Effect of precursor feeding on alkaloid accumulation by a strictosidine synthase over-expressing transgenic cell line S1 of Catharanthus roseus

The transgenic S1 cell line of Catharanthus roseus (L.) G. Don has been used to study possible rate limiting steps in the terpenoid indole alkaloid (TIA) biosynthesis. Line S1 carries a recombinant, over-expressed version of the endogenous Str gene which encodes strictosidine synthase (STR; EC 4.3.3.2). STR catalyzes the stereospecific condensation of tryptamine and secologanin to strictosidine. Various concentrations and combinations of biosynthetic indole precursors L-tryptophan, tryptamine, and iridoid precursors loganin and secologanin were added to the cell suspension cultures of line S1. The largest TIA accumulation occurred when the precursor was supplied at the time of inoculation of the cells into the production medium. Line S1 could supply tryptamine endogenously up to 0.8 mM loganin feeding. The enhancement of the accumulation of TIAs by addition of loganin indicates a limitation in the terpenoid pathway. Supplying tryptamine or tryptophan along with the iridoid precursors resulted in even further increase of alkaloid accumulation. Under optimal conditions, cultures of line S1 accumulated about 600 mol l^–1 of TIAs. Also, the conversion of strictosidine into other TIAs further down the pathway seems to be a limiting step. Considering the mass balance of the intermediates fed and TIAs recovered, several yet unknown pathways must be involved in channeling away intermediates from the TIA pathway and in the breakdown of the TIAs. Our results suggest that high rates of tryptamine synthesis can still take place under conditions of low TDC activity and the flux towards tryptamine is induced by loganin feeding. However, accumulation of tryptamine seems to reduce the flux through feedback inhibition.     

Effect of precursor feeding on alkaloid accumulation by a tryptophan decarboxylase over-expressing transgenic cell line T22 of Catharanthus roseus

To obtain more insight into the regulation of terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus (L.) G. Don cell cultures and particularly to identify possible rate limiting steps, a transgenic cell line over-expressing tryptophan decarboxylase (Tdc), and thus having a high level of tryptamine, was fed with various amounts of precursors (tryptophan, tryptamine, loganin and secologanin) in different time schedules and analyzed for TIA production. When these precursors were added to this culture it was found that the optimal time for supplying the precursors was at inoculation of the cells into the production medium. Alkaloid accumulation by line T22 was enhanced by addition of loganin or secologanin; however, the secologanin feeding was less effective. Tryptamine or tryptophan alone had no effect on TIA accumulation. The over-expression of Tdc causes this cell line to produce quite large quantities of alkaloids after feeding loganin or secologanin. However, in combination with tryptophan or tryptamine, feeding of these precursors resulted in an even further increase of alkaloid accumulation and under optimal conditions line T22 accumulated around 1200 micromol l(-1) of TIAs whereas the control cultures accumulated less than 10 micromol l(-1) TIAs.     

Effects of alkaloid precursor feeding and elicitation on the accumulation of secologanin in a Catharanthus roseus cell suspension culture

In a Catharanthus roseus cell line accumulating secologanin, time-course studies on the uptake of loganin and the in vivo conversion to secologanin were performed. Four-day-old cells converted 100% of the fed loganin to secologanin within 24 hours, showing that this step is unlikely to be limiting for alkaloid accumulation. Thirteen-day-old cells also took up loganin, but only about 25% was recovered as secologanin. A saturation in the uptake of loganin and in the conversion of loganin into secologanin was observed after feeding increasing amounts of loganin. Elicitation by cellulase and pectinase decreased the cellular contents of secologanin and strictosidine whereas it increased the tryptamine content. In addition, the uptake of loganin in elicited cells was blocked. In vitro assays with protein extracts of elicited Catharanthus roseus cells indicated the activation of secologanin degrading enzyme(s). Feeding of tryptophan did not result in any increase in alkaloid contents, despite its complete uptake. Tryptamine feeding led to increased strictosidine contents, but ajmalicine levels remained unchanged. This revised version was published online in June 2006 with corrections to the Cover Date.     

