Determination of gentiopicroside contents during somatic embryogenesis in Gentiana straminea Maxim

A protocol for regeneration of Gentiana straminea Maxim recently established in our laboratory, somatic embryogenesis was obtained from its leaf derived calli. The gentiopicroside contents of embryogenic calli, globular-, heart-, torpedo-, and cotyledon-shaped embryoids as well as regenerated plantlets were determined by high-performance liquid chromatography. Gentiopicroside was detectable in all materials tested. Embryogenic calli showed the lowest gentiopicroside content. The changes of gentiopicroside contents were not significant (P < 0.05) with the development of somatic embryos. The highest gentiopicroside content (30.7 mg g^?1 dry weight) was achieved in regenerated plantlets. The contents of gentiopicroside were not significant (P < 0.05) differences between control plants and embryogenic calli, different stages of somatic embryos and regenerated plantlets. This protocol could be employed for producing gentiopicroside or other medicinal compounds.     

藏药麻花艽愈伤组织的诱导及植株再生

1植物名称麻花艽（Gentiana straminea Maxim．）,又名麻花秦艽,藏药中称为“解吉嘎保”。 2材料类别幼嫩叶子。     

白花龙胆花抗炎作用研究

为了研究白花龙胆花的抗炎作用,本文对白花龙胆花水提物和70%乙醇提取物的抗炎活性进行了试验,并测定了水提物和70%乙醇提物中龙胆苦苷的含量.试验采用二甲苯所致小鼠急性耳肿胀试验和二甲苯所致小鼠腹部毛细管通透性试验.将昆明种小白鼠分为7组,分别为空白对照组,白花龙胆花水提物高、中、低剂量组和70%乙醇提取物高、中、低剂量组,灌胃(ig)给药14 d后,用二甲苯分别于小鼠右耳及腹部致炎.测定左右耳重量,计算肿胀度及肿胀抑制率,测定吸光值.结果表明:白花龙胆花水提物和乙醇提取物的中、高剂量组对于二甲苯所致的小鼠急性耳肿胀和小鼠腹部毛细管通透性都有显著的抑制作用.从而表明:白花龙胆花具有显著的抗炎疗效.     

RP-HPLC法测定青海产四种秦艽中獐牙菜苦苷的含量

建立了秦艽中獐牙菜苦苷含量测定的RP-HPLC方法。色谱条件：采用ZorbaxEclipseXDB-C18柱（150mm×4.6mm,5μm）,流动相：甲醇-0.03%磷酸水溶液梯度洗脱（0～25min：15%～23%;25～30min：23%～30%）,流速：1mL/min,柱温：25℃,检测波长240nm,獐牙菜苦苷在0.20～1.8μg范围内成良好的线性关系,r=0.9999,回收率RSD=1.32%。对青海产4种秦艽中獐牙菜苦苷的含量进行了定量分析,测定结果：小秦艽中獐牙菜苦苷的含量最高为：0.62%,麻花秦艽与管花秦艽的含量没有显著差异,分别为：0.43%,0.45%,簧管秦艽的含量最低为：0.32%。     

High-frequency plant regeneration from immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis

This study reports high-frequency plant regeneration from immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis. Numerous green globular structures were directly formed on the surfaces of cotyledons and radicles from 2-week-old immature zygotic embryos at a frequency of 42.1 % when cultured on Murashige and Skoog (MS) medium supplemented with 2 mg l^?1 of α-naphthaleneacetic acid (NAA) and 1 mg l^?1 of 6-benzyladenine (BA). In comparison, white globular structures and pale-yellow calluses were formed simultaneously at a frequency of 28.3 % when cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). The pale-yellow calluses were transferred to MS liquid medium supplemented with 2,4-D to establish embryogenic cell suspension cultures consisting of round, isodiametric cells that formed cell aggregates. Upon plating of these cell aggregates on half-strength MS medium without growth regulators under light conditions, cell aggregates gave rise to numerous globular embryos at a frequency of 56 %. Of the globular embryos, 15 % were successfully converted into cotyledonary embryos when cultured on half-strength MS medium under light conditions. The plant regeneration system of H. cordata established in this study will be useful for the selection, genetic transformation, and mass proliferation of elite clones with medicinal potential.     

