白花泡桐优树组织培养及产业化快繁技术

以自选育的白花泡桐优树茎段为外植体,进行种苗组培快繁技术研究。结果表明:其最佳的外植体灭菌方法是以0.1%升汞处理7 min;合适的初代诱导培养基为MS+6-BA 2.0 mg·L~(-1)+IBA 0.2 mg·L~(-1)+糖30 g·L~(-1)+琼脂3.5 g·L~(-1)(pH 5.8),培养30 d,芽诱导率70%;合适的继代增殖方法为在高浓度植物生长物质培养基MS+6-BA 4.0 mg·L~(-1)+IBA 0.4 mg·L~(-1)+蔗糖30 g·L~(-1)+琼脂3.5 g·L~(-1)(pH 5.8)和低浓度植物生长物质培养基MS+6-BA 0.4 mg·L~(-1)+IBA 0.04 mg·L~(-1)+蔗糖30 g·L~(-1)+琼脂3.5 g·L~(-1)(pH 5.8)中交替培养,获得的丛生芽长势良好,玻璃化率低于5%,增殖系数大于6.0/25 d;最适的生根培养基为1/2MS+NAA0.2 mg·L~(-1)+蔗糖20 g·L~(-1)+卡拉胶3.4 g·L~(-1)(pH 5.8),培养14 d,得到白花泡桐生根苗,每株长根5~10条,根长3~5 cm,生根率98%,根系洁白、根毛少而短,易于清洗。将生根苗按照常规方法炼苗后移栽于温室大棚中,50 d后即可出圃,此时平均苗高1.0 m、地径1.0~2.0 cm,成活率在90%以上。     

马兜铃不含腋芽茎段不定芽的诱导

为建立马兜铃(Aristolochia debilis Sieb.et Zucc)不含腋芽茎段的不定芽诱导体系,采用正交设计方法研究植物生长调节剂、预培养方式和AgNO3对不定芽诱导的影响。结果表明:植物生长调节物质对不定芽诱导的影响以TDZ6-BAIAA,其中TDZ的影响极显著(P0.01),6-BA的影响显著(P0.05)。不定芽诱导的最适培养基为MS+0.5 mg L–1 TDZ+0.1 mg L–1IAA+0.5 mg L–1 6-BA+2 mg L–1 AgNO3+3%蔗糖+0.6%琼脂(pH 5.8);预培养方式为在MS+0.1 mg L–1 2,4-D+3%蔗糖+0.6%琼脂培养基上暗培养2 d。马兜铃不含腋芽茎段的不定芽诱导率最高可达37.5%。     

Production of the new antimalarial drug artemisinin in shoot cultures of Artemisia annua L.

From aseptically grown Artemisia annua plantlets, shoot cultures were initiated. Using different concentrations of auxine, cytokinine and sucrose, a suitable culture medium was developed, with respect to the growth of the shoots and their artemisinin accumulation. Nitrate concentration and conductivity appeared to be suitable growth parameters. The artemisinin content was measured gas chromatographically. The shoot cultures were maintained in the developed standard medium, consisting of a half concentration of MS-salts with vitamins, 0.2 mg l^-1 BAP, 0.05 mg l^-1 NAA and 1% sucrose. The growth of the shoots and the artemisinin content remained stable for a longer period. They showed considerable photosynthetic activity and generally contained ca. 0.08% artemisinin on a dry weight basis. The highest artemisinin content found was 0.16% in the above mentioned standard medium, but also on the same medium with 0.5% sucrose. Attempts were made to further improve the artemisinin production by varying the medium composition through addition of gibberellic acid or casein hydroly-state; by omitting plant growth regulators; by precursor feeding, i.e. mevalonic acid; by influencing the biosynthesis routing through inhibition of the sterol synthesis by miconazole, naftifine or terbinafine; by changing gene expression with 5-azacytidine or colchicine; and by elicitation, using cellulase, chitosan, glutathione or nigeran. Enhanced artemisinin production was found with 10 mg l^-1 gibberellic acid, 0.5 g l^-1 casein hydrolysate, 10 mg l^-1 or 20 mg l^-1 naftifine. Relative increases of 154%, 169%, 140% and 120% were found, respectively. Other additions caused the growth to cease and the artemisinin contents to drop.Abbreviations BAP benzylaminopurine - DW dry weight - FW fresh weight - GA₃ gibberellic acid - MS Murashige & Skoog basal medium - NAA naphthaleneacetic acid     