通过组织培养筛选高含量喜树碱细胞系

测定不同生长期喜树苗各器官中喜树碱的含量。其中新生叶和根中的喜树碱的含量较高,然而,喜树碱的含量随叶和枝条生长期的延长而逐渐下降。以喜树碱高含量的幼叶为材料,进行了愈伤组织的诱导和培养,并通过优良细胞系的筛选,使愈伤组织中喜树碱的含量提高到0．02％。     

喜树愈伤组织的诱导及诱导子对愈伤组织中喜树碱含量的影响

通过组织培养的方法分别对喜树营养器官进行愈伤组织的诱导和继代,筛选红色、黄色和褐色3个不同的细胞系;运用HPLC法测定各愈伤组织中喜树碱的含量,并在培养基中外加诱导子,分析其对愈伤组织中喜树碱含量的影响。结果表明,经幼叶所诱导的愈伤组织中喜树碱含量相对高于其它,红色和黄色的细胞系在不同的外植体中其喜树碱含量均高于褐色的细胞系。但从整体看来,愈伤组织中喜树碱的含量远远低于原植物中的含量。外加诱导子对愈伤组织中喜树碱的含量有影响,浓度为0.1 mg·L^-1,1 mg·L^-1的水杨酸和茉莉酸甲酯处理的愈伤组织中喜树碱含量提高,0.1 mg·L^-1的水杨酸对喜树碱的积累有明显的促进作用,茉莉酸甲酯的影响不大;浓度为3 mg·L^-1,5 mg·L^-1的水杨酸处理的愈伤组织中,喜树碱含量降低;3 mg·L^-1的茉莉酸甲酯处理的愈伤组织中,喜树碱含量降低,5 mg·L^-1处理的愈伤组织中未检测到喜树碱。     

酸性培养基对喜树叶片细胞壁降解的影响

在组织培养过程中对喜树叶片外植体进行解剖学观察。发现在组织培养条件下,喜树叶片在培养基的酸性环境中细胞壁呈现模糊微弱降解、明显降解和完全降解直至消失的解剖学特征。在同样的组培条件和相同的时间内,同一喜树叶片不同部位出现细胞壁程度不同的降解和消失现象,可能是喜树叶片因上表皮凸凹不平进而导致其不同部位与酸性培养基接触的程度不同,因而使培养基中的酸性物质对喜树叶片上表皮的不同部位影响出现异质化。本文对培养基中的酸性成分对喜树叶片细胞壁降解的影响有了进一步的认识和理解。     

Effects of Chemical and Physical Conditions on Growth of Camptotheca acuminata Cell Cultures

Callus was induced from Camptotheca acuminata, which produces an antitumor alkaloid, carnptothecin. Using the Murashige and Skoogs’ medium as the basal, cultural conditions were examined for C. acuminata suspension cultures. As a result, a medium, containing 0.1 mg/liter 2,4-D, 3 mg/liter kinetin and 0.05 mg/liter GA₃, was established as a medium that gave the best cell growth in suspension cultures. In addition, conditioning of medium and addition of 0.115 mm l-Trp and l-Phe to medium promoted remarkably growth of cell suspensions.     

伤害及诱导子对喜树幼苗中喜树碱含量的影响

通过伤害处理和外施诱导子——水杨酸(SA)、茉莉酸甲酯(MeJa)和乙烯利(Ethrel)以及伤害和诱导子复合处理,研究不同处理对喜树中喜树碱(camptothein,CPT)含量的影响。结果表明:(1)4种单独诱导处理均能在一定程度上增强喜树碱的合成,其中水杨酸处理和机械伤害诱导效果最明显,较对照组的喜树碱含量分别提高了47.7%和27.2%,而机械伤害和诱导子复合处理均产生拮抗作用,喜树碱含量降低;(2)诱导作用受诱导剂浓度的影响,SA、MeJa和Ethrel的适宜质量浓度分别为50~100 mg.L-1、5~10 mg.L-1和5~10 mg.L-1。     

