PLANT PHYSIOLOGY

In addition, damage by the harmful homogenizingtreatments and damage by sonication are not addi-tive; chloroplasts prepared at pH 7.3 are inactivatedfurther only a very small amount by sonication. Thismakes it appear that the harmful homogenizingtreatments, and sonic oscillation, attack at primarilythe same site.

These facts lead us to suggest that there may betwo pathways for dye reduction in the Hill reactionof red kidney bean chloroplasts. One of these path-ways is susceptible to damage by sonic oscillation, orby a pH below 8 during homogenization; the secondpathway is resistant to these treatments. The veryhigh rates of dye reduction which can be observedunder optimal conditions represent the sum of re-duction along both pathways.

A similar conclusion as to the complex nature ofthe reducing pathway in the Hill reaction has beenarrived at independently by T. Punnett on the basisof the pH optimum of different kinds of chloroplasts.

SUMMARYThe Hill reaction rates of red kidney bean chloro-

plasts prepared by usual procedures were found to below compared to those of spinach. Active chloroplastscould be prepared only if Versene was added to thehomogenizing buffer. Raising the pH of the grindingbuffer to 8 or over could substitute in part for thepresence of Versene.

Once the leaves were ground, no further changesin chloroplast activity could be induced by recipro-cation of the above treatment. Chloroplasts with lowactivity have apparently been damaged in a dark step,not in the light dependent part of the Hill reaction.

Sonic oscillation has only a slight effect on chloro-plasts already damaged by a harmful homogenizing

medium. On the other hand, it causes a rapid loss of80 % of the activity of highly active chloroplasts.After brief sonication both types of chloroplasts arereduced to the same activity which is then resistantto extended additional sonication.

Consideration of these phenomena leads to a sug-gestion that there may be two pathways for dye re-duction in the Hill reaction of red kidney bean chloro-plasts, one sensitive, and one resistant to these inacti-vating conditions.

LITERATURE CITED1. ARNON, D. I. Copper enzyemes in isolated chloro-

plasts. Polyphenol oxidase in Beta vulgaris.Plant Physiol. 24: 1-15. 1949.

2. GREENFIELD, ROBERT E. and PRICE, VINCENT E. Livercatalase III. Isolation of catalase from mito-chondrial fractions of polyvinylpyrrolidone-sucrosehomogenates. Jour. Biol. Chem. 220: 607-618.1956.

3. HILL, R. Oxygen evolved by isolated chloroplasts.Nature 139: 881. 1937.

4. HOLT, A. S. and FRENCH, C. S. Oxygen productionfrom chloroplasts. Arch. Boichem. 19: 368. 1948.

5. LUMRY, RUFUS, SPIKES, JOHN D. and EYRING, HENRYPhotosynthesis. Ann. Rev. Plant. Physiol. 5: 271-340. 1954.

6. RABINOWITCH, E. I. Photosynthesis. Vol. II, pp.1594-1595. Interscience Publishers, New York1956. t

7. STOCKING, C. P. Precipitation of enzymes duringisolation of chloroplasts in carbowax. Science 123:1032. 1956.

8. THOMAS, J. B., BLAAUW, 0. H. and DUYSENS, L. N. M.On the relation between size and photochemicalactivity of fragments of spinach grana. Biochim.Biophys. Acta 10: 230-240. 1953.

EFFECT OF GIBBERELLIN ON GROWTH, FLOWERINGAND FRUITING OF THE EARLYPAK TOMATO,

LYCOPERSICUM ESCULENTUM 1,2

LAWRENCE RAPPAPORTDEPARTMENT OF VEGETABLE CROPS, UNIVERSITY OF CALIFORNIA, DAVIS, CALIFORNIA

Gibberellin applied to tomato plants has beenshown to induce marked stem elongation (4), to in-crease fresh weight (4, 15), to accelerate floweringand produce greater numbers of flowers per plant(17), and to increase fruit set (15, 17). This paperdescribes more definitively, the influence of gibberel-lins on growth, flowering, and fruit set of the Early-pak tomato.

