J‘H’HHII {‘1 \ HI 1| ‘ A THE {NFLUENCE 0F GSBBERELUC AC“) 3N WE HiGHBUSi-l BLUEBERRY mg 01—) _.{ I (DUI—A Thesis for the Degree of Ph. D. WEE-Elem STATE UNWERSITY RONALD FRED HOOKS 1969 (m 0-169 This is to certify that the thesis entitled The Influence of Gibberellic Acid on the Highbush Blueberry. presented by Ronald Fred Hooks has been accepted towards fulfillment of the requirements for Ph .D . Hort iculture degree in 2 v/ p f4 / [(46 4.4,.(x’la/"’ 5' pr Major professor Date February 3, 1969 Era-0' -vv- v "’ "V' f "'7' I" "3 ‘ ‘l ‘l ' [/15 1“ .1 h 1 Michigan State Universitv ABSTRACT THE INFLUENCE OF GIBBERELLIC ACID ON THE HIGHBUSH BLUEBERRY by Ronald Fred Hooks Over 60% of the acreage of highbush blueberries in Michigan consists of the cultivar Jersey. This variety is noted for an extended ripening period, which has led to a congestion of marketing and handling facilities, and re- duced yield, small berry size, and late maturing fruit resulting from poor pollination. Growth regulators have shown some influence on the ripening processes by delaying senescence of plant organs or speeding up the ripening. Exogenous GA3 has been shown to set some fruit partheno- carpically without pollination. In 1966 and 1967, a randomized block design was used with 2 replications of 5 bushes per treatment on the variety Jersey. Treatments were single applications of GA3 applied as a solution sprayed to the drip point 2 weeks after full bloom. Concentrations used were 25, 75, and 150 ppm. In order to study the effect of GA3 on fruit set an experiment was set up under different pollinating in- tensities. Field plot design was a randomized block Ronald Fred Hooks consisting of a factorial arrangement of 3 pollinating in- tensities and 3 levels of GA3 with 3 replications of 2 bushes each. Some bushes were caged with and without bees, while others were left to be pollinated in the open. The GA3 was applied at full bloom at concentrations of 250 ppm and 500 ppm. Response parameters measured were berry size, sugar content, titratable acidity, seed weight, and total yield. In 1966 and 1967, berry size was significantly in- creased with GA3 applications. Different responses to con- centrations were observed each year. Concentrations of 25 ppm and 75 ppm increased berry size. Berry growth rate was enhanced by GA3 at 75 ppm, whereas 25 ppm and 150 ppm delayed the rate of growth. GA3 reduced the percentage of blue fruit and in- creased the percent citric acid in 1967 at concentrations of 75 ppm and 150 ppm. In 1967, GA3 increased the citric acid content of the canned product with no effect on drained weight, soluble solids, pH, and fruit firmness. GA3 had no effect on berry firmness, sugar content, respiration, leaf or fruit nutrient content, vegetative growth, floral ini- tiation, and total yield. When GA3 at 25 ppm was applied at full bloom in 1967, it enhanced the total yield, the loss of citric acid, and increased the percentage of blue fruit. Ronald Fred Hooks From these 2 years research, it was found that GA3 applied at full bloom may cause fruit to ripen earlier than if it is applied after full bloom. High concentrations delayed ripening and low concentrations enhanced ripening. In 1968, GA had no effect on final berry size, 3 but there was an indication that GA3 did cause the fruit to be larger early in the growing season. Seed weight was reduced by GA3 treatment under all pollinating intensities. No effect of GA3 on sugar content or percent citric acid of the fruit was noted under any of the pollinating intensities. In bushes caged with bees, GA3 reduced yield, while in those caged without bees or in the Open, GA3 increased the yield. It was found that by combining GA3 and a small number of honeybee hives per acre near optimum yields could be achieved. THE INFLUENCE OF GIBBERELLIC ACID ON THE HIGHBUSH BLUEBERRY BY Ronald Fred Hooks A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1969 ACKNOWLEDGMENTS The author wishes to express his sincere thanks and appreciation to Dr. A. L. Kenworthy for his assistance in carrying out the experimental work and preparing the manuscript. Grateful acknowledgment is expressed to Drs. Jerome Hull, D. R. Dilley, C. L. Bedford, and H. M. Sell for their suggestions in editing the manuscript. The author is grateful to Mr. John Nelson for his enthusiastic encouragement and help, and to the Michigan Blueberry Growers Association for providing financial as- sistance, and to the growers of the association on whose plantations the research was carried out. The author also wishes to express thanks to his fellow graduate students for their untiring help in applying the treatments and collecting data. Special appreciation is due my family for their patience and sacrifices during the course of graduate study. ii TABLE O ACKNOWLEDGMENTS. . . . . . . TABLE OF CONTENTS. . . . . . LIST OF TABLES . . . . . . . LIST OF FIGURES. . . . . . . LIST OF APPENDICES . . . . . INTRODUCTION . . . . . . . . REVIEW OF LITERATURE . . . . MATERIALS AND METHODS. . . . RESULTS. 0 O O O O O O O O O 1966 StUdy O O O O O I O 0 Experimental design. . . Berry size . . . . . . . Fruit firmness . . Percent sugar. . . . . . Seed weight. . . . Respiratory Gas Analysis Nutrient analysis. . . Vegetative growth and £10 Discussion . . . . . 1967 study 0 O I O O O O 0 Experimental design. . . Berry size . . . . . . . Berry growth . . . . . . Fruit firmness and sugar Total yield per bush . . Percent blue fruit . . . Titratable acidity . . . Canned product analysis. Time of Application Study. Citric acid and blue frui Total yield. . . . . . . Discussion . . . . . . . F CONTENTS Page . . . . . . . . . . . . . 11 . . . . . . . . . . . . . iii a o o o o o o o o o o o 0 V . . . . . . . . . . . . . vii O O O O O O O O O O O O O Viii . . . . . . . . . . . . . l . . . . . . . . . . . . . 3 . . . . . . . . . . . . . ll . . . . . . . . . . . . . 20 . . . . . . . . . . . . . 20 . . . . . . . . . . . . . 21 . . . . . . . . . . . . . 21 . . . . . . . . . . . . . 21 . . . . . . . . . . . . . 22 . . . . . . . . . . . . . 23 . . . . . . . . . . . . . 23 . . . . . . . . . . . . . 25 ral initiation. . . . . . 25 . . . . . . . . . . . . . 25 . . . . . . . . . . . . . 27 . . . . . . . . . . . . . 28 . . . . . . . . . . . . . 28 . . . . . . . . . . . . . 28 content . . . . . . . . . 29 . . . . . . . . . . . . . 32 . . . . . . . . . . . . . 32 . . . . . . . . . . . . . 33 . . . . . . . . . . . . . 33 . . . . . . . . . . . . . 34 t . . . . . . . . . . . . 34 . . . . . . . . . . . . 34 . . . . . . . . . . . . . 36 iii 1968 Study. . . . . . . . Experimental design . Mechanical Harvesting Total yield . . Pollination Study . . . . Berry size. . . . . . . Sugar content . . . . . Titratable acidity. . . Seed weight . . . . . . Total Yield . . . . . . Discussion. . . . . . . DISCUSSION. 0 O O O O O O 0 Berry size. . . . . . . Titratable acidity an Seed weight . . . . . . Total yield . . . . . . No response parameters. Residue . . . . . . . . FUTURE RESEARCH . . . . . . SUMMARY 0 O O O I O O I I 0 LITERATURE CITED. . . . . . APPENDIX. C O O O I O O O O U) r.- (3 Q: ‘< iv percent blue fruit Page 37 37 38 38 39 39 42 43 43 43 45 46 46 47 48 48 49 49 SO 51 55 59 Table 10. 11. LIST OF TABLES Effect of various GA3 concentrations on berry size in size category No. 6 (0.9 cm or greater) in 1966 at Grand Junction, Michigan. . . . Firmness of fresh fruit as influenced by GA3 concentrations in 1966 at Grand Junction, MiChigan O O O O O O O O O I I O O O O O 0 Percent sugar in fresh fruit treated by GA3 at various concentrations in 1966 at Grand Junction, Michigan . . . . . . . . . . . . Effect of GA3 at various concentrations on the seed weight of 25 uniform size berries 1966 at Grand Junction, Michigan . . . . . Effect of GA3 on fruit respiration in 1966 Grand Junction, Michigan (cc/Kg/hr). . . . Leaf nutrient content as affected by GA3 at various concentrations in 1966 at Grand Junction, Michigan . . . . . . . . . . . . Vegetative growth and floral initiation as affected by various concentrations of GA3 in 1966 at Grand Junction, Michigan . . . . . Effect of various GA3 concentrations on berries in size category No. 6 (0.9 cm or greater) in 1967 at Grand Junction, Michigan Percent blue fruit as affected by various concentrations of GA3 in 1967 at Grand Junction, Michigan . . . . . . . . . . . . Effect of GA3 at various concentrations of titratable acidity, expressed as percent citric acid, in 1967 at Grand Junction, Michigan. The effect of various concentrations of GA3 on canned products prepared from harvest 2 8/9 in 1967 at Grand Junction, Michigan. . on Page 22 22 23 24 24 26 27 29 32 33 34 Table Page 12. Effect of GA at 25 ppm on percent citric acid and percent lue fruit when applied at full bloom, 2 weeks after full bloom, and 7 weeks after full bloom in 1967 at Grand Junction, Michigan. . . . . . . . . . . . . . . . . . . . 35 13. Effect of GA at 25 ppm applied at different times on total yield at the last harvest in 1967 at Grand Junction, Michigan. . . . . . . . 35 14. Yield as affected by GA treatment at various concentrations applied weeks after full bloom in 1968 at Grand Junction, Michigan . . . 39 15. Effect of GA3 at different concentrations on total yield using only 1 late picking in 1968 at Grand Junction, Michigan. . . . . . . . 39 16. Berry size as affected by GA3 applications and different pollinating intensities in 1968 at South Haven, Michigan . . . . . . . . . 