Protoplast Technology Research Articles

Optimization of protoplast isolation protocols from callus of Eurycoma longifolia

Protoplast technology offers a unique single cell system that facilitates several aspects of modern biotechnology. In this study, an efficient protocol to isolate the protoplast from callus culture of a valuable medicinal plant in Southeast Asia,Eurycoma longifolia was developed. A range of parameters which influence the isolation of E. longifolia protoplasts were investigated by using “change-one-factor-at-a-time” method. From the results obtained, callus fresh weight (FW) of 0.2 g produced the highest number of viable protoplasts, which was 1.58 ± 0.36 × 104protoplasts/gFW. The highest amount of viable protoplasts (1.75 ± 0.68 × 104protoplasts/gFW) was obtained when the sorbitol concentration was maintained at 0.5 M. The optimum enzyme concentration was found to be 1.5% (w/v) of cellulase and pectinase in which 2.75 ± 1.04 × 104 protoplasts/gFW were isolated. Meanwhile, an incubation period of 3 h with enzyme solution resulted in the maximum yield of protoplasts (5.58 ±1.46 × 104 protoplasts/gFW). Key words: Eurycoma longifolia, callus culture, protoplast, osmoticum, viability.

Arabinogalactan-proteins stimulate the organogenesis of guard cell protoplasts-derived callus in sugar beet

Arabinogalactan proteins (AGPs) represent a class of proteoglycans implicated in the development and differentiation of cells and tissues both in planta and in vitro. Here we report that AGP-rich extracts isolated from media of embryogenic and non-embryogenic suspension cultures of sugar beet (Beta vulgaris L.) are able to enhance the organogenesis of guard protoplast-derived callus and to increase the number of shoots formed, in comparison to control cultures. Immunocytochemical detection of carbohydrate antigens in the extracts revealed the presence of epitopes that typify both AGP and pectin, the latter being frequently bound to AGPs or, in some cases, even contributing to the polysaccharide structure of proteoglycan molecules. The most abundant epitopes proved to be those recognized by the JIM13, LM2, and MAC207 antibodies, whereas some others could be found only in relatively small or trace amounts--these included epitopes recognized by JIM16, JIM5, and LM6. Surprisingly, the JIM4- and JIM8-binding epitopes that are expressed in the course of in vitro morphogenetic processes of many species could not be detected at all in sugar beet AGPs. This is the first report of the improvement of sugar beet protoplast-derived callus organogenesis by exogenous AGP-rich extracts, an achievement that will have great impact on the biotechnological applications of protoplast technology in this species.

From protoplasts to gene clusters

Advances in protoplast technology underpinned many crucial developments in our understanding of the molecular biology of filamentous fungi. This review follows one of these developments, namely the discovery and analysis of difuran toxin gene clusters. Our understanding of the biosynthetic pathway of the agriculturally important toxin, aflatoxin, has been dramatically enhanced by the use of protoplasts and protoplast-based gene transformation methods. Since the identification of the first pathway genes by complementation of mutants with transforming DNA, transformation has continued to play a critical role in the elucidation of gene function and regulation. But despite the wealth of knowledge accumulated so far some fundamental questions remain to be answered. How did these gene clusters evolve? What is the biological role of aflatoxin? The discovery of homologues of aflatoxin genes in other fungal species such as the pine needle pathogen Dothistroma septosporum may help to shed some light on these questions.

Spices biotechnology

In recent times, biotechnological tools have supplemented various conventional approaches in conservation,characterization, improvement and utilization for increasing production and productivity of spices. In manyspices, viable micropropagation technologies are available for commercial production and generation of disease - free planting material. Somaclonal variation is important in crops where natural variability is low and a few useful somaclonal variants have been identified in ginger, turmeric and vanilla. Protoplast technology is also available for capsicum, black pepper, fennel, fenugreek, garlic, saffron and peppermint. In vitro cryopreservation, Synseed and Micro-rhizome technologies are available for safe propagation, conservation, movement, and exchange of spices germplasm. Studies are in progress for in vitro production of flavour and colouring compounds like capsaicin, vanillin, anethole, crocin, picrocrocin, saff'ranal, etc. using immobilized and transformed cell cultures. Use of molecular markers for crop profiling, fingerprinting, molecular taxonomy, identification of duplicate hybrids, estimation of genetic fidelity and tagging of genes for marker aided selection (MAS) is gaining importance. Isolation of important and useful genes and development of transgenics is in the preliminary stage.