青藏高原高山植物麻花艽的传粉生态学研究

对青藏高原东部麻花艽 (Gentiana straminea)海北居群的传粉生态学进行了 3a连续的观察和实验。试验表明自然去雄、人工自交和杂交处理均结实 ,而人工去雄套袋和自然套袋不结实。麻花艽自交亲合 ,但必须依赖传粉媒介才能完成授粉过程 ,不存在无融合生殖。野外捕捉到 14种访花昆虫 ,它们分别属于 2个纲 ,7个目 ,8个科。观察和分析了各种昆虫的访花行为后 ,认为苏氏熊蜂 (Bombus sushikini)是麻花艽有效而稳定的传粉者。测量表明麻花艽花蜜通道的深度和苏氏熊蜂的舌长基本吻合。苏氏熊蜂的访花频率在 10 :0 0～ 12 :0 0 ,13:0 0～ 15 :0 0和 16 :0 0～ 18:0 0时间段没有差别 ,单花的访花频率为 0 .0 0 5次 / (花· min)。和其它高山植物相比 ,青藏高原高山植物麻花艽的访花频率较高。熊蜂传粉和高频率的访花维持了麻花艽在极端寒旱的青藏高原环境下的有性生殖。此外 ,高频率的访花对于维持该地区高山植物的生殖保障具有重要的现实意义 ,但是否具有普遍性 ,还有待研究更多的高山代表类群。     

Somatic embryogenesis and plant regeneration from immature embryos of saw palmetto,an important landscape and medicinal plant

Somatic embryogenesis and plant regeneration from immature zygotic embryos was achieved for saw palmetto (Serenoa repens (Bartr.) Small). Embryos, isolated from immature fruit of native-grown plants, were cultured on Murashige and Skoog medium plus 0.15% (w/v) activated charcoal and supplemented with 452 M 2,4-dichlorophenoxyacetic acid (2,4-D) and 14.7 M N⁶-(2-isopentenyl)adenine (2iP). Clusters of somatic embryos developed from all immature zygotic embryos 5 weeks after culture initiation. After 12 weeks, explants were transferred to the same medium with the amount of 2,4-D reduced to 90.4 M which resulted in somatic embryo proliferation. Somatic embryos were then transferred to the basal medium containing 0.9, 9 M thidiazuron (TDZ), or no growth regulator for conversion into plantlets. The 9 M TDZ treatment was ineffective for plant regeneration. However, 12% of the embryos subcultured on 0.9 M TDZ were able to produce complete plantlets. Shoot production was obtained from 35% of the embryos subcultured in the absence of growth regulators. Rooting (100%) was achieved when these shoots were transferred onto medium containing 22.2 M -naphthaleneacetic acid (NAA).     

水母雪莲体细胞胚胎发生及其植株再生

水母雪莲（Saussurea medusa Maxim.)茎和叶片的切段接种于MS＋2mg/L NAA 0.5mmg/L 6-BA的培养基上,20d后产生黄褐色的愈伤组织,经过几个月的继代培养,愈伤组织仍保持旺盛的增殖能力,但部分由黄褐色逐渐变为红色,将红色愈伤组织转到MS＋0．1mg/L NAA＋0．2mg/L 6-BA 5mg/L GA3的培养基上,30d后可分化出大量的体细胞胚,体细胞胚成熟后转到1／2MS＋0．2mg/L IAA 0.5%活性炭的培养基上,30d后可长出2－4cm的根,带根的小苗经锻炼后移栽到土壤中,成活率达76％,细胞组织学观察表明,发育成熟的体细胞胚具有胚根,胚轴和胚芽的完整结构,具有独立的维管系统。     

秦艽提取物的高效液相色谱指纹图谱研究

本文采用梯度洗脱法建立了秦艽提取物的HPLC指纹图谱。流动相：甲醇-0．1％冰醋酸梯度洗脱（甲醇浓度变化：0.12min,20～45％;12～45min,45～100％）,检测波长：260nm,并采用国家药典委员会出版的“中药色谱指纹图谱相似度评价系统2004A版”软件进行谱图比较。结果10批次提取物指纹图谱相似度均大于0．90,建立了一种方便、准确、可靠的提取物指纹图谱质量控制方法。     