42 17. The effect of GA applications on berry sugar content in 1968 at South Haven, MiChiganO O O O O O O O O O O O O O O O O O O O 42 18. Percent citric acid as affected by GA3 applications in 1968 at South Haven, MiChiganO O O O O O O O O O O O O O O O O O O O 43 19. Effect of GA at various concentrations on seed weight of 25 uniform size berries in 1968 at South Haven, Michigan . . . . . . . . . 44 20. The effect of GA applications on total yield per bush in 1968 at South Haven, Michigan . . . 44 vi Figure 1. LIST OF FIGURES Page The effect of GA at various concentrations on berry growth in I967 at Grand Junction, Michigan. . . . . . . . . . . . . . . . . . . . 30 The effect of GA3 at various concentrations on berry growth under different pollinating intensities in 1968 at South Haven, Michigan. . 40 vii Table 1A. 2A. 3A. 4A. 5A. 6A. 7A. LIST OF APPENDICES Page Fruit nutrient content as affected by GA3 at various concentrations in 1966 at Grand Junction, Michigan. . . . . . . . . . . . . . . 59 Effect of GA at various concentrations on berry diameter (cm) in 1967 at Grand Junction, Michigan. . . . . . . . . . . . . . . 6O Firmness of fresh fruit as influenced by GA3 at different concentrations in 1967 at Grand Junction, Michigan. . . . . . . . . . . . 60 Percent sugar in fresh fruit treated with GA3 at various concentrations in 1967 at Grand Junction, Michigan. . . . . . . . . . . . . . . 61 The effect of GA3 at various concentrations on yield per bush in 1967 at Grand Junction, MiChiganO O O O O O O O O O O O O O O O O O O O 61 Effect of GA3 applications on residual gib- berellins in the fruit in 1968 at South Haven, MiChigan O O O O O O O O O O O O O O O O 62 Effect of GA3 applications on endogenous gibberellins in 1968 at Grand Junction, MiChiganO O O O O O O O G O O O O O O O O O O O 62 viii INT RODUCT ION The highbush blueberry is an economically important horticultural crop in Michigan and any cultural practices that would facilitate mechanical harvesting and handling, or improve fruit-set would be commercially important. This thesis concerns the effects of gibberellic acid (GA3) on the fruit of the highbush blueberry under field conditions in Michigan. Presently the highbush blueberry is harvested over a period of four to six weeks depending on location (39). Over 60% of the acreage in Michigan consists of the cultivar Jersey, with Rubel a distant second. These cultivars are noted for their extended ripening period. With the Jersey cultivar, 70% of the ripe fruit are picked during the second and third weeks of the harvest, with the other 30% being harvested during the first and fourth weeks or later. Although new varieties with short periods of ripening have been introduced to replace these older varieties, it will take 15 to 20 years for these to become commercially impor- tant. A congestion of marketing facilities has also been observed, because of the large acreage planted to a single variety. The Michigan Blueberry Growers Association has become interested in finding some way of lengthening the marketing period of varieties now in production so they can use their facilities more economically. Pollination has also become a point of concern for blueberry growers. A lack of adequate pollination causes reduced yield, small berry size, and late maturing fruit. The number of wild bees working many Michigan plantations is inadequate. This has led to the expensive practice of placing honeybee colonies in the plantation during bloom to increase the population of pollinating insects. REVIEW OF LITERATURE The highbush blueberry, Vaccinium corymbosum, Lamrach, is an important economic crop in Michigan. Michigan has over 8,000 harvested acres of blueberries. The leading state in highbush blueberry production fluctuates between New Jersey and Michigan from year to year. Some of this fluc- tuation may be due to a lack of labor for harvesting and/or inadequate pollination. Robbins (29) classified the inflorescence of the blueberry as a raceme, a simple inflorescence of pediceled flowers upon a common elongated axis. Young (39), studying the fruit growth of the highbush blueberry, found a double- sigmoid growth pattern. Three stages of growth were ob- served, two rapid growth rates with a slow growth rate in between. The first stage, in the Jersey cultivar, lasted from 28 to 30 days. The second stage lasting from 14 to 56 days and determines the length of time from flower to fruit maturity. The final stage lasted from 16 to 26 days and the increase in size was greater than either of the other stages (1, 19). In order to cause a more concentrated fruit ripening, a means of regulating this second stage of growth must be found. The difference of 42 days during the second stage makes a difference in whether the fruit is harvested during the first or third week of the harvest period. In an extensive study of the highbush blueberry, Woodruff (38) attempted to determine the proper time for harvesting for optimum dessert and keeping quality. He found that the sugar-acid ratio gave a good indication of maturity. The sugar content would increase for the first eight or nine days after a color change then would remain constant. The acidity would decrease continuously through- out the harvest period, so the sugar-acid ratio increased with ripening. Growth regulators have shown some influence on the ripening processes by delaying senescence of plant organs or enhancing the ripening. Effect of Gibberellic Acid (GA3) on Floral Initiation and Fruit Set Flower formation and fruit-set are phenomena of great significance in crop production. Gibberellins con- stitute the only group of chemical substances where con- sistent effects on flowering are found in large segments of the plant kingdom (24, 36). Exogenous gibberellins delay flowering in tomatoes and peas by increasing the node number subtending the first inflorescence (4, 37). High concentrations of GA3 resulted in absence of flower buds on spurs of the almond, apricot, cherry, and plum during the season following application, and buds of peach were unaffected (5). Hull and Lewis (20) reported that gibberellin applied 3 1/2 weeks after full bloom to Redheaven, or 2 1/2 months after full bloom to Halehaven peaches at a concen- tration of 100 ppm resulted in complete absence of flowers the following year. Partial absence of flowers was reported in Montmorency cherry trees as a result of post-bloom appli- cation of gibberellin at 100 ppm concentration the previous season. No affect on flowering or vegetative growth was reported on mature bearing apple trees after gibberellin treatment at 100 ppm (20). Recently, workers have found an inhibition of flowering in apple due to gibberellin treatment (10, 16, 17, 27). Edgerton (14) has reported a reduction in flower buds the following spring after gibber- ellin treatment at a concentration of 50 ppm and 200 ppm in the peach cultivars Redhaven and Golden Jubilee. Main- land and Eck (26) and Devlin and Demoranville (ll) demon- strated a decrease in floral initiation in cranberries as a result of gibberellic acid treatment. Sparks (33) showed a partial inhibition of male flowers, catkins, in pecans due to one application on June 1 with gibberellic acid at 200 ppm. If a second application of gibberellic acid was made 23 days later complete inhibition was observed. He Speculated that the GA3 interferred with floral differen- tiation rather than floral initiation. His first application of GA3 was made about 1 1/2 months after the start of catkin differentiation. The second application may have covered the entire catkin formation interval resulting in total catkin inhibition. Fall applications of GA3 had no effect on catkin initiation. Essentially, no pistillate flowers were found in the spring following previous GA3 treatment. Bradley and Crane (5) suggested that in Prunus, gibberellin blocks floral initiation rather than floral differentiation. In the highbush blueberry, Mainland and Eck (25) reported that under greenhouse conditions GA3 reduced the number of flower buds formed the year after treatment. The GA3 was applied at concentrations of 5 ppm, 50 ppm, 200 ppm, and 500 ppm at full bloom. These papers would suggest that gibberellin can be expected to affect floral initiation and/or differentiation depending on plant species and time of application. The capacity of gibberellin for promoting fruit- set among different types of fruit crops has been elucidated since 1960. Some of these fruits are the tomato, fig, pear, apple, grape, almond, peach, apricot, and citrus (9). Bar- ker and Collins (3) reported that gibberellic acid at con- centrations as low as 20 ppm set fruit parthenocarpically in the lowbush blueberry. They stated that the chemical must be in direct contact with the pistil for a period before the blossom opens until petal fall. The GA3 was applied as a lanolin paste to emasculated blueberry flowers. Field experiments did not show the compound as being eco- nomical in fruit production. However, Smith (32) reported an increase in set in the highbush blueberry cultivar Coville of 10% to 30% after application of gibberellic acid at 100 ppm at late bloom. This increase in set was still considered to be too low for commercial acceptance. Effect of GA3gon Fruit Growth and Maturation Jackson (22) reported that activity of endogenous GA3 in the ovary of peach and apricot was low at full bloom, and immediately after full bloom activity was found in the seed, and later in the mesocarp and endocarp. The amount