Plant protoplast technology: Current status

Robust and reproducible protoplast-to-plant systems are crucial for underpinning genetic manipulation technology involving somatic hybridisation and transformation. Novel and effective approaches for maximising the efficiency of such protoplast cultures include supplementation of media with surfactants and artificial gas carriers, such as perfluorochemicals and haemoglobin. Physical parameters, particularly electrostimulation, also enhance the development of protoplasts and protoplast-derived cells in culture. DNA uptake into protoplasts is now a routine and universally accepted procedure in plant biotechnology for introducing and evaluating both short-term (transient) and long-term (stable) expression of genes in cells and regenerated plants. Importantly, protoplast fusion overcomes pre- and post-zygotic sexual incompatibility barriers and generates novel germplasm through new nuclear-cytoplasmic combinations. In this respect, considerable progress has been made in generating somatic hybrid plants, particularly in citrus, brassicas and potato. Isolated protoplasts are also a unique single cell system for evaluating aspects of ultrastructure, genetics and physiology, with potential for the biosynthesis of novel secondary products, including commercially-important recombinant proteins (e.g. antibodies), and as systems in toxicity screening. Recent advances in protoplast technology have benefited from advances in animal and microbial cell culture, with interesting parallels existing between these systems. Further innovations will necessitate the strengthening of interdisciplinary links in these research fields and the requirement for continued dialogue and co-operation between workers with diverse but complementary skills.

693 Practical Applications of Citrus Protoplast Technology

Citrus protoplast technology has advanced to where several practical applications in variety improvement and plant pathology are routine. We will report on progress in the following areas: somaclonal variation—`Valencia` and `Hamlin' sweet orange protoclones have been selected for improved juice color, higher soluble solids, seedlessness, and altered maturity dates; somatic hybridization for scion improvement—allotetraploid breeding parents have been created from numerous combinations of elite parental material, and are now being used as pollen parents in interploid crosses to produce seedless triploid varieties; somatic hybridization for rootstock improvement—numerous somatic hybrids combining complementary rootstock germplasm are under commercial evaluation and several look promising for wide adaptation, improved disease resistance, and tree size control; transformation—an alternative protoplast-based transformation that utilizes EGFP for selection has been developed; virus resistance assays—a protoplast-based assay is being used to screen varieties and candidate sequences for resistance to citrus tristeza virus at the cell level, saving time and greenhouse space.

Protoplast technology for the breeding of top-fruit trees (Prunus, Pyrus, Malus, Rubus) and woody ornamentals

Protoplast technology for the breeding of top-fruit trees (Prunus, Pyrus, Malus, Rubus) and woody ornamentals

Conditions for isolation of regenerating protoplasts from Pleurotus sajorcaju

AbstractLow rate of protoplast regeneration of Pleurotus spp. is one of the limiting factor for strain improvement through protoplast technology. A method for overcoming this barrier and isolation of higher frequency of regenerating protoplasts both from mycelium and sporophore capable of producing fruit bodies is reported in this communication.

WOODY PLANT PROTOPLAST TECHNOLOGY REVISITED

Woody plant protoplast technology revisited. II International Symposium on In Vitro Culture and Horticultural Breeding

Regeneration from protoplasts-a supplementary literature review

Protoplast technology is important in, on the one hand, the application of various cellular methods in plant breeding (i.e. somatic hybridization, cybridization, direct DNA transfer via polyethylene glycol (PEG), electroporation, micro-injection), and various fundamental studies (e.g. on membrane transport, cell compartmentation, the cytoskeleton in relation to the cell cycle and cell division) on the other hand. For practical applications, plant regeneration from protoplasts is a prerequisite. In 1989, regeneration from protoplasts was listed (Roest & Gilissen 1989) for 214 higher plant species (Spermatophyta), representing 97 genera and 31 families. Since then, regeneration procedures for more than 100 higher plant species have been reported. These include many economically important agricultural and horticultural crops, as well as woody plant species. In this paper these new species are listed, supplemented with specific information on the donor tissue used, the culture technique applied, and the type of development of the regenerants. In addition, recent achievements in fundamental aspects of protoplast research, which gradually provide further insight into the genetical, physiological and ultrastructural background of the phenomenon of totipotency of plant cells, will be briefly reviewed.

