Protoplasts are cells of plants, fungi, or bacteria in which the cell wall has been removed, but the plasma membrane is intact. The invention a novel and efficient method for protoplast culture comprising the steps of isolating the protoplasts from a cell suspension, mixing the protoplasts with equal volumes of alginate solution, placing 4050. The enzymes for this purpose are preparations which are commercially available. A cell of a plant, fungus, bacterium, or archaeon from which the cell wall has been removed, leaving the protoplasm and plasma membrane. Transient gene expression systems using protoplasts have been widely used for rapid functional characterization of genes and highthroughput analysis in many model and crop species. Here, we describe a simplified and highly efficient root protoplast isolation and transient expression system in the model legumes lotus japonicus and medicago truncatula. Rice oryza sativa protoplast isolation and its application for transient expression analysis feng he. Cassava, manihot esculenta, protoplast isolation, transient expression. Compared with the stable gene expression in transgenic plants, transient gene expression represents a fast, convenient and efficient alternative system with higher expression levels. Protoplast isolation protoplasts are cells which have had their cell wall removed, usually by digestion with enzymes.
Protoplasts underwent further division and formed cell clumps that consisted of two to 10 cells 1015 d after culture on liquid medium and 20 d after culture on agarosesolidified medium. Establishment of method for protoplast fusion with pegmediated between jatropha curcas l. View the article pdf and any associated supplements and figures for a period of. On the other hand, attempts at a similar approach with cereal leaves failed completely because the cell walls were modified, in response to even 1 days preculture, such that protoplast isolation was no longer possible. Jun 10, 2016 rice oryza sativa protoplast isolation and its application for transient expression analysis feng he state key laboratory for biology of plant diseases and insect pests, institute of plant protection, chinese academy of agricultural sciences, beijing, china. Protoplast isolation this protocol describes the process of isolating protoplasts from plant cells and then fusing the protoplasts in order to carry out further investigations. An idealized example of protoplast isolation will show the steps involved in protoplast isolation and culture. For best yield of protoplasts, cassells and burlass 1978 suggested growth under low light intensity 2. Present chapter describes the methods of protoplast isolation, purification, culture techniques as. Overall, the complete protoplast isolation protocol is completed in 3 hr and 22. Highly efficient mesophyll protoplast isolation and peg. This chapter presents the techniques and approaches used in most studies of protoplast isolation and culture.
The optimum combination of enzymes for protoplast isolation was 1. Pdf microalgae protoplasts isolation and fusion for. Feeder layers consist of protoplasts or cells plated separately in an independent layer usually below the protoplast layer. In fact, the various enzymes for protoplast isolation are commercially available. The different enzymes for protoplast isolation and the corresponding sources are given in table 44. The task scheduling program integrates the microplate mover with the other equipment to enable transfer of plates between each piece of equipment. Rice oryza sativa protoplast isolation and its application. The successful isolation of protoplasts depends on table 1. Protoplast isolation and fusion protoplasts are naked plant cells without the cell wall, but have. The isolation of bacterial1 and fungal protoplasts2,3 following the use of enzymes digesting the cell wall suggested that protoplasts might be obtained from plant cells by treatment with cellulase. A robotic platform for highthroughput protoplast isolation.
Establishment of method for protoplast fusion with peg. Isolation and manipulation of protoplasts from the. Agarose is most frequently used to solidify protoplast culture media. Protoplast isolation was optimal when the tissue was incubated in enzymolysis solution composed of 1. Protoplast suspension is taken at double density and mixed with melted agar medium at 45c and mixed well and plated in small petridish. The first protoplast division occurred within 34 d, and small colonies were observed microscopically after 2 wk of protoplast isolation fig.
The success of protoplast isolation depends especially on the condition of the tissue and the combination of enzymes being used. Isolation and culture of protoplast from leaves of lactuca sativa. Compared to previously reported protocols, our protoplast isolation method is markedly faster saving 4. Protoplast fusion would continue to be an existing area of research in modern biotechnology 11. Protoplast isolation from shoots of asparagus cultures core.
Frontiers a protoplast transient expression system to. Previous reports on asparagus protoplast isolation have used callus or cell. Optimization conditions of wheat mesophyll protoplast. Isolation and fusion of protoplasts from mesophyll cells of. Plants from distantly related or unrelated species are unable to reproduce sexually as their genomes are incompatible. A general section will discuss the parameters involved in the developmental sequence from protoplast to plant. Pdf protoplast isolation, culture, and regeneration researchgate. Isolation of mesophyll protoplasts from leaves of dalbergia sissoo. Highly efficient protoplast isolation and transient. Protoplasts are used in a number of ways for research and for plant improvement. Protoplast isolation has been reported from mesophyll cells of in vivo and in vitro growing plantlets, aseptic seedlings, microspore mother cells, young microspores, pollen grain calli and embryogenic and nonembryogenic suspension cultures. Strain improvement of pleurotus species by protoplast fusion. Protoplast surfaces bear strong negative charges, and intact protoplasts in suspension repel each other, hence fusion is accomplished by the addition of calcium ions or polyethylene glycol peg, or by using electric fields.