of gibberellin was closely correlated with cell expansion in each tissue but not cell division. Hayashi, gt al. (18) found the presence of GA3 in apple fruit tissue and sug- gested that fruit tissue other than seeds may participate in the biosynthesis of GA. The effect of exogenous GA3 on parthenocarpic fruit growth has been reviewed by Crane (9). Some of the fruit crOps he reported on were tomato, lemon, navel orange, grapefruit, madarin, apple, and peach. In general, size of fruit was usually inferior to pollinated fruit. Wittwer and Bukovac (36) found that the size of parthenocarpic tomato fruits was inferior to that of pol- linated fruits. They presented results showing differences in the growth of parthenocarpic tomato fruits induced with GA, through GA9 (36). The greatest increase in fruit diam- eter occurred with GAS, although GA3, GA6 and GA7 were only slightly less effective. The GAZ and GAB were least active in this respect. The response of citrus fruits to GA application varies according to concentration and time of application. Valencia orange fruits sprayed with GA at the mature-green stage were smaller at maturity than controls; they develOped pointed stylar ends, big shoulders, thick rinds, less juice, and colored poorly (21). Several of these fruit attributes were linearly related to log concentration of GA. Fruits sprayed at a later stage of deve10pment responded by delaying maturity, enhanced regreening. The rind was found to contain lower concentrations of carotenoid pigments and higher levels of chlorophyll pigments than controls (7). Lisbon lemon fruits sprayed with GA failed to grow to normal size and they were later in maturing, as measured by rind color and juice determinations (8). Proebsting and Mills (28) reported that the early Italian prune had less internal browning, longer shelf life and was firmer when treated with 100 ppm GA3 after bloom. Little delay in color or soluble solids was noted in GA3 treated fruit. Size increases greater than those of controls have occurred only in Black Corinth and Thompson Seedless grapes, both naturally seedless cultivars, as a result of GA treat- ment (30). Sachs and Weaver (30) stated that increase in berry size was the result of water uptake accompanied by solute storage and synthesis of cell components. Histo- logical studies revealed that berry enlargement was the result of tissue growth in a region of the pericarp between the locule and peripheral vascular tissues. This tissue increase was noted 48 hours after treatment with GA. Ber- ries treated with GA showed a 10-fold increase in parenchyma cell diameter in this tissue between anthesis and maturity. Applications of GA3 at the beginning of pit-hardening to individual apricot or peach fruits increased the growth rate (22). Jackson (22) suggested that the presence of gibberellin increased the ability of a tissue to compete for metabolites, and thus, an increased growth rate. In the highbush blueberry, cv. Coville, Mainland and Eck (25) showed a reduction in berry size when GA3 was applied at full bloom. The reduction in size was dependent on the concentration of GA3 applied. Coggin, gt E£° (7, 8) showed that gibberellin sup- pressed fruit ripening in citrus and suggested this was an Opposite effect from that of ethylene (ripening hormone). Scott and Leopold (31) reported opposing effects of GA3 and ethylene on lettuce seedlings. The lettuce seedlings treated with GA3 exhibited no growth stimulation when ethylene at 10 ppm was added. Ethylene depressed invertase activity at each concentration of GA3 in sugar beet disc tissue. In barley endosperm, a-amylase activity was reduced by the presence of ethylene in the system. The synthesis of a-amylase and ribonuclease was enhanced in isolated aleurone layers in barley endosperm (6). It was shown 10 that GA was needed for continued d-amylase synthesis. Valdovinos, 22 El. (34) found that auxin synthesis was con- trolled by ethylene and GA3 in pea seedlings. Ethylene decreased the amount of auxin, and GA3 had no affect on auxin synthesis. Summary In summation, the following general responses can be expected when gibberellic acid is applied to horticul- tural crops. 1. GA3 may be expected to affect floral initiation and/or differentiation depending on plant species and time of application. GA3, when applied at full bloom, may set many fruit parthenocarpically. GA3 set fruit may be inferior in size in some fruit crops. GA3 may delay maturation in some fruit when applied at full bloom or before harvest. GA3 may have an effect on fruit ripening opposite to that of ethylene. MATERIALS AND METHODS Chemical Properties Name: Gibberellic acid or Gibberellin A3 Abbreviation: GA3 Structure: C19H2206 HO HO 1 CH CH3 coon Molecular weight: 346 Commercial name: Pro/Gibbl-2% active ingredient in solution. 1966 Studies In 1966, an experiment was carried out to determine the effects of gibberellic acid (GA3) on the highbush blue- berry, Vaccinium corymbosum, L. cv. Jersey. The GA3 was applied as a foliar spray two weeks after full bloom. Jer- sey bushes, approximately 20 years old, were selected for lPro/Gibb supplied by the Agricultural Division, Abbott Laboratories, N. Chicago, Illinois. Pro/Gibb con- tains a composit of gibberellins. ll 12 uniform vigor and size in a commercial plantation located at Grand Junction, Michigan. All sprays were applied to the drip point with a high pressure sprayer using a single nozzle gun. The concentrations used were 25, 75 and 150 ppm GA3. Field plot design was a randomized block with two replications. There were five bushes per treatment and only the middle three bushes were sampled to decrease the possible error of spray drift. 1967 Studies In 1967, an experiment was set up to try to further substantiate the 1966 findings and determine the effects of repeated application. The same bushes were again sprayed two weeks after full bloom with GA3 at the same concentra- tions as the previous year. To determine the effect of time of application on yield, a preliminary study was set up. Gibberellin A3 was applied to 20 year old Jersey bushes in the same plantation as the other experiments. The con- centration used was 25 ppm. One application was made at full bloom, 4 weeks after full bloom or 7 weeks after full bloom. The application 7 weeks post-bloom.was selected to coincide with the earliest change of color of the fruit from.green to pink. 13 1968 Studies Mechanical harvesting study.--In 1968, the GA3 was applied at concentrations of 150 and 300 ppm 2 weeks after full bloom to 8 year old Jersey bushes located in a plan- tation near Grand Junction. Twenty bushes were sprayed per treatment, and only 10 bushes were harvested at the first harvest and all bushes were harvested at the last harvest, so that half the bushes were harvested one time and the other half two times. Pollination study.--An experiment was designed to determine the effects of GA3 on fruit-set and yield under different pollinating conditions. Ten-year-old Jersey bushes were selected for uniformity and vigor in a planta- tion located near South Haven, Michigan. Field plot design was a randomized block consisting of a factorial arrangement of 3 pollinating intensities and 3 levels of GA3 with 3 replications of 2 bushes each. Pollinating intensities were bushes caged (1) without bees, (2) caged with l honey- bee hive for 2 bushes, and (3) open pollination (wild bees plus 3 honeybees hives/acre). Cages to exclude bees, were placed over the bushes at the start of bloom, but the bees were not placed in the cages until about 25% of the flowers had.0pened. GA3 at concentrations of 0, 250, and 500 ppm was applied at about 75% of full bloom. Cages were not removed for spraying, but were removed at petal fall. 14 Laboratory Analysis Woodruff (38) used the sampling technique of re- moving all ripe fruit from tagged clusters, and then har- vesting the remaining fruit at intervals to give a range of fruit maturity from unripe to overripe. He found that fruit harvested 6 to 16 days after red coloration gave the best quality characteristics. For this reason the first harvest of each year was selected to be about 10 days after red coloration of the berries. The sampling procedure each year was picked to give an average value of fruit maturity for each bush at every harvest. Sampling procedure in l966.--Sampling consisted of removing 1 quart of blue fruit from the middle 3 bushes per treatment at weekly intervals starting on August 10. The sampling coincided with the commercial harvests as closely as possible. The quart samples were then placed in a styro- foam lined iced box to remove field heat and to prevent heating while being transported to East Lansing. Total yield from 1 bush per treatment was determined by harvesting with hard vibrators 1 month after the last commercial har- vest. Sampling procedure in l967.--At each harvest all fruit removed from 1 bush per treatment and then a quart sample was removed for laboratory analysis. The harvesting was done using hand vibrators plus hand picking at weekly intervals. Cooling and transport was the same as in 1966. 15 Samplinggprocedure in l968.--At the Grand Junction location, the straddle row picker was used at 2 harvests. The first harvest was taken at the largest commercial har- vest and the second, about 2 weeks later. At the South Haven location, all mature blue fruit was removed from the bushes at three harvests using hand vibrators. Quart samples were then taken for further mea- surements. Berry size.