Genetic transformation protoplast

One of the exciting features of the direct DNA delivery system is that it does not rely on an infection. The limited host range of other vector delivery systems is therefore irrelevant, and the way is opened for genetic engineering of cereals. Cereal protoplasts are equally amenable to uptake of foreign DNA after electroporation and the system therefore has potential for use with the major crop species. However, there is at present one drawback, namely that for cereals it has not yet proved possible to grow fertile whole plants from the genetically transformed cells. 

A number of different methods for stable or transient genetic transformation of plants or plant cells have been developed [13-15]. These comprise particle bombardment, Agro actermm-mediated transformation, floral dip transformation, agrodrench, viral vectors, protoplast transformation and ultrasound. These are the main techniques for the genetic transformation of plants, and many of them have also been applied for the transformation of secondary metabolite pathways in an attempt to alter the metabolic pathways of target... 

Takeno, M., Taguchi, H., and Akamatsu, T. (2011) Essential involvement of the Bacillus subtilis ABC transporter, EcsB, in genetic transformation of purified DNA but not native DNA from protoplast lysates. /. Biosci. Bioeng., 112 (3), 209-214. 

Vardi A, Bleichman S, Aviv D (1990) Genetic transformation of Citrus protoplasts and regeneration of transgenic plants. Plant Sci 69 199-206... 

Finally, we tested the particle bombardment method, a biolistic approach that was shown before to work when other approaches have failed. In particular, particle bombardment has allowed the generation of transgenic plants such as crop species, which are not susceptible to Agrobacterium tumefaciens or cannot be regenerated from protoplasts. Furthermore, particle bombardment has facilitated organelle transformation in intact cells and the genetic modification of cultured cells that were not accessible to other transfection techniques. 

Chloramphenicol acetyltransferase is used as a genetic marker system to study transformation and gene expression in plant protoplasts, cells, and tissues. 

Heterokaryotic hybrids also can be produced by fusing protoplasts to overcome genetic barriers to anastomosis (33. 34. Plasmid-mediated transformants have been successfully produced (TeBeest D.O., unpublished data) in this fungus and with several other Colletotrichum species (35-37). To date, genetic exchange appears limited in Colletotrichum and hybrid isolates often exhibit reduced fitness characterized by reduced growth and sporulation and decreased virulence. 

Cellulases and other carbohydrases are being used to isolate higher plant cells and to produce protoplasts (67, 68), A new era in plant breeding is envisioned as cell lines are manipulated genetically by mutagens, protoplast fusion, or possibly transformed by DNA transfer. 

Protoplasts are osmotically weak plant cells dispossessed of their cell wall, also referred to as naked cells. Upon removal of the cell wall, protoplast can behave like animal cells which by nature have no cell wall barrier. Protoplasts have the potential ability to redifferentiate into whole plants due to their totipotency, and this has been exploited for genetic modification of plants however, there exist many recalcitrant plant species which have not been able to be transformed following this method, either because of failure to isolate viable protoplasts or impossibility of regeneration. 

Campoy, S., Perez, F., Martin, J. F., Gutierrez, S., and Liras, P. 2003. Stable transformants of the azaphilone pigment-producing Monascus purpureus obtained by protoplast transformation and Agrobacterium-mediated DNA transfer. Curr. Genet. 43(6), 447-452. 

Cell culture-based methods suitable for obtaining chloroplast-encoded herbicide resistance in crops will be reviewed. These will Include mutant selection in cultured cells, transfer of chloroplasts via protoplast fusion, and selection of recombinant chloroplast genomes. Examples will be limited to resistance to triazine herbicides. Chloroplast transformation involving introduction of novel DNA into chloroplasts, will not be covered. Preliminary data indicate that chloroplast transformation may be feasible (4) but have not been confirmed. The genetics, physiology... 

Several techniques are now available to change the genetic information in plant cells transformation with Agrobacterium spp., direct DNA injection, and protoplast fusion. Novel techniques in molecular biology have found rapid application in plant cell biotechnology, as, for example, the use of antisense DNA 9,120,121). 

The protoplasts used in the experiments just mentioned above were isolated from the mesophyll of leaves, i.e. from highly specialized cells. The result of the experiments constitute a further piece of evidence for totipotency. However, protoplasts are of the greatest interest for entirely different reasons which ought at least to be mentioned here they promise to be useful objects for genetic manipulation, for somatic hybridization as well as transformation (p. 7). 

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