Characterization of callus formation and camptothecin production by cell lines of Camptotheca acuminata

Both incubation temperature and photosynthetic radiation affected morphogenesis, callus culture and plantlet culture of sweet orange (Citrus sinensis) cultured in vitro. Bud culture from nodal stem segments, regeneration of shoots and buds from internode stem segments and induction of primary callus were near optimal at incubation temperatures between 21–30°C. The optimal temperature for root formation was 27°C with temperatures above and below being clearly deleterious. Incubation in the dark or under low photosynthetic photon flux density (PPFD) was beneficial for callus induction and growth and also favored the production of rooted plantlets from bud cultures. Incubation in the dark improved considerably the regeneration of shoots and buds from internode segments and the recovery of whole plants. No off-types, as determined by protein and isoenzyme analysis, were observed among plantlets recovered from bud cultures or from regeneration of shoots from internode stem segments.     

Cu2+对喜树愈伤细胞中喜树碱合成的影响

喜树碱是一种从木本植物喜树(Camptothecaacuminata)中分离得到的抗癌活性物质,通过细胞培养合成喜树碱是喜树碱生产的一条重要途径。研究Cu~(2 )对喜树愈伤细胞培养中喜树碱积累的影响,结果表明,在B5培养基中添加0.008mg/mLCuCl2时,对喜树愈伤细胞的生长没有明显影响,但是对喜树愈伤细胞合成喜树碱的促进作用最强,喜树碱含量比未诱导前增加了约30倍。Cu~(2 )阻止培养细胞后期过氧化物酶活力的下降,并抑制花青素的生成。与黑暗培养相比,光照条件下添加Cu~(2 )更利于喜树愈伤细胞诱导合成喜树碱。     

影响喜树组织培养苗离体生根的因素

为了建立有效的喜树(Camptotheca acuminata)组培苗生根系统,提高其移栽成活率及适应性,用不同生长素种类及浓度、不同蔗糖浓度及不同培养基对喜树组培苗不定根形成影响以及移栽初期根系发育状况进行了研究.结果发现:1)生长素种类和浓度明显影响喜树组培苗不定根形成,在含有IBA0.5 mg·L-1培养基中取得了最佳生根效果,生根率达到了98%,外植体平均生根数为5.9条/株;2)不同浓度蔗糖对喜树组培苗生根也有一定影响,在10～30 g·L-1范围内,随着蔗糖浓度增加,生根百分率和生根数量都有增加,蔗糖浓度达到30 g·L-1时,生根百分率为95%,外植体平均生根数为5.4条/株;蔗糖浓度在40 g·L-1时,表现出对生根抑制作用;3)在基本培养基对喜树组培苗生根影响研究中发现,MS培养基对根形成表现出一定抑制作用;1/2MS和WPM培养基均适合喜树组培苗生根;4)根系发育正常的喜树组培苗移栽后成活率可达96%,但组培苗根系根毛系统发育较差.组培苗单位叶面积根尖数量显著低于对照实生苗,而且此参数与叶片气孔导度呈显著正相关.这种较差根系发育导致叶片气孔导度过低可能是组培苗叶片光合能力较低的重要原因.     

