L KT. Under the culturing conditions above-mentioned, suspended cells went into logarithmic phase in 6th day, the cell biomass reached its maximum in the 21th day,the dry weight reached 21.68g / L. After 21 th day the cell biomass began to decline into the growth of quiescent phase. At this stage Flavonoids content produced by secondary biological cell metabolites reached to the peak value of 69.05mg / g. pH value changed in the cultivating process, first decrease, then the trend of rising became apparent.
The amount of flavonoids was measured by spectrophotometric method, content of the pomegranate callus and the cells in liquid culture in regular collection of pomegranate leaves, pomegranate leaves and solid cultivation, and analyzed of trends at different times. The results showed that: the content of total flavonoids megranate skin over time and showed a gradual downward trend, by the end of June the flavonoid content in pomegranate skin reached a peak value of 124.7mg / g, When the fruits mature from the end of September to early October, the Flavonoids content in pomegranate skin stabilized basically to 64.8mg / g. The flavonoids content in Pomegranate leaves changed little, there is a peak period in the late September, the concentration of 30.40mg / g. Pomegranate leaves were 73.13mg / g and 69.05mg / g, induced through tissue culture of callus and cell suspension cultures .Their total flavonoids was equal to the average level of megranate skin, but was higher than the pomegranate leaves.
愈伤组织的诱导阶段,研究了不同外植体类型、不同灭菌条件、不同激素种类和水平对石榴愈伤组织诱导率、疏松程度和褐化率的影响。结果表明:靠近顶端的成熟叶片更容易诱导为疏松愈伤组织,体积分数为75%酒精消毒5s和质量分数为0.1%的升汞消毒4min组合的消毒效果较好,石榴愈伤的最佳诱导培养基激素配方为:MS + 1.0 mg/L 2,4-D + 1.0mg/L 6-BA+1.0mg/LNAA。
石榴愈伤组织的继代过程中,出现愈伤组织生长过于致密,不利于实现悬浮培养,因此获得疏松、生长快速的愈伤组织是成功实现石榴细胞悬浮培养的前提。研究不同激素种类和水平对疏松愈伤组织的影响。结果表明石榴最佳继代培养配方为:MS + 1.0mg/L2,4-D +0.1mg/LKT。同时确定了石榴愈伤组织在36天的动态生长曲线,伤组织在这个培养阶段内,呈现出“S”型生长周期,第12d进入对数生长期,在培养的第30d,达到细胞生物量的最大值,为6.48g,增长了5.34倍。
建立石榴细胞悬浮培养体系,研究接种量、摇床转速、蔗糖浓度、装液量、激素配方、培养过程中pH的变化等因素对细胞悬浮培养体系的影响。同时还对悬浮培养中细胞动态生长曲线的测定和黄酮类化合物的积累做了部分研究。
本文得出石榴细胞悬浮培养的最佳条件如下:以MS基本培养基的成分和含量为标准,配制成液体培养基,调节蔗糖浓度为3%,即30g/L,培养瓶的装液量为25mL/150mL,每瓶接种3g愈伤组织,以110rpm的摇床转速,最佳激素配方为:MS+1.0mg/L2,4-D+0.1 mg/L KT。在以上条件下进行培养,悬浮细胞在6d后进入对数生长期,在21d细胞生物量达到最大值,干重达到21.68g/L。21d后细胞生物量开始下降,进入生长静止期,次级生物代谢产物细胞总黄酮含量在此阶段出现高峰值,含量为69.05mg/g。在培养过程中pH值有变化,先降低,后有升高趋势。
用分光光度法,对定期采收的石榴皮、石榴叶和固体培养中的石榴愈伤组织、液体培养中的细胞,测量其总黄酮含量,并分析在不同时间的变化趋势。结果表明:石榴皮的总黄酮含量随时间变化而呈逐步下降趋势,在六月份底石榴皮黄酮含量达到高峰值,为124.7mg/g,到九月底十月初果实成熟时,石榴皮总黄酮含量基本稳定,为64.8mg/g。石榴叶的总黄酮含量变化不大,在九月下旬有一个高峰期,浓度达到30.40mg/g。石榴叶片通过组织培养诱导的愈伤组织和悬浮培养的细胞组织,分别为73.13mg/g和69.05mg/g,它们的总黄酮含量和石榴皮的平均水平相当,高于石榴叶的总黄酮含量。
In this paper,the different organs tissues of pomegranate(Punica granatum L) plant are selected as the material, plant tissue and cell culture technology were used to induce callus and establish cell suspension culture system. The callus inducement and regulation , and the characteristics and growth regularity of differentiated loose callus suspension particles in cultural system, were studied systematically. The amount of flavonoids from different sources of pomegranate tissues was measured by spectrophotometric method, to provide the experimental and theoretical basis for the industrial production of flavonoids.
