Variability of azadirachtin inAzarirachta indica (neem) and batch kinetics studies of cell suspension culture

Seeds of neem were collected from different parts of India and analyzed for their azadirachtin content by High Performance Liquid Chromatography (HPLC). In order to assess the effects of genotypic and geographical variation on azadirachtin content in cell cultures, callus development was attempted in the seeds containing high and low concentration of azadirachtin. The concentration of azadirachtin in callus cultures was significantly affected by the explant source. Seed kernels with higher azadirachtin content produced higher azadiractin content in callus cultures and lower azadirachtin content was seen in callus cultures produced from seed kernels with low azadiractin content. The protocol for development of elite stock culture ofAzadirachta indica was established with the objective of selecting a high azadirachtin-producing cell line. The highest azadirachtin-producing cell line was selected and the effects of different media and illumination conditions on growth and azadirachtin production were studied in shake flask suspension culture. Detailed batch growth kinetics was also established. These studies provided elite starter culture and associated protocols for cultivation ofA. indica plant cell culture in the bioreactor.     

Production of 8-epi-tomentosin by plant cell culture ofXanthium strumarium

This study was conducted to establish a plant cell culture system for the production of medically important secondary metabolites fromXanthium strumarium. The effects of plant growth regulators including NAA, 2,4-D, kinetin, and ABA were examined in terms of callus induction, maintenance of callus and suspension cultures. It was shown that callus was induced upon treatment with NAA while embryo was induced after treatment with 2,4-D. Callus formation was further improved by treatment with ABA and NAA. The level of callusing increased by 17–29% for the seed case, cotyledon, leaf, and hypocotyl and by 96% in the case of the root. Suspension cell lines were established using calli produced from cotyledon, hypocotyl and root and cultured at 25°C under light conditions. The cells grew up to 15 g/L with NAA 2 ppm, BA 2 ppm, and ABA 1 ppm treatment. Supernatants of suspension cultures of cell lines derived from coyledon and hypocotyl produced some distinctive secondary metabolites, one of which was identified as 8-epi-tomentosin, which belongs to the xanthanolides. The amounts of 8-epi-tomentosin produced by the cotyledon-and hypocotylderived cell lines were 13.4 mg/L and 11.0 mg/L, respectively.     

Production of saponins from <Emphasis Type="Italic">Panax ginseng</Emphasis> suspension and adventitious root cultures

Biomass growth and ginsenoside production in cell suspension and adventitious roots of Panax ginseng C.A. Meyer cultures cultivated both in Erlenmayer flasks and a 3 dm³ bioreactor were studied. The maximum content of ginsenosides was found in the suspension culture cultivated in the bioreactor (4.34 % dry mass), however the saponin content was limited to two major ginsenosides, Rb₁ and Rg₁. The production of ginsenosides in adventitious roots was lower (1.45 or 1.72 % dry mass), nevertheless, the full range of ginsenosides was detected.This work was supported by 521/02/P064, COST 843.10, ME671 and Z4 055 905 projects.     

植物组织培养生物反应器技术研究进展

从植物大规模组织培养的特点、反应器类型和反应器中微环境等方面介绍了生物反应器技术在药用植物微繁殖和天然产物细胞生产中的研究进展。     

Hormonal mechanisms for differentiation in plant tissue culture

Summary Cells possess extraordinary powers to organize their molecular processes not only to maintain a cell in a given steady state but also to recognize that state during differentiation. Regulation of these organizational forces appears to be under the control of chemical factors, and a hormonal concept of regulation has evolved. Hormones have been considered to act by reacting with a specific target site. This may be part of their mode of action, but I would like to suggest that a hormone enters and becomes part of a total molecular resonance system. In so doing, the entire molecular system of the cell is modified. Of the known plant hormones, the cytokinins, because of their role in experimentally induced cell division and differentiation, serve as a probe of hormonal involvement in differentiation. Cultured somatic cells of tobacco plants can be induced to undergo differentiation by addition of cytokinin and auxin to the medium. Studies of the cytokinin hormones show a series of diverse molecular involvements. The archetype cytokinin, N⁶-(Δ²-isopentenyl) adenosine (i⁶Ado), occurs in some molecular species of tRNA where it plays a vital role in the codon-anticodon interaction of tRNA and m-RNA. i⁶Ado under-goes extensive metabolism in the tobacco tissue. It is either degraded to adenosine or converted to derivatives that possess biological activity. It is perhaps, therefore, more correct to consider the hormone function as being derived from this total metabolic web. The normal somatic cells of tobacco cultures spontaneously change occasionally into an autonomous form that requires no external growth factors. This line of cells synthesizes i⁶Ado. The metabolic web of the hormone-dependent strain can be perturbed by added auxin but such is not the case in the autonomous strain. These data provide some insight into the altered state of cytokinin activity in which a cell line changes into an autonomous form. Curiously, in become independent of the requirement for exogenous cytokinin, the autonomous tissue becomes sensitive to added cytokinin. i⁶Ado also inhibits the growth of lines of mammalian cancer cells grown in culture. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Bioreactor-based advances in plant tissue and cell culture: challenges and prospects

