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An Overview of LifeLineTM Products for Plant Tissue Culture
Plant tissue culture is a miraculous technology with the potential to solve many of
the world's most pressing problems of providing food for a burgeoning population,
and otherwise influencing our surroundings. But the key to realizing this potential
lies in the combined efforts of all members of the plant biotechnology community in
achieving production costs of final plants which are in reasonable economic proportion
to other alternatives.
The strategy to achieve this goal can be copied from that of the microelectronics
industry. Just as that industry used micro technology to increase performance and
cut cost, so the plant tissue culture industry must cut its costs by:
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Minimizing infrastructure costs (both of investment in buildings and overhead maintenance)
by restricting the use of clean room space only to critical operations, and using
low cost means to otherwise control contamination.
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Significantly raising the density of production (ie the amount of culture produced
per sq. meter of lab space/unit time).
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Reducing labor costs, especially in cutting and handling, through moderately priced
technological aids.
Implementing the three principles listed above needs to be the concern of everyone
connected to plant biotechnology, and not only those actively managing commercial
micropropagation laboratories. Academic and industrial workers must strive not only
to achieve the most sophisticated gene transformation work, but to marry that research
to the most advanced and economic means of tissue culture propagation. Doing so will
save many months of costly R&D when the project reaches the stage of preparation
for mass plant production.
The LifeLineTM product family is designed to provide the latest in micropropagation
technology to all workers in plant tissue culture. The products are designed to address
the key technical culture problems of the academic or commercial lab, while simultaneously
constituting a complete system for economically efficient plant culture:
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Reduce the clean room to the size of the growth vessel with LifeGuard vessels and
aseptic vented lids.
-
Increase plant production density and reduce handling costs using liquid culture medium
and membrane rafts (LifeRaftTM) or disposable airlift bioreactors (LifereactorTM).
-
Improve cutting efficiency (reduce labor costs) by a random mechanical separator (VitrocutTM).
Vented Vessels Aseptically Controlling the Culture's Micro-atmosphere
No matter what kind of media or type of culture, the LifeGuardTM family
of vented culture vessels and lids help you:
-
SAVE MONEY
With an integrally sealed, submicron microporous membrane vent in the
lid of vessels which are easily and securely closed, both aeration efficiency and
freedom from contamination are completely assured over long periods of time. This
minimizes:
-
Losses and costs due to contamination outbreaks.
-
the need for expensive growth rooms (use space with much lower investment requirements).
-
GROW BETTER PLANTS
- vents of different size for a given vessel volume allows the user
control over the microatmosphere in the vessel. This can be used to optimize the number
of gas exchanges to:
-
eliminate negative phenomenon such as hyperhydricity (vitrification), and,
-
increase rates of growth and plant quality, especially as regards plant survival in
the field (see Application Notes and References on venting).
The LifeGuard TM family includes
Avoid falling into the trap of using inexpensive, easily contaminated vessels housed
in a very expensive clean room. This pennywise pound foolish approach incurs:
-
Excessive costs of investment in clean room infrastructure, including the air circulation
and filtration systems, and quality of floor and wall finish for contamination control.
-
Excessive costs of maintenance for contamination control (periodic spraying, clean
room maintenance costs, etc.) and energy costs for air circulation systems.
-
Excessive loss of valuable plant material due to periodic outbreaks of contamination.
All of the above problems are cured by the use of LifeGuardTM vessels and
vented lids.
Liquid Medium Culture - LifeRaftTM Membrane Rafts and LifeReactorTM Airlift
bioreactors:
LifeRaftTM membrane rafts, floats, and culture holder accessories,
contained in LifeGuardTM vented vessels, constitute a complete, very small
batch mode liquid culture growth system. This flexible system is so easy to use that
it is even part of a student course on micropropagation available from the Carolina
Biological Supply Company (see pages 192-3 of their 2001 catalog).
LifeReactorTM sterile disposable bioreactors make it possible to carry
out pilot and commercial high production density culture (both multiplication and
elongation and growth) at a small fraction of the price normally associated with such
processes. The system is available both as a kit and individual reactors, and includes
a full line of accessories for efficiently handling the culture and avoiding contamination.
The specific features of these systems are explained in the section on Product- Line.
To understand their relevance in your work, we need to first examine the many economic
and technical advantages of liquid culture medium over those of semi-solid media such
as agar or other gels.
Why Liquid Medium?
The Positive Effects of Liquid Medium on Plant Growth:
Rates of Growth
Compared to growth on gels, plants as varied as orchids, Spathiphylum, Syngonium,
potatoes and Cucumis meuliferus, grown on liquid medium have shown faster and
better rates of growth, at lower concentrations of plant growth regulators. Why should
this be the case? An examination of the fundamental physical processes at work shows
that this result was to be expected.
