ways in the laboratory, they are most often used either for DNA introduction or to generate
fusion hybrids.
The two main methods used to introduce DNA into protoplasts are electroporation and
polyethylene glycol (PEG) treatment. Forelectroporation, protoplasts are placed in a DNA
solution between two electrodes and exposed to brief pulses of high-voltage current. The
pulses cause pores to form in the membrane and the DNA then enters the cells.PEG treat-
mentsare also performed in the presence of DNA and probably also result in the formation
of pores, from membrane destabilization.
With their cell walls removed, protoplasts can be manipulated in additional ways that are
not possible with intact plant cells. DNA can be introduced into protoplasts using microin-
jection, which is the most common method used for transformation of animal cells.
Microinjection utilizes precisely drawn and cut glass needles, which will shatter if
pushed into an intact plant cell. Surprisingly, protoplasts can also be very efficiently trans-
formed usingAgrobacterium. The bacteria are able to very effectively adhere to and trans-
form protoplasts as the protoplasts are regenerating new cell walls.
Although protoplast transformation can be extremely efficient with more than 50% of the
cells receiving DNA, tremendous problems are encountered when attempting to recover
whole plants from these single cells. Whole transgenic plants have been recovered from
a variety of plants using protoplast transformation, but this method is seldom used today
for generation of transgenic plants. Because DNA introduction efficiency can be very
high, transient expression in protoplasts is routinely used for analysis of factors that modu-
late gene expression (Sheen 2001).
10.5.3 Whole-Tissue Electroporation
Although electroporation can be used for very efficient transformation of protoplasts,
application of electric pulses to whole tissues can also result in DNA introduction, although
at reduced rates of efficiency (D’Halluin et al. 1992). With the cell wall intact, formation of
pores in the cell membrane is of limited value for DNA introduction. Whole-tissue electro-
poration has been successfully used with rapidly growing tissues which contain thin, newly
formed cell walls. Partial enzymatic digestion of whole tissues using cellulases and pecti-
nases can remove enough of the cell wall to allow DNA introduction using electroporation
of “intact” tissues.
10.5.4 Silicon Carbide Whiskers
Developed originally for DNA introduction into insect eggs, use of silicon carbide whiskers
have been successfully applied for DNA introduction into plant cells (Kaeppler et al. 1990).
Silicon carbide whiskers are long, rigid two-pointed microscopic “spears” that are added to
plant cells and DNA and then vortexed. The spears or whiskers are approximately 1mm
thick and 15–50mm long. Although the analogy of “being in a Jacuzzi with porcupines”
has been used to describe this technology, the shaking motion is much more violent and is
probably more closely akin to a paint mixer found in hardware stores. It is unclear whether
the whiskers enter the cell with DNA as they are thrown about, or whether they penetrate the
cells after being wedged between two cell clusters as they collide. The low efficiency of
transformation using silicon carbide whiskers along with disposal under conditions
similar to those for asbestos render this method unsuitable for most laboratories.
262 TRANSGENIC PLANT PRODUCTION