Bioreactors are engineered systems capable of supporting a biologically active situation for conducting aerobic or anaerobic biochemical processes. Stability, operational ease, improved nutrient uptake capacity, time- and cost-effectiveness, and large quantities of biomass production, make bioreactors suitable alternatives to conventional plant tissue and cell culture (PTCC) methods. Bioreactors are employed in a wide range of plant research, and have evolved over time. Such technological progress, has led to remarkable achievements in the field of PTCC. Since the classification of bioreactors has been extensively reviewed in numerous reviews, the current article avoids repeating the same material. Alternatively, it aims to highlight the principal advances in the bioreactor hardware s used in PTCC rather than classical categorization. Furthermore, our review summarizes the most significant steps as well as current state-of-the-art of PTCC carried out in various types of bioreactor.     

适合于悬浮培养的枇杷愈伤组织诱导及状态调控

以枇杷不同品种和不同器官为外植体,通过对愈伤组织诱导率、诱导状态、熊果酸（UA）和齐墩果酸（OA）含量的综合考虑,筛选适合于悬浮培养的枇杷愈伤组织,并通过调整2,4-D、6-BA浓度,同时加入酸水解酪蛋白（CH）、谷氨酰胺（Gln）、KCl等物质对花丝愈伤组织状态进行调控。结果表明：枇杷‘早钟6号’幼胚诱导的愈伤组织最适用于枇杷悬浮培养,愈伤诱导率为83.70%,愈伤组织嫩黄、疏松、颗粒明显,UA、OA含量分别为6.90、2.34 mg/g dw。在MS中附加2,4-D 6.0 mg/L、6-BA 0.8 mg/L、CH 200 mg/L、KCl 500 mg/L,花丝诱导的坚硬愈伤出现细软、嫩黄的愈伤组织,但多次继代后水渍化,状态不佳。     

茅苍术愈伤组织诱导及其细胞悬浮培养研究

采用正交试验法研究了不同的植物生长调节剂对茅苍术叶柄、叶片和根茎愈伤组织诱导的影响,结果表明,不同外植体在各自的最佳培养条件下,叶柄、叶片和根茎愈伤组织的诱导率分别为99.0%、83.5%和71.5%,以叶柄的培养效果最好,其中2,4-D对茅苍术愈伤组织的诱导具有极显著的效果,在各种植物生长调节剂组合中,诱导叶柄愈伤组织形成的最佳组合为0.4mg·L-1NAA、4.0mg·L-12,4-D和0.4mg·L-1KT,培养20d左右,诱导率达到99.0%。此外,将茅苍术叶柄细胞悬浮培养至18d时,细胞量、多糖和苍术素的含量均达到最大值,分别为9.07g·L-1、15.68mg·L-1和19.62ug·L-1。     

Tissue culture of ornamental pot plant: a critical review on present scenario and future prospects

Recent modern techniques of propagation have been developed which could help growers to meet the demand of the horticultural industry in the next century. An overview on the in vitro propagation via thin cell layer, meristem culture, regeneration via organogenesis and somatic embryogenesis is presented. Available methods for the transfer of genes could significantly simplify the breeding procedures and overcome some of the agronomic and environmental problems, which other wise would not be achievable through conventional propagation methods. The development and remarkable achievements with biotechnology in ornamental pot plants made during the three decades have been reviewed. The usefulness of the pot plants in commercial industry as well as propagation techniques, screening for various useful characteristics and selection of somaclonal variation is also discussed.     

辣木资源利用中的悬浮细胞培养体系研究

辣木富含多种营养成分,在食品和药物开发方面有巨大的潜在开发价值。本文提供了一种可行的辣木细胞悬浮培养技术。由辣木的根诱导形成愈伤组织和叶诱导形成愈伤组织的合适细胞悬浮培养条件分别为MS培养基(MS)+1.0mg/L 2,4-二氯苯氧乙酸(2,4-D)+1.0mg/L激动素(KT)和MS+0.5mg/L 2,4-D+0.5mg/L KT,摇床转速均为50~100r/min,将愈伤组织添加到液体悬浮培养基中20d左右可得到大量悬浮细胞。本研究为辣木细胞水平的培养和研究提供了一条途径,为辣木潜在价值的开发利用提供新的思路。     

