Surface immobilization of plant cells

A novel technique has been developed to immobilize plant cells. The cells are deposited on a surface of manmade fibrous material that provides for strong binding of the plant tissue biomass growing in the submerged culture. The immobilized plant cells remain fully viable. Relatively uniform biomass loadings of up to 20 mg d.w. plant cells/cm(2) support material have been attained. All plant cells from the inoculum suspension became attached within the first 24-48 h depending on the support matrix configuration and hydraulic culture conditions. The advantages and scale-up potential of this technique are discussed and compared to other culturing modes.     

细胞培养技术在植物抗性生理研究领域中的应用

本文综述了近10年来有关利用细胞培养技术所进行的抗性生理领域的研究成果,包括在培养过程中植物细胞对外界胁迫的反应、有关利用细胞培养技术研究植物的抗逆性反应,以及植物抗逆性的理论在实际中的应用。大量的实验证明,细胞培养技术在植物抗逆性研究领域具有广阔的应用前景。     

细胞培养技术在植物抗性生理研究领域中的应用

本文综述了近10年来有关利用细胞培养技术所进行的抗性生理领域的研究成果,包括在培养过程中植物细胞对外界胁迫的反应、有关利用细胞培养技术研究植物的抗逆性反应,以及植物抗逆性的理论在实际中的应用。大量的实验证明,细胞培养技术在植物抗逆性研究领域具有广阔的应用前景。     

Application of pressure probe techniques in studies of plant water relations]

This review introduces the pressure probe technique that was originally designed to detect the turgor of a giant algal cell, then adapted to measure the turgor and other water-relations parameters of higher plants, and now has developed into a diverse tool on researches of plant physiology and eco-physiology. This technique can be used to measure in situ the permeability of cell membranes to water and solutes at the resolution of single cells, and hence is a useful tool to study function and regulation of water channels (aquaporins) of intact plant cells. The recently developed xylem-pressure probe technique is the only way to directly measure the negative pressure in xylem conduits. In this review we introduce the basic principles and the theoretical backgrounds underlying the pressure probe. Finally some important achievements and applications of the pressure probe in studies of plant water relations are reviewed and discussed.     

Homokaryons from animal and plant cells generated by electrofusion

A new apparatus was constructed which enables the use of the electrofusion method to obtain polynuclear cells of various mammalian cell lines, erythrocytes and plant protoplasts. This technique was applied to both suspensions and monolayers. Electrical and other physical parameters were monitored to find optimal conditions for mutual contact of cells (dielectrophoresis) and subsequent fusion. In the suspension technique, dielectrophoresis of mouse erythrocytes occurred at a field frequency of 20 kHz and a strength of 500 V.cm-1, whereas cultured mammalian cells and plant protoplasts required a frequency of 1-1.4 MHz and a strength of 250-800 V.cm-1. Fusion of cells was induced after the application of 1 to 10 high-voltage pulses of 1-5 kV.cm-1, 10-36 microseconds duration. After these high-voltage pulses were to the monolayer of mouse L cells, about 12% viable homokaryons were obtained.     

植物细胞和器官大规模培养研究的进展

植物细胞,组织培养技术的发展,使得许多在实验室进行的研究已向工厂化生产过渡,除了植物细胞培养技术以外,近年来植物器官（茎,芽,根,胚和毛状根等）培养也得到迅速发展,建立了许多培养体系并在各种反应器中进行了探索性的培养实验,尤其毛状根培养越来越受到人们的瞩目,大规模培养技术的日趋完善,为植物生物技术的产业化发展带来巨大的动力。     

Production of indole alkaloids by surface immobilized C. roseus cells

A technique was developed to surface immobilize plant cells and was scaled up in laboratory size bioreactors. This technique was shown not to hinder the biosynthetic potential of Catharanthus roseus immobilized cells and to induce a partial release (300 mug/L) of serpentine into the culture medium contrary to suspension cultured cells. The release pattern seemed to follow the biosynthesis trends of the product. This release mechanism could be stimulated by a factor of 10 within 2 h by increasing the pH of the culture from 5.0 to 5.5.     

A Technique for Continuous Observation of the Infection Process in Cucumber Anthracnose

A new light microscope preparation technique for high magnification observation of living plant tissue and fungal penetration is described. Agar immersion is used in differential interference contrast microscopy (DIC) instead of coverslips (lens 40) or instead of coverslips and oil (lens 100).
This technique is suitable for
(a) longtime observation of living tissue, because the tissue to be observed remains on the plant, and for
(b) thick and uneven samples, as no coverslips are required.
With this technique it was possible to observe the dynamics of penetration of Colletotrichum lagenarium into epideral cells of cucumber cotyledons for 72 hours.
A time lapse film using this technique is in preparation.     

