Evaluation of a Simple Automated Bioreactor for the Production of Pelargoniums 1993 Proposal
Production of Pelargoniums
Justification:
The worldwide demand for virus-indexed Pelargonium cuttings is increasing
at a very rapid pace. In order to meet this increasing demand, rapid efficient
production of Pelargonium plants is necessary. All of the woric on the
propagation of Pelagoniums has relied on conventional methods of propagation.
The feasibility of using tissue culture for the production of Pelargoniums
has intrigued researchers and growers for many years. However, the use
of standard shake culture methods was not economically feasible. Today
with new bioreactor technology available the use of tissue culture can
become a reality. The growth as well as handling of the cultures is simpler
and more efficient by using modern bioreactors. In fact, a 10-50 L bioreactor
will do the same work as 100-500 individual culture flasks thereby, saving
space, time and labor. Accurate monitoring and control of temperature,
pH, carbon dioxide, oxygen, and ethylene in bioreactors provide more uniform
growth conditions and also better flexibility for optimizing conditions.
Since the bioreactor is a single, closed, sterile system it minimizes contamination
problems which can be very costly. Production in a bioreactor is more easily
automated than it is in small individual culture vessels. It is easier
to automatically transfer cultures or media in and out of a large vessel
than from many individual flasks. The use of conventional tissue culture
for the production of Pelargoniums would be labor intensive and not economically
feasible. Interestingly, it has been established that the greatest operational
expense of commercial plant tissue culture propagation as a whole is labor.
Reliable figures for the actual cost of labor are unavailable for proprietary
reasons but is estimated to account for 40 to 60 percent of the total production
cost (Constantine, 1986). Today with the advent of biotechnology there
are many types of bioreactors commercially available which have the potential
for automation thereby reducing costs. We will initially use an airlift
design bioreactor on a small scale with the ultimate goal of using this
design to scale-up for commercial production.
Over the past decade there have been reports on the regeneration of
Pelargionums from callus (Brown and Charlwood, 1986) and protoplasts (Yarrow
et al., 1987). These reports show that the growth of callus and suspension
cultures of a number of Pelargonium species are very rapid, in fact, one
report shows that the fresh weight of suspension cultures increased four
fold within an eight day period (Yarrow et al., 1987). In addition, the
yields of protoplasts from this callus are very high. There have been no
reports on the growth of Pelargoniums in bioreactors which has potential
commercial value. Additional benefits of callus cultures are the ability
to produce large numbers of a particular stock in a very short period of
time and they can be frozen and stored for use at a latter date. Although
tissue culture has a great deal of potential only conventional methods
have been used for the propagation of Pelargoniums requiring large amounts
of time and manpower, by using bioreactors this problem can be overcome.
Objectives: The overall goal of the proposed research is to develop
an automated bioreactor which can be used to increase the commercial production
of selected Pelargoniums with desired characteristics such as new genotypes
or disease-free plants rapidly and efficiently. The availability of such
inethods will also lead to increased breeding and selection efforts which
in turn will promote increased quality of Pelargoniums at a reduced cost.
This will be accomplished by the following:
1 . Produce callus to Pelargonium leaves, maximize growth rates while
maintaining chromosome stability and develop methods for cryopreservation
of cells; 2. Develop hormonal treatments to induce regeneration of plantlets;
3. Seed an airlift bioreactor with cells grown in suspension culture and
do the following: - treat with hormones to promote maximum growth of callus,
- induce plantlet production with treatments developed earlier and transfer
plantlets to soil and grow; 4. Design a larger bioreactor and evaluate
how initial studies work on a commercial scale.
Procedures: Production of Callus: Pelargonium x hortorum cv. Sincerity
will be obtained from Oglevee Associates culture-virus-indexed stock. Fully
expanded leaf tissue will then be surface sterilized by immersing in 70%
ethanol for 15 seconds, rinsing 2 times with sterile water, then incubating
in a 10% bleach solution containing a drop of Tween 20 for 5 minutes and
rinsed 3 times with sterile water (this procedure has been successful in
our laboratory for Pelargoniums). The distal and proximal regions of the
leaf will be discarded and the remaining portion will be bisected longitudinally.
These explants will then be transferred to Murashige and Skoog (1962) salt
media plus vitamins supplemented with sucrose (3%), BAP (1 ppm) and 2,4-D
(1 ppm) for the initiation of callus. Callus cultures will be grown at
25′C in the light at an irradiance of 100uE*m-2*s-1 with 12 hour days and
12 hour nights. Callus tissue will be subcultured every 2 weeks to fresh
medium. Once callus is established (approx. 5-6 weeks) on solid medium
it will be used to produce suspension cultures which will be grown in the
same medium without agar and shaken on a rotary shaker at 125 rpm under
the same growth conditions outlined above. Cell suspensions will be subcultured
every week to fresh medium. Once cell suspensions have been established
different hormone combinations (BAP and 2,4-D) will be tested to optimize
regeneration conditions. Chromosome stability will be tested after each
subculture period according to the procedure of Simmonds and Cummings (1976).