GENERAL PROCEDURETomato seed (var. Earlypak, Ferry-Morse Seed

Company, Salinas, California) was germinated inflats of vermiculite. Before the first true leaf ex-

1 Received revised manuscript April 30, 1957.2 This paper is based on Project 1175 D.

panded, uniform plants were transferred to flats orcans and grown in a greenhouse held at 18° to 210 Cor in an outdoor lath house. Plants were grown tofruit maturity in soil benches or in large cans of soil.

A mixture of gibberellic acid ([a]D + 92°) andgibberellin A (Ga) ([a]D + 360) (9) was dissolved in0.5 % ethyl alcohol or in water. In the experimentdealing with growth response 0.05 ml of the mixtureof gibberellins was applied with a micropipette to theyoungest developing leaves (when about 2 cm inlength) and to the adjacent vegetative apex. Simi-lar applications were made during later growth. Anatomizer was used to spray flowers and developingfruits.

In the experiments concerned with stem heights,

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measurements were taken at the appearance of thefirst flower clusters, and the distance between thecotyledonary node and the node below the first flowercluster is reported.

EXPERIMENTALSTEM ELONGATION: The influence of repeated ap-

plications of Ga at specific intervals during growthwas investigated. Using tomato plants grown in cansof soil, 25 ,ugm of Ga was applied to developing leavesat each of certain nodes as they appeared (table I).The final application coincided with anthesis of thefirst flower cluster. In plants treated at the firstnode increases in stem elongation were observedwithin a week. However, treatment of the succeedingnodes produced no observable stem height increases,and by anthesis of the first flower cluster no signifi-cant differences in stem elongation had resulted.

TABLE IIEFFECT OF GIBBERELLIN * ON ELONGATION AND FLOWERING

OF EARLYPAK TOMATO PLANTS

GIBBERELLIN,1AGM/PLANT

0

2550100200

L.S.D. at 1 %

HT, CM

47.049.054.658.770.510.4

NO. OF NODESPRECEDING1ST FLOWERCLUSTER

88888

-N.S.**

DAYS TOFLOWERING

58545352593.7

* Application made twice weekly from the appear-

ance of the first expanding true leaves until the appear-ance of the first flower cluster.

** N.S. = Not significant.

TABLE ISTEm ELONGATION AND FLOWERING OF EARLYPAK TOMATOPLANTS AS INFLUENCED BY SINGLE OR REPEATED APPLI-

CATIONS OF GIBBERELLIN AT THE INDICATED NODES

NO. OF TIMETOTAL NODES FROM

NODES TIMES GIBBER- ToTAL PRECED- SEEDINGTREATED TREATED ELLIN HT ING 1ST TO

APPLIED FLOWER FLOWER-CLUSTER ING

no. no. /Agm/plant cm days... 0 0 45 7 53

1 1 25 45 8 491,4 2 50 48 9 481,4,8 3 75 46 8 481,4,8,12 4 100 51 8 48L.S.D. at 1 %O N.S.* N.S.* 0.7

* N.S. = Not significant.

The effect of higher cortcentrations and repeateddosages was determined by treating tomato plantstwice weekly with 0, 25, 50, 100 or 200 ,ugm per plantuntil the first flower cluster appeared. Repeatedtreatment with 25 ,ugm and 50 ,ugm per plant did notsignificantly affect stem elongation; with 100 and 200ugm per plant, however, slight and marked differ-ences, respectively, were obtained (table II).

The surprising lack of response by anthesis tosingle or repeated applications of 25 and 50 /Agm perplant (tables I and II) and the longer stems result-ing from repeated applications of 100 and 200 ligmper plant of Ga indicated a further study of thegrowth responses of young Earlypak tomato plants.Single applications of 2.5, 25, and 50 ,ugm of Ga perplant to the first expanding leaves stimulated steinelongation markedly. In contrast to elongation(measured at anthesis of the first flower cluster) ofplants treated repeatedly until anthesis (tables I andII), significant effects resulted from single applica-tions with 2.5 or 25 ,ugm per plant when plants weremeasured during early growth (table III). Stem

height increased no more with 50 ,ugm per plant,however, than with 25 ,ugm per plant.

