Proc. Fla. State Hort. Soc. 92tl40-143. 1979.

PROTEIN EXTRACTION FROM WATER SPINACH (IPOMOEA AQUATICA)

J. H. Bruemmer and B. Roe cucumber leaves or about 5 to 9% of their DM (14). Par- U. S. Citrus and Subtropical Products Laboratory1 ticulate proteins are more than twice as abundant as soluble P{. O. Box 1909, proteins, but cannot be solubilized and extracted unless Winter Haven, Florida 33880 the lipoprotein and glycoprotein complexes in the leaves are ruptured. Lipid solvents dissociate lipid/protein bonds Abstract. Extractability of protein in water spinach (Ipo- and are used routinely in the treatment of biological tissue moea aquatica Forsk., I. reptans Poir), a possible source of for extraction of enzyme protein (12). Because plant pig food protein concentrate was examined. Fresh and fresh ments are soluble in lipid solvents, treatment of water frozenleaves were extracted by the conventional buffer- spinach with solvents should remove color as well as in homogenate method at room temperature and by a method crease the extractability of protein. We report on screening involving pre-treatment of the leaves with polar and non- of a variety of organic solvents for increasing extractability polar solvents. Pre-treatment of leaves with dimethylsul- of protein from water spinach and on the discovery that foxide (DMSO) increased total yield of extractable N slightly one of the solvents is effective for extracting protein. and increased extractability of TCA-precipitable protein to 1% of the dry matter (DM). Hot DMSO extracts of fresh Materials and Methods water spinach contained protein equivalent to about 8% of the DM. Also, hot DMSO extracted more protein from ace Fresh water spinach was obtained from the Agricultural tone-dehydrated leaf meal than from oven-dried meal. Hot Research and Education Center (AREC), Belle Glade, FL. (284° F) DMSO extracts, upon dilution with 4 volumes of Leaves were washed, air-dried to remove adherent moisture, ethanol, yielded a protein precipitate that dried to a light and frozen for storage. These are referred to as fresh-frozen tan powder. leaves. Fresh leaves were also harvested from plants grown at our laboratory. Dimethylsulfoxide (DMSO) was obtained from Aldrich Chemical Company, Milwaukee, WI; other Water spinach (Ipomoea aquatica Forsk., /. reptans Poir) chemicals and reagents were from Fisher Scientific Company, is an aquatic plant found in marshy or wet sandy areas or Pittsburg, PA. floating on water in many parts of tropical Asia, Africa, Moisture content was determined on 3.5 oz (100 g) Oceania, and the Americas. It is also known as swamp samples of water spinach leaves by drying at 140°F (60°C) cabbage (18), ung choi in Hawaii, can cong in the Philli- in vacuum to constant dry weight. pines, and ung ts'oi in China (17). It is cultivated in south Protein was determined by the biuret method (7) and by east Asia where it is an important summer vegetable, and Potty's method (13). Amino acids and small peptides were constitutes 15% of Hong Kong's vegetable output (5). determined by the method of Spies and Chambers (15). Morton and Snyder (11) suggested that, as an alternative to sugar cane and other terrestrial crops, water spinach be planted in the Everglades Agricultural Area for use as a Kjeldahl-N vegetable and for preventing organic soil subsidence. We A 3.5 x 10~3-oz (100 mg) sample was digested with 0.14 oz became interested in water spinach as a possible source of (4 ml) cone H2SO4. The digest was diluted to 3.5 oz (100 food protein concentrate. The edible portion can contain ml) with ammonia-free water, and the ammonia content 29% protein on dry matter (DM) basis (18) and may be determined with Orion electrode Model 95-10, Orion as suitable a source of food protein as alfalfa leaves (22.3% Company, Cambridge, MA. An aliquot of diluted digest crude protein) (10). Although alfalfa contains about 7% [0.35 oz (10 ml)] was added to a pre-read standard contain water-soluble protein (DM), only about 2% of the DM ing 0.035 oz (1 ml) of 100 ppm ammonia N in 3.5 oz (99 was recovered as a "white" food grade concentrate in ml) 0.4 M NaOH. Several readings were taken for each pilot-plant studies (6). Water spinach has a lower fiber sample according to the procedure in Orion Company's content than alfalfa (18, 10). Fiber decreases protein ex Application Bulletin 17: low level Kjeldahl analysis. tractability by lowering the effectiveness of grinding to rup ture cells and release protein (8). It also filters out protein- Soluble Cytoplasmic Protein rich chloroplasts from the liquor. Soluble cytoplasmic proteins constitute about 30% of A 3.5 oz (100 g) portion of fresh leaves was frozen in the total protein inspinach, tobacco, tomato, gherkin, and liquid N2 [-320°F (-196°C)] and milled to a fine powder in an analytical micromill (Scientific Products, Inc., Mc- Graw Park, IL) until no intact cells were observed in thawed iSouthern Region, Science and Education Administration, U. S. Department of Agriculture. samples viewed under the microscope. The frozen powder We thank Dr. G. H. Snyder of the University of Florida for provid was thawed in 18 oz (500 ml) of 0.1 N Tris buffer, pH ing fresh water spinach, and A. W. Griffin for technical assistance. 7.4, at 4°C containing 75 mM ascorbic acid, 6.6 mM cysteine Mention of a trademark or proprietary product is for identification HC1 and 142 mM mercaptoethanol. The suspension was only and does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture and does not imply its approval kept at 4°C during the extraction. After blending for 1/2 to the exclusion of other products which may also be suitable. min at high speed in a household blender, the homogenate

