is “bigger, better, stronger, faster.” Certainly, plant transformation is achievable, and
transgenic plants have been obtained using all of the plants of major economic importance.
But efficiencies of existing methods can always be increased, and new methods may yield
even higher transformation rates. Floral dip ofArabidopsisis very straightforward and
efficient, but there is room for improvement in the recovery of more transgenics, and
more importantly, application of this method to other plants would be quite useful. In
addition, all of the methods that have been presented, including those that will be presented
below, are protected by patents. The status of intellectual property drives much of plant
biotechnology, and the methods for transgenic plant production are no exception. New
transformation technologies will probably be protected by patents, but the availability of
more choices is always beneficial (see Chapter 14). The additional technologies presented
here do not represent a complete or thorough list. The methodologies are presented to
provide a sampling of the types of ideas that have been generated since the dawn of
transgenic plant production in the mid-1980s.
10.5.2 Protoplasts
For DNA introduction into plant cells, the cell wall represents the major barrier. When the
plant cell wall is enzymatically or physically removed, protoplasts (Fig. 10.10) are the end
result. Protoplasts are very fragile single cells that must be maintained in a osmotically and
nutritionally balanced medium to prevent lysis. They are typically generated using enzyme
mixtures of cellulases and pectinases to digest cell walls, and mannitol is often used to
maintain the osmotic integrity of these naked cells. Protoplasts can be generated from
many different types of tissue, but young leaf mesophyll tissues and embryogenic
cultures are the most common. Although protoplasts can be manipulated in a number of
Figure 10.10.Maize protoplasts, electroporated with agfpgene, showing brightfield (a) and with
GFP filters (b). (Illustrations provided by Pei-Chi Lin and JC Jang, Department of Horticulture and
Crop Science, OARDC/The Ohio State University.) See color insert.
10.5. OTHER METHODS 261