Progress 10/01/04 to 09/30/10
Outputs
Significant finds over life time of this project include: (1) finding of anti-cancer drug "Taxol" in Filbert (Hazelnut) trees; (2) two patents for imaging spectrophotometer/fluorimeters; (3) storage and recovery of digital information in living leaf; (4) a large number of applications of in vivo spectroscopy of plants; (5) demonstration of orientation of plant tissues in magnetic fields (NMR); (7) quantification of extent of diversity by acrylamide gel electrophoresis in Cydonia, Pyrus and Corylus; (8) changes in chloroplast structure of strawberries with june yellows; (9) demonstration of thionin-like proteins in tomato, mango, papaya, and walnut; (10) in vivo characterization of chloroplast-protein complexes; (11) others as described in papers found at site indicated below. The corresponding scientific papers for these and other findings are listed and can be accessed at: http://oregonstate.edu/Dept/hort/faculty/DaleyPublications.html
Impacts
The impact of these studies is expected to improve ways to determine basic properties of plants and their application to aagriculture in general and to human well being.
Publications
- Total publications are found at: 2006-1983 http://oregonstate.edu/Dept/hort/faculty/DaleyPublications.html
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Progress 01/01/05 to 12/31/05
Outputs
The chloroplast division work arose out of observation of anomalies in photosynthetic efficiencies found during in vivo fluorescence imaging spectroscopy of leaves. Using plant germplasm with characteristic spatial zonation of chloroplast division, such as found in Amaryllis leaves, and by use of confocal microscopy, we are making considerable progress in the elucidation of this phenomena. Taxol work arose in a similar fashion. In these studies, we were using mass spectroscopy investigating the resistance of accessions of Corylus to the pathogen Anisograma anomala Eastern Filbert Blight). Characteristic mass/charge fragments corresponding to this very medically significant metabolite showed up in these scans. This was very surprising since the phylogeny of Corylus an angiosperm is far distant from the gymnosperms where this compound was originally found. The interest in the strange evolution of Crassulacean Acid Metabolism (CAM) arose from a series of experiments
using microcalorimetry, which can be considered as time dependent spectroscopy of infrared radiation emission. In this work CAM, plants were demonstrated to have potentially useful chill tolerant carbon fixing enzymes. This work introduced the laboratory to CAM species in the genus Cissus, often lumped into the genus Vitis. These Cissus species are dramatically different morphologically, physiologically, and biochemically and yet so closely related to more common C-3 Vitis species such as domestic grapes. This case alone, and there are many such cases e.g. orchids, Oxalis, and among the filicales, suggest basic unexplained germplasm phenomena in the discontinuous evolution of the physiological manifestation of CAM. As can been seen from PI citations, our interest in CAM is not new. We hope that the result of our theoretical work may help make the difficult tasks of crop breeders a little easier.
Impacts
Chloroplast division is a crucial step, since seeds have very few chloroplasts, and the mature plant requires many times more. Contrary to what is often assumed, chloroplasts do not function continuously but require rest and recovery. In our work, we have found that in order to get highest productivity, plants need high levels of functional chlorophyll and thus chloroplasts. Such could lead to better understanding of how to enhance photosynthetic properties that influence crop yield. CAM evolution investigations appear to open a window on the puzzle of cryptic genes, which may well help crop breeders plan experiments to re-express "lost" or hidden traits of use in agriculture. Taxol investigations mass spectroscopy studies show that extraction of paclitaxel (generic technical term for Taxol) is dependent on pH and the formation of ammonium ion and other adducts and dimers. It is highly probable that paclitaxel exists in plants as an ammonium ion dimer with a little
more than twice the expected molecular weight. The array of paclitaxel adducts and degradation products found is most complex and theoretically interesting. We have proposed at the NC-1132 meetings (see ORE00488A report) the novel concept that that Taxol has a regulatory role in plant growth.
Publications
- Zhang, J. 2005. Degradation kinetics of Taxol using mass spectroscopy. MS thesis, Department of Horticulture, Oregon State University.
