Cryopreservation of Plant Cell Lines Using Alginate Encapsulation.

Plant cell cultures consist of single cells or cell clusters growing as callus or suspension. Such cell cultures may be able to produce secondary metabolites and/or possess embryogenic potential. Therefore, they can be used for very different purposes in research, biotechnological applications, as well as for plant propagation. Cryopreservation is the only practical method to preserve such cultures until they are needed. Different cryopreservation approaches that have been developed for differentiated plant tissues including slow freezing, vitrification, and encapsulation/dehydration have also been applied to plant cell cultures. The controlled rate or slow-freezing approach, however, remains to be the gold standard for cell cultures. In this chapter, a standard slow-freezing cryopreservation procedure in combination with alginate immobilization is presented for long-term preservation of plant cell cultures.

ATP Content and Cell Viability as Indicators for Cryostress Across the Diversity of Life.

In many natural environments, organisms get exposed to low temperature and/or to strong temperature shifts. Also, standard preservation protocols for live cells or tissues involve ultradeep freezing in or above liquid nitrogen (-196°C or -150°C, respectively). To which extent these conditions cause cold- or cryostress has rarely been investigated systematically. Using ATP content as an indicator of the physiological state of cells, we found that representatives of bacteria, fungi, algae, plant tissue, as well as plant and human cell lines exhibited similar responses during freezing and thawing. Compared to optimum growth conditions, the cellular ATP content of most model organisms decreased significantly upon treatment with cryoprotectant and cooling to up to -196°C. After thawing and a longer period of regeneration, the initial ATP content was restored or even exceeded the initial ATP levels. To assess the implications of cellular ATP concentration for the physiology of cryostress, cell viability was determined in parallel using independent approaches. A significantly positive correlation of ATP content and viability was detected only in the cryosensitive algae Chlamydomonas reinhardtii SAG 11-32b and Chlorella variabilis NC64A, and in plant cell lines of Solanum tuberosum. When comparing mesophilic with psychrophilic bacteria of the same genera, and cryosensitive with cryotolerant algae, ATP levels of actively growing cells were generally higher in the psychrophilic and cryotolerant representatives. During exposure to ultralow temperatures, however, psychrophilic and cryotolerant species showed a decline in ATP content similar to their mesophilic or cryosensitive counterparts. Nevertheless, psychrophilic and cryotolerant species attained better culturability after freezing. Cellular ATP concentrations and viability measurements thus monitor different features of live cells during their exposure to ultralow temperatures and cryostress.

Cryopreservation of plant cell lines.

Plant cell cultures may consist of dedifferentiated cells as well as of cells showing embryogenic potential. They can be used for very different purposes in research and biotechnology as well as for plant propagation. For such cell cultures, cryopreservation is the only means for long-term preservation. Most of the different cryopreservation approaches, which are generally used for plant tissues, have also been applied to plant cell cultures; they include slow freezing, vitrification, and encapsulation/dehydration approaches. The controlled-rate slow freezing approach which is described here, however, remains to be the gold standard for cell cultures. In this chapter, a standard cryopreservation procedure is presented for plant cell cultures.

Impact of pr-10a overexpression on the cryopreservation success of Solanum tuberosum suspension cultures.

Although many genes are supposed to be a part of plant cell tolerance mechanisms against osmotic or salt stress, their influence on tolerance towards stress during cryopreservation procedures has rarely been investigated. For instance, the overexpression of the pathogenesis-related gene 10a (pr-10a) leads to improved osmotic tolerance in a transgenic cell culture of Solanum tuberosum cv. Désirée. In this study, a cryopreservation method, consisting of osmotic pretreatment, cryoprotection with DMSO and controlled-rate freezing, was used to characterize the relation between cryopreservation success and pr-10a expression in suspension cultures of S. tuberosum wild-type cells and cells overexpressing pathogenesis-related protein 10a (Pr-10a). By varying the sorbitol concentration, thus modifying the strength of the osmotic stress during the pretreatment phase, it can be shown that the wild type can successfully be cryopreserved only in a relatively narrow range of sorbitol concentrations, while the pr-10a overexpression leads to an enhanced cryopreservation success over the whole range of applied sorbitol concentrations. Together with transcription data we show that the pr-10a overexpression causes an enhanced osmotic tolerance, which in turn leads to enhanced cryopreservability, but also indicates a role of pr-10a in signal transduction. An increased cryopreservability of the transgenic cell line occurs for pretreatments longer than 24 h. Since both genotypes, characterized by distinct baseline levels of expression, exhibited similar patterns of expression induction, the induction of pr-10a appears to be a key step in the stress signal transduction of plant cells under osmotic stress.

Over-expression of PR-10a leads to increased salt and osmotic tolerance in potato cell cultures.

The PR-10a protein (formerly STH-2) is known to be induced by biotic stress in potato. The present study demonstrates that transgenic suspension cells of the potato cultivar Desiree over-expressing the PR-10a protein exhibit significantly increased salt and osmotic tolerance compared to the respective wild type cells. A comparison of the proteome pattern of Solanum tuberosum suspension cultures cv. Desiree before and after the treatment with NaCl or sorbitol under equiosmolar conditions (740mOs/kg) revealed the pathogenesis related protein PR-10a to be one of the predominant differentially expressed proteins in potato cell cultures. The pr-10a mRNA was confirmed to be present by RT-PCR from salt challenged suspension cells and was transcribed into cDNA. For PR-10a over-expression Agrobacterium tumefaciens mediated transformation of the potato cells and a dicistronic vector harboring the cDNA of the pr-10a gene linked to a luciferase gene by an IRES (Internal Ribosome Binding Site) was used. The IRES mediated translation leads to co-expression of PR-10a and luciferase in a fixed ratio. By non-invasive luciferase assay homologous PR-10a over-expressing callus was identified after selection on phosphinothricin supplemented medium. This callus was used for the setup of a transgenic suspension culture. Along with increased salt and osmotic tolerance the transformed culture showed changed proline and glutathione levels under abiotic stress conditions in comparison to the wild type.

Dicistronic binary vector system-A versatile tool for gene expression studies in cell cultures and plants.

Dicistronic binary vector constructs based on pGreenII vectors for Agrobacterium mediated gene transfer alleviate the translational expression monitoring of a target gene in plants. The functionality of the transformation vectors was proven by marker gene constructs containing a mannopine synthase promoter (p-MAS) fused to a beta-glucuronidase (gus) gene followed by an internal ribosome entry site and a firefly luciferase (luc) gene. The cap-dependent translation of a physically independent target protein can be monitored by the cap-independently co-translated luciferase, because both mRNAs are located on the same strand. Among three different IRES elements, the tobamo IRES element showed highest activity in transient expression. As a proof of principle for physiological studies the gus gene was replaced by a sodium antiporter gene (Atnhx1). Comparative studies with Atnhx1 transgenic luc expressing tobacco cell cultures and pea plants (Pisum sativum L.) showed improved salt tolerance in relation to their wild type counterparts grown under corresponding conditions. A coincidence of the luc gene expression and increased sodium chloride tolerance is demonstrated by measurement of luminescence and cell growth.