毛刺槐花药培养及再生植株的获得

以毛刺槐的花药为材料,开展其组织培养和植株再生系统的研究。结果显示：将毛刺槐的花药接种在MS附加2,4—D0．1mg／L和BA3．0mg／L的培养基上,20d时花药愈伤组织诱导率可达41．5％。花药愈伤组织在MS附加BA5．0mg／L的分化培养基上继代培养2个月后,可分化出许多绿色的芽点,待不定芽长至2—3cm高时将其切下,转入MS附加IBA1．0mg／L的生根培养基上,2周后即可得到完整的再生植株。同时,研究就4℃低温预处理和蔗糖浓度对毛刺槐花药培养的影响进行了研究和讨论。     

Development of a helical-ribbon impeller bioreactor for high-density plant cell suspension culture

A double helical-ribbon impeller (HRI) bioreactor with a 11-L working volume was developed to grow high-density Catharanthus roseus cell suspensions. The rheological behavior of this suspension was found to be shear-thinning for concentrations higher than 12 to 15 g DW . L(-1). A granulated agar suspension of similar rheological properties was used as a model fluid for these suspensions. Mixing studies revealed that surface baffling and bottom profiling of the bioreactor and impeller speeds of 60 to 150 rpm ensured uniform mixing of suspensions. The HRI power requirement was found to increase singnificantly for agar suspensions higher than 13 g DW . L(-1), in conjunction with the effective viscosity increase. Oxygen transfer studies showed high apparent surface oxygen transfer coefficients (k(L)a approximately 4 to 45 h(-1)) from agar suspensions of 30 g DW . L(-1) to water and for mixing speeds ranging from 120 to 150 rpm. These high surface k(I)a values were ascribed to the flow pattern of this bioreactor configuration combined with surface bubble generation and entrainment in the liquid phase caused by the presence of the surface baffles. High-density C. roseus cell suspension cultures were successfully grown in this bioreactor without gas sparging. Up to 70% oxygen enrichment of the head space was required to ensure sufficient oxygen supply to the cultures so that dissolved oxygen concentration would remain above the critical level (>/=10% air saturation). The best mixing speed was 120 rpm. These cultures grew at the same rate ( approximately 0.4 d(-1)) and attained the same high biomass concentrations ( approximately 25 to 27 g DW . L(-1), 450 to 500 g filtered wet biomass . L(-1), and 92% to 100% settled wet biomass volume) as shake flask cultures. The scale-up potential of this bioreactor configuration is discussed.     

Suspension culture of hematopoietic stem cells in stirred bioreactors

Hematopoietic stem cells have applications in bone marrow transplantations for the treatment of hematopoietic disorders. When murine hematopoietic stem cells were cultured in 50 ml stirred bioreactors for 14 d, stem-cell-antigen-1 positive cells (hematopoietic primitive progenitor cells) and long-term culture-initiating cells (hematopoietic stem cells) grew by 5-fold and 4-fold, respectively. These results show the possibility of growing hematopoietic stem cells using a stirred bioreactor.     

盾叶薯蓣组织培养技术的优化

以盾叶薯蓣的根状茎、茎段、叶柄、幼叶为材料,进行愈伤组织诱导、分化及再生植株形成的研究。结果表明：盾叶薯蓣不同外植体均能诱导出愈伤组织,其中茎段愈伤组织的诱导率最高;不同激素配比的培养基对愈伤组织的形成有很大的影响：以LS为基本培养基,2,4-D浓度为4．0mg／L、6-BA浓度为1．0mg／L的激素配比诱导率最高,达62．5％;以改良MS为基本培养基,2,4-D浓度为2．0mg／L、6-BA浓度为0．5mg／L的激素配比诱导率最高,达71．4％。筛选到优化的分化培养基为改良MS附加2．0mg／L的6-BA和0．5mg／L的Vc,且能直接诱导出根,并形成完整植株。     

Toward the production of artemisinin through tissue culture: Determining nutrient-hormone combinations suitable for cell suspension cultures

