There are only-two examples of isolated corn protoplasts undergoing sustained divisions to give cell cultures (callus or suspension): 1) protoplasts from corn stem tissue (Potrykus et al., 1977, MGG 156:347) which, however, has not proved repeatable, and 2) protoplasts isolated from established cell lines (those originating from corn stem protoplasts) (Potrykus et al., 1979, TAG in press). The cell lines can be routinely used for establishing protoplast-derived secondary cell cultures, and they have been distributed to various laboratories. In view of our aim--the genetic modification of corn plants through protoplast technology--both systems lack a very important feature: the capacity to regenerate plants.
We are now trying to establish a morphogenic corn protoplast system, but as cereals have proven to be extremely recalcitrant in vitro (King et al., 1978, Physiol. Veg. 16:381) we expect this to be a very long term project. For example there has been no report to date of cereal plant regeneration from cultured single cells or protoplasts.
We are isolating and culturing protoplasts from tissues or cells which have either expressed totipotency or which can safely be assumed to be totipotent: 1) very young embryos--2-3 days post-pollination, 2) immature embryos--12-18 days, 3) immature embryos undergoing multiple shoot formation (Green et al., 1975, Crop Sci. 15:417), 4) meristems (vegetative shoot tip, tassel and ear primordia, nodes, leaf base, 5) sporogeneous tissues, 6) pollen mother cells and 7) microspore tetrads. As there is no method available to detect or select for totipotent cells, even here we deal with mixed populations of protoplasts. In addition, we are continuing our trials with protoplast populations which may or may not include totipotent cells: internode, leaf sheath, leaf blade, crown roots, nucellus and endosperm. Our renewed interest in culture of protoplast from these organs stems from the observation that differentiating cells in some of these organs can be induced to embark upon additional divisions in vivo.
I. Potrykus, D. Hanold, C. Harms, P. Larkin and H. Lorz
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