To reduce the time taken from seed to generate protoplasts, 14dold plantlets have also been used for protoplast isolation. Application of protoplast technology to crisprcas9. In 10 of the 12 reports listed in table 1, section d, the protoplast source was leaves from shoot cultures. Petal protoplasts were successfully isolated from petals collected 115 days after full bloom. It is necessary to have higher than 50% transfection efficiency for a reliable analysis. Selecting leaves from the correct growth stage is critical for ensuring a. Tapearabidopsis sandwich a simpler arabidopsis protoplast.
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. Isolation of protoplast from cell suspension culture 3. In arabidopsis research, the methods of protoplast isolation and transient expression have been wellstudied 4,16,17. Protoplast isolation science and plants for schools. Overall, the method was found to be a simplified and effective alternative to those previously described for pineapple protoplast isolation, obtaining the highest protoplast yield 3. A simple protoplast isolation protocol that was designed to recover totipotent plant protoplastswith relative een describedease has the key elb e mthe ents of protocol are, tissue digestion at slightly elevated temperatures and use of protoplast releasing enzythat are stable and efmes ent at hficiigher t empbesides enzyeratures mes. Nov 24, 2009 in this protocol, fiveweekold plants are used for protoplast isolation. Protoplasts regeneration and isolation plant tissue culture. A transient expression assay using maize mesophyll. Protoplast isolation and culture plant tissue culture. In this report, we further develop protoplast isolation protocols for several crop and ornamental species, and the model plant arabidopsis. Protoplasts are used in a number of ways for research and for plant improvement.
The following protocol is suitable for protoplast isolation of plant parenchymatic and meristematic tissues. Wall formation, cell division and callus formation 7. B newly isolated protoplasts plated in liquid medium with a polyester screen disc for support openings in the screen are 150 11m. Remove the main vascular bundles from the leaves of lambs lettuce and cut them into pieces if the leaves are large. Pdf protoplast isolation, culture, and regeneration. Wash the pieces with wi and remove by pipet, then add 5ml fresh enzyme solution 5ml.
We describe a simple singleprotoplast isolation protocol and use this protocol to edit the tobacco ntpds gene. Isolation of protoplasts from tissues of 14dayold seedlings. Fully open flower petals were used for protoplast isolation. Although rice genes have been extensively investigated in the last two decades, the functions of many genes in the rice genome are still not known. Protoplast isolation protoplasts are cells which have had their cell wall removed, usually by digestion with enzymes. The petal strips were transferred to a petri dish containing freshly prepared enzyme. Efficient protocol for protoplast isolation and plant. An efficient protocol for model legume root protoplast isolation and transformation ning jia 1, yali zhu 1,2 and fang xie 1 1 national key laboratory of plant molecular genetics, cas center for excellence in molecular plant sciences, shanghai institute of plant physiology and ecology, chinese academy of sciences, shanghai, china. However, a step towards the plant genetic manipulation and integrated approach of breeding programs is primarily laid on an efficient protocol in protoplast isolation, culture, and regeneration. Incubation time protoplast yields x105 per g f wt h light dark. Handle the protoplasts carefully since they are fragile and will easily rupture. To develop novel hybrid plant through protoplast fusion, genetic engineering would continued to be an exciting area of research in modern plant. We used these protoplasts to evaluate crisprcas9 mutagenesis ef. Similar to the protoplast isolation protocol, 72% of the transformation procedure is spent incubating the reaction.
To establish an efficient protocol for pineapple protoplast isolation, we pooled together leaves from plants grown in liquid rooting medium for 1 month as the source material fig. Pdf protoplasts of arabidopsis thaliana have been isolated from a. Protoplast are purified by sucrose density gradient centrifugation and collected at the interface of w5 and enzyme solutions fig. After completion of the protoplast isolation protocol, the transformation protocol is initiated, and completed within 27. Protocol for the preparation of metabolically competent protoplasts. A transient expression assay using arabidopsis mesophyll. Suspensioncultured alex cells of arabidopsis thaliana l. A robotic platform for highthroughput protoplast isolation. Frontiers a protoplast transient expression system to. A simple method for the isolation of plant protoplasts. Protoplast transfection efficiency is approximately 6090% with this protocol in general. However, a step towards the plant genetic manipulation and integrated approach of breeding programs is primarily laid on an efficient protocol in protoplast isolation, culture, and regeneration duquenne et al. A protoplast efficiency of 70% is needed for transformation.
A simple and efficient method for isolation of pineapple. However, this method consumes many seeds, which is a disadvantage for the study of mutants that have a low seed production phenotype. Optimization of isolation and culture of protoplasts in. Isolation of protoplasts methods for the isolation of protoplasts from leaf mesophyll tissue, root and cell suspension cultures are described in chapter 6. The success of protoplast isolation depends especially on the condition of the tissue and the combination of enzymes being used. The age of leaves more than 30 days was not suitable for protoplast isolation due to yield and viability of these protoplast were very low. Rice oryza sativa protoplast isolation and its application. Dec 12, 2017 in this report, we further develop protoplast isolation protocols for several crop and ornamental species, and the model plant arabidopsis. Highly efficient protoplast isolation and transient expression. 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.