菜心组织培养技术初探

为建立菜心(Brassica campestris ssp.chinensis var.utilis)的快繁技术体系,以花药和子叶-子叶柄为外植体进行组织培养研究。结果表明,花药培养以选取未开放的花蕾为宜,且花柱略高于花瓣,此时小孢子多数处于单核靠边期。菜心花粉的萌发率不高,且秋冬季的花粉比夏季的萌发率高。菜心花药愈伤组织诱导培养基为:MS+1.0 mg L–1 KT+1.0 mg L–1 2,4-D+3%糖+6 g L–1琼脂+8%椰乳,不定芽诱导培养基为:MS+2.0 mg L–1 6-BA+0.5 mg L–1 NAA+1.0 g L–1活性炭+2%糖+6 g L–1琼脂或MS+2.0 mg L–1 ZT+0.5 mg L–1 IAA+0.5 g L–1 AgNO3+1.0 g L–1活性炭+2%糖+6 g L–1琼脂。花药培养的不定芽诱导率为36.7%,不定芽培养出现褐化现象,不能形成再生植株;而以子叶-子叶柄为外植体培养获得的植株再生率可达80%。     

Optimization of culturing conditions for the production of biomass and phenolics from adventitious roots ofEchinacea angustifolia

We investigated different concentrations of auxins (1AA, IBA, NAA), the strength of the MS medium, sucrose and ammonium/nitrate contents, initial medium pH, and inoculum size to determine their effects on biomass increase and the accumulation of total phenols and flavonoids in adventitious roots ofEchinacea angustifolia. These roots were cultured under darkness in shake flasks for 4 weeks. IBA proved the best auxin for inducing root proliferation. Root growth was inhibited when the initial pH was maintained below 5.0 or above 6.0. Nitrate, rather than ammonium, was more necessary for root growth and phenolics accumulations. Overall, biomass increased and total phenol and flavonoid contents were maximized under the following conditions: half-strength MS medium supplemented with 2 mg L^-1 IBA, 5% (w/v) sucrose, 5:25 (mM) ammonium/nitrate ratio, pH adjusted to 6.0 before autoclaving, and an inoculum size of 10 g L^-1 FW. These results indicate that the type ofin vitro environment strongly affects growth and the accumulation of phenolics from adventitious root cultures ofE. angustifolia. Such optimization is beneficial to large-scale production of biomass and secondary metabolites in that species.     

Production of diosgenin by hairy root cultures ofTrigonella foenum-graecum L.

Hairy root cultures ofTrigonella foenum-graecum L. were established withAgrobacterium rhizogenes strain A₄. The hairy roots produce diosgenin, an important spirostanol for the semi-synthesis of steroid hormones. Fourteen different liquid media were investigated. The fastest growth was obtained in McCown's woody plant (WP) medium supplemented with 3% sucrose; the highest diosgenin content was observed in half-strength WP medium with 1% sucrose (0.040% dry weight), which represents almost twice the amount detected in the 8-month-old non-transformed roots (0.024%). A time-course study in WP liquid media supplemented with 3% sucrose was undertaken. In these conditions, 17 g diosgenin/g fresh weight were produced. The influence of cholesterol, medium pH and chitosan on diosgenin production was tested. The addition of 40 mg/l chitosan elevated the diosgenin content to three times that found in non-elicited hairy roots.Abbreviations MS Murashige and Skoog (1962) medium - WP McCown's woody plant medium     