- Zheng, P. 2005. Imaging fluorescent determination of energetics of chloroplast division and related matters. PhD thesis, Department of Horticulture, Oregon State University.
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Progress 01/01/04 to 12/31/04
Outputs
Work continues on three principal fronts: 1) The important implications to agriculture of the monophyletic theory of CAM evolution. In this theory it is postulated that the more than forty different reported 'evolutions' of CAM in plants from the most primitive to the most advanced are based upon hidden (cryptic) information conserved in most plant specie's germplasm. If this theory proves correct, as it seems in all likelihood to be, the cryptic coding of DNA in the germplasm of plants, may well contain far more information than is generally believed. This circumstance not only provides a basis for research but in retrospect explains the enormous success of applied empyrical plant breeders. 2. Our discover of paclitaxel (Taxol) in hazel trees not only provides a potential alternate source for this important anticancer medicine, but suggests that plant growth may well be regulated by this substance. One mechanism of action may be the control of the activity of
mitochondria in the plant cytoskeleton. 3. The in vivo measurement of chloroplast division is expected to become useful in crop development studies.
Impacts
We expect that the three main finds above will impact the thoughts and work of young plant crop investigators in numerous and useful ways.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Three main research topics are underway: 1) The imaging fluorometric detection of chloroplast division. A program to automatically quantify the phenomena from confocal data is the last part of Ping Zheng's PhD thesis and MS degree in Computer Science. 2) Theoretical considerations for mono-phyletic Crassulacean Acid Metabolism (CAM) evolution are being developed. The present orthodox interpretation is that independent polyphyletic (multiple) evolution took place. This polyphyletic theory postulates numerous independent evolutions throughout the plant kingdom from most primitive to most advanced higher plants. As more and more CAM species are found in diverse families, the theory of polyphyletic evolution of CAM view is beginning to lose adepts. In the mono-phyletic theory, CAM is coded in the genome of all or most all plant species, and this property can be expressed or not depending on environmental conditions and expression or reorganization of 'controller' genes.
Were this theory to prove correct, considerable gene manipulation of CAM to better fit agricultural needs may well be possible. A paper in preparation on the evolution of Crassulacean Acid Metabolism suggests that germplasm genetic DNA codes may contain sequences which, if activation were possible, would allow drought resistance of a high order. While this is very far from practical use, it may have eventual and significant crop applications. 3) Research on on the presence of Taxol in Corulys angiosperm continues as we try to increase synthesis of this highly valued medicine in hazelnut trees. We are also trying to develop a track record of publications to support grants to seek genes useful to Oregon from other areas of the world.
Impacts
We have developed a method of determining chloroplast division in vivo which has promise of a more sensitive and immediate measure of crop productivity. Our work very strongly suggests that we can determine the presence of chloroplast division in vivo using our patented imaging fluorometer. Although still in the testing stage, this method promises to be a sensitive indicator of the vigor of crop plants at critical points in their development. Basic biophysics of germplasm holds many surprises. Our method of determining chloroplast division in vivo has promise of a more sensitive and more immediate measure of crop productivity. Plant breeders have long taken an empiric approach to crop improvement. Our work on CAM evolution suggests that this is based upon cryptic codes commonly unexpressed and unused in the genome which are turned off and on by epigenetic processes. Our objective is to develop technologies to facilitate Taxol production from hazelnut (filbert) trees.
The reason for this is that Taxol is a very important cancer fighting drug. Taxol is expensive to make from Yew. However, some years back we also found Taxol in filbert. No industry support developed, so we did more work. For growers to sell filbert Taxol, we need appropriate technologies. Thus we need to know something about best growth conditions and best part of plant to extract. We chose to do this by precise and sensitive mass spectroscopy. We are working out methods to facilitate extraction of Taxol from filbert tissues.
Publications
- Zheng, P., Thomas, D.R., and Daley, L. 2003. Tests of in vivo method to detect chloroplast division in crop plants: Part 3. Statistical methods used to define multiple populations of chloroplast efficiencies Spectroscopy 17.
- Daley, L. 2003. Restoration of Cuban gallery forests, especially on the banks of the Bayamo and other rivers of the Cauto Basin Cuba. Transition 12:225-232.