--The berries were mechanically sized on a cross sectional diameter basis into the following size categories in 1966 and 1967: (1) less than 0.5 cm, (2) 0.5 to 0.6 cm, (3) 0.6 to 0.7 cm, (4) 0.7 to 0.8 cm, (5) 0.8 to 0.9 cm, and (6) 0.9 to 1.0 cm. Size category No. 6 would correspond to the "Great Lakes" commercial grade of berries. In 1968 a random sample of berries was sized by use of hand calipers. The number of berries in each category was re- corded and expressed as a percent of the total sample. Firmness.—-Ten berries were randomly selected from the quart samples for firmness measurement in 1966 and 1967. The firmness measurements were made with a Shore Durometer,2 an instrument designed for measuring the resiliency of foam rubber and other materials. The instrument has a 3/32 inch diameter probe, which protrudes 0.1 inch from the flat 2The Shore Instrument and Mfg. Co., Jamaica, New York. ‘ 16 surface of the instrument. The probe was pushed against the fruit until the fruit comes in contact with the flat surface. The resistance of the fruit to this probe was read on a graduated scale from 0 to 100. Firmness measure- ments were made with the Type 00 Durometer where a scale reading 100 = 114 grams. §2g35.--Twenty berries were randomly selected from each sample, and were crushed in a small bottle and the refractive index of the extracted juice measured on a Bausch and Lomb Abbe-3L-refractometer. The refractive index was converted to percent sucrose. Titratable acid.--Twenty-five grams of berries were randomly selected and 25 grams distilled water was added to give a 1/1 ratio. This mixture of water and fruit was then placed in a Waring blender and a puree prepared. A uniform aliqust (If the puree was then titrated to a pH of 8.1 with 0.1 N_NaOH. Percent total acid was expressed as percent citric acid. Respiratory gas analysis.--Quart samples were har- vested at the first 2 harvests in 1966 for respiratory gas analysis. The samples were cooled and not analyzed for 24 hours. Carbon dioxide evolution and oxygen consumption of 3 the samples were measured at 20°C in the "APRIL" system (12). 3Automatic Photosynthetic Respiration Intergrating Laboratory, Horticulture Department, M.S.U., E. Lansing, Mich. 17 A total of six 12 hour cycles were determined for each sample. Residue analysis.—-Samples of each treatment were harvested for residue analysis in 1968. Five samples con- sisting of 4 lbs each for each treatment were analyzed by Abbott Laboratories. These samples consisted of fresh fruit and were analyzed by the procedure reported by Gordon and Pankratz (15). Nutrient analysis.--Leaf samples were collected from bushes in the 1966 study in July for elemental analysis. Nitrogen determinations were made by macro-Kjeldahl, potas- sium by flame photometry, and the remaining elements by spectrographic analysis (23). Canned product analysis.--12 oz. of fruit from the main harvest was placed in No. 303 cans and processed in 1966. After a 9 month storage period, 2 cans for each treatment were evaluated as follows: Drained weight: The contents of the cans were emptied on a No. 8 circular mesh screen set at an angle on a white enamel pan, and allowed to drain 2 minutes and the fruit weighed. Sugar: The refractive index of the juice was de- termined on a Bausch and Lomb Abbe refractometer and con- verted to percent sucrose. 18 Total acidity: 5 milliters (ml) of juice was diluted with 50 ml of distilled water and titrated to pH 8.1 using 0.1 N NaOH. Firmness: Firmness or character of the canned prod- uct was measured by use of an Instron Shear Press model TTBM4. A sample of approximately 150 grams of berries was used in a Kramer Shear Box, No. C332. Field Measurements Berry growth.--In 1967 and 1968, berry diameter measurements were taken at weekly intervals starting shortly after full bloom and continuing until harvest. The third berry from the basal part of the inflorescence was measured with calipers. Ten berries per treatment were measured. In 1967, the inflorescence was tagged so the same berry was measured each week, but in 1968, a random selection of inflorescence was used. Vegetative growth and floral initiation.--Measure- ments were made in the spring of 1967 following treatments applied in 1966. Shoot length: The 1966 growth of 10 shoots per treatment was measured in inches. Total buds: The total number of buds on the above shoots were counted and used to calculate the internode length. 4Manufactured by Instron Corp., Canton, Mass. 19 Flower buds: The total number of inflorescenses on the above 10 shoots was determined and the percent of the total buds that were flower buds was calculated. Statistical Analysis Analysis of variance was run for all data except the residue data. A randomized block design was used for all data that did not involve harvests. Measurements in- volving harvests were analyzed as a split-plot. The Duncan Multiple Range Test or orthogonal comparisons were used to separate the individual means or groups of means. RESULTS The experimental design, concentrations of GA3 applied, and parameters evaluated varied each year. There- fore, the results will be presented on an individual year basis. This will permit the development of the final year program, which should help to elucidate the effects of gib- berellic acid on the highbush blueberry. 1966 Study The growing season of 1966 was a very poor production year in Michigan. The highbush blueberry yields were low due to a severe cold winter and poor weather during pollination. Many bushes in the Grand Junction area showed signs of die- back, because of the extremely low winter temperatures. The weather prior to and during pollination was cool and rainy. This, combined with the fact that no bees were brought into the plantation, limited bee activity to a very minimum. The large amount of rain resulted in water stand- ing in the plantation even 2 weeks after full bloom. Cold weather, also, destroyed some of the early opening blooms in the spring. One indication of poor bee activity was the large number of seedless fruit found in the plantation. Such fruit, of the Jersey variety, will set parthenocarpically 20 21 to a limited extent, but usually remain small and seldom reach the size of pollinated fruit. Experimental design.--A randomized block design composed of 2 replications of 5 bushes per treatment was used. Parameters for measuring responses were berry size, fruit firmness, percent sugar, seed weight, respiratory gas analysis, nutrient analysis, vegetative growth, and floral initiation. Berry size.--The percent of berries in the largest size category, 0.9 - 1.0 cm, was used as an index of berry size. As shown in Table 1, an application of GA3 at 150 ppm significantly increased average berry size. A normal decrease in berry size with each harvest was shown for the zero concentration or control. Although 25 and 75 ppm GA3 resulted in somewhat larger berries, the overall effect was not significant. This was probably due to the decrease in percentage of larger berries observed for 75 ppm during the second harvest. During the second harvest, there was a significant increase in percent of large fruit for 25 ppm and a significant decrease for 75 ppm. For the third har- vest, there was a significant increase for 75 ppm while 25 ppm did not have a significant effect. Fruit firmness.--The firmness of the fresh fruit was not changed significantly by treatment with GA3 (Table 2). A slight increase in firmness was observed for GA3 22 Table 1. Effect of various GA concentrations on berry size in size category No. 6 (0.9 cm or greater) in 1966 at Grand Junction, Michigan. GA3 Percent of Berries in Size No. 6 Concentrations - Harvest Dates (ppm) 8/10~ 8/17 8/24 Average 0 29.3 22.3 15.4 22.3 in“ 25 27.9 27.0* 17.2 24.0 75 29.2 15.6* 27.9** 24.2 150 32.5 27.9* 32.8** 31.1* *Means significantly different from 0 ppm at the 5% level. a Err-v **Means significantly different from 0 ppm at the 1% level. Table 2. Firmness of fresh fruit as influenced by GA3 con- centrations in 1966 at Grand Junction, Michigan. GA 3 Firmness as Measured With the Concentrations Durometer Harvest Dates (ppm) 78710 8717 8/24 Average 0 76.1 71.7 73.0 73.6 25 74.7 72.5 73.9 73.7 75 75.0 71.0 73.1 73.0 150 74.2 73.5 73.2 73.6 NOS. NOS. NOS. NOS. N.S. Means not significantly different from 0 ppm. treated fruit at the last 2 harvest dates, but this did not differ significantly from the control. Percent sugar.--The percentage of sugar in the fresh fruit was not altered significantly by treatment with GA3 23 (Table 3). In general, a reduction in sugar content was observed, but the differences between the control and the GA3 treated berries were not large enough for significance. Table 3. Percent sugar in fresh fruit treated by GA3 at various concentrations in 1966 at Grand Junction, Michigan. GA3 Percent Sugar Concentrations Harvest Dates (ppm) 8/10 8/17 8/24 Average 0 16.9 14.1 14.2 15.0 ;j 25 15.5 16.2 14.1 15.2 ‘ 75 14.8 14.0 13.3 14.0 150 15.4 13.3 13.4 14.0 NOSO NOSO NOSO NOSO N.S. Means not significantly different from 0 ppm. Seed weight.--The weight or amount of seeds found in 25 uniform sized berries was influenced significantly by GA3 treatment (Table 4.). At the first harvest, 25 ppm caused an increase in seed weight, whereas, 75 ppm decreased the seed weight, and no difference was observed for 150 ppm. No explanation for the difference in response to concentra- tions can be given. On 8/24, all concentrations reduced the weight of seeds significantly. Respiratory gas analysis.