Protoplast isolation, culture and callus regeneration of. C a snapshot of cell viability test after resuspending cells in wi0. In this process some of the plasmolyzed cells were cut only through the cell wall, releasing intact protoplasts. Microalgae protoplasts isolation and fusion for biotechnology research. Isolation and culture of protoplast from leaves of lactuca. Protoplast isolation the source tissue is a common and important factor in successful poplar protoplast culture. Molding division shower head example of a 3 plate mold protoplast inc.
The petal strips were transferred to a petri dish containing freshly prepared enzyme. Fully open flower petals were used for protoplast isolation. Rishikesh meena assistant professor department of botany university of rajasthan jaipur. Disadvantages include limited plant species that are amenable to regeneration from protoplasts, and among those that are amenable, the technique is time consuming and labor intensive. The isolation and culture media used vary with the species and with the tissue from which the protoplasts were isolated. The isolation of bacterial1 and fungal protoplasts2,3 following the use of enzymes digesting the cell wall suggested that protoplasts might be obtained from plant cells by. The protoplast isolation, yield and viability assessment were performed as described above using 0. A mixture of carbohydrases can be used to degrade plant tissue, producing a protoplast suspension which can easily be seen under a.
Protoplast systems have been proven powerful tools in modern plant biology. Development of a rapid, lowcost protoplast transfection system for. Protoplast definition of protoplast by the free dictionary. Jan 02, 2014 importance of protoplast isolation and culture the isolation, culture and fusion of protoplast are one of the most fascinating fields of research. Protoplast isolation science and plants for schools.
Protoplast definition and meaning collins english dictionary. Protoplast isolation and transient gene expression in switchgrass. Importance of protoplast isolation and culture the isolation, culture and fusion of protoplast are one of the most fascinating fields of research. However, successful preparation of abundant viable protoplasts remains a challenge for cymbidium orchids. Transient gene expression in plant protoplasts is a powerful technique for studying subcellular localization of proteins, gene and promoter activities, proteinprotein interactions, and signal transduction 17. Based on these results, 88% of the protoplast isolation protocol is spent digesting. Isolation of protoplast from leaves leaves are most commonly used step are 1 sterilization of leaves 2 removal of epidermal cell layer 3 treatment with enzymes 4 isolation of protoplast purification of protoplast viability of protoplast 6. For protoplast isolation, it is always desirable to grow plants under controlled growth conditions such as 0 lx light intensity16 h at 2225 c. State key laboratory for biology of plant diseases and insect pests, institute of plant protection, chinese academy of agricultural sciences, beijing, china. Protoplast isolation an overview sciencedirect topics. The question whether the cell cycle phase plays a role, which phase is optimal for protoplast isolation. Pdf protoplasts of arabidopsis thaliana have been isolated from a variety of explant sources with varying degree of. A a flowchart showing the procedure of protoplast isolation.
Stages in the culture of protoplasts through plant regeneration for populus alba x p grandidentata. Whole plants can be regenerated from protoplasts grown on solid or liquid media. Mechanical method klecker in 1892 has first initiated the protoplast isolation by mechanical means the cells were kept in a suitable medium plasmolyticum and cut with a fine knife. Protoplast isolation protocol is based on the method developed by sheen and colleagues sheen, j. Protoplast fusion protoplast fusion was performed according to a modified method of 1, 2. Isolation and fusion of protoplasts from mesophyll cells. Pegmediated protoplast transformation with naked dna. There is no standard method for the isolation and culture of protoplasts. Protoplast isolation the highest yield of protoplasts was obtained from 30 dayold leaves of l. Download pdf abstractexcerpt full text pdf protoplast isolation and culture protocols were developed for leaf tissue from 6 kenaf cultivars everglades 41 e41, e71, guatemala 4 g4, g45, g51, and tainung 1. Ijms free fulltext highly efficient protoplast isolation.