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Progress 01/01/02 to 12/31/02
Outputs
Two main research topics are underway: 1. The imaging fluorometric detection of chloroplast division (see references) is being verified by confocal microscopy. This requires development of a de novo program to automatically quantify the phenomena from confocal data. A very well qualified PhD student Ping Zheng (see references) is undertaking a additional Masters degree in Computer Science to carry out this purpose. 2. Theoretical considerations for mono-phyletic CAM evolution are being developed. The present orthodox interpretation is that independent polyphyletic (multiple) evolution took place. This polyphyletic theory postulates numerous independent evolutions throughout the plant kingdom from most primitive to most advanced higher plants. As more and more CAM species are found in diverse families, the theory of polyphyletic evolution of CAM view is beginning to lose adepts. In the mono-phyletic theory, CAM is coded in the genome of all or most all plant species,
and this property can be expressed or not depending on environmental conditions and expression or reorganization of "controller" genes. Were this theory to prove correct, considerable gene manipulation of CAM to better-fit agricultural needs may well be possible. Research on Taxol production in Corylus continues, increased growth effects ginkgolide content is being written up and germplasm research among tropical gallery forests has been initiated.
Impacts
Basic biophysics of germplasm holds many surprises. We are developing a method of determining chloroplast division in vivo which has promise of a more sensitive and more immediate measure of crop productivity. Plant breeders have long taken an empiric approach to crop improvement. Our work on CAM evolution suggests that this is based upon cryptic codes commonly unexpressed and unused in the genome.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
PHOTOSYNTHESIS. Our work very strongly suggests that we can determine the presence of chloroplast division in vivo using our patented imaging fluorometer. Although still in the testing stage, and only basic science publications have resulted so far, this method promises to be a sensitive indicator of the vigor of crop plants at critical points in their development. GERMPLASM. A paper in preparation on the evolution of Crassulacean Acid Metabolism suggests that germplasm genetic DNA codes, putatively present but not expressed by most plants, may contain sequences which, if activation were possible, would allow drought resistance of a high order. While this is very far from practical use, it may have eventual and significant crop applications. NATURAL PRODUCTS. Work on the presence of taxol in angiosperm continues as we try to increase synthesis of this highly valued medicine in hazelnut trees. A model system using ginkgo plants shows that simple manipulation of
fertilizer levels can change ginkgo metabolites (a matter in itself of some commercial value); and implies that such treatments may be able to increase taxol in these trees to levels that will attract commercial interest.
Impacts
This research is expected in the short-term to increase interest in 'medical crops' in Oregon. In the long-term, it is expected to increase the quality of these very valuable products of Oregon agriculture.
Publications
- Zheng, P., K. Ammar, A-M.M. Girard, C. Wetzel, S. Rodermel, D.R. Thomas, L. Ning, J.B. Callis, G.E. Edwards, and L. Daley. 2002. Tests of in vivo method to detect chloroplast division in crop plants: Part 2. Verification of phenomena by germplasm methods and confocal microscopy. Spectroscopy (in press).
- Ning, L., G.E. Edwards, and L. Daley. 1999. Oregon State University Patent. U.S. Patent No. 5,981,958 . For: METHOD AND APPARATUS FOR DETECTING PATHOLOGICAL AND PHYSIOLOGICAL CHANGE IN PLANTS. Issued Nov. 9, 1999.
- Ning, L., G.E. Edwards, and L. Daley. 1998. Oregon State University Patent. U.S. Patent No. 5,854,063. For: METHOD AND APPARATUS FOR SPECTROPHOTOMETRIC OBSERVATION OF PLANTS. Issued: Dec. 29, 1998.
- Hoffman, A., W. Khan, J. Worapong, G.Strobel, D.Griffin, B. Arbogast, R.B. Boone, L. Ning, P. Zheng, and Larry Daley. 1998. Bioprospecting for taxol in angiosperm plant extracts. Spectroscopy 13(6):22-32.