--Samples were taken from 'the first and second harvest to determine respiratory activ- ity of the fresh fruit. No significant effect of GA3 on 24 Table 4. Effect of GA3 at various concentrations on the seed weight of 25 uniform size berries in 1966 at Grand Junction, Michigan. GA3 Weight of Seeds in Grams Concentrations Harvest Dates (ppm) 8/10 8724 0 0.3313 0.1002 25 0.4302** 0.0775* 75 0.0880** 0.0202** 150 0.3015 . 0.0197** * Means significantly different from 0 ppm at the 5% level. ** Means significantly different from 0 ppm at 1% level. respiration was found (Table 5). A slight reduction in respiratory activity was noted, but this reduction did not differ significantly from the control. Table 5. Effect of GA on fruit respiration in 1966 at Grand Junction, Michigan (cc/Kg/hr). — _ GA3 Carbon Dioxide Evolution Concentrations Harvest Dates (ppm) 8/10 8/I7 0 29.5 28.4 25 25.8 26.5 75 27.2 26.1 150 31.1 27.1 NOSO NOSO ‘ N.S. Means not significantly different from 0 ppm. 25 Nutrient analysis.--GA3 did not alter either the leaf nutrient content (Table 6), or fruit nutrient content (Appendix Table 1A). Vegetative growth and floral initiation.--GA3 ap- plications at 2 weeks after full bloom had no effect on vegetative growth or number of flower buds (Table 7). These measurements were taken in early spring of 1967, so that the flower buds could easily be counted on the 1966 growth. A slight reduction in the percent of flower buds, and an increase in internode length was observed due to GA3 treat- ment. Discussion.--The GA3 concentrations used had no significant effect on fruit firmness, sugar content of fruit, analysis of respiratory gases, leaf or fruit nutrient analysis, vegetative growth, and floral initiation. However, weight of seed per fruit was reduced and the percentage of fruit in the largest size category was increased with 150 ppm GA3. Considering the possibility that fruit firmness may not have been a good index of maturity because only blue fruit were harvested and that the effect of GA3 on certain of the other parameters evaluated may be detectable ‘with a larger sample size, it was decided to continue the ‘treatments on the same bushes another year and vary the guarameters of evaluation but use the same concentrations. :. Ilaj 0N4” HN m.mm v33” mm. mm. mH.o Hm.o 0mm v0.0 mmH.o «ml—n omH MNH NH m.mm blnH mm on NH.O 0N.o mmN mo.o omH.o mat—n mm % @NH NH m.mN O.NH on. Nb NH.O om.o mNm H©.o v¢H.o mm.H mm MNH . ma v.vm m.NH Nb Nu. 0H.o Hm.o hem mo.o mini—”.0 No...” 0 6mm He Ema Gm and m Ema 90 Egg mm Ema s2 w m: w mu Ema mz wx wm wz “Emmy mucmEon ucofluusz mood mcoflumuucoocoo m «U um mom CH mCOaflMHUCOOGOO H H . . mSO Hw> um M40 ha Umuow m o M“ .ammseoaz .cofluocse mango o beefinusa mama .0 magma 27 Table 7. Vegetative growth and floral initation as affected by various concentrations of GA3 in 1966 at Grand Junction, Michigan. Measurements Taken in Spring of 1967 GA 3 Length Concentrations Flower Buds Total Buds Shoot Length Internode (ppm) (percent) (number) (inches) (inches) I 0 54.0 283 152.6 .539 E 25 49.5 306 171.8 .561 75 52.0 275 157.6 .573 150 49.3 272 160.5 .590 N.S. N.S. N.S. N.S. 5 N-S. Means not significantly different from 0 ppm. 1967 Study Nineteen sixty-seven was a good production year in the Grand Junction area for highbush blueberries. Ideal weather conditions occurred during pollination. Bee activ- ity was high due to the weather and the increased concen- tration of honeybee hives in the area. Honeybee hives were placed at the ends of each row of bushes used in the 1967 study. It has been shown that large concentrations of bees during pollination will increase yields under ideal weather conditions (13). Bees will not work in cool, rainy weather. Very little die-back was noted in the spring, because of a very mild winter. This led to increased growth and flowering potential for the treated bushes. 28 Experimental design.--A randomized block design composed of 2 replications of 5 bushes per treatment was again used. Parameters for measuring GA3 responses were berry size, berry growth, fruit firmness, percent sugar, total yield per bush, percent blue fruit, titratable acidity (citric acid), and canned product analysis. In the time of application study, a randomized block design was used, which consisted of 2 replications of 5 bushes per time of application. GA3 was applied as a foliar IKE-5.. . I" spray at a concentration of 25 ppm. Response parameters were titratable acidity, percent blue fruit, and total yield per bush. Sampling in both studies consisted of removing all fruit from 1 bush per treatment, then removing a 1 quart sam- ple for laboratory analysis. These samples contained both mature and immature fruit. Berry size.--Gibberellin A3 significantly altered the percentage of fruit found in size category No. 6 (Table 8). All concentrations of GA3 resulted in increase in size of berries for the first and second harvests and the average of all harvest dates. A slight decrease in berry size was found at the third harvest at concentrations of 25 ppm and 75 ppm. Berry growth.--Gibberellin A3 affected the growth of the third berry from the basal end of the cluster of 29 Table 8. Effect of various GA concentrations on berries in size category No. 6 (0.9 cm or greater) in 1967 at Grand Junction, Michigan. Percent of Berries in Size No. 6 Concentrations Harvest Dates (ppm) 8/2 8/9 8/16 9/13 Average 0 48.8 60.6 57.3 54.9 55.4 25 61.8* 77.8** 46.7* 59.9 61.6 75 61.6* 77.1** 48.1* 71.9** 64.7 150 70.1** 77.4** 63.6 58.2 67.6* * Means significantly different from 0 ppm at 5% level. ** Means significantly different from 0 ppm at 1% level. 10 different clusters per treatment, measured at weekly intervals throughout the growing season (Figure 1 or Appen- dix Table 2A). GA3 at 75 ppm enhanced the growth of the fruit, whereas, 25 ppm and 150 ppm GA3 delayed growth. The effect of GA3 was immediate on berry growth. One week after application, the effect of GA3 was noted whether it was an increase or decrease in berry growth. The GA3 effect held until the first harvest (8/2), when size of the tagged ber- ries showed very little difference. Fruit firmness and sugar content.--No effect of GA3 was found on fruit firmness or sugar content (Appendix Tables 3A and 4A). These data further substantiated the findings of 1966. Figure 1. 30 The effect of GA at various concentrations on berry growth in 1967 at Grand Junction, Michigan. «>00 no on a? N? mm mm —N 31 can gum—<01 Ema ms. (OI Ema nu (Gallo Emu O (Golo a ousmflm (ma) telewoga 32 Total yield per bush.--No significant difference was found in total yield per bush due to GA3 treatment (Appendix Table 5A). A slight reduction in total yield was noted on 8/2 and 8/16, but this reduction was not signifi- cant. Percent blue fruit.--GA3 150 ppm reduced the per- cent of blue fruit significantly at the first and second harvests (Table 9). At 25 ppm GA3 significantly increased the amount of blue fruit in the first harvest. GA3 at 150 ppm significantly reduced the percent of blue fruit on 8/2 and 8/9. No differences in fruit color were observed between any treatments on 8/16. Table 9. Percent blue fruit as affected by various con- centrations of GA in 1967 at Grand Junction, Michigan. (Complete harvest of one bush/replicate on each date). GA3 Percent of Blue Fruit by Number Concentrations Harvest Dates (ppm) 872 8/9 8/16 0 40.7 76.7 86.0 25 52.6* 65.4 81.2 75 48.1 67.5 91.9 150 25.5** 52.5** 84.6 *Means significantly different from 0 ppm at 5% level. '**Means significantly different from 0 ppm at 1% level. 33 Titratable acidity.--A significant increase in per- cent citric acid was found on 8/2 and 8/9 due to GA3 treat- ment (Table 10). On 8/2, 25 ppm and 75 ppm GA3 were sta- tistically the same as the control in percent citric acid, but 150 ppm was higher than the control. All GA3 treat- ments were more acid than the control on 8/9, but the same . on 8/16. If these data are compared with the findings of Woodruff (38) the berries would be considered to be im- mature. fl.“fl_ . Table 10. Effect of GA3 at various concentrations on . titratable acidity, expressed as percent citric acid, in 1967 at Grand Junction, Michigan. GA3 Percent Citric Acid Concentrations Harvest Dates (ppm) 8/2 8/9 8/16 0 1.45 .83 .44 25 1.35 l.l9** .32 75 1.59 1.12** .32 150 1.68* l.l7** .54 * Means significantly different from 0 ppm at 5% level. ** Means significantly different from 0 ppm at 1% level. Canned product analysis.--A11 GA3 treatments sig- nificantly increased the percent citric acid of the juice (Table 11) of canned blueberries. Drained weight, soluble solids, and pH were not affected by GA3 treatment. The canned fruit firmness followed closely the fresh fruit firmness and no significant difference was found. 34 Table 11. The effect of various concentrations of GA3 on canned products prepared from harvest 2 on 8/9 in 1967 at Grand Junction, Michigan. GA3 Drained Soluble Citric Fruit .Firmness Concentrations Weight Solids Acid Fresh Canned (ppm) (oz) (%) pH (%). 0 8.75 14.6 3.4 .16 73.0 28.3 25 8.25 14.7 3.1 .40** 73.9 28.0 75 8.75 14.5 3.3 .48** 73.1 26.5 150 9.25 14.5 3.1 .34** 73.2 30.0 ** Means significantly different from 0 ppm at 1% level. Time of Application Study Citric acid and blue fruit.