高山红景天致密愈伤组织颗粒内氧传递特性与细胞活性的研究

实验测定高山红景天致密愈伤组织颗粒(CCA)内部有效氧扩散系数很小,其值在O．34×10^-10～5．4×10^-10m2／s之间,且随粒径的增大而增加;理论计算结果表明CCA颗粒内部处于严重氧枯竭状态。但实际测定CCA颗粒内部细胞活性很高,表明CCA颗粒内部存在着高效氧传递机制。颗粒内出现的管状分子等分化结构以及胞间连丝可能与氧在颗粒内部的传递机制有关。     

培养基成分和培养时间对匍匐翦股颖植株再生的影响

以匍匐翦股颖成熟种子为外植体,研究了培养基2,4-D浓度、2,4-D和6-BA组合配比、蔗糖浓度对匍匐翦股颖愈伤组织诱导的影响以及愈伤组织再生过程中继代时间、6-BA浓度、蔗糖浓度对愈伤组织分化的影响。结果表明:在MS培养基上,2 mg·L^-1 2,4-D和0.1 mg·L^-1 6-BA的组合最利于愈伤组织的诱导,诱导率高达94%。蔗糖浓度为30 g·L^-1时愈伤组织诱导率最高,为82%; 在再生过程中,当6-BA浓度为1 mg·L^-1时分化率最高(62%),蔗糖浓度为40 g·L^-1时,愈伤组织分化率最高(52%)。经过2次继代培养的愈伤组织(外植体放到培养基后40天)的分化率为最高(71%),随着继代次数增多,分化率逐渐降低,在经过5次继代后(培养100 d)分化率仅有18%。     

滇紫草培养细胞中色素合成的代谢调节

培养基中分别加入浓度为10^-5mol／L的铜离子,滇紫草愈伤组织中色素含量提高了5．5倍,悬浮细胞中色素含量提高8．1倍。细胞培养第21天,加入浓度为10^-5mol／L的L-Phe,色素的合成量最大。浓度为10^-6mol／L的抗坏血酸,能明显地促进培养细胞中色素的合成。     

裸果木再生体系建立与不定芽发生研究

为探究裸果木再生体系建立的影响因素,确定其不定芽发生的起源,该研究以裸果木健壮植株的茎段为外植体,采用6 BA和IBA不同浓度组合,筛选愈伤增殖及不定芽再生的最佳浓度组合,确定生根诱导的关键影响因素,建立再生体系,并对其不定芽分化进程进行解剖结构分析,以确认其起源。结果表明：(1)裸果木茎段的最佳愈伤增殖培养基为MS+1 mg·L^-1 IBA+1 mg·L^-1 6 BA+30 g·L^-1蔗糖+7 g·L^-1琼脂,主体间效应分析表明IBA为关键影响因素;愈伤大小随IBA浓度增加呈现先升高后下降的趋势。(2)最佳不定芽诱导培养基为MS+0.5 mg·L^-1 6 BA+30 g·L^-1蔗糖+7 g·L^-1琼脂,诱导不定芽数量为4.9个/块,生芽率达92.3%。(3)生根诱导中,SH基本培养基和蔗糖浓度为关键因素,最佳生根培养基为SH+0~10 g·L^-1蔗糖+7 g·L^-1琼脂,生根率达91.3%。(4)解剖结构观察发现,不定芽起源于愈伤表层的分生细胞,为外起源。该研究通过器官发生途径建立了裸果木的再生体系,确定了不定芽为外起源,为裸果木这一珍稀濒危的林木种质资源保护及可持续利用奠定了研究基础,并为其未来的发展利用提供了有效途径。     

狭叶红景天愈伤组织中红景天甙含量及相关代谢酶活力的研究

室温下分别对狭叶红景天的茎和叶进行愈伤组织诱导,在24C和4C进行继代培养,测定其苯丙氨酸解氨酶、肉桂酸-4羟化酶和酪氨酸解氨酶的活性,并利用高效液相色谱法测定其红景天甙含量。结果表明：培养温度和愈伤组织的外植体来源均影响红景天甙的含量和3种代谢酶的酶活力;在24C和4C温度条件下,相同外植体来源的愈伤组织叶中和相同培养温度条件下的茎和叶2种不同来源外植体的愈伤组织中,红景天甙含量和3种代谢酶活力之间均存在显著性差异。     

