Limitations associated with conductivity measurement for monitoring growth in plant tissue culture

The suitability of conductivity measurement for monitoring growth in plant cell culture has been tested using suspended cells and genetically-transformed hairy roots of Atropa belladonna, and aggregated cells of Solanum aviculare. Other researchers have proposed that a constant ratio exists between increase in cell concentration (x) and decrease in medium conductivity (C). In all cases studied in this work, x/C was not constant over a wide range of cell densities tested in batch culture. With cell suspensions, x/C decreased continuously during the growth phase from 3.4 to 2.5 g cm l^–1 mS^–1. For the hairy roots, the ratio between x and C varied by as much as 4-fold during growth. The relationship between conductivity and growth for S. aviculare aggregates was found to vary depending on inoculum density. No simple correlation between conductivity change and cell growth was apparent for the plant-cell systems studied.     

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。     

Comparison of tissue culture and whole plant responses to salinity in potato

Salt sensitivities of six potato cultivars using six levels of sodium chloride (0.0 to 0.25M) were studied in a greenhouse. Responses of these cultivars were also determined in tissue culture by studying rooting of stem segments, increase in length of cultured roots and inhibition of growth of cell suspension cultures using similar salt concentrations. Responses of cultured stem segments and cell suspensions differed from those expressed by whole plants. A close similarity was observed between the salt stress response of whole plants and of cultured roots. The latter technique may provide a preliminary screening method for assessing salt tolerance in potato genotypes.     

Karyotype evolution in a tumour derived plant tissue culture analysed by Giemsa C-banding

Summary A hyperdiploid aneuploid karyotype, consisting of 7 chromosomes, has been found in a tumorous suspension cell culture ofCrepis capillaris (2 n=6). Giemsa C-banding has revealed that these 7 chromosomes show consistent patterns of differential staining in all dividing cells. This stable karyotypic situation has persisted during 18 months of cytological monitoring of the culture. Comparison with the diploid C-banded complement of the root tip indicates that numerous structural rearrangements must have occurred during the formation of the aneuploid complement. A likely pathway for evolution of this karyotype involves initial tetraploidy followed by chromosome loss. Such a mechanism has previously been proposed for a plant tissue culture system (Bayliss andGould 1974) and commonly occurs in animal systems, particularly in animal tumours (Terzi andHawkins 1975). An alternative mechanism, which does not necessarily involve tetraploidy, is also proposed.     

Sugarcane tissue and protoplast culture

Experiments are described which improve the protocols for initiating in vitro cultures of sugarcane and allowing efficient regeneration of plants even after 30 months of callus proliferation. Procedures adopted included use of leaf base explants, CS medium with 3 mg/l 2, 4-D and 0.25 mg/l kinetin for callus initiation and growth, MS medium with 0.5 mg/l IAA and 1 mg/l BAP for shoots, MS medium with 5 mg/l NAA and 7% (wt/vol) sucrose for rooting of shoots. Casein hydrolysate (N-Z amine) significantly shortened the lag period in the growth of sugarcane suspension cultures, but did not increase the rate of growth following the lag phase. Protoplasts isolated from two types of cultures could be grown to re-establish cell cultures but no plants have yet been regenerated derived from isolated protoplasts.     

Raman spectroscopy as a method to replace off-line pH during mammalian cell culture processes

Raman spectroscopy is a robust, well-established tool utilized for measuring important cell culture process variables for example, feed, metabolites, and biomass in real-time. This study further expands the functionality of in-line Raman spectroscopy coupled with partial least squares (PLS) regression modelling to develop a pH measurement tool. Cell line specific models were developed to enhance the robustness for processes with different pH setpoints, deadbands, and cellular metabolism. The modelling strategy further improved robustness by reducing the temporal complexity of pH shifts by splitting data sets into two time zones reflective of major changes in pH. In addition, models were developed to assess if lactate and partial pressure of carbon dioxide (pCO₂) could be used in a PLS model for pH. Splitting the data sets into early and late for the process resulted in errors of 0.035 pH and 0.034 pH for the two respective Raman cell lines models which was within acceptance criteria. The lactate and pCO₂ PLS model with values provided by Raman models had a further 0.001 pH error reduction. This study illustrates the potential to eliminate off-line samples to correct for in-line measurements of pH and further illustrates the capabilities of Raman to measure additional process variables.     

High density cell culture of Panax notoginseng for production of ginseng saponin and polysaccharide in an airlift bioreactor

High cell density of Panax notoginseng in a 17 l airlift bioreactor was achieved in batch cultivation using a modified MS medium. The dry cell weight, ginseng saponin and polysaccharide reached 24, 1.7 and 2.8 g l^–1, respectively, after 15 d. A strategy of sucrose feeding based on changes in the specific O₂ uptake rate was applied to the cell cultures, which increased these respective yields to 30, 2.3 and 3.2 g l^–1.     