Optimization of Factors Influencing Adventitious Rooting in Hybrid Larch

Optimization of in vitro adventitious root induction contributes to the development of a large-scale production system of hybrid clone seedlings of larch (Larix spp.). We used orthogonal testing to investigate the factors that affect the rooting of hybrid larch shoots–activated carbon, vitamin B1, sucrose, glycine, glutamic acid (Glu), cysteine, pH, and induction time in the dark and indole-3-butyric acid (IBA). Variance analysis showed that the effects of sucrose concentration on rooting rate, survival rate, and average root number, pH on the average number of roots; and Glu on survival rate were all significant (p < 0.05) when compared to the mock treatments. Among all of the 45 treatments, the highest rooting rate was 90%, the highest survival rate was 100%, and the highest average root number was 11.8. The theoretically optimal procedure of in vitro root induction of hybrid larch shoots is shoot induction on L9 modified medium (pH 6.7) containing 2 mg.L^–1 naphthalene acetic acid, 0.25 mg.L^–1 IBA, 1/3 macroelements, 7.5 g.L^–1 sucrose, and 5 mg.L^–1 Glu for 8 days, and then root induction on L9 modified medium without auxin.     

Accumulation of anthraquinones in Morinda citrifolia cell suspensions

Cell suspensions of Morinda citrifolia were cultivated in a B5-medium containing 4% sucrose as the sole carbon source and 1 mg l^-1 naphthyl acetic acid (NAA) or 1 mg l^-1 2,4-dichloro-phenoxyacetic acid (2,4-D). Both auxins were able to support growth but only in the presence of NAA anthraquinone production was observed. 2,4-D inhibited the production in NAA cultures. Anthraquinone synthesis took place in the growth and the stationary phase and amounts of 0.2–0.4 mmol (about 100–200 mg) g^-1 dry weight could be reached.Under both growth conditions sucrose was hydrolyzed extracellularly by invertase. From the resulting monosaccharides, glucose was taken up preferentially and an appreciable uptake of fructose only took place when medium glucose was exhausted. Sugar uptake rates were similar when cells were grown in NAA and in 2,4-D medium but the intracellular sugar contents (expressed on a dry weight basis) differed considerably. The presence of sucrose, glucose and fructose was demonstrated under both growth conditions. The amounts of sucrose and glucose were much lower in the 2,4-D cells than in the NAA-cells especially during the growth phase. Fructose contents were low and comparable, while in NAA cells an unknown sugar (possibly the sugar moiety of the glycosylated anthraquinones) was observed especially at the end of the growth phase and in the stationary phase. The differences in sugar concentrations were even larger due to the lower water contents of the NAA cells.Respiration of 2,4-D cells was much higher than that of NAA cells during the growth phase. A sharp increase in sugar contents (mainly sucrose) occurred in the 2,4-D cells at the end of the growth phase and corresponded with the fall in respiratory activity.A possible correlation between the lack of production of anthraquinones in 2,4-D cells and a less efficient growth metabolism in these cells is discussed.Abbreviations AQ anthraquinones - 2,4-D 2,4-dichloro-phenoxy-acetic acid - DW dry weight - FW fresh weight - NAA naphthyl acetic acid - pCPO p-chloro-phenoxy-acetic acid     

Effects of humic acid-herbicide interactions on the growth of Pisum sativum in nutrient solution

Slight increases in root length and dry matter production were measured in roots and shoots of Pisum sativum seedlings, when germinated seeds were grown for eight days in a controlled environment chamber, on Nitch nutrient solution to which 10 mg L^-1 soil humic acid (HA) had been added. A concentration of 100 mg L^-1 of HA produced a small reduction in dry matter production of shoots. In the presence of herbicides, 10 mg L^-1 cycluron or 100 mg L^-1 prometone, root elongation and shoot growth was reduced by varying degrees with respect to the control. Nutrient solutions containing 100 mg L^-1 cycluron, 10 or 100 mg L^-1 alachlor, or as little as 0.1 mg L^-1 of 2,4-D resulted in an almost complete suppression of the growth of pea seedlings. The addition of 10 or 100 mg L^-1 of HA to nutrient solution containing the herbicide was found to be either ineffective in enhancing growth, or even further reduced growth, compared to samples grown in the presence of the herbicide alone. The results of this preliminary study suggest that the generally accepted view that humic substances exert a positive effect on plant growth may not occur when humic substances interact with herbicides.     