--Gibberellin A3 at a concentration of 25 ppm applied at full bloom enhanced the loss of citric acid and increased the percentage of blue fruit at each harvest (Table 12). When GA3 at 25 ppm was applied 2 weeks and 7 weeks after full bloom, citric acid was significantly lower on 8/2 and 8/16. The percentage of blue fruit was increased at all harvest dates, but sig— nificantly only on 8/2. Total yield.--Gibberellin A3 at a concentration of 25 ppm applied at full bloom significantly increased the total yield per bush on 8/16 (Table 13). Yield data was not taken at earlier harvests. II- 35 Table 12. Effect of GA at 25 ppm on percent citric acid and percent Blue fruit when applied at full bloom, 2 weeks after full bloom, and 7 weeks after full bloom in 1967 at Grand Junction, Michigan. Harvest Dates . 8/2 8/9 8/16 T12: Of Citric Blue Citric Blue Citric Blue 3 Acid Fruit Acid Fruit Acid Fruit E Application (%) (e) (%) (%) (%) (s) i 3 None 1.45 40.7 .83 76.7 .44 86.0 | Full Bloom .56** 75.5** .34** 86.1 .23** 92.8 p a 2 Wk After f.b.a 1.05** 54.8* .71 79.0 .27** 91.1 5 7 Wk After f.b. 1.05** 54.1* .68 78.6 .23** 92.6 i a f.b. - Full bloom. * Means significantly different from 0 ppm at 5% level. ** Means significantly different from 0 ppm at 1% level. Table 13. Effect of GA3 at 25 ppm applied at different times on total yield at the last harvest in 1967 at Grand Junction, Michigan. Time of Total Yield Per Bush Application (lbs) Control 18.00 Full bloom 24.00* 2 wk after full bloom 20.50 7 wk after full bloom 18.00 * Means significantly different from 0 ppm at 5% level. 36 Discussion.--The percentage of fruit in the largest size category was increased with 150 ppm GA3. GA3 concen- trations used had no significant effect on fruit firmness, sugar content, or total yield. The growth of fruit was enhanced by GA3 at 75 ppm, whereas, 25 ppm and 150 ppm GA3 delayed berry growth. GA3 at 150 ppm reduced significantly the percent of blue fruit, and increased the percent citric acid at the first and second harvest. At the second har- vest, 25 ppm and 75 ppm GA3 increased citric acid content of the fruit. In fruit canned from the 8/9 harvest, the percent citric acid was significantly enhanced by GA3 treat- ment. Drained weight, soluble solids, pH, and fruit firm- ness was not affected by GA3. GA3 at 25 ppm applied at full bloom, 2 weeks and 7 weeks after full bloom enhanced the loss of citric acid and increased the percentage of blue fruit. Total yield per bush was increased significantly only by the full bloom application. The increase in bee population, combined with good conditions for fruit set, resulted in a marked increase in yield. This would be an important variable when trying to elucidate the effects of GA3 on the highbush blueberry. It was decided to alter the 1968 treatments to include a study of this. Also, higher concentrations, 250 ppm and 500 ppm, should be studied. Unpublished results from 37 New Jersey5 indicate a beneficial effect at these concen- trations. GA3 at 150 ppm gave the most consistent delay in maturity for 2 years, therefore 150 and 300 ppm GA3 were chosen to determine the effects of GA3 in conjunction with mechanical harvesting. 1968 Study Nineteen sixty-eight was a year of good production on late blooming varieties of the highbush blueberry. A late spring freeze destroyed most of the early blooming flowers. Early blooming varieties were especially affected in the Grand Junction area, so it was decided to use another location for the pollination study. A plantation near South Haven was selected, because the freeze damage appeared to be only slight. Another plantation was selected at Grand Junction for the mechanical harvesting study. The plantation con- tained Jersey bushes that had been adapted for mechanical harvesting. Little freeze damage was observed in this plantation. Experimental design.--In the mechanical harvesting study, the field plot design was a randomized block con- sisting of 2 replications of 20 bushes per treatment with 5Personal communication, P. Eck, Rutgers University, New Brunswick, N. J. 1' ’m ”2“; 38 a split for harvest date. The response parameter consisted of total yield for each 10 bushes. Severe pruning 2 years before in order to facilitate mechanical harvesting was expected to reduce yields per bush. Sampling consisted of removing all fruit from the bushes and obtaining total weight. The GA3 was applied 2 weeks after full bloom at concentrations of 150 ppm and 300 ppm. In the pollination study, the field plot design was a randomized block consisting of a factorial arrangement of 3 pollinating intensities and 3 levels of GA3 with 3 repli- cations of 2 bushes each. Parameters for measuring responses were berry size, sugar content, titratable acidity, seed weight, and total yield. Only the average of the different harvesting periods will be presented. Sampling consisted of removing all blue fruit from each treatment at weekly intervals starting on 8/2. The GA3 was applied at concen- trations of 250 and 500 ppm at full bloom. Mechanical Harvesting Study Total yield.--GA3 at the various concentrations applied 2 weeks after full bloom produced no significant effect on total yield (Table 14). A slight decrease in yield was noted at the first picking, but a slight increase in yield occurred at the last picking due to GA3 treatment. GA3 significantly reduced yield when only 1 late picking was used (Table 15). All harvesting was done using the straddle row mechanical picker. 39 Table 14. Yield as affected by GA3 treatment at various concentrations applied 2 weeks after full bloom in 1968 at Grand Junction, Michigan. Yield Per Ten Bushes GA 3 First Picking Second Picking 7Total Yield Concentrations (7/31) (8/28) (PPm) (lbs) (lbs) (lbs) 0 39.50 8.87 48.38 150 33.37 9.87 42.75 300 36.80 10.12 47.00 N’s. N.S. N.S. N.S. Means not significantly different from 0 ppm. Table 15. Effect of GA at different concentrations on total yield using only 1 late picking in 1968 at Grand Junction, Michigan. = GA3 Yield Per Ten Bushes Concentrations Late Picking (8/28) (ppm) (lbs) 0 23.00 150 lS.00* 300 15.87* * Means significantly different at 5% level. Pollination Study Berry size.--GA3 had no effect on the final size <3f the ripe fruit (Table 16 and Figure 2). Fruit sprayed (with 500 ppm GA3 and without bees were significantly larger 'than those without GA3 and no bee pollination (Figure 2). . ‘ 0 .‘.. U'I~ 40 Figure 2. The effect of GA at various concentrations on berry growth under different pollinating inten- sities in 1968 at South Haven, Michigan. 41 > a on 3.2..” no on 0% at mm mm a 3 k _ _ _ _ _ _ _ _ . 0 «can as! I‘m a I I. o 2.. 2... a... a I N «can 2! Ifimgm I l V. IO. 0 w .l .0 q m n: .. a. .w I o.— I u.— .. t— N musmflm 42 GA3 at 250 ppm gave the same results and neither 250 ppm nor 500 ppm were different from the 0 ppm GA3 and bees. Table 16. Berry size at harvest as affected by GA3 appli- cations and different pollinating intensities in 1968 at South Haven, Michigan. m Berry Size GA3 Caged With Caged.without Open Concentrations Bees Bees Pollination (ppm) (cm) (cm) (cm) 0 1.30 1.30 1.40 250 1.30 1.30 1.30 500 1.40 1.30 1.30 N.S. N.S. N.S. N.S. Means not significantly different from 0 ppm. Sugar content.--No effect of GA3 was found on sugar content under any of the pollinating intensities (Table 17). Table 17. The effect of GA3 applications on berry sugar content in 1968 at South Haven, Michigan. f GA Sugar Content of Berries 3 Caged With Caged Without Open Concentrations Bees Bees Pollination (Ppm) (%) (%) (%) 0 14.9 15.2 15.3 250 15.1 15.2 15.2 500 15.2 14.9 15.1 N.S. N.S. N.S. N.S. Means not significantly different from 0 ppm. 43 Titratable acidity.--No effect of GA3 was found on the percent of citric acid present in the fresh fruit (Table 18). Although a slight increase in citric acid con- tent was noted when GA3 was used to produce parthenocarpic berries. Table 18. Percent citric acid as affected by GA3 applica- tions in 1968 at South Haven, Michigan. Citric Acid GA3 Caged With Caged Without Open Concentrations Bees Bees Pollination (ppm) (%) (%) (%) 0 .39 .36 .36 250 .40 .39 .39 500 .39 .45 .39 N.S. N.S. N.S. N.S. Means not significantly different from 0 ppm. Seed weight.--Gibberellin A3 significantly reduced the weight of seeds found in 25 uniform size berries (Table 19.) Gibberellin A3 in some way interfered with seed develOpment. Total yield.--GA3 at various concentrations applied at full bloom significantly influenced the total yield per bush (Table 20). Bushes caged with one hive of honeybees per 2 bushes showed a significant reduction in total yield when sprayed with GA3. On bushes caged without bees, an increase of 2.96 lbs and 5.19 lbs per bush for applications 44 Table 19. Effect of GA at various concentrations on seed weight of 25 uniform size berries in 1968 at South Haven, Michigan. Seed Weight GA 3 Caged With Caged'Without Open Concentrations Bees Bees Pollination (ppm) (grams) (grams) (grams) .3 0 .409 .073 .434 250 .319* .045* .338* . 500 .310* .067 ' .316* ‘ * Means significantly different at 5% level. Table 20. The effect of GA3 applications on total yield per bush in 1968 at South Haven, Michigan. Total Yield Per Bush GA3 Caged With Caged Without Open Concentrations Bees Bees Pollination (PPm) (lbs) (1bS) (lbs) 0 15.16 3.04 8.75 250 10.24* 6.00* 11.55* 500 9.41* 8.23** 14.34** * Means significantly different at 5% level. ** Means significantly different at 1% level. of 250 ppm and 500 ppm GA3, respectively, was noted. This fruit was seedless or parthenocarpic. Open pollinated bushes showed an increase of 2.80 lbs and 5.59 lbs per bush when 250 ppm and 500 ppm GA3 was used respectively. 