Sep 28, 2015 a presentation covering the process of protoplast culture including protoplast isolation, protoplast fusion, culture of protoplast, its application, factors af slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. A simple method for isolation, liquid culture, transformation and. Protoplast isolation an overview sciencedirect topics. An efficient method for protoplast isolation from micropropagated pineapple leaves. Isolation and fusion of protoplasts from mesophyll cells. A transient expression assay using maize mesophyll protoplasts. An efficient procedure for protoplast isolation from. Protoplast isolation from shoots of asparagus cultures core. Protoplasts are cells of plants, fungi, or bacteria in which the cell wall has been removed, but the plasma membrane is intact. Isolation and culture of protoplast from leaves of lactuca sativa. The different enzymes for protoplast isolation and the corresponding sources are given in table 44.
Rashid 1988 for additional discussion of protoplast fusion methods and the possible results of somatic hybridization. Importance of protoplast isolation and culture the isolation, culture and fusion of protoplast are one of the most fascinating fields of research. 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. The below mentioned article provides an overview on the isolation and culture of protoplast. The successful isolation of protoplasts depends on table 1. Electrofusion is preferred over chemical fusion, as fusion conditions are much easier to control duquenne et al. Previous reports on asparagus protoplast isolation have used callus or cell. Feb 12, 2020 your overall aspect of standardizing a common protocol for protoplast isolation is good, but in my point if your samples are from distinct places at first biological parameters like ph, salinity. The enzymes for this purpose are preparations which are commercially available. In this protocol, fiveweekold plants are used for protoplast isolation. Protoplast preparation recipes serva electrophoresis gmbh.
To protoplast the remaining yeast, at ml either lyticase or zymolyase to their respective tubes and incubate for the determined time to reach 70% protoplast efficiency. In protoplast technology,two genetically different protoplast isolated from the somatic cells and are experimentally fused to obtain parasexual. A leaves from shoot cultures are the source tissue. We used these protoplasts to evaluate crisprcas9 mutagenesis efficiency. In fact, the various enzymes for protoplast isolation are commercially available. Pdf optimisation of protoplast isolation protocols using in vitro. Pdf protoplast isolation from in vitro leaves of dendrobium crumenatum was carried out. Jun 21, 2007 protoplast transfection efficiency is approximately 6090% with this protocol in general. Hypocotyl and mesophyll materials are treated identically forprotoplast isolation. Jun 10, 2016 rice oryza sativa is not only the staple food for half of the worlds population but also a model monocot plant for molecular biology studies. Pegmediated protoplast transformation with naked dna. Protoplast isolation, culture, and regeneration article pdf available in methods in molecular biology 82.
There is no standard method for the isolation and culture of protoplasts. Simple protoplast isolation system for gene expression and. Transfer immediately these pieces into a petri dish or a bottle containing 15 ml of digestion solution 1. The isolation and culture media used vary with the species and with the tissue from which the protoplasts were isolated.
Molding division shower head example of a 3 plate mold protoplast inc. Stages in the culture of protoplasts through plant regeneration for populus alba x p grandidentata. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast releasing enzymes that are stable and efficient at higher temperatures. Ultrastructural and hisochemical analysis of cell wall. Protoplasts can be isolated from a range of plant tissues. This protocol is optimized to produce a high yield of pure chloroplasts from isolated arabidopsis protoplasts. The protoplast protocol was applicable to different landraces of arabidopsis thaliana l. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum. Prepare protoplast and isolate nuclei from arabidopsis.
Resuspend the purified protoplasts gently in cell wall regeneration medium at a final density of about 105 protoplasts ml1, and incubated at 25c to regenerate cell walls. Protoplast isolation the highest yield of protoplasts was obtained from 30 dayold leaves of l. A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The protocol established in this study will assist researchers in the isolation and culture of protoplasts in alfalfa and will accelerate the research processes like protoplast fusion and genetic engineering. Protoplasts regeneration and isolation ptc protoplasts are cells that have had their cell walls removed, and possess plasma membrane and all other remaining cellular components. Isolation and fusion of protoplasts from mesophyll cells of. Protocol for isolation and culture of protoplast 2. Protoplast isolation the source tissue is a common and important factor in successful poplar protoplast culture. Pdf protoplast isolation, culture, and regeneration researchgate. Cellulase enzymes digest the cellulose in plant cell walls while pectinase enzymes break down the pectin holding cells together. Further, most of the shoot cultures were from nonseedling trees, which allowed researchers. The twopart method is a simple, scaleddown, and lowcost procedure that readily provides healthy mesophyll protoplasts, which are then ruptured to release intact chloroplasts. The techniques are important for the following reasons. That is, it contains all the functional part of the cell without the barrier of the cell wall.
691 1392 235 551 334 178 1127 1436 482 305 990 1233 436 561 1191 1563 1517 1340 457 1244 937 239 1504 539 353 848 946 1240 343 1131 1397 1095 264 647 825 405 1098 273 572 72 115