Evaluation of a Simple Automated Bioreactor for the Production of Pelargoniums 1992 Proposal
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 methods 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 100 uE 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 fi-oin 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 are 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. In: 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
American Florists Endowment December 1, 1991 - November 30,1992
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 4,625
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 Born: 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 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
PUBLISHED PAPERS — 1985-PRESENT Tsai, D. S. and R. N. Arteca. 1985.
Effects of root applications of gibberellic acid on photosynthesis and
growth in C3 and C4 plants. Photosynthesis Res. 6:147-157. Arteca, R. N.,
J. M. Bachman, J. H. Yopp, and N. B. Mandava. 1985. Relationship of steroidal
structure with a stimulation in ethylene production by etiolated mung bean
segments. Physiol. Plant. 64:13-16. Schlagnhaufer, C. and R. N. Arteca.
1985. Brassinosteroid induced epinasty in tomato plants. Plant Physiol.
78:300-303. Arteca, R. N., D. S. Tsai, and C. Schlagnhaufer. 1985. Root
applications of abscisic acid: A possible method to reduce shipping injury
in geranium cuttings. HortScience 20:370-372.
Arteca, R. N., E. J. Holcomb, C. Schlagnhaufer, and D. S. Tsai. 1985.
Effects of root applications of gibberellic acid on photosynthesis and
growth of geranium plants grown hydroponically. HortScience 20925-927.
Schlagnhaufer, C. D. and R. N. Arteca. 1985. Inhibition of brassinosteroid-induced
epinasty in tomato plants by aminooxyacetic acid (AOA) and cobalt Co2+.
Physiol. Plant. 65:151-155.
Arteca, R. N. and J. M. Bachman. 1987. Light inhibition of brassinosteroid-induced
ethylene production. J. Plant Physiol. 129:13-18.
Arteca, R. N. and D. S. Tsai. 1988. Effects of abscisic acid on photosynthesis,
transpiration and growth of tomato plants. Crop Research 27:91-96.
Tsai, D. S., R. N. Arteca, J. M. Bachman, and A. T. Phillips. 1988.
Purification of 1- aminocyclopropane-1-carboxylate synthase from etiolated
mung bean hypocotyls. Arch. Biochem. Biophys. 264:632-640.
Arteca, R. N., J. M. Bachman, and N. B. Mandava. 1988. Effects of indole-3-acetic
acid and brassinosteroid on ethylene biosynthesis in etiolated mung bean
hypocotyl segments. J. Plant Physiol. 133:430-435.
Arteca, R. N., J. M. Bachman, D. S. Tsai, and N. B. Mandava. 1989. Fusicoccin…
An inhibitor of brassinosteroid-induced ethylene production. Physiol. Plant.
74:631-634.
Arteca, R. N. 1989. Hormonal induction of ACC synthase. In: Advances
in Agricultural Biotechnology. Biochemical and Physiological Aspects of
Ethylene Production on Lower and Higher Plants. Kluwer Academic Publishers,
pp. 119-133.
Clark, W. S., D. J. Beattie, and R. N. Arteca. 1989. A growth inhibitor
in Clematis viticella seed. J. Plant Physiol. 134:492-495.
Arteca, R. N. and C. D. Schlagnhaufer. 1989. Effects of the antitranspirant
Vapor Gard on photosynthesis, transpiration and growth of soybean and maize
plants. Life Science Advances in Plant Physiology, 8:55-58.
Arteca, R. N. and J. M. Arteca. 1990. Use of a monoclonal antibody for
the determination of free indole-3-acetic acid. J. Plant Physiol., 135:631-634.
Tsai, D. S., R. N. Arteca, J. M. Arteca, and A. T. Phillips. 1991. Characterization
of 1- aminocyclopropane-1-carboxylate synthase. J. Plant Physiol., 137:301-306.
Arteca, R. N., C. D. Schlagnhaufer and J.M. Arteca. 1991. Effects of
root applications of gibberellic acid on growth of seven different Pelargonium
cultivars. HortScience, 26,(5):555-556.
Schlagnhaufer, C. D. and R. N. Arteca. 1991. Immunoassay of brassinosteroids.
J. Plant Physiol., in press.
Schlagnhaufer, C.D. and R.N.Arteca. 1991. The uptake and metabolism
of brassinosteroid by tomato (Lycopersicon esculentum) plants. J. Plant
Physiology, in press.
Arteca, R.N. and D.S. Tsai. 1991. The inhibition of BR-induced biosynthesis
in etiolated mung bean hypocotyl segments by 2,3,5-triiodobenzoic acid
and 2-(p-chlorophenoxy)-2- methylpropionic acid. J. Plant Physiology, in
press.
Wang, T-W. and R. N. Arteca. 1991. Anaerobic root stress effect on ethylene
biosynthesis in tomato plants (Lycopersicon exculentum, Mill). Plant Physiology,
in press.
Guo, L. and R. N. Arteca. 1991. Hormonal induction of ACC malonyltransferase
in etiolated mung bean hypocotyl segments. Plant Physiology, submitted.