High-frequency embryogenesis and plantlet regeneration from isolated microspores of indica rice

We report high-frequency embryogenesis and plantlet development from microspores isolated from anthers of two indica (IR-43, IR-54) and a japonica (T-309) rice cultivars, without prior nutrient preculture of anthers. Pretreatment stress of anthers with mannitol or a sugar-starvation medium, and use of maltose as the carbohydrate source in the microspore culture medium were found to be critical. Co-culture of microspores with rice ovaries was found beneficial but not essential. More than 60% of the microspores of the japonica variety Taipai-309 and 25–45% of the indica cultivars IR-54 and IR-43 showed induction of non-gametophytic development. Consequently, in the best treatments for IR-43 and T-309, more than 500 microspore-derived embryos could be obtained from a single dish (35 mm) containing about 80,000 microspores. Among the indica cultivars, the maximum response was obtained in the basal medium M-019. Plantlet regeneration occurred in about 9% (T-309), 7% (IR-43) and 2% (IR-54) of the transferred embryo-like structures. Received: 6 November 1996 / Revision received: 18 June 1997 / Accepted: 20 August 1997     

Plant regeneration through direct somatic embryogenesis from immature zygotic embryos of the medicinal plant,Paris polyphylla Sm.

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant Paris polyphylla Sm. has been developed for the first time. Immature zygotic embryos (IZEs) were cultured on different media namely Gamborg (B5), ½ B5, Murashige and Skoog (MS), ½ MS, Chu et al. (N6), ½ N6, Schenk and Hildebrandt (SH) and ½ SH. Highest frequency of somatic embryogenesis (32.6 %) and mean number of somatic embryos (SEs) per explant (28.7 ± 1.7) were obtained on ½ MS medium directly without an intermediate callus phase. The frequency of SE induction was significantly increased to 40.7 % when ½ MS medium was solidified with gelrite compared to agar (32.6 %). Secondary somatic embryos (SSEs) appeared on the primary SEs in a repetitive way on plant growth regulator-free ½ MS medium but with a gradual decrease in embryogenic potential during subsequent subcultures. Plasmolyzing pre-treatment of SSEs with 1.0 M mannitol for 12 h effectively maintains its embryogenic capacity. Primary embryos at the elongated dimpled and early cotyledonary stage displayed the highest embryo forming capacity of 26.94 and 27.87, respectively. High frequency of SE germination (94.0 %) occurred on ½ MS medium with 0.5 mg/l gibberellic acid. Highest percentage of seedling to plantlet conversion was observed in the medium supplemented with 0.05 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthalene acetic acid. Regenerated plants displayed morphological characteristics similar to that of the wild plants. Flow cytometry analysis showed ploidy stability of the regenerated plants.     

西宁和海北麻花艽净光合速率和叶绿素荧光参数的日变化比较

以青藏高原药用植物麻花艽为材料,研究了西宁和海北两个地区麻花艽叶片的净光合速率和叶绿素荧光参数的日变化进程。结果表明：在中午太阳辐射较强时两地麻花艽叶片的净光合速率（Pn）均下降,下午随日间光强的减弱逐渐上升,形成双峰曲线;海北麻花艽叶片的净光合速率（Pn）及其日变幅均低于西宁。随日间光强的增加麻花艽叶片的PSⅡ最大光化学效率（Fv／Fm）、PSⅡ的潜在活性（Fv/Fo）下降,非光化学猝灭系数（NPQ）则上升,黄昏各参数都恢复到接近早晨的水平,表明未发生光合机构的破坏;一天中海北麻花艽叶片的Pn、Fv／Fm、Fv／Fo均低于西宁,表明随海拔的升高、光强的增加,海北麻花艽热耗散增多,午间光抑制加重。     

青海秦艽高效液相色谱指纹图谱的研究

利用高效液相色谱法建立了青海秦艽的色谱指纹图谱.色谱条件:K rom as il C18柱(250 mm×4.6 mm×5μm),甲醇∶磷酸水(0.04%)梯度洗脱,流速为1 mL/m in,检测波长222 nm.通过比较发现秦艽样品的13个主要共有峰,可作为鉴别秦艽药材的主要依据.对12批秦艽药材进行了相似度计算和聚类分析,结果表明,所建立的秦艽指纹图谱可用于秦艽药材的真伪鉴别和质量评价.     