In the Callus inducement period, the effects of different explants types,different sterilization conditions, different hormones and hormones levels on pomegranate callus induction rate, the raritas rate and the browning rate were studied.The results showed as follow: raritas callus were more easily induced from the mature leaves near the top, the combination of alcohol with a volume fraction of 75% 4 s and mercuric chloride with a mass fraction of 0.1% 4 min has a better effect for disinfection. The best callus inducement medium hormone prescription of pomegranate callus was: MS + 1.0 mg / L 2,4-D + 1.0mg / L 6-BA +1.0 mg / LNAA.
In the continuing generation process of Pomegranate callus, as it appeared that the callus grew excessively dense and it was not conducive to achieve suspension culture, so the premise of successfully accomplishing pomegranate cell suspension culture was to obtain loose, rapidly growing callus. The effects of raritas callus by different types and different levels of hormone were studied. The results showed that The best secondary culture prescription for Pomegranate was:MS + 1.0mg/L2 ,4-D +0.1 mg / LKT. At the same time, developmental growth curve in 36 days of the pomegranate callus was determined. In this cultivating stage, injured tissues showed a "S"-type growth cycle, entered to logarithmic phase in the 12th day, and in the 30th day the cell biomass reached its maximum of 6.48g, which has increased 5.34 times.
The cell suspension culture system of pomegranate was established, the effects of cell suspension culture systems by the inoculation capacity, shaker speed, sucrose concentration, liquid volume, hormone prescriptionss, such factors as pH change in the culture process were studied. At the same time the determination of developmental growth curve in the cell suspension culture and the accumulation of flavonoids were partly researched.
In this paper, the optimum conditions of pomegranate derived cell suspension culture are as follows: with the composition and content of standards of MS basic medium, liquid medium was prepared , the sucrose concentration was regulated to 3%, that is, 30g / L, the liquid volume of the culture bottles is 25mL/150mL, 3g calli were inoculated in every bottle. The shaker speed was 110rpm, the best prescription for hormones was:MS +1.0 mg/L2 ,4-D +0.1 mg / L KT. Under the culturing conditions above-mentioned, suspended cells went into logarithmic phase in 6th day, the cell biomass reached its maximum in the 21th day,the dry weight reached 21.68g / L. After 21 th day the cell biomass began to decline into the growth of quiescent phase. At this stage Flavonoids content produced by secondary biological cell metabolites reached to the peak value of 69.05mg / g. pH value changed in the cultivating process, first decrease, then the trend of rising became apparent.
The amount of flavonoids was measured by spectrophotometric method, content of the pomegranate callus and the cells in liquid culture in regular collection of pomegranate leaves, pomegranate leaves and solid cultivation, and analyzed of trends at different times. The results showed that: the content of total flavonoids megranate skin over time and showed a gradual downward trend, by the end of June the flavonoid content in pomegranate skin reached a peak value of 124.7mg / g, When the fruits mature from the end of September to early October, the Flavonoids content in pomegranate skin stabilized basically to 64.8mg / g. The flavonoids content in Pomegranate leaves changed little, there is a peak period in the late September, the concentration of 30.40mg / g. Pomegranate leaves were 73.13mg / g and 69.05mg / g, induced through tissue culture of callus and cell suspension cultures .Their total flavonoids was equal to the average level of megranate skin, but was higher than the pomegranate leaves.
引文
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