Isolation and transient transformation of stem and leave protoplasts from sweet potato (Ipomoea batatas L.)
Garcia, R., Pimentel, E., Somonte, D., Mena, J., Zaldua, Z., Lopez, A., Moran, R., and Garcia*, M.
Centro de lngenieria Genetica y Biotecnologia. P.O. Box 387, C.P. 70100. Camaguey, CUBA
(Received 2 February 1998 / Accepted 16 July 1998)
Abstract.
Isolation of sweet potato protoplasts from different organs has been very difficult to develop, due to the recalcitrant behavior of this crop when it is manipulated in-vitro. An efficient protocol for protoplast isolation from leaf and stem tissues was established. Different enzyme combinations and concentrations were tested, as well as several digestion conditions. A hard enzymatic treatment, combined with mechanical separation of the cells from the digested tissue, led to a high protoplast yield. Other factors, such as the osmotic solution used in the different washing steps, and the starch concentration in the medium, were evaluated for their influence on the final protoplast recovery and viability. The transformation of isolated protoplasts was mediated by polyethylene glycol (PEG). The plasmid transient expression was detected by a spectrophotometrical P glucoronidase (GUS) assay.
Keywords: sweet potato, protoplast isolation, PEG mediated transformation
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