Plant protoplasts, fusion

Pioneering experiments were carried out in the field of protoplast fusion (using bacterial and fungal protoplasts) leading to internationally recognized outstanding results. Significant advances were also made in the field of plant protoplast fusion and practical application has been initiated. 

Kumar, A. Cocking, E.C. (1987). Protoplast fusion A novel approach to organelle genetics in higher plants. American Journal of Botany, 74, 1289-303. 

Physical methods of gene transfer. Genes can often be transferred without the use of a cloning vehicle. This is especially important for certain plant cells, such as those of cereal grains, for which transfer of genes via the Ti plasmid has been difficult.167 If DNA, which may be in a plasmid, is coprecipitated with calcium phosphate, it can often be taken up directly either by animal cells or by plant protoplasts.168 169 Polycations also facilitate DNA uptake cationic liposomes seem to be especially effective.170 In the widely used electroporation technique a short electrical pulse of a few hundred volts / cm is applied to create transient pores in the plasma membrane through which the DNA can enter a cell.111 8,171 175 Chromosomes can be transferred by cell fusion and either... 

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. 

Alloplasmlc conversion Is a strategy which emerged from capabilities developed with plant protoplasts. Originally touted as a method for the recovery of exotic Interspecific hybrids (12), protoplast fusion has proven to be an excellent way to approach the wholesale manipulation of mitochondrial genes (13). The predominant trait of note Is cytoplasmic male sterility (cms), which Is of Interest to the seed Industry as a tool to facilitate hybrid seed production. Little or no Impact on possible value-added traits Impacting on food quality, however, Is anticipated from alloplasmlc conversion. 

Genetic techniques for obtaining chloroplast-encoded herbicide resistance in crop plants are reviewed. These are based on the use of cell culture methods and Include mutant selection in cultured cells, chloroplast transfer via protoplast fusion, and genetic recombination between chloroplast genomes. The first two methods are described in detail using trlazine resistance as an example. 

The chloroplasts of triazine-resistant weed species are a potential source of herbicide resistance for treinsfer into crop plants. Chloroplast transfer, that is the combination of desirable chloroplasts and nuclei, can be achieved by crossing or by protoplast fusion. Chloroplast transfer by crossing is carried out using the line with the desirable chloroplasts as the maternal parent and the line with the desirable nuclear background as the pollen parent. Repeated crossing results in substituting the chromosomes (nucleus) of the chloroplast source with those of the recurrent pollen parent as chloroplasts in crops are only inherited by the maternal parent. This strategy has been used to introduce the triazine-resistant chloroplasts from bird s rape (a weed, Brassica campestris) into cultivated B. campestris and B. napus... 

Combination of atrazine resistance and cytoplasmic male sterility in rapeseed. Transfer of atrazine resistant chloroplasts from bird s rape (B. oapestris) into oilseed rape has been accomplished by back-crossTng (s this volume). The purpose achieved by somatic cell fusion was to combine the atrazine-resistant chloroplasts carried by a B. napus line with cytoplasmic male sterility (CMS), a mitoohondrlal tralt (l9). The CMS trait was carried by a B. napus line with the cytoplasm of radish (Raphanus sativus). Since both traits, atrazine resistance and CMS, are maternally inherited in sexual crosses, the only way to combine the traits was by protoplast fusion. The desired atrazine-resistant cybrids were identified in a randomly regenerated sample of plants by the presence of the CMS flower morphology and their darker green color at low temperature. Somatic hybrids rather than cybrids were obtained in each clone since no irradiation had been used to facilitate the elimination of one of the parental nuclei. 

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). 

Sumitomo Chemical. Jpn. Kokai Tokkyo Koho. JP 55096096, 21-07-1980. JP 79-4275 16-01-1979. Enhancement of protoplast fusion in plant cell cultures (Rauvolfia serpentina). Chem. Abstr. 93, 218142q. 

Figure 2.5 Trichomes of mint plants (a) and resulting phenotype of a somatic hybridization (b, hybrid) between mint varieties (b, E33, E87 leaves of parent lines used for protoplast fusion, unpublished results E. Baumann, Phytowelt GreenTechnologies GmbH).

A CaMV-GUS fusion in pUC19 making use of the observation that the pRAJ275 initiator context gives enhanced translational yields, e.g., a "consensus" CaMV-GUS-NOS cassette. This works well in transient expression in a variety of monocotyledonous and dicotyledonous plant protoplast systems. 

Fusion of Plant Protoplasts after Dielectrophoretic Collection... 

E. Jacob, W. Forster, and H. Berg, Microbiological implications of electric field effects. II. Inactivation of yeast cells and repair of their cell envelope, Z.Allgem.Mikrobiol., 21 225-233 (1981). Senda, J. Takedo, Sh. Abe, and T. Nakamura, Induction of cell fusion of plant protoplasts by electrical stimulation. Plant Cell Physiol., 20 144 (1979). 

Barsby, T.L., Yarrow, S.A., Kemble, R.J. and Grant, I. (1987) The transfer of cytoplasmic male sterility to winter-type oilseed rape (Brassica napus L.) by protoplast fusion. Plant Sci. 53, 243-248. 

Narasimhula, S.B., Kirti, P.B., Bhatt S.R., Prakash, S., Chopra, V.L., 1994. Intergeneric protoplast fusion between Brassica carinala and Camelina saliva. Plant Cell Rep. 13,657-660. 

Ovcharenko, O., Momot, V., Cherep, N., Sheludko, Y., Komamitsky, 1., Rudas, V., Kuchuk, N., 2011. Transfer of transfwmed Lesquerella fendleri (Gray) Wats, chloroplasts into Orychophragmus violaceus (L.) O.E. Schulz by protoplast fusion. Plant Cell Tissue Organ Cult. 105, 21-27. 

Between the methods of Agrobacterium and microprojectile transfer, nearly every plant species can be transformed effectively [35]. However, these methods are covered by patent claims and may result in limited transformation efficiency for some cell types. For this reason, the use of alternative gene-transfer methods is an active area of research for all cell types. Alternative gene-transfer techniques include electroporation, microinjection, liposome fusion, direct transfer into protoplasts, and laser treatment [38]. In electroporation, DNA is transferred into the cell using a high-voltage electrical pulse [39]. Standard... 

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