Plant cells regeneration from protoplasts

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. 

The cell and tissue culture of the major tropane alkaloid-producing species does not apparently offer any special problems. The regeneration of plantlets from callus and tissue cultures seems to be routine (286,307,309,323,325,332,350-352). Plants have also been regenerated from protoplasts of Atropa belladonna (353), Duboisia myoporoides (354), and Hyoscyamus muticus (355,356). Cryopreservation has been reported for Anisodus and Datura species (349,357). 

Attree, S.M Bekkaoui, F. Dunstan, D.L. Fowke, L.C. (1987). Regeneration of somatic embryos from protoplasts isolated from an embryogenic suspension culture of white spruce (Pkea glauca). Plant Cell Reports, Vol. 6, pp. 480-483. 

Indeed one can go a step further. Cell walls may be removed from plant cells by treatment with appropriate enzymes (pectinases, cellulases, etc.) and protoplasts are then obtained. Isolated protoplasts such as these can also be grown into whole plants. This was achieved not only with standard test objects, such as tobacco plants and carrots, in regeneration experiments but also with petunias. 

Salisbury, P.A., 1987. Blackleg resistance in weedy crucifers. Cruciferae News Lett. 12, 90. Sigareva, M.A., Earle, E.D., 1999. Camalexin induction in intertribal somatic hybrids between Camelina sativa and rapid-cycling Brassica oleracea. Theor. Appl. Genet. 98,164-170. Sikdar, S.R., Chatterjee, G., Das, S., Sen, S.K., 1987. Regeneration of plants from mest hyll protoplasts of the wild crucifer Eruca sativa Lam. Plant Cell Rep. 6, 486-498. 

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. 

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