45 Discussion.—-The study using a commercial straddle row mechanical harvester showed that GA3 when applied 2 weeks after full bloom had no effect on total yield. A significant reduction in yield was noted when only 1 late picking on 8/28 was made. In the pollination study, GA3 had no effect on the final berry size, sugar content, and percent citric acid under different pollinating intensities. However, the size of parthenocarpic berries set with GA3 were equal in size to those set with pollination. Gibberellin A3 showed a de- crease in seed weight with increasing concentrations under all pollinating intensities. Total yield was reduced by GA3 on bushes caged with a high population of bees. The total yield was increased by GA3 on bushes caged without bees and open pollinated bushes. DISCUSSION Berry size.-—Gibberellin A3 at 150 ppm applied 2 weeks after full bloom increased the percentage of berries in the largest size category (0.9 - 1.0 cm diameter) in 7 both 1966 and 1967. These data were collected using the ; mechanical sizer, which measured minimum diameter of the berry. Berries in this size category would be placed in the premium grade of "Great Lakes" by the Michigan Blue- L berry Growers' Association. To be placed in this grade, no more than 175 berries must be used to fill a standard cup. Gibberellin A3 has been shown to increase cell en- largement (30), and to enhance the release of diffusible auxin, which could affect fruit growth. Rate of berry growth was altered by GA3. Gibber- ellin A3 at 75 ppm enhanced the growth of the fruit, whereas, GA3 at 25 ppm and 150 ppm delayed the growth. A possible explanation for the different effect on berry growth of GA3 at various concentrations may be the effect of exogenous GA3 on the natural levels of gibberellins. It was found by Mainland and Eck (25) that exogenous GA3 decreased the lev- els of natural gibberellins. Gibberellin A3 had no effect on final berry size when applied at full bloom to bushes caged with and without bees, or open pollinated. 46 47 However, when GA3 was applied at concentrations of 250 ppm and 500 ppm, there was an indication that GA3 without bees caused the fruit to be larger early in the growing season. These data showed that GA3 does not affect final berry size, but can substitute for the bee effect. Titratable acidity and percent blue fruit.-fThe a effect of GA3 on acidity and percent blue fruit was depen- dent on time of application and concentration. When GA3 was applied at full bloom at concentrations up to 500 ppm, the percent of citric acid not affected and percent blue in . fruit was enhanced. GA3 applied at a concentration of 150 ppm or above as a post-bloom spray delayed the loss of citric acid and decreased the percent blue fruit early in the harvest season. Endogenous production of gibberellins is known to begin at full bloom (35). Therefore, if exog- enous gibberellins are applied at full bloom, an enhance- ment of fruit maturity could be expected. Later in stage I of fruit growth, endogenous gibberellins begin to de- crease. Perhaps if gibberellins are applied post-bloom, the fruit may remain in stage I longer, thus, a delay in maturity. The higher level of citric acid resulting from GA3 applications could be of great importance to blueberry marketing associations. Ballinger, 22 31. (2) has shown blueberry keeping quality to be correlated with fruit acid- ity, with high acidity giving longer keeping quality to 48 the fresh product. With this longer keeping quality, blue- berries could have a longer shelf life or they could be shipped greater distances and new markets developed. Also, higher percent of citric acid carried over into the pro- cessed product could mean that other processors, such as pie and jelly manufacturers would have to use less artifi- cial acid to prepare their product. Seed weight.--Gibbere11in A3, whether applied at full-bloom or post-bloom, decreased the amount of seeds per fruit. GA3 at high concentrations could cause the seeds to fail to develop, or cause them to abort. Seeds from GA3 fruit were examined under a microscope and very few brown viabile seeds were found. The seeds from GA3 treated fruit were white and clear, which is considered to be non-viabile. GA3 could, also, decrease seed weight at full-bloom by decreasing pollen viability. Total yield.--Under controlled pollination, GA3 increased yield per bush if bee concentrations were not too high. When a honeybee hive was placed in a cage for each 2 bushes, a decrease in yield was noted for GA3 treat- ments. This was probably due to the extremely high concen- tration of bees. The pollinator intensity of 1 hive/2 bushes is approximately 100x normal commercial practice. Under commercial conditions GA3 increased the total yield an additional 2.80 lbs to 5.59 lbs per bush. With a 49 planting of 1,000 bushes per acre, this would amount to about 5,590 lbs per acre increase in production. The opti- mum yield per bush under these conditions would be about 15 lbs, and over 14 lbs per bush was obtained with open pollination and GA3 at 500 ppm. Thus, it would appear that 500 ppm GA3 at full-bloom would assure optimum yields. No response parameters.--GA3, regardless of time [I of application or concentration, had no effect on sugar content, leaf or fruit nutrient analysis, respiratory gas analysis, fruit firmness, vegetative growth, and floral fl initiation. Residue.--Analyses were made by the Wisconsin Alumni AssociationeiJ11968 to determine the residual amounts of gibberellins found in the fruit after GA3 treatment. Less than 0.02 ppm gibberellins were found in the fresh fruit regardless of concentration of GA3 applied or time of application (Appendix Tables 6A and 7A). An experimen- tal label for blueberries with a tolerance of 0.15 ppm gibberellins is being requested by Abbot Laboratories for 1969. 6Analysis made at request of Abbott Laboratories, N. Chicago, Illinois. FUTURE RESEARCH In order to substantiate these findings, these studies should be continued. Additional information is needed on: (1) methods of application, (2) effect of high concentrate sprays, (3) other varietal responses, (4) loca- tion responses, (5) long term effect of repeated application, (6) further studies at different concentrations, and (7) time of application studies. 50 f”. 3': H X4 ' SUMMARY Field experiments were conducted during the summers of 1966, 1967, and 1968 to evaluate the effect of gibberel- mm lic acid (GA3) on the highbush blueberry. The blueberry plantations used were located at Grand Junction and South Haven in southern lower Michigan. In 1966 and 1967, a randomized block design was Er} used with two replications of five bushes per treatment of the variety Jersey. Treatments were single applications of GA3 applied as a solution sprayed to the drip point two weeks after full bloom. Concentrations used were 25, 75, and 150 ppm. In 1967, treatments were applied to the same bushes used in 1966. In 1966 and 1967, GA3 significantly altered the percentage of fruit found in size category No. 6 (0.9 cm or greater). In 1966, berry size was enhanced at the later two harvest periods. In 1967, the percentage of fruit in size No. 6 was increased at the early harvests and at a late harvest. Differences in size due to concentrations occurred both years. Data suggests that in 1966, 25 ppm and 150 ppm increased average berry size. In 1967, 25 ppm and 75 ppm increased average berry size, depending on har- 'vest period. In 1967, berry growth was enhanced by GA3 at 75 ppm, whereas 25 ppm and 150 ppm delayed berry growth. 51 52 Berry firmness and sugar content was not signifi- cantly altered in either 1966 or 1967. Seed weight was significantly reduced in 1966 at all concentrations. In 1966, no effect of GA3 was observed on fruit respiration rate, leaf or fruit nutrient content, and veg— etative growth and floral initiation the season following treatment. In 1967, no significant difference was found in total yield per bush due to GA3 treatment. A slight reduc- tion in total yield was noted at the first and third har- L Q! J’ .37.“— ii“ vest periods. Gibberellin A3 reduced the percentage of blue fruit at the first and second harvests, and increased the percent citric acid in 1967. In the canned product, 1967, no effect of GA3 was observed on drained weight, soluble solids, pH, and fruit firmness. Total acidity of the juice was increased at all concentrations. In 1967, when GA3 at 25 ppm was applied at full bloom, it enhanced the total yield, the loss of citric acid, and increased the percentage of blue fruit. From these two years research, it was found that GA3 applied at full bloom may cause fruit to ripen earlier than if it is applied after full bloom. High concentrations delayed ripening and low concentrations enhanced ripening. In 1968, two experiments were established to study further the delay in ripening and the effect on yield. 