Genotype and plant growth regulator-dependent response of somatic embryogenesis from Gentiana spp. leaf explants

Gentiana kurroo (Royle), Gentiana cruciata (L.), Gentiana tibetica (King. ex Hook. f.), Gentiana lutea (L.), and Gentiana pannonica (Scop.) leaves derived from axenic shoot culture were used as explants. For culture initiation, leaves from the first and second whorls from the apical dome were dissected and cultured on Murashige and Skoog (MS) basal medium supplemented with three different auxins: 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid (NAA), or 3,6-dichloro-o-anisic acid (dicamba) in concentrations of 0.5, 1.0, or 2.0 mg/l; and five different cytokinins: zeatin, 6-furfurylamonopurine (kinetin), N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ), N-(2-chloro-4-pyridyl)N′-phenylurea, or 6-benzylaminopurine (BAP). The cytokinin concentrations used were dependent on the type of cytokinin and varied between 0.25 and 3.0 mg/l. After 2 mo. of culture, the morphogenic response of explants was assessed. Frequency of embryogenesis was the highest for G. kurroo (54.7%) and dependent on plant growth hormones (PGRs). This gentian was the only species showing morphogenic capabilities on media supplemented with all applied combinations of PGRs, while none of the 189 induction media permutations stimulated somatic embryogenesis from G. lutea explants. G. tibetica and G. cruciata both produced an average of 6.6 somatic embryos per explant, while G. pannonica and G. kurroo regenerated at 15.7 and 14.2 somatic embryos per explant, respectively. Optimum regeneration was achieved in the presence of NAA combined with BAP or TDZ. This auxin also stimulated abundant rhizogenesis. Somatic embryos were also regenerated from adventitious roots of G. kurroo, G. cruciata, and G. pannonica. Somatic embryos converted into plantlets on half strength MS medium.     

Repetitive somatic embryogenesis from leaves of the medicinal plant Petiveria alliacea L.

Leaf segments from in vitro-grown shoot cultures of Petiveria alliacea were incubated on Murashige and Skoog (MS) medium supplemented with different concentrations of zeatin, thidiazuron, 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram (PIC). Direct somatic embryogenesis was induced in response to all tested concentrations of 2,4-D and PIC. Primary somatic embryos displayed highly repetitive embryogenesis, both on the induction medium and in liquid hormone-free MS medium. Plantlets were obtained from these secondary embryos at an estimated frequency of 5 %, after 180 days of culture on half-strength MS medium gelled with 0.2 % Phytagel. Simultaneous development of friable non-embryogenic callus was also observed on media containing PIC or 2,4-D at different concentrations. Cell suspension cultures initiated from these callus tissues did not show an increase in biomass. The embryogenic portions formed at the surface of the explants in response to 20.0 μM PIC were inoculated in hormone-free full-or half-strength liquid MS medium (MS0) and showed high rates of secondary embryogenesis, resulting in the production of a mean of 35 embryos for each embryo inoculated at the culture initiation. Embryos that started the conversion process in the liquid MS0 medium originated whole plants at a frequency of 100 % when transferred to MS0 medium solidified with 0.7 % agar. Acclimatization was achieved in 90 % of the converted plantlets, with the production of phenotypically normal plants. This system is potentially useful for the micropropagation of this species, as well as for the production of substances with pharmacological interest, such as dibenzyl trisulfide.     

Somatic embryogenesis and plant regeneration from embryonic tissues ofCamellia japonica L.

Somatic embryogenesis and plantlet regeneration were achieved from immature and mature zygoticCamellia japonica embryos cultured on Murashige & Skoog's mineral medium without growth regulators or with various combinations of IBA and BAR The dependence of embryogenesis rates on growth regulator levels was not clear, though high concentrations such as 4 mg 1^-1BAP plus 2 mg 1^-1IBA were definitely inhibitory. BAP at 1 or 2 mg 1^-1 did appear to determine the formation of bud-like embryos. By far the most responsive initial explants were immature embryonic axes collected in September, 94% of which produced somatic embryos as against only 20% for embryonic axes from mature seeds collected in October. Cotyledon explants were also embryogenic. Somatic embryos differentiating directly on the hypocotyl of the embryonic axes or the surface of cotyledons passed through typical stages of embryogenesis. Indirect somatic embryogenesis via callus was also evident. Embryogenic potential was maintained by secondary embryogenesis through the successive generations of embryos.     