不同理化因子对雪莲培养细胞中黄酮类形成的影响

研究了不同理化因子对水母雪莲(Saussurea medusa Maxim)愈伤组织生长及黄酮类化合物生物合成的影响。结果表明,有利于细胞生长及黄酮形成的合适温度为25℃。白光对愈伤组织生长无促进作用,但有利于黄酮的形成。培养基中添加1mg／L NAA和O．2mg／L的KT组合对细胞的生长较有促进作用。5％蔗糖和1％葡萄糖的组合有利于细胞的生长和黄酮的形成。用⁶⁰C0-γ射线辐照愈伤组织,在剂量为4000Gy的条件下,获得一个合成黄酮能力高于原愈伤组织70％的细胞系。用高效液相和紫外分光光度法,测定离体培养光照条件下干细胞总黄酮的含量为3．2％,是暗培养的4．4倍。培养温度25℃时干细胞黄酮的含量为2．02％,分别为20℃,35℃时的5倍和3．2倍。     

黑果枸杞原生质体培养及植株再生

该研究以黑果枸杞(Lycium ruthenicum)无菌苗为材料,建立了愈伤组织来源的原生质体再生体系,采用ISSR和FCM技术对再生植株进行了遗传稳定性分析。结果表明：(1)黑果枸杞叶片愈伤组织是产生原生质体的最好材料,在含0.5 mg·mL^-1甘露醇的酶液中,继代1次的叶片愈伤组织中原生质体产量为7.77×10⁶个·g^-1,活力为92%。(2)改良MS培养基 固体液体双层培养(MS₂ 固液双层)是培养原生质体的最好方式,培养10 d的原生质体分裂频率为45.9%,培养20 d的细胞团形成频率为22.9%。(3)在1.5 mg·mL^-1 6 BA+0.1 mg·mL^-1 IBA+MS培养基中,叶片愈伤组织产生的原生质体可分化获得再生植株。(4)ISSR分析显示,再生植株的平均遗传相似系数为0.88;FCM显示再生植株为二倍体,与亲本植株一致。该研究结果为进一步研究枸杞体细胞杂交技术转移野生植物抗逆遗传性状提供科学依据,为枸杞优良品种的选育奠定了基础。     

广藿香原生质体制备、培养与融合技术优化研究

为建立高效稳定的广藿香原生质体培养与融合技术体系,该研究以广藿香愈伤组织悬浮细胞为材料,研究了原生质体制备的酶解条件和培养方法、细胞密度、激素种类和浓度等因素对原生质体培养的影响,并通过测定融合产物直径确立融合细胞筛选范围,进一步研究聚乙二醇浓度、细胞密度、融合时间及融合液加入量等因素对原生质体融合的影响。结果表明:制备原生质体的适宜条件为pH5.8,酶解温度25 ℃; 原生质体培养以铵盐减半的MS₁培养基进行海藻酸钠包埋、激素选用0.2 mg·L^-1 NAA、2.0 mg·L^-1 6-BA,培养密度2.0×10⁵个·mL^-1、蔗糖添加量1.0%、酸水解酪蛋白500 mg·L^-1的条件下原生质体分裂频率、植板率均较高,且开始分裂时间和细胞团形成时间都较短; 双细胞融合产物筛选范围为69.33～87.35 μm; 以40% PEG 6000化学促融30 min、加入0.5倍体积的融合液、细胞密度2.0×10⁵个·mL^-1的条件进行原生质体融合,聚合率可达57.19%; 获得的融合产物经海藻酸钠包埋培育2个月后可观察到再生愈伤组织。     