Pre-analytical method for metabolic profiling of plant cell cultures of Passiflora garckei

Passiflora garckei cell cultures were used as a model to describe a reproducible sample preparation method. Solid phase extraction (SPE) was employed to isolate the plant metabolites for nuclear magnetic resonance (NMR) analysis and to subsequently detect the differences between yeast extract elicited and control cells. Compared with previous results obtained by using a Sephadex LH-20 column, SPE coupled with NMR spectroscopy improves the analysis of aromatic compounds e.g.: trans-feruloyl derivatives and trans-coumaroyl derivatives. Moreover, it decreases the concentration of sugars that usually overlap with many plant metabolite signals.     

Application of Raman spectroscopy in monoclonal antibody producing continuous systems for downstream process intensification

Monoclonal antibodies (mAbs) are biopharmaceuticals produced by mammalian cell lines in bioreactors at a variety of scales. Cell engineering, media optimization, process monitoring, and control strategies for in vitro production have become crucial subjects to meet increasing demand for these high value pharmaceuticals. Raman Spectroscopy has gained great attention in the pharmaceutical industry for process monitoring and control to maintain quality assurance. For the first time, this article demonstrated the possibility of subclass independent quantitative mAb prediction by Raman spectroscopy in real time. The developed model estimated the concentrations of different mAb isotypes with average prediction errors of 0.2 (g/L) over the course of cell culture. In situ Raman spectroscopy combined with chemometric methods showed to be a useful predictive tool for monitoring of real time mAb concentrations in a permeate stream without sample removal. Raman spectroscopy can, therefore, be considered as a reliable process analytical technology tool for process monitor, control, and intensification of downstream continuous manufacturing. The presented results provide useful information for pharmaceutical industries to choose the most appropriate spectroscopic technology for their continuous processes.     

Commercialization of plant tissue culture in India

Commercial application of plant tissue culture started in USA with micropropagation of orchids in 1970s. It has seen tremendous expansion globally from 1985 to 1990 in the number of production units as well as the number of plants produced. With an estimated global market of 15 billion US dollars per annum for tissue cultured products, even with exponential expansion in the industry, the demand far exceeds production, leaving enough scope for expansion. This industry appears to be undergoing a pause in growth presently in developed countries as it is finding difficult to remain cost–effective. In US, only half the production capacity is being utilized currently due to high labour costs. In developing countries, with lower wage scales, plants are being produced at much cheaper rates. Indian micropropagation industry, though a late starter by almost a decade, compared to its western counterparts, has expanded exponentially from 5 million annual capacity in 1988 to 190 million in 1996. The facilities now created are at par with the best in leading countries like the Netherlands and USA. To remain in profitable business and to earn the much needed foreign exchange, Indian units need to judiciously mix steady revenue generating items with unique speciality items based on demand in domestic and international markets. This revised version was published online in June 2006 with corrections to the Cover Date.     

A plant cell bioreactor with medium-perfusion for control of somatic embryogenesis in liquid cell suspensions

The Braun Biostat BF2 bioreactor system employs a novel aeration and agitation system, designed to enhance gaseous exchange and reduce shear stresses on submerged cell suspension cultures. The Biostat BF2 bioreactor employs a central pivoting spindle, around which the aeration tubing is wound forming a large paddle-type structure suspended from the top-plate and swung in a circle by a solid-state magnetic stirrer.The aeration tubing is a polypropylene capillary membrane, which has a unique microporous structure and is ideal for aeration, permitting two-way, bubble-free, gaseous exchange of the medium. This tubing can be rendered porous and can be used in the perfusion of aqueous solutions, enabling cell-free media exchange to be conducted. Thin-walled silicone rubber tubing, although gas permeable to a degree, cannot be made porous to aqueous solutions.The bioreactor was inoculated with a suspension culture of Sitka spruce (Picea sitchensis [Bong.] Carr.) known to be embryogenic and capable of maturing to plantlets on solidified medium. The perfusion capability of the bioreactor was employed to replace the inital proliferation medium with maturation medium in order to induce the development of the somatic embryos in submerged cell culture. The size ratio of the somatic embryo heads was monitored over 7 weeks. This cell line was found to mirror just the initial elongation, previously observed in shake-flask culture.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - SSPM Selby Sitka proliferation medium - SSMM Selby Sitka maturation medium The following was presented at the NERC TBLG '95 Meeting as the Bioreactor Workshop     