Summary Artemisia annua L. is the source of a potent antimalarial, artemisinin. As part of a program to produce artemisinin through tissue culture, a series of 14 multifactorial experiments were conducted to determine suitable conditions for initiating and maintaining friable callus fromA. annua. In the first six experiments, three different nutrient formulations [Gamborg B5 (B5), Murashige and Skoog (MS), and Whetmore and Rier (WR)], each with 32 combinations of auxins and cytokinins [2,4-dichlorophenoxyacetic acid (2,4-D) with benzyladenine (BA), or 1-naphthaleneacetic acid (NAA) with 6-furfurylaminopurine (kinetin)], were tested. Both B5 and WR nutrients supported friable callus formation from leaf explants with some combinations of auxin and cytokinin. Inasmuch as friable callus seemed to be produced over a wider range of auxin and cytokinin concentrations in combination with B5, the remaining experiments were conducted solely with this nutrient formulation. In the remaining eight experiments, it was determined that friable callus formed when combinations of NAA with kinetin or 2,4-D and BA were used with B5 medium. Lighter colored, more friable callus formed in response to 2,4-D and BA than with NAA and kinetin. No single combination of concentrations of auxin and cytokinin seemed to be &#x201C;ideal&#x201D; for producing friable callus. Ranges of 2,4-D from 0.5 to 2.0 with BA between 0.025 and 0.1, or NAA between 0.5 and 2.0 with kinetin between 0.5 and 1.0 mg/liter, produced acceptable results.     

骆驼蓬的组织培养及植株再生

以骆驼蓬(Peganum harmala L)无菌苗下胚轴切段为材料,在不同的培养基上进行愈伤组织的诱导,发现在MS基本培养基附加2．0mg/L 2,4—D、0．5mg／L 6—BA和3％蔗糖时,可100％的诱导出愈伤组织。愈伤组织在附加2．0mg／L 6—BA、0．5mg／L NAA、500mg／L CH和3％蔗糖的MS培养基上诱导出丛生芽,进而发育成苗,苗的分化频率在30％左右。分化苗或其茎切断在附加0．2mg／L IBA、0．2mg/L NAA和3％蔗糖的l／2MS培养基上出现根的分化,分化频率在90％以上。再生植株经炼苗后移栽成活,成活率在80％以上。     

灰绿黄堇愈伤组织培养及生物总碱含量的初步研究

将灰绿黄堇(Corydalis adunca Maxim．)无菌试管苗的茎段接种于附加不同BA和NAA浓度组合的MS培养基上,诱导出愈伤组织。实验表明较高浓度的BA和较低浓度的NAA组合有利于灰绿黄堇愈伤组织的诱导和生长以及愈伤组织中生物碱的合成。在附加BA2．00mg／L NAA0．2mg／L的MS培养基上,灰绿黄堇愈伤组织生长快,诱导率较高,合成的生物总碱含量较高,可达0．312％。     

Isolation and culture of protoplasts from cell suspension cultures of Duboisia myoporoides with subsequent plant regeneration

Protoplasts were isolated from suspension cultures of various cell lines of Duboisia myoporoides R. Br. There were differences among cell lines with respect to optimal conditions for protoplast isolation including the amount and kind of enzymes and the osmoticum concentration. Protoplasts isolated from one cell line were successfully cultured and induced to form cell colonies in liquid modified B5 medium. Addition of conditioned medium, coconut milk and glucose as an osmoticum to protoplast culture medium as well as maintenance of high protoplast density in culture (> 10⁵/ml) were essential to obtain protocolony formation. Reduction of osmoticum concentration and deletion of coconut milk and conditioned medium from the culture medium were necessary to allow further colony development leading to cellus formation. Intact plants regenerated from calli derived from protoplasts were successfully transferred to pots.     

Application of imaging tools to plant cell culture: Relationship between plant cell aggregation and flavonoid production

Summary Image analysis tools were developed to measure biomass concentration, aggregate size and distribution, and pigmentation from anthocyanin-producing cell suspension cultures of ohelo (Vaccinium pahalae). The ex situ imaging system could image cell aggregates from 30 μm to 2 mm in diameter. The image analysis algorithm was based on extracted geometric features and morphological methods for biomass volume estimates, and hue, saturation, and intensity color characteristics for pigmentation estimates. Detailed information available from sampled cell culture images was validated by comparison to standard destructive manual measurements. Image analysis measurements revealed that pigment accumulation was negatively correlated with aggregate size. Although a substantial proportion of small aggregates remained colorless, the highly-pigmented small aggregates, 18 to 238 μm in breadth, contributed over 70% of the culture anthocyanin production (mg L⁻¹), despite their minor contribution to the overall biomass. The relative frequency of pigmented aggregates was higher in large-size aggregate classes; however, the pigmented sectors were mostly confined to only the periphery of the aggregates. As a result, large aggregate classes had only a minor contribution to overall culture anthocyanin yield.     

Sex differentiation: state of the art and future prospects

Our knowledge on sex differentiation in mammals has considerably progressed during the last decennials, beginning with the discovery of the testis-determining factor. Here, the morphogenetic processes involved in the early gonadic switch will be presented, together with the major genes involved in testis and ovary formation. Existing differences between the widely used mouse model and other mammals, such as human and goat, will be highlighted.