Protoplast isolation, culture, and regeneration article pdf available in methods in molecular biology 82. Pdf protoplast isolation from hibiscus sabdariffa l researchgate. Asterisks mark the petals used for protoplast isolation. Cellulase enzymes digest the cellulose in plant cell walls while pectinase enzymes break down the pectin holding cells together. May 15, 2019 the protoplast isolation, yield and viability assessment were performed as described above using 0. The protoplast isolation and dna transfection procedures take 68 h, and the results can be obtained in 224 h. In this paper, a protocol was conducted for protoplast isolation from roselle explants using enzymatic approach.
Protoplast isolation was most efficient when fec was cultured under vacuum for 5 min in an enzyme solution consisting of 4% cellulase, 0. The unicellular charophycean green alga penium margaritaceum has emerged as an appealing experimental organism in plant cell wall and cell biology research. B protoplast morphology remained intact in enzyme solution supplemented with 0. Protoplast fusion enables organelle transfer between. Us6569680b2 efficient method of protoplast culture. For protoplast isolation, the best combination of hydrolytic enzymes was cellulysin 1% wv. The following protocol is suitable for protoplast isolation of plant parenchymatic and meristematic tissues. The total time from initiation of the procedure to complete loading of the enzymes by the multimode dispenser is 2. A leaves from shoot cultures are the source tissue. Isolation of protoplast separartion of protoplasts from plant tissue 1.
Further, most of the shoot cultures were from nonseedling trees, which allowed researchers. Simple protoplast isolation system for gene expression and. Incubation time protoplast yields x105 per g f wt h light dark. Petal protoplasts were successfully isolated from petals collected 115 days after full bloom. The disadvantage of this method is that it does not permit the isolation of single colonies derived from one parent cell. Protoplasts can be isolated from a range of plant tissues. Unfortunately, the mesophyll protocol failed to release protoplasts from the cell culture, leaving predominately intact cells. To develop novel hybrid plant through protoplast fusion, genetic engineering would continued to be an exciting area of research in modern plant. A method for the isolation of plant protoplasts and vacuoles. Pdf protoplast isolation, culture, and regeneration. Updated protoplast preparations for gene transfection 109 protoplast isolation protocol is based on the method developed by sheen and colleagues sheen, j. Rice oryza sativa protoplast isolation and its application for transient expression analysis feng he state key laboratory for biology of plant diseases and insect pests, institute of plant protection, chinese academy of agricultural sciences, beijing, china. Protoplast isolation and culture plant tissue culture. In 10 of the 12 reports listed in table 1, section d, the protoplast source was leaves from shoot cultures.
Herein, we established an efficient protoplast isolation protocol from orchid petals through optimization of enzymatic conditions. French protoplaste, from old french, the first man, from late latin protoplastus, from greek protoplastos. Effect of different cellulase and pectinase enzyme. In the process of protoplast isolation, the yield of free protoplast was significantly different under the conditions of adding different mannitol concentration 0. Highly efficient mesophyll protoplast isolation and pegmediated. Protoplast fusion has been used as a method to create mushroom hybrids especially when conventional method cannot be achieved. Turn on all components of the robotic system figure 1a and open the plate mover task scheduling software. Cell wall formation and colony formation were better in a liquid medium than on a semisolid agarose medium. An efficient protocol for model legume root protoplast. B newly isolated protoplasts plated in liquid medium with a polyester screen disc for support openings in the screen are 150 11m. Among the many uses for these naked cells is transient or permanent genetic transformation by introduction of transgene dna, somatic hybridization by protoplast fusion of species or subspecies resistant to traditional crossbreeding, and isolation of sub.
The media were as described in table 1 and they were supplemented with pgr according to table 2. The techniques are important for the following reasons. Here we report the first protocol to isolate large numbers of viable protoplasts from both leaves and roots of two switchgrass genotypes. More recently, viable protoplasts have been obtained from male and female gametes. Electrofusion is preferred over chemical fusion, as fusion conditions are much easier to control duquenne et al. Protoplast isolation and culture from kalanchoe species. Nootjaree tudses1, siripong premjet,2,3 and duangporn premjet1,3. The protocol establsihed for our lab can be downloaded from here. Jul 31, 2014 isolation of protoplast from leaves leaves are most commonly used step are 1 sterilization of leaves 2 removal of epidermal cell layer 3 treatment with enzymes 4 isolation of protoplast purification of protoplast viability of protoplast 6. Jun 21, 2007 the protoplast isolation and dna transfection procedures take 68 h, and the results can be obtained in 224 h.
144 1391 1407 1401 302 378 1170 763 783 1330 1025 1373 806 373 1111 869 1449 1200 1364 1236 545 726 669 380 482 530 1387 369 150 1533 1487 520 242 1177 1054 1388 1173 1032 787 597 652 1049 1355 1340 1102