140 Proc. Fla. State Hort. Soc. 92: 1979. was centrifuged at 50,000 G for 30 min. The supernatant [0.0088 oz (250 mg)] was suspended with stirring in 0.5 oz was decanted and the residue was washed once by blending (15 ml) hot DMSO. After 3 or 30 min the suspension was in 7 oz (200 ml) of 0.1 M Tris buffer, pH 7.4, and then cooled and centrifuged at 50,000 G for 10 min. The super- centrifuging as before. The wash buffer and initial super nate was decanted and 4 vol of 95% ethanol were added to natant were combined and 50% trichloroacetic acid (TCA) precipitate the proteins. The ethanolic mixture was stored was added so that its final concentration was 10%. Protein for 18 hr at 40°F (4°C) and the protein was pelleted by precipitated by the TCA was pelleted by centrifugation centrifugation, recovered, and dissolved in 0.1 N NaOH (50,000 G, 15 min) and dissolved in 0.1 N NaOH for pro for analysis. tein assay.

Results and Discussion Protein Extraction at 20° C Water spinach grown in tanks at AREC-Belle Glade A 3.5-oz (100 g) portion of fresh or fresh frozen leaves contained 83.2 dz 1.4% moisture and 0.68 ± 0.05% was passed through a manually operated meat grinder with Kjeldahl-N or 25.1% crude protein (N x 6.25) on DM the aid of 1.8 oz (50 ml) of 0.1 M carbonate-bicarbonate basis. Tris buffer extract of 3.5 oz (100 g) of fresh (frozen- (CB) buffer, pH 9.5 (4). Another 1.8 oz (50 ml) of the milled) leaves contained 30 x l(H-oz (780 mg) TCA-pro- buffer was used to wash the adhering leaf pulp from the tein. Thus, only 4.8% of the DM was recovered as cyto- grinder. The ground pulp and wash water were combined plasmic, soluble protein. This value is lower than the and homogenized in a household blender at high speed for average amount reported for terrestrial plant leaves (14). 2 min. The homogenate was centrifuged at 50,000 G for 30 Low recovery of protein is not peculiar to water spinach. min, and 50% TCA was added to the supernatant so that Boyd (1) examined 22 species of aquatic plants for protein its final concentration was 10%. Protein precipitated by the extractability and obtained satisfactory results with only TCA was pelleted by centrifugation (50,000 G, 15 min) 6 species. and redissolved in 0.1 N NaOH for protein assay. Tempera ture of the extraction procedure was controlled at 68°F Pre-treatment of Leaves With Organic Solvents (20°C). Protein was extracted from 35 x 10~3-oz (1 gm) samples Pre-treatment of fresh-frozen water spinach leaves with of leaf meals by homogenizing the meals with 10 ml CB organic solvents at 77°F (25°C) did affect protein extract- buffer for 2 min at high speed in a household blender and ability in aqueous buffer (Table 1). All solvents suppressed then centrifuging the homogenate at 50,000 G for 30 min aqueous extraction of Kjeldahl-N, but only DMSO in-

to obtain the extract. TCA (50% sol) was added to the Table 1. Aqueous extraction of Kjeldahl-N from solvent treated leaves.^ extract so its final concentration was 10%. Protein pre cipitated by the TCA was pelleted by centrifugation (50,000 G, 15 min) and dissolved in 0.1 N NaOH for protein assay. Nitrogen distribution (% total) Total N Water Pulp Solvent extracted