- Bowyer, W.J., L. Ning, L.S. Daley, G.A. Strobel, G.E. Edwards, and J.B. Callis. 1998. In vivo fluorescent imaging for detection of damage to leaves by fungal phytotoxins. Spectroscopy 13(11):36-44.
- Ning, L., B.E. Petersen, G.E. Edwards, L.S. Daley, G.A. Strobel, and J.B. Callis. 1997. Recovery of digital information stored in living plant leaf photosynthetic apparatus as fluorescence signals. Applied Spectroscopy 51(1):1-9.
- Ning, L., W.J. Bowyer, A.M. Chozinski, T. Buban, A. Azarenko, G.E. Edwards, L.S. Daley, G.A. Strobel, and J.B. Callis. 1997. Five novel applications of imaging visible and short near infrared spectrophotometry and fluorometry in the plant sciences. Part II. Non-invasive in vivo applications. Spectroscopy 12(1):37-46.
- Zheng, P., K. Ammar, A-M.M. Girard, C. Wetzel, S. Rodermel, D.R. Thomas, L. Ning, J.B. Callis, G.E. Edwards, and L. Daley. 2002. Tests of in vivo method to detect chloroplast division in crop plants: Part 1. Discovery of the phenomena. Spectroscopy (in press).
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Progress 01/01/94 to 12/30/94
Outputs
ORE00322 The work we do describes plant germplasm biochemically and biophysically. There were three main topics in the present report period. 1) We found a small molecular weight protein with oral toxicity for insects suitable for bioengineering. We are exploring mode of action to distinguish this protein from BT toxins. We have been advised to withhold details pending possible patent procedures. 2) We are using a bacterial organism capable of provoking brown stain symptoms in hazelnuts (filberts) to explore hypersensitive defense mechanism in hazel varieties. 3) We have developed several instruments to determine germplasm differences with absorption, fluorescence, and interferometry. Patent procedures have been initiated for one instrument, another is being developed to support models of remote sensing of sites of plant germplasm diversity.
Impacts
(N/A)
Publications
- GARDEA, A. A., DALEY, L. S., KOHNERT, R. L., SOELDNER, A. H., NING, L., LOMBARD,P. B., and AZARENKO, A. N. 1994. Proton NMR signals associated with eco- and en.
- NING, L., DALEY, L. S., LU, Z., and CALLIS, J. B. 1994. Imaging of the interior of Trandescantia zebrina Bosse leaves by optical cross correlation interferometry. Biochem. Biophys. Res. Communic. 205(1):638-644.
- GARDEA, A. A., MORENO, Y. M., AZARENKO, A. N., LOMBARD, P. B., DALEY, L. S., andCRIDDLE, R. S. 1994. Changes in metabolic properties of grape buds during development. J. Amer. Soc. Hort. Sci. 119:756-760.
- NING, L., OZANICH, R., DALEY, L. S., and CALLIS, J. B. 1994. Construction of an imaging spectrophotometer and its application to plant sciences. Spectroscopy 9(7):41-48.
- ESKELSEN, S. R., CRABTREE, G. D., BOONE, R. B., VOLK, G. M., NING, L., and DALEY, L. S. 1994. Visible spectroscopy of herbicides: Monitoring in vivo effects and modeling off-target movement. Spectroscopy 7(2):34-40.
- FENG, W., NING, L., DALEY, L. S., MORENO, Y., AZARENKO, A., and CRIDDLE, R. S. 1994. Determination of effective temperature minima for CAM carboxylation in diverse plants by scanning microcalorimetry. Plant Physiol. Biochem. 32:319-330.
- FENG, W., NING, L., DALEY, L. S., MORENO, Y., AZARENKO, A., and CRIDDLE, R. S. 1994. Theoretical fitting of energetics of CAM path to calorimetric data. Plant Physiol. Biochem. 32(4):591-598.
