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Protoplast Cultures and Protoplast Fusion Focused on Brassicaceae – a Review

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Protoplast Cultures and Protoplast Fusion Focused on Brassicaceae – a Review Protoplast cultures and protoplast fusion focused on Brassicaceae – a review B. NAVRÁTILOVÁ Faculty of Science, Palacký University in Olomouc, Olomouc, Czech Republic ABSTRACT: The subjects of this article are protoplast isolations and protoplast fusions, in particular their history, a review of factors influencing the protoplasts isolation and fusion, selection of hybrid plants and utilization of somatic hybrids in plant breed- ing. Somatic hybridization through protoplast fusion can overcome sexual incompatibility among plant species or genera; transfer genes of resistance to diseases (viral, bacterial, fungal), pests, herbicides and others stress factors; obtain cybrid plants; transfer cytoplasmic male sterility or incease content of secondary metabolites in hybrid plants. The article is focussed mainly on the family Brassicaceae because among representatives are significant crops for the human population. Various successful combination of intraspecific, interspecific and intergeneric protoplast fusion were reported between representatives of the family Brassicaceae with the genus Brassica which belonged to the first agricultural crops used for the isolation of protoplast. Keywords: Brassicaceae; Brassica; protoplast isolation; protoplast fusion; somatic hybridization; resistance to diseases and abiotic factors Isolated protoplasts are a unique system for studying interspecific hybrid obtained by the protoplast fusion of the structure and function of cell organelles, cytoplasmic Nicotiana glauca + Nicotiana langsdorffii appeared in membrane transport in plants and cell wall formation. 1972 (CARLSON et al. 1972), but this hybrid could also Another possibility of their use is protoplast fusions, ge- be produced by sexual crossing. The finding was that the netic manipulations or experimental mutagenesis. hybrids were characterized by spontaneous emergence Somatic hybridization through plant protoplast fu- of tumours without any need to add growth regulators sion enables not only to combine parent genes in higher into the culture medium. plants but also to overcome barriers existing between The real breakthrough came in 1978 when, through plant species or genera, it is possible to obtain asym- the protoplast fusion of Solanum tuberosum L. and metric hybrids and plants that are heterozygous in extra- Solanum lycopersicum L. from the Solanaceae family, nuclear genes. hybrid cells were formed and plants were regenerated Protoplast fusion enables to transfer desirable quali- from them which could not be produced by sexual hy- ties, e.g. resistance to pathogens or stress factors, even bridization (MELCHERS et al. 1978). Two types of hy- between the genotypes that cannot be hybridized in a brids were produced, one type with the chloroplasts of traditional way. potato (Solanum tuberosum L.) and the other type with the chloroplasts of tomato (Solanum lycopersicum L.), HISTORY they made shoots similar to potatoes but they did not flower. Klerceker (BAUER 1990) carried out the first me- chanical isolation of protoplasts in 1892 from the tissue ISOLATION OF PROTOPLASTS of Stratiotes aloides L. Because of the very low density of isolated protoplasts, the isolation of protoplasts was Enzymatic isolation of protoplasts is limited to paren- considered as an unsuitable method until the time of chymal cells with unlignified cell walls, as cells with chemical digestion of the cell wall by the enzymes pec- lignified cell walls are resistant to enzymes. By apply- tinase, hemicellulase and cellulase isolated from fungi ing enzymatic preparations with cellulase and pectinase (Trichoderma viride, Rhizopus sp.). activity to tissues and cells it is possible to remove the In 1960, Cocking obtained for the first time a high cellulose cell wall of the plant cells and to obtain spheri- number of protoplasts at the root tips of tomato Lyco- cal protoplasts. persicon esculentum Mill. by applying enzymes with There exist standard and general methods of isolation cellulase and pectinase activity. The first plants rege- and cultivation of protoplasts, but the details must be nerated from protoplasts were grown by NAGATA and modified for specific species or even variety. In a ma- TAKEBE (1971) in tobacco (Nicotiana tabacum L.). An jority of cases the optimum conditions and enzymatic Supported by the Ministry of Agriculture of the Czech Republic, Grant QD 1356. 