Solvent extract residue extract Protein Extraction After Solvent Treatment %

Fresh frozen water spinach leaves [0.88 oz (25 g)] were O 17 83 0 17 homogenized with solvent [0.44 oz (12 ml)] in a household DMSO 15 76 9 24 blender at high speed for 1 min. The leaf pulp was recovered Ethanol 5 89 6 11 Propanol 8 84 8 16 from the homogenate after centrifugation (50,000 G, 30 2-Propanol 6 86 8 14 min), and then homogenized with 0.35 oz (10 ml) 0.1 M CB Butanol 6 86 8 14 buffer, pH 9.5, in a household blender at high speed for 1 Acetone 6 87 7 13 min. The homogenate was centrifuged (50,000 G, 30 min), Hexane 12 84 4 16 and the supernatant decanted from the pulp residue and lyophilized. The residue and lyophilized extract were z0.88 oz (25 g) leaves blended with 0.44 oz (12 ml) solvent, 1 min high speed blender. Solvent separated by centrifugation and the residue analyzed for Kjeldahl-N and after dissolving in 0.1 N NaOH extracted with 0.35 oz (J10 ml) 0.1 N CB buffer pH 9.5 to obtain water for protein. All procedures were carried out at room extract. Residues and water extracts were analyzed fbr Kjeldahl-N. temperature [77°F (25°C)]. Solvent extract N was obtained by difference [N in 0.88 oz (25 g) leaves minus N in water extract and in residue].

Dry Leaf Meals creased total N extracted from the leaves. When the ratio of solvent to leaf was 1:2 (v/w), all solvents removed Fresh-frozen leaves [1.8 oz (50 g)] were homogenized with Kjeldahl-N from the leaf pulp, thereby decreasing the 21 oz (600 ml) acetone in a household blender for 1 min. amount of N extractable with buffer. The aqueous extract The homogenate was heated to 122°F (50°C) for 10 min, after DMSO treatment contained almost as much N as cooled, and filtered. The residue was reextracted with 10 oz the extract from untreated pulp. At least part of the N (300 ml) acetone by the same procedure of homogenization, in both solvent and aqueous extracts was due to amino acids heating and filtration. The acetone-dehydrated leaf meal and small peptides because we obtained a positive test for was then dried at 77°F (25°C). For the preparation of oven- their presence. Plant leaves are assumed to contain about dried meal, fresh frozen leaves were chopped into approxi 30% N as non-protein N (6). mately 0.4-in.2 (1 cm2) pieces and dried in a vacuum oven DMSO was more effective as a pre-treatment for at 140°F (60°C) for 18 hr. The dried pieces were then milled (Wiley Intermediate Lab Model, Arthur H. Thomas aqueous extraction of N when the ratio of solvent to leaves (v/w) was 1:1 or greater father than 1:2 or lower (Table Company, Philadelphia, PA) through 60 mesh screen [0.1 x 10"3 in. (250 micron openings)] and stored in a 2). As much as 30% of leaf N was extracted from leaf pulp with DMSO (2:1 v/w) and 0.1 M CB buffer, pH 9.5. desiccator. DMSO extracted more N at 122°F (50°C) than at 77°F Hot DMSO Extraction (25°C) (Table 3). Duration of incubation was not an im portant factor in the extraction. At 122°F (50°C) DMSO Fresh chopped leaves [0.035 oz (1 gm)] or leaf meal removed almost 20% of the N content of fresh leaf pulp.