高山红景天细胞悬浮培养中pH值对红景天甙胞外释放及细胞活性…

高山红景天细胞悬浮培养中,通过降低培养基ＰＨ值能有效地诱导培养细胞中红景天甙的胞外释放。红景天甙的跨膜运输是一个有与Ｈ对运的动态过程,培养基ＰＨ值决定了红景天甙在胞内外含量的分布。细胞组织在ＰＨ值大于３的培养基中处理３ｈ以内,对细胞活性的影响不大。将诱导释放处理过的细胞转入到新鲜的生产培养基中,细胞仍具有合成红景天甙的能力。     

杜仲叶片愈伤组织诱导的激素优化研究

以杜仲优树L33的幼叶为材料,在B5培养基上添加不同浓度的生长素与细胞分裂素进行愈伤组织诱导研究,结果表明：无激素的培养基上不能诱导出愈伤组织,单独加入2,4-D、NAA和IBA均可诱导出愈伤组织,以0.5 mg·L^-1 2,4-D、0.5～1.0 mg·L^-1 NAA、1.0 mg·L^-1 IBA出愈率最高,达100%,且愈伤组织生长较好;在适宜浓度的生长素的培养基上加入细胞分裂素时,KT的加入对愈伤组织的诱导及生长起抑制作用;1.0 mg·L^-1 NAA+0.3～0.5 mg·L^-1 BA与1.0 mg·L^-1 IBA+0.3～0.5 mg·L^-1 BA 的组合可明显促进愈伤组织的诱导和生长,适合进一步继代培养。     

高山红景天细胞悬浮培养中红景天甙生物合成代谢的调控 Ⅰ:前体的影响

探索了高山红景天(Rhodiola sachalinensis A.Bor)细胞培养中红景天甙生物合成的途径,认为甙元酪醇是经由莽草酸途径生成的。在此基础上研究了酪醇、L-酪氨酸与L-苯丙氨酸三种前体加入对红景天甙生物合成的调控作用。结果表明,酪醇、酪氨酸等前体易被多酚氧化酶氧化成褐色,用与前体浓度为1:1的V。来防止褐化效果显著;浓度为0.5mmol/L的酪醇,酪氨酸及苯丙氨酸在细胞培养15d时添加,使红景天甙含量由0.336％分别提高到1.43％、1.11％、0.85％。     

杜仲细胞悬浮培养产黄酮及其动力学研究

本文应用正交设计对杜仲细胞悬浮培养的基本培养基和植物生长物质浓度进行了筛选,并对影响杜仲细胞悬浮培养和总黄酮含量的不同因素进行了考察。结果表明,B5培养基+0.5mg/L NAA+0.6mg/L 6-BA、蔗糖30g/L、初始pH 5.0-5.5、接种量20g(FW)/L以及摇床转速110r/min为杜仲细胞悬浮培养的适宜条件。通过对杜仲悬浮细胞生长和代谢动力学的分析表明：杜仲细胞悬浮培养生长符合Logistic生长模型,最大比生长速率( m)为0.417d-1;细胞基于蔗糖的真正比生长得率(YG)与维持系数(m)分别为0.619g/g和0.0206g/(g·d-1);黄酮合成属部分生长耦联型,可用Luedeking-Piret模型进行描述。研究结果为杜仲细胞大规模悬浮培养生产天然活性成分奠定了基础。     

高山红景天细胞悬浮培养中红景天甙生物合成代谢调控Ⅱ:真菌诱导物的影响

考察了6种真菌诱导物对高山红景天(Rhodiola sachalinensis A. Bor)细胞生长与红景天甙积累的影响。其中以黑曲霉诱导物效果最好。在细胞培养初期添加浓度为10mg(carbohydrate)/L的黑曲霉诱导物能使培养细胞中红景天甙含量提高到0.995％。前体与诱导物调控组合运用最终使红景天甙产量达到167.4mg/L,是对照培养的3.5倍。另外,对真菌诱导物的作用机理也进行了探讨,真菌诱导物添加促进红景天甙的积累应该与激活培养细胞中的苯丙烷类代谢途径有关。     