140 HORT. SCI. (PRAGUE), 31, 2004 (4): 140–157 processing for the combination of genotype/explant transferred to the culture medium, the plants are subcul- must be defined empirically (BLACKHALL et al. 1994a). tured regularly and uniform clones can be obtained by Plant protoplasts may be isolated mechanically or their micropropagation. enzymatically. Mechanical isolation, cutting parts of Frequent objects of protoplast cultures in dicotyle- the plant and releasing protoplasts from the cut sur- donous plants are Nicotiana tabacum, Lycopersicum face, is a historically important technique, but it is used esculentum, Datura stramonium, the representatives of scarcely as the number of isolated protoplasts is insuf- the genus Brassica. ficient. However, its advantage is the elimination of the unknown influence of enzymes on protoplasts (AHUIA Enzymes 1982; BAUER 1990). Enzymatic isolation is advantageous because proto- Density and viability of isolated protoplasts depend on plasts are gained at a high quantity, cells are not dam- the concentration of used enzymes, the period of enzy- aged and the osmotic conditions may be influenced. matic action, pH of the enzymatic solution, temperature, Enzymatic isolation may be carried out in two different the ratio of the enzymatic solution to the volume of the procedures: two-step or one-step procedure. plant tissue. In the first step of the two-step procedure, individual Enzymes may be divided into two categories: cells are released with the help of commercial enzymat- – pectinase dissolving the middle lamella and separat- ic preparations (e.g. macerozyme, macerase). The cells ing individual cells, are released by degradation of the middle lamellas and – cellulase and hemicellulase decomposing the cell the decay of tissue to individual cells. The free cells are wall and releasing the protoplast. then processed in the second step with the help of cellu- Enzymes are then purified and filtrated. To increase lases (cellulase Onozuka R-10, cellulysin) to protoplasts the stability of protoplasts, inorganic salts (Ca2+) and by dissolving the cell wall. The cells are exposed to the organic buffer (e.g. morpholinoethane sulphonic acid) enzymes for a shorter time than at one-step isolation. are added that minimize the changes of pH during incu- One-step isolation is used more often, during which bation. Osmotic values of the environment into which the mechanically loosened tissue (e.g. by cutting strips) the protoplasts are released are critically important. Sea- is put into a mixture of enzymes (pectinase and cellula- water is used as an osmoticum or the tissues are slightly se, commercial preparations). For each plant object the plasmolyzed prior to the isolation, e.g. in sorbitol solu- optimal composition of enzymatic mixture is necessary tion. Movement and slight shaking of the mixture dur- (POWER, CHAPMAN 1985; ONDŘEJ 1985) as well as the ing enzymatic action increases the number of isolated optimal pressure of extraction media. protoplasts (UCHYMIYA, MURASHIGE 1974; DĚDIČOVÁ 1995). FACTORS INFLUENCING THE ISOLATION Pre-action (pre-enzymatic action) prior to enzymatic OF PROTOPLASTS action may lead to increased metabolic activity and stimulation of division after the isolation of protoplasts A successful isolation of protoplasts depends on many from the tissue. The period of action of the enzymes or factors such as the source of tissue (leaves, cell suspen- enzymatic mixture is varying, short-term action (2 to sion), plant species and cultivar, physiological condition 6 hours) or slower long-term action (16–24 hours) in the of the donor plant, the composition of the enzymatic dark at room temperature. mixture and the period of enzymatic action, the osmotic The purification of protoplasts and perfect removal of characteristics of the extraction mixture and the indi- the residues of cell walls, damaged protoplasts and iso- vidual steps during the isolation of protoplasts. lation enzymes are the condition of further cultivation of protoplasts and are done by repeated centrifugation Plant material (LANDGREN 1978). The protoplasts may be isolated from different tissues Osmotic conditions and organs including leaves, shoot apices, roots, coleop- tiles, hypocotyls, petioles, embryos, pollen grains, calli, In isolated protoplasts, the pressure of the missing cell cell suspensions. A reliable source of protoplasts, e.g. in wall on the protoplast is replaced by suitable osmotic the genus Brassica, is the cells of leaf mesophyll. The values of used solutions and media. The osmotic poten- leaves are a good source of protoplasts enabling the iso- tial is adjusted by adding mannitol, sorbitol, glucose or lation of a high number of relatively uniform cells. sucrose into the enzymatic mixture, washing solution Material may be taken from field plants, plants grown in and culture medium. The stability, viability and further greenhouse or in vitro. The physiological condition of the growth of protoplasts are connected with the appropriate plant influences the success of the isolation of protoplasts, osmotic conditions of isolation and subsequent cultiva- therefore the plants are grown under
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