Proc. Fla. State Hort, Soc. 92: 1979. 141 Table 2. Effect of solvent-to-leaf ratio on aqueous extraction of Protein Extraction With Hot DMSO Kjeldahl-N.* DMSO is an effective solvent for the water-soluble pro tein albumin at 320°F (160°C); solutions of 5% albumin Nitrogen distribution (% total) in DMSO have been prepared (2). This observation and our Water Solvent detection of protein in the 122°F (50°C) DMSO extract DMSO/leaf (v/w) ratio extract Residue extract of water spinach prompted us to examine DMSO as a solvent for extracting protein from water spinach. 1:3 16 78 6 1:2 16 78 6 DMSO extraction of protein from water spinach was 1:1 20 72 8 time and temperature dependent (Table 4). After 3 and 30 2:1 20 70 10 Table 4. Effect of time and temperature on protein extraction with hot DMSO.z ^Conditions described in Table 1, except solvent-to-leaf ratio was varied. Values are means of three analyses. Protein; % of DM

Table 3. -Time and temperature effects on DMSO extraction of Acetone-dehydrated Kjeldahl-N. Fresh leaves meal Oven-dried meal Temp, °C 3 min 30 min 3 min 30 min 3 min 30 min

Nitrogen distribution (% total)5 5 Equilibration time 25°C 50°C 160 3.1 4.6 4.6 4.8 2.8 3.4 160 6.2 6.5 5.4 6.1 3.2 3.6 hr Residue Solvent Residue Solvent 180 7.9 8.1 6.5 7.7 4.3 4.5

0.1 94±8 6 ±0.5 84±7 16±2 *8.8 x 10-3 oz (250 mg) leaf meal or 35 x 10-3 oz (1 g) fresh chopped 1 93 ±7 7 ±0.5 86±9 14±3 leaves was suspended with stirring in 0.52 oz (15 ml) DMSO at tempera 2 92±8 8 ±0.5 82±8 18±2 ture indicated for 3 or 30 min. Then the suspension was cooled to 77°F 4 94±8 6 ±0.5 81±8 19±2 (25°C) and centrifuged to separate extract. Protein was precipitated from extract with 4 vol ethanol (80% ethanol, final cone), separated by z3.5 oz (100 g) of fresh leaves was blended with 3.5 oz (100 ml) DMSO; centrifugation and dissolved in 0.1 N NaOH for protein assay. Values and the homogenate transferred to 16 tubes to contain 0.35 oz (10 ml). are means for three samples. Series A of 8 tubes was incubated at 77 °F (25 °C). Series B of 8 tubes was incubated at 122°F (50°C). Two tubes of each series were removed min at 284°F (140°C), 3.1 and 4.6% of the DM of fresh after 0.1, 1, 2 and 4 hr and centrifuged. Residues were analyzed for Kjeldahl-N. Solvent values were obtained by difference: N in 0.35 oz leaves was extracted as protein; after 30 min at 356°F (10 ml) homogenate - N in residue. Values are means ± SD of 3 (180°C), 8.1% of the DM was extracted. DMSO extracted analyses of two tubes. more protein from fresh leaves than from the leaf meals, even though the leaf meals were finely milled and the fresh Some of this N was protein-N because the 122°F (50°C) leaves were chopped. DMSO extracted more protein from DMSO extract gave a positive test for protein. Most of the acetone-dehydrated leaf meal than from the oven-dried meal, N extracted with DMSO at 77°F (25°C) was probably which had been heated to 140°F (60°C). Drying at this amino acid-N and other non-protein N, because no protein temperature apparently decreased protein extractability. was detected in those extracts. These data indicate that These data show that hot DMSO extracted about 32% the DMSO pre-treatment should be applied at room temper (8-J% -r- 25.1% x 100) of the crude protein as true protein ature or below to prevent loss of protein in the pre-treat- (Biuret). Since only 19% (4.8% -f- 25.1% x 100) of the ment. crude protein was identified as cytoplasmic soluble, a large When 3.5 oz (100 g) of fresh water spinach was pre- portion of the protein in the DMSO extract came from treated with DMSO (1:1 v/w) at 68°F (20°C), the subse particulate protein. Thus, DMSO could be more effective quent extraction with 0.1 M CB buffer, pH 9.5, yielded than aqueous systems for extraction of plant proteins. 6 x 10~3 oz (160 mg) TCA-protein, an amount equal to Heating fresh leaves in DMSO at 320°F (160°C) and about 1% of the DM. This procedure represents a slight im 356°F (180°C) caused a noticeable browning reaction that provement over direct extraction of leaves with buffer darkened the protein concentrate. The protein concentrate [5 x 10~3 oz (130 mg)], but considerably less than the avail from 284°F (140°C) extraction dried to a tan, off-white able cytoplasmic soluble protein (780 mg). Apparently, powder, whereas the concentrates from 320°F (160°C) and homogenization in a household blender at 68 °F (20° C) 356°F (180°C) extractions yielded progressively darker without safeguards against enzymic degradation and de- powders. We plan to determine the conditions that optimize naturation decreased extractability of protein. DMSO pre- yield and improve appearance of the concentrate and to treatment removed some color from the pulp residue such evaluate the nutritional value of the protein. that the aqueous extract contained less color than extracts Our finding that hot DMSO could extract 8% of the DM of leaves without pre-treatment. of water spinach as protein should be encouraging to agri culturists and sanitary engineers interested in the economic Protein Extraction From Dry Leaf Meal aspects of using aquatic plants grown to control nutrient run-off into lakes from agricultural lands and from waste Aqueous extracts (pH 9.5) removed about 2 x 10~3 oz treatment plants. Water spinach grows prolifically and acts (62 mg) of TCA-protein from 0.6 oz (16.8 g) of oven-dried as a living sponge for water soluble nutrients. Beds of water meal prepared from 100 g fresh frozen leaves. Thus, protein spinach immediately lowered NO3-N content of NO3-N- was only about 50% as extractable from dried leaf meal as enriched water passed through the beds (16). Although cat from fresh leaves. Lu and Kinsella (9) recovered about one- tail (Typha spp.) and water hyacinth (Eichhornia crassipes) third as much protein in extracts of alfalfa meal dried at are currently being used in field tests to remove nutrients 167°F (75°C) as the amount was obtained from extracts from drainage water, water spinach should have higher of fresh alfalfa leaves. In our work, pre-extraction with economic value on protein basis. Water spinach has much DMSO did not improve extractability of protein in the dry more DMSO-extractable protein than both of these aquatic leaf meal by pH 9.5, CB buffer (data not presented). plants (3).