Monitoring the rate of oxygen consumption in plant cell suspensions

A method is described that allows the rate of oxygen consumption to be monitored in plant cell suspensions. The method utilized oxygen electrodes placed in beakers of plant cells subjected to various treatments. The voltage readings from calibrated electrodes were converted to % oxygen (100% equals air equilibration) and the rate of oxygen consumption was estimated by calibration graphs made with no cells present. This system simultaneously monitors one to sixteen or more samples, allowing comparison of treatments on identically treated cells. We have used this method to study the respiratory burst of plant cells produced in response to viable or heat-killed bacteria. Because the system was computer-monitored and open to the atmosphere, data could be collected over several hours. Various factors that affected the measurement of dissolved oxygen concentration with this technique were explored and considered. This revised version was published online in June 2006 with corrections to the Cover Date.     

High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of Phloem development and structure in Arabidopsis

Currently, examination of the cellular structure of plant organs and the gene expression therein largely relies on the production of tissue sections. Here, we present a staining technique that can be used to image entire plant organs using confocal laser scanning microscopy. This technique produces high-resolution images that allow three-dimensional reconstruction of the cellular organization of plant organs. Importantly, three-dimensional domains of gene expression can be analyzed with single-cell precision. We used this technique for a detailed examination of phloem cells in the wild type and mutants. We were also able to recognize phloem sieve elements and their differentiation state in any tissue type and visualize the structure of sieve plates. We show that in the altered phloem development mutant, a hybrid cell type with phloem and xylem characteristics develops from initially normally differentiated protophloem cells. The simplicity of sieve element data collection allows for the statistical analysis of structural parameters of sieve plates, essential for the calculation of phloem conductivity. Taken together, this technique significantly improves the speed and accuracy of the investigation of plant growth and development.     

Influences of plant hormones on photoperiodic flowering in Pharbitis nil: re-evaluation by the perfusion technique

Influences of plant hormones on photoperiodic flowering in Pharbitis nil, var. Violet was re-evaluated by assaying them with a newly developed perfusion technique which can directly treat mesophyll cells with sample solution. Gibberellin A₃ promoted the flowering response and indole-3-acetic acid, trans-zeatin and abscisic acid inhibited it when they were perfused immediately before an inductive dark treatment. The promotion or inhibition of flowering was not or hardly observed when solutions containing these plant hormones were applied by the dropping method to surface of cotyledons or plumules of the assay plants. The detection of clear flower-promoting and -inhibiting effects of the plant hormones may be due to the improved efficiency of incorporation of applied substances into plant tissue in the perfusion technique.     

Application of vanadate-induced nucleotide trapping to plant cells for detection of ABC proteins

The vanadate-induced nucleotide trapping technique, which has been conventionally used to characterize mammalian ATP-binding cassette (ABC) proteins, was applied to berberine-producing plant cell cultures, Thalictrum minus and Coptis japonica. One membrane protein at ca. 180 kDa was photoaffinity-labeled with 8-azido-[alpha-(32)P]ATP in the T. minus cells in the presence of vanadate, which was specifically induced by the addition of benzyladenine in a similar manner as the induction of berberine biosynthesis in these cell cultures, whereas three bands were observed in the C. japonica cells in the size region between 120 and 150 kDa corresponding to full-sized ABC protein. The benzyladenine-induced band in T. minus showed properties similar to those of human MDR1, including the recognition of berberine, which suggests that the ABC protein detected in T. minus takes this endogenous alkaloid as a putative substrate for transport. This is the first application of this technique to plant cells.     

Direct cell to cell transfer of organelles by microinjection

Summary A new technique for transfer of organelles to plant cells is presented. The organelles are removed from the donor protoplast by micromanipulation and microinjected directly into the acceptor cells. First results obtained by this technique for transfer of chloroplasts and fluorescently labelled mitochondria are presented.     

T-DNA transfer in meristematic cells of maize provided with intracellular Agrobacterium

Agrobacterium has been established as a tool for gene delivery to most dicotyledonous plant species. However, it is not generally efficient in monocotyledonous plant species, especially not in Graminae . In maize, Agrobacterium -mediated DNA transfer has been detected but early developmental stages in the plant proved incompetent as recipients. This research tests whether the lack of competence in young immature embryos of maize could be overcome by providing Agrobacterium in the interior of the plant cell. A microinjection technique was used to target single meristematic cells and prove competence to Agrobacterium . This response is dependent on the maize plant genotype.     

Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants

Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of basic concepts in cell biology. The protocol presented here has underpinned much of the in vivo results highlighting the dynamic nature of the plant secretory pathway. Transient transformation of tobacco leaf epidermal cells is a relatively fast technique to assess expression of genes of interest. These cells can be used to generate stable plant lines using a more time-consuming, cell culture technique. Transient expression takes from 2 to 4 days whereas stable lines are generated after approximately 2 to 4 months.     

Detection of in vivo protein interactions between Snf1-related kinase subunits with intron-tagged epitope-labelling in plants cells

Plant orthologs of the yeast sucrose non-fermenting (Snf1) kinase and mammalian AMP-activated protein kinase (AMPK) represent an emerging class of important regulators of metabolic and stress signalling. The catalytic alpha-subunits of plant Snf1-related kinases (SnRKs) interact in the yeast two-hybrid system with different proteins that share conserved domains with the beta- and gamma-subunits of Snf1 and AMPKs. However, due to the lack of a robust technique allowing the detection of protein interactions in plant cells, it is unknown whether these proteins indeed occur in SnRK complexes in vivo. Here we describe a double-labelling technique, using intron-tagged hemagglutinin (HA) and c-Myc epitope sequences, which provides a simple tool for co-immunopurification of interacting proteins expressed in Agrobacterium-transformed Arabidopsis cells. This generally applicable plant protein interaction assay was used to demonstrate that AKINbeta2, a plant ortholog of conserved Snf1/AMPK beta-subunits, forms different complexes with the catalytic alpha-subunits of Arabidopsis SnRK protein kinases AKIN10 and AKIN11 in vivo.     

Digitonin-aided loading of Fluo-3 into embryogenic plant cells

This paper describes a method to load embryogenic plant cells with Fluo-3 in its cell impermeant form with the aid of digitonin. Attempts to load cells with Fluo-3/AM were all unsuccessful. Presumably the indicator is cleaved outside the cells and cannot penetrate in its acidic form. At a low pH, Fluo-3 enters the plant cells but normal Ca2+ homeostasis seems to be disturbed. Successful loading of Fluo-3 was achieved by adding 0.1% digitonin during incubation with the Ca(2+)-indicator. A bright fluorescence was observed in the epidermal layer of heart and torpedo shaped somatic embryos of carrot with confocal scanning laser microscopy. Vacuoles were always without fluorescence which indicates that the dye, after loading, remains in the cytosol and does not leak out. The fluorescence intensity was sensitive to treatments with A23187 and EGTA. We conclude that Fluo-3 can be effectively loaded, with the aid of digitonin, into plant embryogenic cells in liquid culture. Therefore, we expect this technique to be very useful for the study of changes in cytosolic free Ca2+ levels during plant growth and development.     

拟南芥悬浮细胞系的玻璃化法超低温保存

悬浮培养细胞系是植物生理生化研究的好材料之一。为了保持细胞系的遗传稳定性,需要采用超低温保存技术。玻璃化法是一种不用程序降温仪的超低温保存技术。本文报道了从模式植物拟南芥建立悬浮细胞系并对其进行玻璃化法超低温研究。细胞经过合理的预培养处理和保护剂处理,直接投入液氮贮存。复温后的细胞能恢复生长,恢复生长的细胞保持着植株再生能力。国外,拟南芥悬浮细胞系的程序降温法保存和包埋脱水法保存已经报道,玻璃化法保存尚未见报道。     

Agrobacterium tumefaciens-mediated transformation of recalcitrant crops

The most widely used technique for the introduction of new genetic information into plant cells is based on the natural gene transfer capacity ofAgrobacterium tumefaciens. Currently, this technique is routinely applicable in just a few model species, like tobacco and petunia. Thus far, the numerous efforts to apply the technique to crop species have had limited success. In this review, an attempt is made to survey all the research experience onAgrobacterium tumefaciens-mediated transformation of recalcitrant crops and to highlight the problems generally encountered. The main difficulty appears to be directing the gene transfer towards those plant cells that are amenable to regeneration. The various ways to reduce stress during the transformation and regeneration process are often beneficial. The influence of the developmental stage of the plant material and the host range of theAgrobacterium strain depends largely on the plant species used, which hampers the formulation of common procedures. However, some general guidelines for the development of a transformation protocol are discussed.