Bio-Rad PDS-1000 / He™ and Hepta™ Systems - Manual (Page 37)

Recommended starting particle size/type for bombardment of various cell types is
Bacteria
0.7 µm (M5) tungsten
Yeast 0.6 µm gold
Algae 0.6 µm gold
Plant cells/tissue 1.0 µm gold
Animal cell cultures 1.6 µm gold
Sub-cellular organelles 0.6 µm gold
Table 5.1 and bulletins 1688 and 2015 give a detailed discussion of optimization parameters.
5.10 Preparation of Biological Material for Bombardment
Plant Cells
Many factors contribute to optimum performance in any system for plant gene transfer.
Transformation procedures employing microcarrier bombardment are no exception, but the improved
PDS-1000/He apparatus lends itself to rapid and easy modification, to tailor its performance to a par-
ticular application. Microcarrier bombardment also allows exploitation of a very broad range of
explant tissue types. This permits optimization of a crucial aspect of the transformation system
which usually is difficult to alter when transforming by other means. The following is a discussion
of some of the critical parameters in the microcarrier transformation system, together with a brief
examination of explant characteristics which affect transformation efficiency. This is not intended
as a “how to” manual. Because of the extreme diversity of applications possible with the system, and
the various types of tissues which can be employed, it is not possible to produce a universal proto-
col for all applications. Rather, this discussion is intended to aid the new user to design experiments.
Note: If you are a first-time user, it is important to be certain that your PDS-1000/He
apparatus has been assembled correctly and is operating according to specifications. We
suggest that you use the Yeast Optimization Kit (catalog number 170-3100) to be sure that
microcarrier preparation has been carried out properly, and that microcarriers are being
delivered uniformly. This also provides an opportunity to examine firsthand the pattern
of particle delivery and gene expression in treated cells. This may save substantial time
as you begin to optimize the system for your own purposes.
Choice of Explant Tissue
Unlike most other procedures for DNA transfer, microcarrier bombardment places few
constraints on the types of tissues which can be treated. DNA can be delivered into essen-
tially any cell or tissue which can be exposed sufficiently to allow particle penetration. Thus
more emphasis usually can be given to issues such as the tissue’s ability to regenerate plants,
or its physiological suitability for gene regulation studies. There are, however, several aspects
of the explant which contribute to the efficiency of the system.
An ideal tissue for microcarrier bombardment could perhaps be described as a cell monolay-
er, all cells of which are capable of expressing introduced genes, and all of which could, indepen-
dently, divide and differentiate into functional plants. Microcarrier bombardment of such a tissue
will likely be efficient because cells can be spread over a large area, thus efficiently capturing a large
proportion of the particles delivered in each bombardment. Particles also have a high probability
of penetrating such cells because they are not covered by overlying cell layers which would very
likely intercept some of the particles delivered by each bombardment. Absolute transient expres-
sion levels are likely to be high, because of the number of cells affected. The low profile of cell clus-
ters reduces the probability that the tissue will be moved about by the gas released in the shockwave
during treatment. Such a tissue would also lend itself well to selection with antibiotics or herbicides,
in part, at least, because it would have a favorable geometry for the establishment of a uniform
concentration of the selective agent throughout the culture.
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