影响喜树组织培养苗离体生根的因素

为了建立有效的喜树(Camptotheca acuminata)组培苗生根系统,提高其移栽成活率及适应性,用不同生长素种类及浓度、不同蔗糖浓度及不同培养基对喜树组培苗不定根形成影响以及移栽初期根系发育状况进行了研究。结果发现: 1)生长素种类和浓度明显影响喜树组培苗不定根形成,在含有IBA0.5 mg.L-1培养基中取得了最佳生根效果,生根率达到了98%,外植体平均生根数为5.9条/株; 2)不同浓度蔗糖对喜树组培苗生根也有一定影响,在10~30 g.L-1范围内,随着蔗糖浓度增加,生根百分率和生根数量都有增加,蔗糖浓度达到30 g.L-1时,生根百分率为95%,外植体平均生根数为5.4条/株; 蔗糖浓度在40 g.L-1时,表现出对生根抑制作用; 3)在基本培养基对喜树组培苗生根影响研究中发现,MS培养基对根形成表现出一定抑制作用;1/2MS和WPM培养基均适合喜树组培苗生根; 4)根系发育正常的喜树组培苗移栽后成活率可达96%,但组培苗根系根毛系统发育较差。组培苗单位叶面积根尖数量显著低于对照实生苗,而且此参数与叶片气孔导度呈显著正相关。这种较差根系发育导致叶片气孔导度过低可能是组培苗叶片光合能力较低的重要原因。     

Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

光强对喜树幼苗叶片次生代谢产物喜树碱的影响

喜树碱是我国特有树种——喜树中所含的重要次生代谢产物 ,在人工控制条件下观察了光强对喜树幼苗叶片喜树碱含量的影响。喜树幼苗叶片的喜树碱含量随着遮荫程度的增加 (光照强度降低 )而增加 ,但严重遮荫的 (光强为全光照的 2 0 % )在处理后期 (75 d)喜树碱含量降低。叶片的喜树碱产量 (喜树碱含量与叶片生物量乘积 )在处理初期 (30 d)随光强减弱而缓慢地略有增加 ,处理后期 (45 d以后 )随光强的减弱而有明显增加 ,但光强低于全光照的 6 0 %以后喜树碱产量迅速下降。喜树碱的增加可能是喜树幼苗通过次生代谢过程对不良环境 (遮荫 )的一种适应性反应     

The effect of growth regulators and sucrose on anthocyanin production in Camptotheca acuminata cell cultures.

The effect of different concentrations of growth regulators and sucrose on anthocyanin production in cell suspension cultures of Camptotheca acuminata Decaisne (Nyssaceae) was described for the first time and qualitatively and quantitatively evaluated. Anthocyanin production was significantly greater in the presence of kinetin, compared to benzyladenine, with the greatest concentration observed in the presence of 2 microM kinetin. No significant differences in anthocyanin production were observed when comparing 2,4-dichlorophenoxyacetic acid to alpha-naphthaleneacetic acid, except when using 2 microM, 2,4-dichlorophenoxyacetic acid, which resulted in greater anthocyanin production. High sucrose concentration enhanced the production of anthocyanins. Based on the absence of anthocyanin production in the dark, we concluded that light was essential for stimulating anthocyanin production. The optimised medium consisted of: 2 microM kinetin, 2 microM 2,4-dichlorophenoxyacetic acid and 292 mM sucrose. HPLC/DAD and HPLC/MS analyses revealed that the main anthocyanin was Cy 3-O-galactoside and that the minor derivative was Cy 3-O-glucoside.     

喜树内生菌与喜树碱的关系

通过对喜树幼苗中喜树碱含量的分析,发现不同生长期、不同器官中喜树碱的含量不同,幼叶和根中喜树碱的含量较高。虽然喜树中含有对真核细胞具有毒性作用的喜树碱,但仍有12种内生菌从喜树的不同器官中分离出来。内生菌对喜树碱的敏感性实验表明,10μg／mL喜树碱对2种内生菌的生长几乎没有抑制作用,即是100μg／mL浓度的喜树碱对它们的生长抑制也是有限的。     