In vitro regeneration of Vigna unguiculata (L.) Walp. cv. Blackeye cowpea via shoot organogenesis

An in vitro regeneration system was developed in cowpea [Vigna unguiculata (L.) Walp.] Blackeye. Among several explants studied, shoot initiation response was observed from shoot apices of 3–5-day-old seedlings. The optimal medium for maximum shoot initiation comprised MS salts, B₅ vitamins, 8.88 μM N ⁶-benzylaminopurine, 1 gl^-1 casein hydrolysate, 342 μM L-glutamine, 3% sucrose, 0.3% phytagel, adjusted to pH 5.8. A shift in pH from 5.8 to 7.0 had no effect on shoot initiation and on number of shoots per explant. The highest shoot initiation frequency (77%) was obtained using this preferred medium, reaching a maximum of eight shoots per explant. For shoot elongation, 14 μM gibberellic acid was supplemented in the shoot initiation medium. Presence of indolebutyric acid in the rooting medium had no effect on root induction. The regenerated plants were fertile and developed normally.     

Indigo production in hairy root cultures of Polygonum tinctorium Lour.

The transformed root culture of Polygonum tinctorium Lour. was established by infecting leaf explants with Agrobacterium rhizogenes A4. These cultures were examined for their growth and indigo content under various culture conditions. Among the four different culture media tested, SH medium showed the highest yield for root growth (28 mg dry wt/30 ml) and indigo production (152 g/dry wt). In SH medium, 30 g sucrose l^–1, 2500 mg KNO₃ l^–1, 300 mg NH₄H₂PO₄ l^–1 were the best conditions for indigo production at pH 5.7. The production of indigo in hairy roots slightly increased with the addition of 200 mg chitosan l^–1 (186 g/dry wt) and 20 U pectinase l^–1 (181 g/dry wt).     

Improved <Emphasis Type="Italic">in vitro</Emphasis> micropropagation method with adventitious corms and roots for endangered saffron

The objective of this study was to investigate development of an efficient in vitro tissue culture system for saffron (Crocus sativus L.) complete with roots and corms. In indirect organogenesis, Murashige and Skoog (MS) media with 3% (w/v) sucrose, 100 mg L⁻¹ ascorbic acid, and the combination of 0.25 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg L⁻¹ 6-benzylaminopurine (BAP) were best for callus initiation and growth while 1.5 mg L⁻¹ BAP was excellent for high rate of adventitious shoot formation. 1 mg L⁻¹ indole-3-butyric acid (IBA) was more preferable for adventitious corm and root initiation as well as growth. Overall, 64% rooting and 33% corm production rates were achieved in indirect organogenesis. In direct organogenesis, MS medium supplemented with 3 % sucrose, 100 mg L⁻¹ ascorbic acid and 1 mg L⁻¹ BAP was optimum for shoot growth. While 1 mg L⁻¹ IBA was best for adventitious corm formation, 2 mg L⁻¹ IBA promoted adventitious root initiation and growth. Overall, 36% and 57% of explants had corm and contractile root, respectively. The high rates suggest that efficient tissue culture system could be achieved for mass propagation and ex situ conservation of threatened saffron genetic resources.     

紫茉莉悬浮细胞培养体系的建立

为建立紫茉莉(Mirabilis jalapa L.)悬浮细胞培养体系,以紫茉莉无菌苗叶片诱导的愈伤组织为材料,筛选紫茉莉悬浮细胞的适宜培养体系。结果表明,紫茉莉愈伤组织在MS+2,4-D 1 mg L-1+KT 0.5 mg L-1的液体培养基中悬浮继代培养3~4次,能得到稳定的悬浮细胞系。培养基的pH值为5.5~5.9,蔗糖浓度为30 g L-1更适合悬浮细胞的生长。紫茉莉悬浮细胞的生长曲线大致呈S型。最佳继代培养时间是10 d,培养液的体积为40 mL时,接种量为7.5 mL,可以较好地保持悬浮细胞系。1 L培养液中可提取分泌蛋白(0.42±0.15) g。这些有助于对悬浮细胞提取分泌蛋白的研究。     