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Progress 01/01/93 to 12/30/93
Outputs
Brown stain of filberts (hazelnuts), reported for over 50 years, is a pathological condition with no known causal agent and a filbert germplasm variable response. Brown stain results in small, 5- 10%, crop losses most years and severe losses, up to 30%, in occasional seasons. We have isolated a gram negative bacteria from early stages of this condition. This bacteria is capable of causing extensive browning when reinoculated into later stage nuts. The bacteria is resistant to thionins (see below) and lysozyme, but is inhibited by EDTA. We cannot yet be sure that this bacteria is the causal agent of the condition; we will attempt to find this out by inoculating into early stage immature nuts this spring. We have been working on extracting gram quantities of thionins from diverse plant germplasm. Thionins are small molecular weight proteins suitable for genetic engineering. Thionins are found in food and are known to be active against some bacteria, fungi, and insects,
and these activities are a germplasm variable. We have had some small success in using thionins to inhibit growth of tobacco budworm. The challenge is to get the thionin through the very alkaline gut of this insect. Visible imaging spectroscopy of in vivo leaves is useful to identify germplasm and physiological parameters. We are working on hardware/software combinations for multiple linear regression analyses and to drive the wavelength scanning motor.
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
It was verified by a hand-held sucrose/glucose monitor specifically adapted for this purpose that sucrose content of filbert stems is related to eastern filbert blight susceptibility. This was found in previous years by mass spectroscopy. However, this year's work allows field testing for this effect. This device has many other potential applications in agricultural field testing. Microcalorimetric evidence very strongly suggests that ornamental CAM plants have enzymes suitable for transgenic passage into C(subscript 4) crop plants. Ginkgo germplasm has active protein suitable for transgenic passage into root weevil susceptible ornamentals.
Impacts
(N/A)
Publications
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Progress 01/01/91 to 12/30/91
Outputs
Eastern filbert blight (EFB) susceptibility was correlated to a stem disaccharide, probably sucrose. This was done using a program written to correlate mass spectra to field tested evaluations of EFB resistance. Theoretical and applied considerations of this data are being considered. Some internal reports are available on request.
Impacts
(N/A)
Publications
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Progress 01/01/90 to 12/30/90
Outputs
Eastern Filbert Blight (EFB) susceptibility/resistance biochemistry. Mass spectroscopy of the ethanol soluble neutral fraction shows promise as a marker (MW 663) for cv. Gasaway. The marker, as in previous work, is not found in Gasaway. Yet it is found in all susceptible cultivars tested. A second marker (MW 514) is found in Gasaway and not in susceptible cultivars. Since the kind of mass spectroscopy used is very simple and rapid, this method holds great promise. In vivo spectroscopy has defined a band for P-700. Electrophoresis has resolved (with a second more rapid method) clones of Pinot noir grapes, in addition, electrophoresis has been used to distinguish mixed breeding lines of phylloxera resistant grapes.
Impacts
(N/A)
Publications
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Progress 01/01/89 to 12/30/89
Outputs
Eastern filbert blight (EFB) susceptibility/resistance biochemistry. Evaluation of our data (see last year's report) suggests that cv. Ennis susceptibility is due, at least in part, to fungal growth promoting substances. Although we have good molecular weights estimates, the nature of these substances is not known. However, this information implies that removal of these substances, may convey resistance to the pathogen. If this is correct, then mutational breeding strategies may be employed. A large amount of material was extracted to investigate the nature of these compounds & their utility as markers for screening resistant cultivars. Other germplasm investigations In vivo analysis of chlorophyll protein spectra has been successfully integrated with in vivo work and a major (22 band) assignment of spectra has been accepted by an international journal. This method permits easy comparisons of our in vivo photosynthetic data on germplasm, nitrogen metabolism and
herbicide action to in vitro work of others.