In a more detailed study, concentrations of 0, 15,150, 300, and 450 ,ugm per plant were applied to thefirst exapnded leaf. Six days after treatment plantsreceiving 150 to 450 ugm, as compared with untreatedplants, elongated significantly. The greatest stemheight increase resulted from applications of 450 /Agmper plant (fig 1).

FRESH AND DRY WEIGHTS: Reports regarding theinfluence of gibberellins on fresh and dry weight areinconsistent (1, 4). Since such increases in responseto an applied chemical would be extremely significant,fresh and dry weights were determined. Ga at 50ugm per plant was applied to the first expandedleaves of Earlypak tomato plants. Ten days later,these plants, together with some which received noGa, were cut at the cotyledonary node and weighed.Fresh weight was 61 % more in the treated plants.

In another study the shoots of plants given 2.5, 25or 50 ugm of Ga were cut and weighed 17 days aftertreatment. Fresh-weight (grams) increases over thecontrol were obtained with all treatments; however,there was no significant difference between treatmentswith 2.5 and 25 /Agm of Ga (table III). The shoots

TABLE IIITHE INFLUENCE OF A SINGLE APPLICATION OF GIBBERELLINAPPLIED TO THE FIRST EXPANDING TRUE LEAVES ON STEMHEIGHTS, FRESH WEIGHTS, AND DRY WEIGHTS OF EARLY-

PAK TOMATO PLANTS

DAYS AFTER FRESH INCREASE INCREASEGIBBERELLIN TREATMENT WT IN FRESH IN DRYPER WT OVER WT OVER

9 13 15 PLANT CONTROL CONTROL

,ugm/plant Height, cm gm % %0 (control) 2.5 4.8 7.5 3.82.5 4.9 8.0 11.3 4.8 26.1 18.5

25 8.3 12.0 15.4 4.5 18.1 14.850 9.2 13.3 16.8 5.3 40.0 40.7L.S.D. at 1 % 2.7 5.9 5.2

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PLANT PHYSIOLOGY

28 u IGA/PLANT26 ,,450

24/

G 0..3-20/

is

Z 16 150

z

-12

lo 10 13 5

6 O ~~~~~~0

2

3 6 8 10 13 5

NUMBER OF DAYS AFTER TREATMENT

FIG. 1. The effects of 0, 15, 150, 300 and 450 /Agm ofgibberellin per plant applied to the vegetative apex onstem elongation of Earlypak tomato plants.

were placed in a 700 F forced-draft oven and driedfor 24 hours. The percentage of dry matter of treatedplants did not vary significantly from that of the con-trols. However, increases in total dry matter (%dry wt x fresh wt per plant) were obtained, especiallywith the 50 ,agm application. The differences infresh and dry weights between treatments at 2.5 and25 ugm were not significant.

MORPHOLOGIC EFFECTS: Repeated foliar applica-tions at concentrations above 25 ,ugm per plant fre-quently resulted in abnormal extension of the leafrachis and leaflet petioles and in chlorosis of theleaves (1). In addition, the normally incised tomatoleaves frequently developed entire margins (fig 2).Leaflets frequently rolled inward along the centralvein after spraying, at high concentrations. Axillarygrowth (tillers) decreased as concentration increasedfrom 50 to 200 ugm per plant. At higher concentra-tions of Ga, spindly, weak plants developed. When

FIG. 2. Morphologic effects of gibberellin at concen-

trations above 500 ,ugm/ml. Notice the continuous mar-

gins, extension of leaflet petioles, and fewer leaflets perblade of treated leaf on the right.

early developing flowers of the first cluster weresprayed before anthesis with concentrations above50 jAgm, pedicels, sepals, petals, and pistils becameenlarged. Fruits from such flowers usually developedabnormally.

FLOWERING: Wittwer and Bukovac (17) showedlthat while flowering in certain determinate tomatovarieties may be hastened by early application ofgibberellins, flowering of indeterminate varieties isrelatively unaffected. Earlypak, like Pearson fromwhich it is derived, is a determinate variety.