Protoplasts as tools for plant genome modifications

We present here the application of protoplast technology in the selection and recovery of rare, spontaneous plant genome alterations. Using protoplasts as a cell cloning system allowed the detection and molecular characterization of intrachromosomal recombination events between genomic repeats. The mechanism, frequencies and the induction of intrachromosomal recombination are discussed as well as its application for genome mutagenesis.

A protoplast to plant system in roses

High yields of protoplasts were isolated from embryogenic suspension cultures of Rosa persica x xanthina and Rosa wichuraiana using an enzyme mixture comprising 20 g l-1 cellulase ‘Onozuka’ R10, 1 g l-1 Pectolyase Y-23 and 10 g l-1 hemicellulase. Agarose-immobilized protoplasts gave the most consistent growth at a plating density of 5×104 protoplasts ml-1 on the basic medium of Kao & Michayluk (KM8p) containing 2 mg l-1 naphthaleneacetic acid and 1 mg l-1 benzylaminopurine. At 25°C in the dark, 0.004% of R. persica x xanthina protoplasts developed into colonies. Using similar culture conditions, but with a plating density of 9×104 protoplasts ml-1, 0.017% of R. wichuraiana protoplasts developed into colonies. On transfer of R. persica x xanthina colonies to Schenk & Hildebrandt's medium containing 3 mg l-1 2,4-dichlorophenoxyacetic acid, globular and later stage embryos were formed. Approximately 30% of these embryos developed into plantlets on transfer to basal Schenk & Hildebrandt's medium. Further development of the plantlets took place on cellulose plugs (Sorbarods) soaked in Murashige & Skoog's medium containing 0.05 mg l-1 naphthaleneacetic acid, 0.05 mg l-1 indole-3-butyric acid and 0.1 mg l-1 benzylaminopurine. Rose breeding is now open to the full range of in vitro genetic manipulation techniques involving protoplast technology.

Plant Regeneration from Protoplasts Isolated from Embryogenic Maize Cell Cultures

Genetic engineering of maize via protoplast technology has been limited due to lack of plant regeneration from maize protoplasts. We have developed a system of protoplast culture that results in high plating efficiency from embryogenic protoplasts and can be followed by plant regeneration. Maize protoplasts were grown on filters directly over a feeder layer of nurse cells in liquid medium. Initial condition and subsequent growth of feeder cells were critical in obtaining a plating efficiency of 10% from protoplasts. Protoplasts were digested from embryogenic cell suspension cultures, and recovered callus retained the morphogenic potential of initial donor cultures. The system has been successfully used with protoplasts of two maize inbreds, one of which is an important commercial line.

Efficient Plant Regeneration from Rice Protoplasts Through Somatic Embryogenesis

Progress in the genetic manipulation of cereals using protoplast technology has been limited by a lack of reproducible plant regeneration from protoplasts of these important species. Here we report that plant regeneration can be achieved efficiently and reproducibly from protoplasts of rice isolated from cell suspension cultures. Regeneration from 10—20% of protoplast–derived colonies of two varieties was obtained rapidly after direct transfer to a hormone–free medium. Green plants were produced at a high frequency through somatic embryogenesis. The protoplast origin of the regenerated plants is unequivocally demonstrated by the inability of contaminating intact cells to divide.

Gewinnung und Regeneration von hefeprotoplasten: Eine Übersicht

The latest advance in protoplast technology results from a long and detailed study of cell wall composition and protoplast systems relating to their isolation and culture. The reports on protoplast preparation and fusion discussed in this review clearly demonstrate the applicability of technique for the genetic manipulation of microorganisms. These points have been demonstrated in Candida strains. The behaviour of protoplasts especially with respect to reversion could be an additional factor that operates during selection. Furthermore the fusion technique, in providing a mechanism which genetic recombination can be readily achieved, should be of great potential in empirical breeding and strain improvement. These aspects are reviewed.