Effect of nitrogen and sucrose on the primary and secondary metabolism of transformed root cultures of Hyoscyamus muticus

Abatract The effect of carbon and nitrogen sources on two well-established hairy root clones, LBA1S and C58A, of Hyoscyamus muticus strain Cairo, were investigated. Both clones exhibited completely different patterns with regards to their growth rate, hyoscyamine accumulation, and fatty acid contents. Clone C58A grew faster and yielded more biomass (17.4 g l^-1, in 21 days), but produced less hyoscyamine. The maximum hyoscyamine content (120 mg l^-1) in clone LBA1S was reached in 28 days. Neither of the clones could use lactose or fructose as the sole carbon source, nor ammonium as the sole nitrogen source. The growth in the medium containing glucose was significantly reduced compared to that containing sucrose. Clone LBA1S was sensitive to the changes in sucrose concentration and an increase in ammonium in the culture medium, whereas C58A tolerated these changes better but was more sensitive to the increase in total nitrogen. Lipid synthesis was active in the exponential growth phase, and the total fatty acid content varied from 5 to 34 mg g^-1 of dry root material. The major fatty acids were linoleic, palmitic and linolenic. There were considerable differences in the total amount of lipids and in their relative ratios when different nutrients were applied.Abbreviations DW dry weight - FA fatty acids - FFA free fatty acids - FW fresh weight     

Growth and steroidal saponin production in hairy root cultures of Solanum aculeatissimum

Summary Hairy root cultures of Solanum aculeatissimum were established by trans-formation using Agrobacterium rhizogenes strain 15834. Root growth and production of steroidal saponin were investigated under various culture conditions. Transformed roots grew better in Gamborg's B5 medium containing 3 % sucrose under continuous light than in the dark. Also, the roots turned light green when cultured under continuous light. Green hairy roots produced aculeatiside A (6.71mg ·) L^–1 and aculeatiside B (6.39mg · L^–1) after 8 weeks of culture, while no steroidal saponin was detected in hairy roots cultured in the dark. Of the three culture media tested, Gamborg's B5 medium was superior for growth and steroidal saponin production. Growth and steroidal saponin production were enhanced when 100g · L^–1 auxin except for 2,4-D was added to the medium. The addition of 2,4-D inhibited growth. Production of steroidal saponin was highest with NAA. Transformed roots used in this experiment were confirmed that hairy roots examined contain both TL-DNA and TR-DNA region of Ri plasmid by PCR amplification analysis of DNA.Abbreviations MS medium Murashige and Skoog's medium (1962) - B5 medium Gamborg's B5 medium (1968) - LS medium Linsmaier and Skoog's medium(1965) - HPLC High performance liquid chromatography - NAA -Naphthaleneacetic acid - IAA Indole-3-acetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - PCR polymerase chain reaction     

Establishment and characterization of Pinus pinaster suspension cell cultures

We report the establishment of a Pinus pinaster (Ait.) cell suspension culture in a modified MS medium supplemented with 2 mg ml⁻¹ 2,4-D and 1 mg ml⁻¹ BA. Calli were obtained from seedling root segments and established a friable isodiametric cell suspension, suitable for in vitro studies of maritime pine at the cellular level. Growth (dry weight), cell viability, pH, and nutrient consumption: carbon source (sucrose, fructose and glucose), nitrogen source (ammonia and nitrate) and phosphate were monitored over 24 h. Suspension cells exhibited a 15-day exponential growth stage, during which a biphasic consumption profile was observed for all nutrients. Phosphate was the first limiting nutrient and preferable consumption was observed for glucose over fructose and nitrate over ammonium.     