Treatment of plants at specific nodes with 295,ugm (table I) or twice weekly with 25, 50, or 100/gm hastened flowering significantly without affectingthe number of nodes below the first flower cluster(table II). No significant differences in flower num-bers in the first two clusters resulted from applica-tions of 0.05, 0.5, 2.5, or 25 ,ugm/ml of Ga made atthe appearance of the first expanded leaf. Appliedto the vegetative apex of tomato plants at first an-

251- U-u 500 gg/ml GA

x o--o ~~50 '

0- 200

w5

15

10

z

0J 5

8/19 9/2 9/9 9/27

DATE COUNTED

FIG. 3. The pattern of fruit set from the appearanceof early developing fruit to the appearance of the firstripe fruit as affected by flower sprays of 0, 1, 10, 50 and500 /,gm/ml gibberellin.

thesis, similar treatments failed to affect subsequentflowering or fruit numbers.

FRUIT SET: Commencing August 4, 1956, the sec-ond and succeeding four flower clusters of tomatoplants were sprayed twice weekly (for a period of 8weeks) with Ga at concentrations of 0, 1, 10, 50, and500 ,ugm/ml. Figure 3 shows the total number ofdeveloping fruits counted (per plant) preceding theappearance of the first ripe fruits. Figure 4 indicatesthe number of ripe normal and parthenocarpic fruitper plant harvested on specific dates. The fruit setof all sprayed flower clusters was rapid and almostcomplete, but in untreated plants fruiting was largelydelayed until the appearance of the 5th or 6th clus-ter. Total fruit set of unsprayed plants neverreached that of plants treated with gibberellin (fig 3).

Statistical analysis revealed that sprays of 1Ugmlmml were as effective as 500 ,ugm/ml in settingEarlypak tomatoes. The decrease in production ofripe normal fruit (fig 4) resulted from the frequencyof harvesting (10/11 and again on 10/14) rather than

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from any apparent differences in the pattern of fruitset. The average numbers of ripe normal fruit har-vested per plant are shown in table IV. Ga spraysfrequently indtuced parthenocarpy (fig 4 and tableIV) with associated underdevelopment and poor rip-ening characteristics. However, when seeds were pres-ent in fruits from sprayed flowers, fruits generallydeveloped normally.

In one experiment, spraying fruits twice weeklydid not appear to affect fruit size, although the vege-tative portions (calyx and pedicel) were character-istically enlarged. The restricted plant-growing areaprovided in 5-gallon cans of soil made the recordingf offruit weights valueless.

DISCUSSION AND CONCLUSIONSRapid stem elongation is seen in many plants after

treatment with extremely minute amounts of the gib-berellins (1, 2, 3, 4, 5, 6, 9, 13, 14, 15, 18). WithEarlypak tomato, although minor differences are ap-parent soon after treatment, pronounced stem heightincreases are obtained primarily with very high con-centrations of Ga within 10 days after application.Stem elongation is marked when the first expandedleaf is treated during early growth (4 to 6 leaves),although by anthesis neither single nor repeated ap-plications of up to 50 /Agm of Ga produced signifi-cant differences in elongation. By anthesis only withrepeated applications at concentrations above 50 ugmdid stem elongation differ significantly from that ofuntreated plants. The results demonstrate the de-creasing responsiveness to Ga as plants develop. A

2

0

PARTHENOCA-RPCphg/nl GA

500/ \'\° s/ \ \ *-. 10

0//\\\\ * * o

£ A

10/2 10/6 10/lI 04 10/21 KIZ07 I3

NORMAL

itex \ \

/11~~~~~~~~~~~~~~

9/29 10/2 0t m 10/1 10/21

DATE HARVESTEDIW7

FIG. 4. The influence of flower sprays containing 1,10, 50 and 500 jugm/ml of gibberellin applied twiceweekly on the numbers of ripe normal and partheno-carpic fruit harvested at the indicated dates.