142 Proc. Fla. State Hort. Soc. 92: 1979. Literature Cited 9. Lu, P. and J. E. Kinsella. 1972. Extractability and properties of protein from alfalfa leaf meal. /. Food Sci. 37:94-99. 1. Boyd, C. E. Leaf protein from aquatic plants. Leaf Protein IBP 10. Morrison, Frank B. 1951. Feeds and Feeding, Morrison Publishing Handbook #20. Ed. by N. W. Pirie, Blackwell Scientific Publica Company, Ithaca, New York, 21st Ed., p 1086. tions, Oxford, p. 9. 11. Morton, J. F. and G. H. Snyder. 1976. Aquatic crops vs organic soil 2. Bruemmer, J. H. 1979. Solubility of proteins in DMSO. (in prepara subsidence. Proc. Fla. State Hortic. Soc. 89:125-129. tion). 12. Morton, R. K. 1955. Extraction of enzymes from animal tissues. 3. and B. Roe. 1979. Protein extraction from aquatic Methods in Enzymology, Ed. by S. P. Colowick and N. O. Kaplan. plants. (In preparation). Vol. 1, p 25. 4. Delory, G. E. and E. J. King. 1945. Sodium carbonate-bicarbonate 13. Potty, V. H. 1969. Determination of proteins in the presence of buffer for alkaline phosphatases. Biochem. J. 39:245-247. phenols and pectins. Anal. Biochem. 29:535-539. 5. Edie, H. H. and B. W. C. Ho. 1969. Ipomoea aquatica as a veget 14. Singer, S. J., Eggman, L., Campbell, J. M. and S. G. Wildman. 1952. able in Hong Kong. Econ. Bot. 23(l):32-36. The proteins of green leaves. IV. A high molecular weight protein 6. Edwards, R. H., Miller, R. E., deFremery, D., Knuckles, B. E., comprising a large part of the cytoplasmic proteins. /. Biol. Chem. Bickoff, E. M. and G. O. Kohler. 1975. Pilot plant production of an 197:233-239. F edible white fraction leaf protein concentrate from alfalfa. /. Agric. 15. Spies, J. R. and D. C. Chambers. 1951. Determination of tryptophan Food Chem. 23:620-626. using photochemical development of color. /. Biol. Chem. 191:787- 7. Gornall, A. G., Bardawill, C. S. and M. M. David. 1949. Determina 789. tion of serum proteins. J. Biol. Chem. 177:751-766. 16. Snyder, G. H. 1977. Personal communication. 8. Kohler, G. O. and E. M. Bickoff. 1971. Commercial production from 17. Takeda, K. Y. 1975. Personal communication. alfalfa in USA. Leaf protein IBP Handbook No. 20. Ed. by N. W. 18. Watt, B. K. and A. L. Merrill. 1963. Composition of Foods, Agri Pirie Blackwell Scientific Publications, Oxford, p 69. cultural Handbook No. 8, USDA, Washington, D.C. p 61.