The influence of ethylene in plant tissue culture

Ethylene produced by plant tissues grown in vitro may accumulate in large quantities in the culture vessels, particularly from rapidly growing non-differentiated callus or suspension cultures, and hence is likely to influence growth and development in such systems. Research into this aspect of tissue culture has been sparse, although it has grown recently with the increasing importance of in vitro regeneration. This review deals with the measurement and relevance of the accumulated ethylene, and the influence of both exogenous and endogenous ethylene in the different types of tissue culture systems. The relationships between ethylene and other growth regulators in tissue culture growth and development are also discussed. Although in some cases its influence seems negligible, in many types of tissue culture ethylene may act either as a promoter or inhibitor depending on the species used. Thus ethylene has an important influence on many aspects of in vitro regeneration, but it is also clear that we cannot at present describe a specific role or roles for ethylene in tissue culture which can be applied at a general, species-wide level. If its effects are to be enhanced or diminished in order to improve the efficiency and range of plant tissue culture, then more research is needed to clarify what its fundamental role might be in in vitro growth and development.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - AOA aminooxyacetic acid - ASA acetylsalicyclic acid - AVG aminoethoxyvinylglycine - BA N⁶ benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid - DNP 2,4-dinitrophenol - GA gibberellin - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - SAM S-adenosylmethionine - STS silver thiosulphate - TIBA 2,3,5-triidobenzoic acid     

Xanthium pith and hypocotyl tissue in culture

A rapid method is described of obtaining callus tissue cultures from hypocotyls of vegetative and flowering Xanthium strumarium L. seedlings. The tissue is grown on Murashige and Skoog medium modified with 1 g/l casein hydrolysate and 5 mg/l each of kinetin and -napthaleneacetic acid.     

Cell Growth and Shikonin Production of Arnebia euchroma in a Periodically Submerged Airlift Bioreactor

Arnebia euchroma was grown in a 2-l periodically submerged, airlift bioreactor (PSAB) in which the non-submerged (immobilization culture) and submerged (suspension culture) operations were controlled automatically. PSAB had advantages in improving cell growth, shikonin content, shikonin production and cell aggregation compared with suspension culture. Under the optimal submerged/non-submerged period of 10 min/15 h, the shikonin content (4.6%, w/w) and, cell dry mass (16.8 g/l) were 229 and 26% higher than those in suspension culture. Revisions requested 31 October 2005 and 6 December 2005; Revisions received 2 December 2005 and 13 January 2006     

Production of azadirachtin from plant tissue culture: State of the art and future prospects

With increasing awareness towards environment-friendly and non-toxic pesticide azadirachtin obtained from neem tree (Azadirachta indica) is gaining more and more importance. Its broad-spectrum activity, peculiar mode of action. eco-friendly and non-toxic action towards beneficial organisms has offered many advantages over chemical pesticides. All currently use commercial formulations based on azadirachtin contains azadirachtin extracted from seeds of naturally grown whole plants which is labour intensive process depending upon many uncontrollable geographical and climatic factors. Plant tissue culture can be a potential process for the production, offering consistent, stable and controlled supply of this bioactive compound, However the research on tissue culture aspects of production are in preliminary stage and requires culture and process optimization for the development of a commercially viable process. This review states the present status and future challenges of plant tissue culture for azadirachtin production.     

Rapid clonal multiplication of Angelonia salicariefolia through tissue culture

Nodal explants of Angelonia salicariefolia were cultured on MS basal medium and induced to form shoots when supplemented with either Kn (1.0 mg/l) or BAP (1.0 mg/l). Rooted shoots were formed in response to Kn+NAA (1.0 mg/l+0.5 mg/l). Subcultures of the shoots of these cultures grown on the same medium supplemented with 0.5 mg/l of NAA, IAA or IBA, together with lowered concentrations of inorganic salts, induced root formation in 20–30 days. Up to 18×10³ plants were produced from one plant in less than a month. Successful transfer of regenerants into soil has been accomplished.     

"植物组织培养"实践操作技能培养与提高

通过运用多媒体辅助教学、规范的演示实验、指导学生善于观察和总结等教学手段,为“植物组织培养”实践操作技能的培养打好基础,同时,重点让学生亲自动手操作实验,通过技能性实践、验证性实践、探索性实践,培养和提高《植物组织培养》实践操作技能.     

Evaluation of automated electrospray-TOF mass spectrometryfor metabolic fingerprinting of the plant metabolome

Metabolic fingerprinting is increasingly employed in microbial and plant metabolomics. Identification and evaluation of analytical factors that influence mass spectra produced with automated electrospray time of flight mass spectrometry to support metabolic fingerprinting are described. Instrument resolution of 4000 (FWHM) at mass 200 Da provided detection of ions of the same nominal mass but different monoisotopic masses. Complex mass spectra were obtained from polar extracts of tomato fruit in positive and negative ion mode. These spectra consist of metabolite ions (molecular, adduct and fragment) and those derived from the extraction medium, largely in the form of [M+H]⁺, [M–H]^–, [M+Na]⁺, [M+K]⁺, [2M+H]⁺, [M+Cl]^– and [2M–H]^–. Ionisation suppression reduced sensitivity, although its effect was consistent for a wide range of metabolite concentrations. Variability in ion signal intensity was lower in analytical (2.2–30.1%) compared to biological (within fruit 9.6–27.6%; between-fruit 13.2–34.4%) replicates. The method is applicable to high throughput metabolic fingerprinting and, with accurate mass measurements, is able to provide reductions in data complexity and preliminary identification of metabolites.