TABLE IVAVERAGE NITMBER OF NORMAL AND PARTHENOCARPIC RIPEEARLYPAK TOMATOES HARVESTED FROM SEPT. 20 TO Nov. 3,1956 FOLLOWING TWICE-WEEKLY SPRAYS OF GIBBERELLIN

GIBBERELLIN, NO. OF FRUIT/PLANTuAGM/ML NORMAL PARTHENOCARPIC

0 3.4 0.41 13.6 4.8

10 11.8 4.650 12.4 4.4

500 15.8 7.4L.S.D. at 1 %O 5. 1.4

possible explanation for the disparity between theseresults and those reported by Bukovac and Wittwer(4) is that they collected samples earlier in growthwhen differences in stem elongation were also appar-ent in the present studies. These differences, however,were negated by the time anthesis of the first flowercluster had occurred, possibly because of the decreas-ing receptiveness of older plants to Ga. In addition,

L important varietal differences occur. In concurrentstudies plants of the Potentate variety were taller byanthesis when treated during early growth with 50ugm per plant of gibberellic acid.

Increases in fresh and dry weights of plants grownwvith a continuous supply of gibberellin in water cul-ture were reported by Brian et al (1) and Marthand coworkers (13). Bukovac and Wittwer reporteda 50 % increase in fresh and dry weight of celery(4). However, working with several tomato varieties,they did not obtain dry-weight increases with asmuch as 20 ugm per plant. In the present experi-ments Ga stimulated consistent stem elongation, leafenlargement (rachis and leaflet petioles), and fresh-weight increases. Dry matter content (% dry wt xfresh wt per plant) was increased, especially whenvery high concentrations were used. The later har-vest of plants in these experiments mav account forthe disparity in results.

Kato (7) reported that gibberellic acid increasedrespiration and water uptake in pea stem segments.Increased cell elongation has also been found follow-ing treatment with gibberellins (8). In Earlypak to-mato fresh-weight increases seem to be coupled withgreater water uptake as well as increases in dry mat-ter content. These increases point to further criticalstudies of gibberellin effects on the mechanism ofwater uptake, cell enlargement, and cell division.

Gibberellin profoundly affects the reproductivedevelopment of Earlypak tomatoes. While floweringwas accelerated only slightly (3 to 6 days) by treat-ment with gibberellins, this and other papers (5, 10,11, 12, 13, 15, 17) suggest further studies of the in-fluence of Ga on flowering of various plants. Thevalue of leaf numbers preceding the appearance offlower parts as an index of accelerated flowering hasreceived considerable emphasis (16). It is of especialinterest, therefore, that although Ga hastened or did

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not affect flowering, it did not influence the numberof nodes preceding the appearance of flowers in theseexperiments. This indicates the need for careful con-sideration of the plant material when leaf numbers areused as an index of flowering. The implication is thatin some plants Ga may accelerate maturation of vege-tative parts preceding flowering. This is further em-phasized by the induction of flowering in biennial (10)and winter annual plants (5) without cold treatment.

The pronounced fruit-setting ability of gibberellinis of interest both physiologically and economically.The search for new growth regulators to control fruitsetting of tomatoes has made fleetingly prominent avariety of chemicals which experimentation and com-mercial use have proved only partially satisfactory.Aside from their typical formative and other unde-sirable effects, these compounds are generally effectivewithin a limited range of concentrations below whichfruit set is inadequate and above which injury to bothfruit and plants frequently results. While it is em-phasized that the quality of fruits harvested fromsprayed flowers in these experiments was not evalu-ated critically, similar responses to sprays of 1 to 500ugm/ml Ga indicate the desirability of continued in-vestigation under temperature conditions unfavorablefor fruit set.

SUMM ARYGibberellin (Ga) stimulated growth, flowering, and

fruit set of Earlypak tomato. Total stem elongationof plants, as measured from the cotyledonary nodeto the node precedingf the first flower cluster, wassignificantly increased by twice-weekly applications ofGa at concentrations of 100 and 200 ugm, but notby concentrations of 25 or 50 /,gm per plant. Neithersingle nor repeated treatments with 25 ugm appliedto the expanding leaves at the first, fourth, eighth,or twelfth nodes produced significant stem heightdifferences. Stem elongation of young plants (4 to 6leaves) was increased, as compared with the control, atconcentrations of 2,5, 15, 25, 50, 150, 300 and 450 ugmper plant. Both fresh-weight and dry-matter contentof young Earlypak tomato plants were significantlyincreased. Leaf enlargement, the development of en-tire instead of incised margins, and an inward rollingof leaflets resulted from treatments with high con-centrations of Ga.