Impacts
(N/A)
Publications
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Progress 01/01/88 to 12/30/88
Outputs
Eastern filbert blight (EFB) susceptibility / resistance biochemistry. Filbert (Corylus) bark/wood was tested for inhibitory (inh)/ stimulatory (stim) activity. Since EFB fungus Anisograma anomala cannot yet be grown rapidly in vitro, tests were done with Rhodoforula rubrum. Specificity was estimated by comparing fungal growth on Tractions (frac) from EFB resistant cv. Gassoway (G) with susceptible cv. Ennis (E) frac. Water, methanol (MeOH), pentane soluble frac were prepared. Mild inh activity found in MeOH frac. Ion exchange treatment of MeOH frac yielded neutral frac with mild inh activity. Neutral frac run on exclusion get molecular weight (MW) calibrated column yielded: 2 growth stim factors (mw 235, 933) in E not G frac, inh factor (mw 213) in G not E frac. Other. Pyrus germplasm electrophoresis complete; data exceeds goals. Video images of all isozyme data and other deliverables to NCGR RL (M. Couey) as scheduled. Work (with T. Righetti and F. Dixon) shows
enhanced isozyme images may identify Pinot noir Vitis clones. Started investigations on microcalorimetry of temperature sensitivity differences of Anjou / Bartless twigs and in vivo spectroscopy of herbicide action on chl-complexes.
Impacts
(N/A)
Publications
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Progress 01/01/87 to 12/30/87
Outputs
Electrophoretic analysis - Chemical fingerprinting of more than 500 pear (Pyrus)and pear relative (accessions by peroxidase, esterase, and acid phosphatase isozymic patterns). Findings include: determination of cultivar duplication, gene pool size estimates, fingerprinting patterns. In vivo spectroscopic analysis - Spectral characterization of strawberry (Fragaria) germplasm with respect to heat tolerance, and specific site of June yellows genetic damage to chlorophyll protein complexes. Determination of genetics of anthocyanins in, and biochemical consequences of, freeze damage to hazelnut (Corylus) tissues. Other analyses - Analysis of soluble solids, titratable acidity, and ascorbic acid content of berries, was performed on over 100 accessions of Fragaria, Rubus, and Ribes. Electron microscopy results suggest pollen micropyle differences may help identification of filbert. Analysis of photosynthetic carbon fixation data from Pyrus species: P. calleryana, P.
koehnei, and P. ussuriensis and from cultivar 'Bartlett' are consistent with RuBPCase as the limiting factor in pear photosynthesis (a presently accepted concept in many other species). This permits use of RuBPCase temperature response to characterize Pyrus germplasm.
Impacts
(N/A)
Publications
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Progress 01/01/86 to 12/30/86
Outputs
Development of the first method of determining chlorophyll-protein complex mutations in intact leaf lamina. This method has demonstrated: a) which protein chlorophyll complex is involved in the recessive field lethal aurea gene of the most common filbert variety Barcelona, and b) that June yellows of strawberry are genetic defects of chloroplast structure, which have different ancestral origins. Produced the first, all season, high resolution, 'fingerprinting' method for hazelnut clones (with R. Menendez and Z. Ahmed). All hazelnut Corylus accessions of sufficient size for analysis have been fingerprinted. None were found to be identical. Chemical fingerprint of Pyrus accessions include: Of 52 groups of suspect duplicated (or triplicated) accessions. 30 groups were identical and 22 (44 accessions were different. All P. calleryana accessions in the Repository collection were examined. Stem isozymes of red fruited pear cultivars of principal Oregon varieties were
found to be identical to those of corresponding non-red cultivar accessions found in the collection. Spectroscopic leaf analysis permits resolution of red- from non-red-fruited varieties.
Impacts
(N/A)
Publications
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Progress 01/01/85 to 12/30/85
Outputs
Identification: A method of plant fingerprinting utilizing gradient polyacrylamide gel electrophoresis has been adapted. This method offers superior resolution and has been applied successfully to about 70 cultivars of Pyrus. Characterization methods have been developed for/to: a) in vivo determination of chlorophyll and anthocyanin, Corylus, Fragaria, b) improve non-radioactive assay for RuBPCase applicable to all genera, c) determine Siegel number, a measure of tolerance to low growing temperatures, Mentha, d) assay for cell wall invertase, putative pollen germination limiting factor, Corylus, e) determination of optimum range of temperature for net photosynthetic carbon fixation, Pyrus. Data collection: Vitamin C, titratable acidity, soluble solids, a total of 600 assays on genera Rubus, Ribes, Vaccinium.
Impacts
(N/A)
Publications
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