From Aggie
Horticulture, Texas A&M AgriLife Extension, Texas A&M
University
System
by R. Daniel Lineberger, Professor of Horticulture
The Methodology Of Tissue Culture
This article first appeared in The Buckeye Nurseryman, September, 1980, Columbus, OH.
Much
has been written and more has been said about the applications of plant
tissue culture to the nursery industry. The subject is still poorly
understood by a majority of plant propagators. Tissue culture is often
looked upon as being practical only for propagation of those plants
which are difficult to propagate by conventional methods. This notion
is quickly being laid to rest. An industry is emerging in various areas
throughout the country which is taking advantage of technological
advances in the realm of tissue culture of woody plants. This
technology suggests that propagation by tissue culture is indeed
applicable to the "difficult to propagate" species, and equally as
important, it may offer economic advantages for some species which are
considered relatively "easy to propagate".
The intent of this
communication is to briefly describe the contemporary techniques of
tissue culture of woody plants, and to review the advantages and
limitations of tissue culture as a propagation method.
The Methodology of Tissue Culture
The
variety of techniques which can be used to get plant development in
vitro (that is, by techniques such as tissue culture) is considerable
and completely dependent upon the species in question. Single cells of
leaf tissue can regenerate whole plants, as can shoot tips, leaf
pieces, root pieces, lateral buds, or stem sections. Not all of these
methods are applicable to woody plants, and indeed, not all have been
applied to a commercial level to any plants.
Shoot tip culture
is the method in widest use for the mass propagation of woody species.
An actively growing shoot tip is surface sterilized and placed on a
defined culture medium under sterile conditions. The culture medium
contains inorganic and organic salts (macronutrients, micronutrients
and vitamins) as well as an energy source (sucrose or table sugar),
growth regulators, and agar )to gel the medium). If the growth
regulators are appropriately balanced, the shoot tip elongates, lateral
buds break and begin growth, and adventitious shoots are also produced
on the stem piece. This rapid proliferation of shoots results in masses
of shoots being produced from a single shoot tip. Up to a hundred
shoots may be produced in as little as eight to twelve weeks from a
single tip. The number of shoots produced and the rapidity of shoot
proliferation varies between species, and in some cases, between
cultivars of a single species. Shoots are removed from the cultures at
regular intervals and a portion of the mass is replaced on fresh media
to continue proliferation. The small shoots which are removed are then
rooted in a separate medium, either a sterile gelled medium or a
peat-perlite medium (in much the same fashion as conventional woody
cuttings).
Shoots produced through tissue culture are generally
easy to root, even though the same cultivar may be difficult to root by
cutting propagation. The resulting rooted shoot is referred to as a
"plantlet" because of its miniature size, but these can generally be
grown in the greenhouse at a rapid rate and with a high degree of
survival. Techniques for rooting tissue cultured shoots are currently
receiving a great deal of research attention as are methods for
establishing these shoots into the greenhouse environment.
The Tissue Culture Laboratory
Most
propagators would probably list the expense of setting up a tissue
culture laboratory among the reasons for not getting involved with this
technology. A lab does cost more than a mist bed! A more severe
limitation is without doubt the absence of personnel skilled in the
techniques of tissue culture. An individual who has had prior
experience with tissue culture methodology can successfully operate a
laboratory with a bare minimum of equipment. An autoclave or
sterilizer, an accurate means of weighing chemicals, a small inventory
of glassware, and chemical stocks or prepackaged media are all that is
required to initiate cultures. The laboratory can be enlarged, more
equipment added, and additional personnel hired as the need for such
arises. The most critical factors are the ingenuity and experience of
the laboratory manager.
As a tissue culture laboratory becomes
operational, procedures other than the actual culturing process become
critical. Maintenance of disease and insect free stock plants with a
low titer of bacterial and fungal contamination is critical. Also
important is the care and handling of the thousands of plantlets prior
to their transfer to the field or containers. The ability to grow off
the plantlets ultimately will limit production, rather than any
limitation due to the propagation phase.
Tissue Culture Propagation of Woody Species
Major
advances in the mass propagation of woody plants have been made over
the past ten years. Because of the somewhat variable response of
species and cultivars to the in vitro environment, much research was
(and still is) needed to define the cultural conditions required by
ornamental species. As an example of the variability encountered, we
have observed that seedling shoot tip cultures of crabapple produce as
few as one or as many as sixty-four shoots per explant after three
months in tissue culture. Similar variations in the tendency for
multiple shoot production have also been noted with adult clonal
material of different apple cultivars.
The number of woody plant
species which have been clonally propagated through tissue culture is
increasing at a rapid rate, and in fact, most commercially important
ornamental species have been studied. Perhaps the best publicized of
the early research on woody plants involved apples, Douglas fir, and
rhododendrons. An extensive commercial scale production program for
apple scion cultivars and rootstocks has developed in England and in
several European countries.
Using the successes of the apple,
rhododendron, and maple programs as a basis, many recent advances have
been made which will facilitate the mass propagation of other woody
species. While it is true that cultivar differences account for lack of
success with some plants, a growing body of evidence suggests that
taxonomic families behave somewhat alike in tissue culture. A large
number of deciduous and evergreen Rhododendron species share common
cultural requirements, the most notable of which is the requirement for
the same cytokinin for adventitious shoot formation. A similar
relationship exists between the trees and shrubs of the Rosaceae
(including crabapples, pears, plums, and hawthorns). Most rosaceous
species will likely be mass propagated on media very similar to that
found to be optimal for apples.
The anomalies alluded to
by the crabapple seedling responses to tissue culture and the emerging
similarities between the requirements of rosaceous trees and shrubs
demonstrate that much research is needed to define the conditions
necessary for commercial scale tissue culture production programs. As
is often found to be the case with propagation systems, those systems
described as optimal in the laboratory would likely need modification
to be considered economical by the industry.
Impact of the Tissue culture Industry
As
is always the case for any emerging technology, the strongest
supporters for the adoption of that technology are likely those
involved in its development. Having acknowledged these biases, one must
now consider the advantages and limitations of tissue culture
propagation, and what its economic impact on the nursery industry will
be.
Not every nursery will OPERATE a tissue culture
laboratory. The trend toward specialization of nursery operations as
either "propagators" or "producers" is likely to continue in the
future. Operation and management of a tissue culture facility requires
personnel and physical facilities which are quite different from those
required by a container or field production nursery. Previously in this
report where the establishment of the laboratory was considered, the
relatively small initial investment in physical facilities and
equipment was discussed. It should be reemphasized at this point, that
the training and quality of the laboratory manager is more important
than the laboratory itself, and that this expertise is not currently
available among contemporary nursery propagators.
Not
every nursery will NEED a tissue culture laboratory. Commercial
operations specializing in mass propagation of nursery crops can likely
produce a sufficient number of plants to satisfy the market.
As
with any other technology, the success of tissue culture within the
nursery industry will be determined by economic factors. Can the
plantlets be produced in sufficient numbers as a cost to compare with
traditional propagation? Will the propagules fit into the established
production sequence in the nursery industry? Are the propagules
true-to-type? These answers must be obtained on a species by species,
cultivar by cultivar basis.
Publication from Aggie Horticulture®
The information, as it is presented on this Website, does not represent
an endorsement
by the State of Texas or any State agency.
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