Hairy root culture of <Emphasis Type="Italic">Plumbago indica</Emphasis> as a potential source for plumbagin

Hairy roots of Plumbago indica were established at high frequency (90 %) by infecting leaf explants with Agrobacterium rhizogenes strain ATCC 15834. The axenic root cultures were established under darkness in hormone-free liquid Murashige and Skoog medium containing 3 % sucrose. The highest plumbagin content was found to accumulate in roots at their exponential phase of growth. A low pH (4.6) and a low concentration of sucrose (1 %) were beneficial for root growth in darkness, while pH 5.6 and 3 % sucrose under continuous irradiance enhanced plumbagin accumulation in roots up to 7.8 mg g⁻¹(d.m.). Direct shoot regeneration from hairy root culture was also achieved under continuous irradiance, thus indicated an easy way of obtaining transformed P. indica plants.     

牛角瓜离体快繁技术

该文选用牛角瓜茎段为外植体,通过组织培养方法探索牛角瓜组织培养和种苗快繁技术。结果表明:最佳外植体表面消毒方法是以0.1%HgCl_2处理7 min,外植体存活率为32.3%;初代培养基为MS+蔗糖30 g·L~(-1)+琼脂3.5 g·L~(-1),培养20 d后形成3～4 cm高的再生芽。增殖培养前期筛选的较为适宜的增殖培养基为MS+6-BA 1.5 mg·L~(-1)+NAA 0.05 mg·L~(-1)+蔗糖30 g·L~(-1)+琼脂3.5 g·L~(-1),增殖系数4.6。但在后续的培养过程中发现,牛角瓜组培苗易玻璃化,且随着世代更迭,玻璃化程度加重,到了第四代几乎全部玻璃化。因此在上述增殖培养的基础上,以AgNO_3作为玻璃化抑制剂,筛选出最终的增殖培养基为MS+6-BA1.5 mg·L~(-1)+NAA 0.05 mg·L~(-1)+AgNO_31.0 g·L~(-1)+蔗糖30 g·L~(-1)+琼脂3.5 g·L~(-1)。用此增殖培养基,培养25 d,苗高5～8 cm,增殖系数5.8,玻璃化率低于10%,且连续培养多代,玻璃化率维持在10%以下。生根壮苗培养基为1/2MS+NAA 1.0 mg·L~(-1)+蔗糖20 g·L~(-1)+琼脂3.6 g·L~(-1),培养14 d,生根率98%;将生根苗移栽于70%遮阴度的大棚中,30 d后,苗高20 cm左右,成活率85%。利用该方法可对牛角瓜优良种苗进行规模化生产。     

Rapidin Vitro propagation and enhanced stevioside accumulation inStevia rebaudiana Bert

A very rapid and efficient regeneration method has been established using mature expiants ofStevia rebaudiana Bert. Adventitious shoots were induced from nodal expiants of field-grown plants on four basal media supplemented with various combinations of auxins and cytokinins. The best performance (23.4 ± 2.1 shoots per expiant) was obtained on a Murashige and Skoog (MS) medium containing 2 mg L^-1 IAA and 0.5 mg L^-1 kinetin. Roots were then produced when thesein vitro- regenerated shoots were transferred to an MS medium supplemented with 3% sucrose and 2 mg L^-1 IBA. When acclimatized to soil, the rooted plants had a 98.4% survival rate. Following transplantation in the field, stevioside contents were similar between the regenerated plants (10.68 mg g^-1 dry weight) and the mother plants (12.01 mg g^-1 dw).     

Hernandulcin in hairy root cultures of Lippia dulcis

The hairy root culture of Lippia dulcis Trev., Verbenaceae, was established by transformation with Agrobacterium rhizogenes A4. The transformed roots grew well in Murashige and Skoog medium containing 2% sucrose. The roots turned light green when they were cultured under 16 h/day light. The green hairy roots produced the sweet sesquiterpene hernandulcin (ca. 0.25 mg/g dry wt) together with 20 other mono- and sesquiterpenes, while no terpenes were detected in the nontransformed root cultures. The growth and hernandulcin production in the hairy root cultures were influenced by the addition of auxins to the medium. The addition of a low concentration of chitosan (0.2 – 10.0 mg / l) enhanced the production of hernandulcin 5-fold.Abbreviations Cht chitosan - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog(1962)