53 Gibberellin A3 was applied two weeks after full bloom in order to use the straddle the row picker. Two harvests were made over the same bushes, one early and one late. Gibberellin A3 did not affect total yield, but a decrease in yield was noted for the first picking. The late picking showed an increase in yield with concentrations. When only ‘:“'L a late picking was used a reduction in total yield occurred due to treatment. In order to study the effect of GA3 on increasing yield an experiment was set up under different pollinating fl All'hull ‘1 n -rur ‘12:. ( intensities. Some blueberry bushes were caged with and without bees, while others were left to be pollinated in the Open. The GA3 was applied at full bloom at concentra- tions of 250 ppm and 500 ppm. GA3 had no effect on final berry size, but there was an indication that GA3 did cause the fruit to be larger early in the growing season. No effect of GA3 on sugar content or percent citric acid of the fruit was noted under any of the pollinating intensities. Seed weight was reduced by GA3 treatment under all pollinating intensities. This would indicate GA3 was either interferring with pollen viability or directly causing embryo abortion. The effect of GA3 on total yield depended on the pollinating agent. In bushes caged with bees, GA3 reduced yield, while in those caged without bees or in the open, GA3 increased the yield. It was found that by combining S4 GA3 and a small number of honeybee hives per acre near Optimum yields could be achieved. r35 LITERATURE CITED 10. LITERATURE CITED Bailey, J. S. 1947. Development time from bloom to maturity in cultivated blueberries. Proc. Amer. Soc. Hort. Sci. 49:193-195. Ballinger, W. E., L. J. Kushman, and G. J. Galletta. 1968. Relationship of fruit acids and sugars to keeping quality of blueberries. North Carolina State University, Raleigh, N. C. Barker, W. G. and W. B. Collins. 1965. Parthenocarpic fruit set in the lowbush blueberry. Proc. Amer. Soc. Hort. Sci. 87:229-233. 2 Bonde, E. K. and T. C. Moore. 1958. Effect of gib- berellic acid on the growth and flowering of Tele- phone peas. Physiol. Plant. 11:451-456. Bradley, M. V. and J. C. Crane. 1960. Gibberellin- induced inhibition of bud development in some spe- cies of Prunus. Science 131:825-826. Chrispeels, M. J. and J. E. Varner. 1967. Hormonal control of enzyme synthesis: On the mode of action of gibberellic acid and abscision in aleurone layers of barley. Plant Physiol. 42:1008-1016. Coggins, C. W. and H. Z. Hield. 1962. Navel orange fruit response to potassium gibberellate. Proc. Amer. Soc. Hort. Sci. 81:227-230. , and S. B. Boswell. 1960. The influence of potassium gibberellate on Lisbon lemon trees and fruit. Proc. Amer. Soc. Hort. Sci. 76:199-207. Crane, J. C. 1964. Growth substances in fruit set- ting and development. Ann. Rev. Plant Physiol. 15:303-326. Dennis, F. G., Jr. and L. J. Edgerton. 1966. Effects of gibberellins and ringing upon apple fruit devel- opment and flower bud formation. Proc. Amer. Soc. Hort. Sci. 88:14-24. 55 11. 12. l3. 14. 15. 16. 17. 18. 19. 20. 21. 22. 56 Devlin, R. M. and I. E. Demoranville. 1967. Influence of gibberellic acid and Gibrel on fruit set and yield in Vaccinium macrocarpon cv. Early Black. Dilley, D. R. 1966. Measuring the respiration of fruits and vegetables. The Analyzer 7(4):3-7. Dorr, J. and E. C. Martin. 1966. Pollination studies on the highbush blueberry. Mich. Agric. Exp. Sta. Quart. Bul. 48(3):437-448. Edgerton, L. J. 1966. Some effects of gibberellin “D and growth retardants on bud development and cold hardiness of peach. Proc. Amer. Soc. Hort. Sci. 88:197-203. Gordon, J. and R. Pankratz. 1968. Fluorometric deter- mination of gibberellic acid. Jour. Agri. and Food Chem. 16:520-522. §,_ Greenhalgh, W. J. and L. J. Edgerton. 1967. Inter- action of Alar and gibberellin on growth and flow- ering of the apple. Proc. Amer. Soc. Hort. Sci. 91:9-17. Guttridge, C. G. 1962. Inhibition of fruit bud for- mation in apple with gibberellic acid. Nature. 196:1008. Hayashi, R. N. F., M. J. Bukovac and H. M. Sell. 1968. Occurrence of Gibberellin A3 in parthenocarpic apple fruit. Plant Physiol. 43:448-450. Hindle, R., Jr., V. G. Shutak and E. P. Christopher. 1957. Growth studies of the highbush blueberry fruit. Uni. of Rhode Island. 69:282-287. Hull, J., Jr. and L. N. Lewis. 1959. Responses of one-year-old cherry and mature bearing cherry, peach and apple trees to gibberellins. Proc. Amer. Soc. Hort. Sci. 74:93-100. Ismail, M. A., R. H. Biggs and M. F. Oberbacher. 1967. Effect of gibberellic acid on color changes in the rind of three sweet orange cultivars (Citrus sinensis Blanco). Proc. Amer. Soc. Hort. Sci. 9I:Iz§-I490 Jackson, D. I. 1967. Gibberellin and the growth of peach and apricot fruits. Aust. Jour. Biol. Sci. 21:209-215. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 57 Kenworthy, A. L. 1967. Plant analysis and interpre- tation of analysis for horticulture crops. Soil Testing and Plant Analysis. Part 11:59-75. Lang, A. and E. Reinhard. 1961. Gibberellins and flower formation. Gibberellins, Adv. Chem. Ser. 28:71-79. Mainland, C. M. and P. Eck. 1966. Use of gibberellin and auxin to set and develop highbush blueberry fruit. Proc. North Am. Blueberry Workers Conf., mm Maine Agri. Expt. Sta., Uni. of Maine. Misc. Rep. 118:117-122. and . 1968. Cranberry fruit set, growth ‘ and yield as influenced by gibberellic acid alone 3 and in combination with Alar. Proc. Amer. Soc. 7 Hort. Sci. 92:296-300. Marcelle, R. and C. Sironval. 1963. Effect of gib- a} berellic acid on flowering of apple trees. Nature 197:405. Proebsting, E. L., Jr., and H. H. Mills. 1966. Effect of gibberellic acid and other growth regulators on quality of early Italian prunes (Prunus domestics L.). Proc. Amer. Soc. Hort. Sci. 89:135-139. Robbins, W. W. 1931. The Botany of Cro Plants. Blakiston's Son and Co., Philadelphia, pp. 517-522. Sachs, R. M. and R. J. Weaver. 1968. Gibberellin and auxin-induced berry enlargement in Vitis vinifera L. Jour. Hort. Sci. 43:185-195. Scott, P. C. and A. C. Leopold. 1967. Opposing effects of gibberellin and ethylene. Plant Physiol. 42:1021-1022. Smith, C. R. 1960. Effect of growth regulators on fruit crops of the Coville blueberry. Blueberry Research Fifty Years of Progress. New Jersey Agri. Expt. Sta., Rutgers Uni. p. 23. Sparks, D. 1967. Effect of potassium gibberellate on fruit characteristics and flowering of the pecan Carya illinoensis Koch cv. Stuart. Proc. Amer. Soc. Hort. Sci. 90:61-66. 34. 35. 36. 37. 38. 39. 58 Valdovinos, J. G., L. C. Ernest and E. W. Henry. 1967. Effect of ethylene and gibberellic acid on auxin synthesis in plant tissue. Plant Physiol. 42:1803-1806. Van Overbeek, J. 1966. Plant hormones and regulators. Science. 152:721-731. Wittwer, S. H. and M. J. Bukovac. 1962. Quantitative and qualitative differences in plant response to the gibberellins. Amer. Jour. Bot. 49:524-529. , and N. E. Tolbert. 1960. 2-Chlorethy1 ’ trimethylammonium chloride and related compounds [7 as plant growth substances. III. Effect on growth and flowering of the tomato. Amer. Jour. Bot. 47:560-565. Woodruff, R. E. 1959. Chemical and physical changes of blueberry fruit associated with ripening and L} deterioration. Ph.D. Thesis, Michigan State Uni- versity. Young, R. S. 1952. Growth and development of the blueberry fruit Vaccinium corymbosum L. and Z. angustifolium Ait. Proc. Amer. Soc. Hort. Sci. 59:167-172. APPENDIX 59 00H nH 0.HH n.NH NN 0N no.0 HH.0 0mm 00.0 0nH.0 n0.0 00H Hma 0H 0.HH n.0 mm mm no.0 no.0 0mm 00.0 mna.o 00.0 0n Hma ma N.0H m.0H om 0H no.0 00.0 m0m 00.0 0na.0 00.0 mm mHH 0H 0.0a m.n mm nH 00.0 00.0 oam n0.0 0nH.0 00.0 o Ema Ema Ema 5mm 8mm Ema m w Ema AEmmv Hfl GN m 50 mm G2 02 mu 02 w& mm wz mGOHuMHquOGOU mucmEmHm ucmflupsz m<0 Gun mCOfl#MHflchCOU .cmmflnosz .aospocsn mango um coma msoflnm> um mdw no pmuommmm mm psousoo ucmwuuss ufisum .4H OHQMB 60 Table 2A. Effect of GA at various concentrations on berry diameter (cm? in 1967 at Grand Junction, Michigan. GA Diameter of Third Berry From Basal Part 3 of Inflorescence Concentrations Measurement Dates (ppm) 6710 6/21 6/28 7/6 7/12. 7/183 7728 872 0 .42 .82 .87 .93 .95 .97 1.07 1.30 25 .39 .61* .67* .71* .75* .76* .82* 1.20 75 .43 .90* .96* .97 .99 1.04 1.12 1.30 150 .37 .73 .80 .81* .81* .83* .85* 1.25 E a Earliest color change. f 1 * Means significantly different from 0 ppm at 5% level. ** Means significantly different from 9 ppm at 1% level. Table 3A. Firmness of fresh fruit as influenced by GA3 at different concentrations in 1967 at Grand Junction, Michigan. GA3 Firmness as Measured With a Durometer Concentrations ' Harvest Dates (Ppm) 8/2 879 8/16 0 71.2 69.6 72.6 25 68.6 66.1 70.3 75 70.4 66.7 74.9 150 63.9 63.6 72.3 N.S. N.S. N.S. N.S. Means not significantly different from 0 ppm. 61 Table 4A. Percent sugar in fresh fruit treated with GA3 at various concentrations in 1967 at Grand Junction, Michigan. .—-—-—-—-1,, :---—= GA3 Percent Sugar Concentrations Harvest Dates (ppm) 872 8/9 8/16 0 15.7 15.3 13.8 77 25 14.9 15.8 14.2 7 75 15.8 14.9 14.0 150 15.5 15.0 14.1 ‘ N.S. N.S. N.S. W) N.S. Means not significantly different from 0 ppm. Table 5A. The effect of GA3 at various concentrations on yield per bush in 1967 at Grand Junction, Michigan. _ t GA3 Total Yield Per Bush Concentrations Harvest Dates (Ppm) 8/2 8/9 8716 0 15.6 lbs 17.1 lbs 18.0 lbs 25 15.0 17.7 15.5 75 11.8 20.2 16.0 150 12.3 18.8 13.4 N.S. N.S. N.S. N.S. Means not significantly different from 0 ppm. 62 Table 6A. Effect of GA3 applications on residual gibber- ellins in the fruit in 1968 at South Haven, Michigan. GA3 Residual. Concentrations Gibberellins (ppm) (ppm) 0 less than 0.02 250 less than 0.02 500 less than 0.02 Source: Wisconsin Alumni Association - for Abbott Labora- tories. Table 7A. Effect of GA applications on endogenous gib- berellins in 1968 at Grand Junction, Michigan. GA3 Residual Concentrations Gibberellins (ppm) (Ppm) 0 less than 0.02 150 less than 0.02 300 less than 0.03 Source: Wisconsin Alumni Association - for Abbott Labora- tories. L