Proc. Fla. State Hort. Soc. 92:143-145. 1979.

PROLINE CONTENT IN FLORIDA FROZEN CONCENTRATED ORANGE JUICE AND CANNED GRAPEFRUIT JUICE1

S. V. Ting and R. L. Rouseff juices, its use as an index has been suggested by several Florida Department of Citrus, authors (1, 5, 6, 8, 15). AREC, P. O. Box 1088, Proline produces a yellow color with ninhydrin buffered Lake Alfred, FL 33850 at pH 5, and this color reaction was used by Moore and Stein (7) in the determination of proline separated by Abstract. Proline is the most abundant amino acid in column chromatography. However, the same reagent pro citrus juices, and has been suggested as one of the indices duces a blue or bluish red color with other amino acids. of orange juice purity by some European drink manufacturers. Ting and Deszyck (13) using a mixed standard of several Using a modified photometric method of acidic ninhydrin re major amino acids of orange juice determined proline along action on proline developed by Chinard, the proline concn of with the other amino acids. Chinard (3) developed a photo Florida frozen concnd orange juice (FCOJ) and canned sinale metric procedure for the estimation of proline using nin strength grapefruit juice were determined. The proline hydrin reagent with concentrated formic acid. Under the content in reconstituted FCOJ varied as much as two and half acidic condition with ninhydrin reagent proline first forms a fold from a low of around 60 mg/100 ml to as high as yellow color, followed by a red color having an absorption 150 mg/100 ml. Canned grapefruit juice was much lower in maximum at 515 nm. Onlv orinthine and hydroxyproline its proline content than orange juice, varying from about 20 interfere in this reaction. None of the other amino acids mg-60 mg/100 ml. With the exception of ornithine, other tested by Chinard produced any significant color with this amino acids found in citrus juices did not interfere in this method. Ouch (9) used this procedure to analyze proline in analysis. grape juice and wine. He found no significant interference Proline, 2, pyrollidine carboxylic acid, is the most from other grace amino acids in amounts normally present abundant of all amino acids in citrus juices (2, 14, 16). in that fruit. He also demonstrated that the red color de Vandercook and Price (14) found an average of 169 mg veloped need not be extracted with a water immiscible of proline in 100 ml of California Valencia orange juice solvent as previously proposed by Chinard (3), but instead but only 94 mg in the same amount of Florida orange juice. that the color may be diluted with isopropanol and Ting and Deszyck (13) using the procedure of Moore and measured directly. Wallrauch (15) found that the use of Stein (7) reported a range of about 45 to 75 mg proline in ethyl acetate to extract the color produced by the acidic 100 ml frozen concentrated orange juice (FCOJ) reconsti ninhydrin reaction was necessary for highly colored juices tuted to 11.8° Brix. Niedermann (8) and Koch (6) both with low proline content. found that proline values in orange juice might vary from The purpose of this paper is to report the use of this 2.5 to 3-fold among the samples. In grapefruit juice, Brenoe acidic ninhydrin method with modification and simplifica (2) reported values varying from 35 to 292 mg per 100 ml. tion to study the range of proline content in Florida FCOJ The use of concentrations of various chemical con and canned grapefruit juice as produced commercially. stituents of fruit juices as indices of juice authenticity or as Variations in proline content of orange juice due to fruit a measure of juice content in a fruit drink has been widely cultivars and maturity were also studied. The simplified accepted. For citrus juice, formol numbers, poly phenols, method could be easily used in most quality control labora some individual amino acids, and several minerals have tories equipped with an inexpensive colorimeter. been proposed either singly or in combinations for this Materials and Methods purpose (10). Since proline is a major amino acid of citrus Samples

iFlorida Agricultural Experiment Stations Journal Series No. 1995. One hundred samples of FCOJ and 80 samples of canned

Proc. Fla. State Hort. Soc. 92: 1979. 143