Let's consider what happens at the plant wall, where uptake of salts, sugar and plant
regulators takes place. Chemicals in the solution immediately next to the wall are
quickly absorbed by the plant, and must be replaced by diffusion from solution further
away. However, in gels, this is a slow process, and in the zone of gel next to the
plant, gradients of concentration develop for each of these chemical species. The
plant's growth needs are now subjugated to the physics of the slow diffusion process.
In liquid medium the rate of diffusion is 10x faster, so that the chemicals in solution
are much more accessible. For this reason lower concentrations of growth regulators
are usually optimal, compared to those used in gel medium formulations.
Overcoming Phenolic Exudate Growth Inhibition
On the other side of the coin, the concentration of phenolic byproducts, which
are growth inhibitors, rises sharply immediately next to the plant wall where it does
the most damage.
Click to Enlarge
This may be seen in Figure 2 , for Aconitum nepallus explants grown on agar.
The explant is surrounded by a dark phenolic ring, which becomes a light brown color
in the rest of the gel.
Due to the 5- 10x faster diffusion rate in liquid systems, exuded growth inhibitors
either quickly diffuse or are washed away. Negative effects on plant growth are thereby
minimized. In addition, liquid systems such as bioreactors may have active mixing
to keep the concentration of the growth nutrients and regulators constant near the
plant wall.
Increased Budding and Reduction of Tetraploid Regenerants
Cucumis metuliferus grown on liquid medium showed 50% more buds per cotyledon
versus those grown on agar, and half the rate of tetraploidy. In Syngonium White
Butterfly, liquid medium showed 35% higher shoot proliferation.
Other Advantages:
-
Low pH Control of Contaminants
:
The growth of many contaminant microorganisms is suppressed at low pH. While plant
cultures may do very well at low pH, this method of control can't be used with most
gels because they require higher pH in order to form a gel. Low pH does not represent
a problem for growth in liquid medium.
-
Ease of Changing Media in the Elongation and Growth Stage:
Plant root systems grown on semi-solid gel media cannot easily be separated from the
gel and placed in fresh medium without causing shock and damage to the plant. The
plant must therefore be left in the gel, which makes either refreshing or changing
the composition of the growth medium a complicated, time-consuming procedure.
With a Liferaft liquid medium system, the vessel is simply opened in a clean bench,
and the raft portion is aseptically transferred to a new vessel containing fresh sterile
medium and a float. This technique is especially important for promoting the growth
of potato microtubers, which must be maintained at high concentrations of sucrose
for extended periods of time. Large microtubers are achieved by first culturing potato
plantlets on a raft with multiplication medium. The raft with the grown plantlets
is then moved to tuberization medium, which is periodically renewed by placing the
raft on fresh medium once per week.
In a Lifereactor bioreactor system, the fresh medium can be added and old medium removed
in sterile fashion, through ports in the cover and the bottom, without opening the
reactor vessel and risking contamination.
-
Secondary Metabolite Production
Many plants are known to produce exudates of useful organic materials during their
growth. With the use of either rafts or bioreactors, it is simple to measure their
rate of production, as well as determine the effects of growth regulators. By coupling
a Lifereactor with an ultrafiltration membrane diafiltration system, a steady-state
bioreactor system can be easily setup to continuously produce the secondary metabolite.
Cutting the Cost of Multiplication The VitroCutTM Random Mechanical
Cutter
The single most expensive, labor intensive part of the conventional plant tissue
culture process is that of multiplying the culture by carefully cutting plant material
on nodes, by hand. To facilitate this process, many cultures have been deliberately
created with long, spindly structures, so that the operator can more easily cut on
the node, pick up the cut material and place it into a semi-solid medium for growth.
For a long while, attempts at automation or mechanization meant attempting to mimic
this procedure with very sophisticated, expensive robotic solutions. The Osmotek approach
is to simplify the process by turning the problem around. If the culture material
possesses a highly compact, branched structure, then it can be cut randomly with a
very high probability that the material cut has cut at a node.
With the patented VitrocutTM device, the culture is cut in totally random
fashion by mechanically forcing the plant material through a grid of knives. Where
necessary, the culture is first modified with specific growth regulators which cause
it to develop the optimal clustered structure. The cut material can be directly collected
either in a vessel containing semi-solid medium, or a membrane raft. The lowest production
costs, however, are achieved when the Vitrocut is used in concert with bioreactors.
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