Cryopreservation of Cells: Suspension cultures of selected clones will
first be hardened according to the procedure of Chen and co-workers (1985).
One hundred milligrams of each of these cell lines will be aseptically
placed in cryogenic plastic vials (1.5 ml capacity) with 0.5 ml of cryoprotectant
(5% DMSO and 0.5 M sorbitol). Five vials per cell line will be frozen.
The vials will be sealed with a screw cap and incubated over ice for 1
hour. The specimens will be cooled in a CryoMed 972 freezer controlled
with a programmable CryoMed 1010 controller. The cooling rate will be 0.5′C/minute
to a terminal temperature of -35′C and the sample will then be placed in
-80′C. Samples will be tested for the ability to regenerate and chromosome
stability following removal from storage.
Establishing a Bioreactor System: Initially, an Airlift Bioreactor (Kontes)
will be used for studies which will include seeding the reactor with cells
grown in suspension culture, the cell suspension will be grown to a maximum
density, the solution will be supplemented with the hormone combination
which promotes plantlet growth, once the plantlets have grown to 1 to 2
cm in length they will be transfered to soil and grown in a growth chamber
grown at 25′C in the light at an irradiance of 100 uE*m-2*s-1 with, 12
hour days and 12 hour nights. After the plants am established they will
be transferred to the greenhouse and evaluated. Chromosome stability will
be monitored at each step as previously mentioned. When studies are complete
with the smaller commercial airlift reactor we will have a larger size
constructed to maximize production utilizing a similar design.
Facilities and Equipment Available: All of the tissue culture facilities
required to successfully complete the proposed experiments are available
to principal investigator.
Literature Cited: Brown, J.T. and Charlwood, B.V. 1986. The accumulation
of essential oils by tissue cultures of Pelargonium fragrans (Willd.).
FEBS Letters 204:117-120.
Chen, T.H.H., Kartha, K.K. and Gusta, L.V. 1985. Cryopreservation of
wheat suspension culture and regenerable callus. Plant Cell Tissue and
Organ Culture 4:101-109.
Constantine, D.R. 1986. Micropropagation in the commercial environment,
pp 175-186. la: Plant Tissue Culture and its Agricultural Applications.
Withers, L.A. and Alderson, P.G. (eds.). Butterworth, London.
Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497.
Simmonds J.A. and Cummings, B.G. 1976. Propagation of Lilium hybrids.
II.Production of plantlets from bulb-scale callus cultures for increased
propagation rates. Scientia Horticulture 5:161-170.
Yarrow, S.A, Cocking, E.C. and Power, J.B. 1987. Plant regeneration
from cell-derived protoplasts of Pelargonium aridum, P. x hortorum and
P. peltatum. Plant Cell Reports 6:102-104.
BUDGET The proposed research will be renewable each year upon satisfactory
progress.
A. Salaries and Wages Prin. Inv. - Arteca 0 Total Category I 0
Technician, wages, 740 hrs @ $6.25/hr 4,625
Total Category II 4,625 Total Salaries and Wages 4,635
B. Fringe Benefits @ 8.1% 375
C. Total Salaries, Wages and Fringe Benefits 5,000
D. Equipment 0
E. Materials & Supplies 2,500
F. Travel 0
G. Publication Costs 0
H. Computer Costs 0
I. Other Direct Costs 0
J. Total Direct Costs 7,500
Investigators Curriculum Vitae and List of Relevant Publications: Richard
N. Arteca Bom: August 23, 1950
ACADEMIC TRAINING:
Utah State University, B.S. in Horticulture, 1972 (minor in Botany)
Utah State University, M.S. in Plant Science, 1976 Washington State University,
Ph.D. in Horticulture, 1979
PROFESSIONAL SOCIETIES: American Association for the Advancement of
Science American Society of Plant Physiologists American Society for Horticultural
Science International Society for Plant Molecular Biology Society of Sigma
Xi Japanese Society of Plant Physiologists Scandanavian Society of Plant
Physiologists Plant Growth Regulator Society of America Tissue Culture
Association Gamma Sigma Delta
PROFESSIONAL EXPERIENCE:
July 92 - Present: Professor, Department of Horticulture The Pennsylvania
State University July 86 - Present: Associate Professor, Department of
Horticulture, The Pennsylvania State University June 86 - Dec.89: Coordinator
of the Centralized Hybridoma Facility, The Pennsylvania State University
Dec. 79 - Present: Assistant Professor, Department of Horticulture, The
Pennsylvania State University Jun. 79 - Dec. 79: Postdoctoral Research
Associate, Washington State Univ. Jun. 77 - Jun. 79: Research Assistant,
Washington State University Dec. 76 - Jun. 77: Teaching Assistant, Washington
State University Sep. 75 - Sep. 76: Research Assistant, Utah State University