Propagation of <Emphasis Type="Italic">Gentiana macrophylla</Emphasis> (Pall) from hairy root explants via indirect somatic embryogenesis and gentiopicroside content in obtained plants

An effective protocol for plant regeneration from hairy root (HR) via indirect somatic embryogenesis was established in medicinal plant Gentiana macrophylla, a perennial herb in Gentianaceae. On the MS medium containing 0.5–2.5 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4-D plus benzylaminopurine (BAP), all the HR explants produced embryogenic calli (ECs). After transfer to plant growth regulator (PGR)-free MS medium, up to 94% of the ECs produced somatic embryos (SEs) of various stages, including cotyledonary SEs. When the calli with cotyledonary SEs were transferred to PGR-free MS medium, the cotyledonary SEs on the calli developed into plantlets (1–12 ones per callus). The cotyledonary SEs showed two types: solitary and fasciculate. The former developed into single plantlets and the latter into fasciculate ones. After transplantation into soil, a half of the plantlets survived, and one of the survivors flowered without fruiting. Morphologically, about 30% plantlets appeared similar to the wild type (WT)-plants, and 70% of them displayed wrinkled dark green leaves with relatively small and dense stomata, long and thick main root with dense lateral roots. The biomass of roots and leaves of the plantlets increased by five- and one-fold, respectively, and the content of gentiopicroside of their roots raised by 72.4%, in comparison with WT-plants. Polymerase chain reaction revealed that the rolC gene integrated into HR genome still existed in the regenerated plants. This study offers us an effective method and material for producing gentiopicroside or other medicinal compounds.     

Somatic embryogenesis and plant regeneration from shoot-tip explants inPhoenix dactylifera L.

For maximum avoidance of somaclonal variation risks, the commonly used medium for somatic embryogenesis inPhoenix dactylifera has been lowered in growth regulators and activated charcoal. When initially cultured on MS basal medium containing only 150 mg dm^?3 charcoal, 5 mg dm^?3 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 mg dm^?3 benzylaminopurine (BAP), 10 to 20% of shoot-tip explants developed into embryogenic calli. The embryogenic potential has been maintained for over 24 months with no decline. In addition, this medium has been found to be more efficient than conventionaly one containing 3 g dm^?3 charcoal, 100 mg dm^?3 2,4-D and 3 mg dm^?3 2-isopentyladenosine (2IP). Plantlet regeneration was achieved when somatic embryos were subcultured to medium with 0.1 mg dm^?3 2,4-D and 0.5 mg dm^?3 BAP or without growth regulators.     

High-frequency plant regeneration from cultured cotyledons of Arabidopsis thaliana

Wild-type plants of Arabidopsis thaliana strain Columbia regenerated at a high frequency from immature cotyledons cultured on a shoot-inducing medium containing 1.0 mg/l 6-benzylaminopurine and 0.1 mg/l 1-naphthaleneacetic acid. Cotyledon segments expanded rapidly and produced numerous shoots after 2–3 weeks in culture. Regeneration occurred in the absence of the original shoot apex. Hypocotyl segments from immature embryos produced root hairs and callus in culture but only rarely developed shoots. Hygromycin, kanamycin and G-418 inhibited cotyledon expansion and shoot formation in culture. Vancomycin was much less toxic to cotyledon segments than either carbenicillin or cefotaxime. Immature cotyledons therefore yield numerous regenerated plants that may be useful in future transformation studies.     

Somatic embryogenesis and plant regeneration inPisum sativum L.

Somatic embryogenesis was induced in immature zygotic embryos of pea (Pisum sativum L.), synthetic auxins α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC) being used. Only one (line HM-6) of 46 genotypes tested exhibited good potential for somatic embryogenesis. 2,4-D was found as the best somatic embryo inductor. Three different ways of somatic embryo conversion have been described. Plantlets from individual somatic embryos were micropropagated as somaclones and subsequently rooted. A sterile morphological mutant has been found within a group of fertile plants of T₀-generation. Sufficient amount of T₁-seeds is available for somaclonal variation studies.