Single or repeated applications of Ga at concen-trations of 25, 50 and 100 (but not 200) ,ugm perplant hastened flowering by 3 to 6 days without af-fecting the number of nodes preceding the first flowercluster. Setting of normal and parthenocarpic fruitwas increased by repeated floral sprays of Ga at con-centrations from 1 to 500 ugm/ml. Spraying thedeveloping fruit did not increase fruit size.

Appreciation is expressed to Dr. F. H. Stodola,Northern Utilization Research Branch, U.S.D.A.,Peoria, Illinois, for supplying gibberellins.

LITERATURE CITED1. BRIAN, P. W., ELSON, C. W., HEMMING, H. G. and

RADLEY, MARGARET The plant growth promotingproperties of gibberellic acid, a metabolic productof the fungus Gibberella fujikuroi. Jour. Sci. FoodAgr. 12: 602612. 1954.

2. BRIAN, P. W. and HEMMING, H. G. The effect ofgibberellic acid on the shoot growth of pea seed-lings. Physiol. Plantarum 8: 669-681. 1955.

3. BRIAN, P. W., HEMMING, H. G. and RADLEY, MAR-GARET A physiological comparison of gibberellicacid with some auxins. Physiol. Plantarum 8:899-912. 1955.

4. BUKOVAC, M. J. and WITTWER, S. H. Gibberellicacid and higher plants: I. General growth re-sponses. Agr. Expt. Sta., Michigan, Quart. Bull.39: 307-320. 1956.

5. HARRINGTON, J. F., RAPPAPORT, LAUWRENCE and HOOD,K. J. The influence of gibberellin on stem elon-gation and flowering of endive. Science 125: 601-602. 1957.

6. HAYASHI, T. and MURAKAMI, Y. Biochemical studiesof bakanae fungus. XXVIII. The physiologicalaction of gibberellin. Jour. Agr. Chem. Soc. Ja-pan 27: 672-675. 1953.

7. KATO, J. Effect of gibberellin on elongation, wateruptake, and respiration of pea stem sections. Sci-ence 123: 1132. 1956.

8. KATO, Y. Responses of plant cells to gibberellin.Bot. Gaz. 117: 16-24. 1955.

9. LANG, A. Stem elongation in a rosette plant, in-duced by gibberellic acid. Naturwiss. 43: 257-258.1956.

10. LANG, A. Induction of flower formation in biennialHyoseyamus by treatment with gibberellin. Natur-wiss. 43: 284-285. 1956.

11. LANG, A. Bolting and flowering in biennial Hyo-scyamus nige1, induced by gibberellin. PlantPhysiol. 31 (Suppl.): 35. 1956.

12. LANG, A. Gibberellin and flower formation. Natur-wiss. 43: 544-545. 1957.

13. MARTH, P. C., AUDIA, W. V. and MITCHELL, J. W.Effect of gibberellic acid on growth and develop-ment of various species of plants. Plant Physiol.31 (Suppl.): 43. 1956.

14. PHINNEY, B. 0. Growth response of single-genedwarf mutants in maize to gibberellic acid. PIoc.Nat. Acad. Sci., U.S. 42: 185-189. 1956.

15. RAPPAPORT, L. Growth regulating metabolites. Cali-fornia Agriculture 10: 4. 1956.

16. RAPPAPORT, L. and WITTWER, S. H. Flowering inhead lettuce as influenced by seed vernalization,temperature and photoperiod. Proc. Amer. Soc.Hort. Sci. 67: 429-437. 1956.

17. WITTWER, S. H. and BUKOVAC, M. J. Some effectsof gibberellin on flowering and fruit setting. PlantPhysiol. 32: 3941. 1957.

18. YABUTA, T. and HAYASHI, T. Biochemistry of thebakanae fungus (2). Isolation of gibberellin, ametabolic product of Gibberella fujikuroi Wr.which promotes the growth of rice seedlings. Jour.Agr. Chem. Soc. Japan 15: 257. 1939.

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