Enhanced salidroside production in liquid-cultivated compact callus aggregates of Rhodiola sachalinensis: manipulation of plant growth regulators and sucrose

Liquid-cultivated compact callus aggregates (CCA) of Rhodiola sachalinensis were initiated from the explant of stems. Microscopic analysis showed that CCA displayed some level of cellular/tissue differentiation, which was perhaps responsible for the increased salidroside production. A higher ratio of cytokinin/auxin was found to be beneficial both for CCA growth and salidroside accumulation. Sucrose (> 100 g l-1) had a positive effect on salidroside synthesis, which was not due to an osmotic effect of the medium. The medium containing 40 g l-1 sucrose, 1.5 μM naphthaleneacetic acid and 15 μM 6-benzylaminopurine yielded ca. 120 mg l-1 salidroside after 24 days of culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Establishment of callus and cell suspension culture of Scrophularia striata Boiss.: an in vitro approach for acteoside production

In the present study, a protocol was optimized for establishment of callus and cell suspension culture of Scrophularia striata Boiss. as a strategy to obtain an in vitro acteoside producing cell line for the first time. The effects of growth regulators were analyzed to optimize the biomass growth and acteoside production. The stem explant of S. striata was optimum for callus induction. Modified Murashige and Skoog medium supplemented with 0.5 mg/l naphthalene acetic acid + 2.0 mg/l benzyl adenine was the most favorable medium for callus formation with the highest induction rate (100 %), the best callus growth and the highest acteoside content (1.6 μg/g fresh weight). Incompact and rapid growing suspension cells were established in the liquid medium supplemented with 0.5 mg/l naphthalene acetic acid + 2.0 mg/l benzyl adenine. The optimum time of subculture was found to 17–20 days. Acteoside content in the cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The maximum content of acteoside (about 14.25 μg/g cell fresh weight) was observed on the 17th day of the cultivation cycle. This study provided an efficient way to further regulation of phenylethanoid glycoside biosynthesis and production of valuable acteoside, a phenylethanoid glycoside, on scale-up in S. striata cell suspension culture.     

高产黄酮苷银杏悬浮培养细胞系选育和继代培养稳定性研究

银杏叶中主要有效成分黄酮苷和萜内酯,具有多种药理作用,银杏叶提取物(EGB)及其加工品具有广阔的市场前景[1,2].为满足市场需要,Carrier等[3]在90年代初就开始细胞培养生产黄酮苷和萜内酯的研究.细胞大规模培养生产黄酮苷的关键技术之一是选育性状稳定、生产能力强的细胞系.     

Studies on Cell Suspension Culture and Plant Regeneration in Rice

The present paper reports the establishment of rice cell suspension culture system, including callus induction and proliferation, isolation of single cells and small aggregates, cell suspension culture and callus re-formation, as well as regeneration of plantlets. The results have been obtained as follows: 1. The compositions of the different media used for callus induction, callus proliferation, cell suspension and plant regeneration are summarized in Table 1.2. Two kinds of disifectants, mercuric chloride and sodium hypochlorite, were used for surface sterilization of brown rice. The percentage of callus formation and callus yields were much higher when sodium hypochlorite was used (Fig. 3). We suggest that the disinfactant is one of the important factors that affect callus formed at the initial stage has an influence upon subsequent isolation of cells and suspension culture and even plant regeneration. 3. Table 3 shows that addition of yeast extract to the medium improves callus yield greatly and the efficiency of callus formation to a lesser extent. 4. Both medium Ⅱ (modified B5 medium) and N6 medium were suitable for cell suspension culture, but medium II was more effective for cell growth and callus re-formation (Fig. 4 and Table 4). 5. Effect of 2, 4-D on cell growth was tested at the concentration range among 0, 10-6, 10-5, 10-4 to 10-3 M. The results indicated that 10-5 M of 2,4-D was most effective for induction of rice callus. It has also been found that absence of 2,4-D increased callus re-formation in suspension culture, but no plant regeneration was observed. 6. By using 7% sucrose in differentiation medium, for all the three varieties, the plant regeneration frequency was raised up to 3 or 4 times than those of the 3% ones (Table 6). Occurrence of albino plants is often reported as one of the problems in rice anther culture. It is, however, no problem in seed-derived rice cell culture.     