滤光膜对喜树幼苗叶片生长和喜树碱含量的影响

喜树 (Camptotheca acuminata)为中国特有树种 ,因其次生代谢产物喜树碱具有抗癌作用而闻名。通过用黄色、红色、蓝色 3种滤光膜对温室栽培的喜树幼苗进行遮光处理 ,研究了不同光照环境下喜树幼苗叶片生物量、叶绿素含量、光合作用和喜树碱含量的差异。结果表明在 30 d的遮光过程中 ,红膜和蓝膜遮光明显导致幼苗叶片生物量降低 ,黄膜遮光下幼苗叶片生物量在处理后 2 5 d才表现明显降低。不同滤光膜下幼苗叶片叶绿素含量先降低然后升高 ,遮光幼苗的叶绿素 a/ b明显低于日光幼苗。幼苗日最大净光合速率的顺序是 :日光 >黄膜 >红膜 >蓝膜。处理后第 2 0天 ,不同滤光膜下幼苗的光饱和光合速率 (Amax)、光饱和点 (Is)、光补偿点 (Ic)、最大表观量子效率 (AQYmax)都不同程度的低于日光幼苗。处理后第 10天至第 30天 ,遮光幼苗叶片喜树碱含量均显著高于日光下幼苗 ,以蓝膜下幼苗的喜树碱含量最高。蓝膜和黄膜下幼苗的喜树碱产量在后期处理中显著高于日光下幼苗 ,蓝膜下幼苗喜树碱产量在第 30天最高 ,是日光下幼苗的 2 .4 9倍。红膜下幼苗的喜树碱产量在第 10天后与日光下幼苗差异不显著。通过滤光膜遮光促进喜树碱在幼苗叶片中的积累 ,提高了叶片喜树碱产量 ,对喜树碱的生产实践有一定的意义     

丛枝菌根对喜树幼苗喜树碱含量的影响

喜树（Camptotheca acuminata）是我国特有的多年生亚热带落叶阔叶树种,因其次生代谢产物喜树碱具有良好的抗肿瘤活性而受到人们的广泛关注.通过温室盆栽接种试验,观察了2属6种丛枝菌根真菌对喜树幼苗喜树碱含量的影响.结果表明,接种的6种丛枝菌根真菌与喜树幼苗均形成了共生体系并且发育良好.透光球囊霉（Glomus diaphanum）、幼套球囊霉（G.etunicatum）、蜜色无梗囊霉（Acaulospora mellea）和光壁无梗囊霉（A.laevis）侵染形成丛枝菌根有利于提高喜树幼苗的喜树碱含量,地表球囊霉（G.versiforme）则影响不大,而木薯球囊霉（G.manihot）却降低了喜树幼苗的喜树碱含量.丛枝菌根形成对喜树幼苗喜树碱代谢的影响还表现在喜树碱的器官分配上,菌根幼苗根中的喜树碱比例均高于无菌根幼苗.     

Effects of Benzyladenine and Naphthalene Acetic Acid on Growth and Camptothecin Accumulation in Camptotheca acuminata Seedlings

The effects of the cytokinin benzyladenine (BA) and the auxin naphthalene acetic acid (NAA) on Camptotheca acuminata Decaisne growth and camptothecin (CPT) accumulation (leaf CPT concentration and total leaf CPT yield) were studied in a hydroponic culture system for three weeks. Increasing BA concentrations from 0 to 3 mg l^–1 in growth medium decreased plant height, stem weight, and leaf weight but increased root weight. High BA levels (1 and 3 mg l^–1) increased leaf CPT concentration (% of dry weight), whereas BA applications had no effect on total leaf CPT yield, the product of leaf CPT concentration and total leaf dry weight per seedling. There was a positive correlation between root weight and leaf CPT concentration under BA treatments. NAA supplementations (from 0.5 to 4 mg l^–1) to growth medium reduced plant height, leaf number, leaf length, specific leaf weight, plant weight, stem weight, and leaf weight compared with the NAA control. Meanwhile, there were no differences in plant height, leaf length, and specific leaf weight among the NAA supplementations. NAA applications had no effect on leaf CPT concentration and NAA applications decreased total leaf CPT yield. There were negative correlations between leaf number and leaf CPT concentration, leaf length and leaf CPT concentration under NAA treatments. Our results suggest that BA applications from 0.3 to 3 mg l^–1 are not helpful for achieving high total leaf CPT yield and NAA applications from 0.5 to 4 mg l^–1 decrease total leaf CPT yield.