Use Of Protoplasts And Separate Cells In Plant Virus Research

Publisher Summary The advancement of knowledge has made protoplasts and plant tissue cultures a multipurpose system and a most workable tool in plant virological investigations, ranging from intricate molecular aspects like the role of a messenger RNA for tobacco mosaic virus (TMV) coat protein to the adaptation of protoplasts and tissue cultures for screening antiviral substances, and from tissue culture as a means for the propagation of viroids to the study of virus resistance. The improvement of the procedure for inoculation of protoplasts with viria, viral nucleic acid, and viroids and the improvement of conditions that promote their replication, as well as the increased knowledge of steps involved in both processes, open an intriguing avenue of investigation in gentechnology (the use of virus as vehicle for genetic information heterologous to the cell). An interesting application of protoplast technology is the investigation of the influence of virus infection on the production of antibiotics by fungi.

Fresh approaches to antibiotic production.

New antibiotics are needed, (a) to control diseases that are refractory to existing ones either because of intrinsic or acquired drug resistance of the pathogen or because inhibition of the disease is difficult, at present, without damaging the host (fungal and viral diseases, and tumours), (b) for the control of plant pathogens and of invertebrates such as helminths, insects, etc., and (c) for growth promotion in intensive farming. Numerous new antibiotics are still being obtained from wild microbes, especially actinomycetes. Chemical modification of existing compounds has also had notable success. Here we explore the uses, actual and potential, of genetics to generate new antibiotics and to satisfy the ever-present need to increase yield. Yield improvement has depended in the past on mutation and selection, combined with optimization of fermentation conditions. Progress would be greatly accelerated by screening random recombinants between divergent high-yielding strains. Strain improvement may also be possible by the introduction of extra copies of genes of which the products are rate-limiting, or of genes conferring beneficial growth characteristics. Although new antibiotics can be generated by mutation, either through disturbing known biosyntheses or by activating 'silent' genes, we see more promise in interspecific recombination between strains producing different secondary metabolities, generating producers of 'hybrid' antibiotics. As with proposals for yield improvement, there are two major strategies for obtaining interesting recombinants of this kind: random recombination between appropriate strains, or the deliberate movement of particular biosynthetic abilities between strains. The development of protoplast technology in actinomycetes, fungi and bacilli has been instrumental in bringing these idealized strategies to the horizon. Protoplasts of the same or different species can be induced to fuse by polyethylene glycol. At least in intraspecific fusion of streptomyces, random and high frequency recombination follows. Protoplasts can also be used as recipients for isolated DNA, again in the presence of polyethylene glycol, so that the deliberate introduction of particular genes into production strains can be realistically envisaged. Various kinds of DNA cloning vectors are being developed to this end. Gene cloning techniques also offer rich possibilities for the analysis of the genetic control of antibiotic biosynthesis, knowledge of which is, at present, minimal. The information that should soon accrue can be expected to have profound effects on the application of genetics to industrial microbiology.

Potentiality of leaf sheath cells for regeneration of rice (Oryza sativa L.) plants

Regeneration of rice plantlets (Oryza sativa L.) from calli originated from leaf sheath cells was made possible. This was possible in tissues initially grown in media containing 2.4-D (2.4-dichlorophenoxyacetic acid) at low temperature and illumination. The slow growing tissues were subsequently subjected to growth conditions at an elevated temperature and higher illumination with addition of kinetin and IAA and without 2.4-D. The suitability of leaf sheath cells for protoplast technology is indicated by this success.

Protoplast technology in relation to crop plants: Progress and problems

The ready availability of isolated plant protoplasts, and the ability of these naked plant cells to fuse together, has greatly stimulated interest in the production of plant somatic hybrids. This new protoplast technology may enable interspecies hybrids to be obtained which are otherwise not possible. Many such interspecies hybrids are required amongst the crop plants, but application of this new technology to these species in presently prevented by our inability to culture cells of many of these species in vitro, and to regenerate plants reproducibly from cultured tissue.

Uptake of the nitrogen fixing blue-green alga Gloeocapsa by plant protoplasts.

In the face of rising world population and diminishing reserves of fossil fuels needed to produce chemical fertilizers, scientists have been motivated to study the process of biological nitrogen fixation. At present nitrogen fixation is limited to certain prokaryotic organisms, which are either free living or are symbionts with plants (chiefly legumes). High demand for chemical fertilizers, and the energy needed to produce them, could be alleviated by introducing the nitrogen fixation process into economically important non-leguminous crop plants. Recent developments in plant protoplast technology have made this goal seem more attainable. Of particular interest was the discovery by Kao and Michayluk (13 in 1974 that polyethylene glycol (PEG) stimulates high frequencies of protoplast fusion.