Production of salidroside and tyrosol in cell suspension cultures of Rhodiola crenulata

Salidroside and its aglycone tyrosol are important compounds found in Rhodiola plants. In this study, callus derived from Rhodiola crenulata was induced and grown when explants were incubated on a Murashige and Skoog (MS) medium containing various concentrations of 6-benzyaldenine (BA), naphthalene acetic acid (NAA) and thidiazuron (TDZ). Callus was easily initiated from juvenile leaves in half strength MS medium supplemented with 0.5 mg/L BA and 3.0 mg/L NAA, while full strength MS containing 0.5 mg/L TDZ and 0.5 mg/L NAA was the best for callus subculture and subsequent cell suspension culture. The activities of l-phenylalanine ammonia lyase (PAL) and β-d-glucosidase, two key enzymes in salidroside synthesis, increased at first and subsequently decreased in cell suspension cultures. The salidroside and tyrosol levels in the cell suspension cultures were determined using high-performance liquid chromatography. High levels of salidroside and tyrosol were detected in cell suspension cultures of R. crenulata extracted with 75 % methanol, demonstrating that the biotechnological production of these compounds using plant cell suspension cultures derived from R. crenulata may be an attractive alternative to harvest-based production.     

高产莪术细胞悬浮系建立及产物前体对挥发油合成的调控研究

研究了高产莪术细胞悬浮系培养的条件及前体物质添加对挥发油合成的调控。结果表明：淡黄色颗粒状愈伤组织是建立高产细胞悬浮系的最佳供试愈伤组织;最佳培养基成分是MS培养基添加葡萄糖与蔗糖各15—30g／L(1：1),氮源为NH4^ 和NO3^-,比例为1：3,总量为80mmol／L;激素组合为6-BA3．0—5．0mg／L、2,4-D1．0mg／L;光下培养10—15天再转入优化条件下的暗培养,可形成稳定的高产细胞悬浮系;其细胞周期中的最大细胞生长量及挥发油含量分别是248g／L和2．28％;前体物质泛酸钙、乙酸铵、乙酸钾的添加均可有效提高培养细胞合成挥发油的百分含量,其中乙酸铵最有效,在指数生长中期添加0．5mmol／L乙酸铵,挥发油的最高含量可达3．11％,产量为8．27g/L,分别是添加前的1.25倍及1.2倍。     

Obtaining and Study of Callus and Suspension Plant Cell Cultures of <Emphasis Type="Italic">Tribulus terrestris</Emphasis> L., a Producer of Steroidal Glycosides

Callus and suspension plant cell cultures of Tribulus terrestris L., a valuable medicinal plant producing steroidal glycosides, were obtained. The seeds from an American population of T. terrestris were used as explants. Regulation of the production and growth of cell cultures, as well as the biosynthetic characteristics of the cell lines, were studied. The combination of phytohormones of 2,4-D (2.0 mg/L) and BAP (1.0 mg/L) was found to be optimal for callus induction and cultivation. Suspension cell culture obtained in liquid medium of the same composition showed such high growth characteristics during prolonged cultivation (more than 2 years) as a maximum accumulation of dry biomass of 13 g/L, specific growth rate at exponential phase of 0.24 day^–1, and economical coefficient of 0.39. A semicontinuous mode of cultivation was used to grow the plant cell suspension in a lab-scale bioreactor. Screening of the steroidal glycosides in the obtained cell cultures was carried out. Steroidal glycosides were not found in the callus cultures. However, as was demonstrated by TLC and UPLC ESI MS methods, the suspension culture contained furostanol glycosides, and their amount increased during the cultivation process. These results support the hypothesis of the autoselection of cultivated cells containing compounds promoting their proliferation in vitro.