Temperature and Genotype Influence Sweet Cherry

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TEMPERATURE AND GENOTYPE INFLUENCE SWEET CHERRY

POLLINATION BIOLOGY

LU ZHANG

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Department of Horticulture

DECEMBER 2014 To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of

LU ZHANG find it satisfactory and recommend that it be accepted.

______Matthew D. Whiting, Ph.D., Chair

______Bhaskar Bondada, Ph.D.

______Preston K. Andrews, Ph.D.

______Roy A. Navarre, Ph.D.

ACKNOWLEDGEMENTS

I would like to express the deepest appreciation to my committee chair, Dr. Matthew Whiting, who showed me the road and always supported me on the path to this degree. Without his encouragement, understanding and patient help this dissertation would not have been possible. I am also thankful for the excellent example he has provided as a kind, bright and enthusiastic professor, and his personality charisma will inspire me to become a better person.

I would also like to express my gratitude to my committee members, Dr. Roy Navarre, Dr. Preston

Andrews and Dr. Bhaskar Bondada for their support, enthusiastic encouragement and invaluable advice for my research.

I would like to express my special thanks to Dr. Caixi Zhang, Mr. Lynn E. Long and Dr. Yiannis

Ampatzidis for their efforts and help on this work. I would also like to thank my friends and everyone in my lab for their help and support in my research and life. Special thanks to Ms. Laura

Wells for valuable assistance and care.

Finally, but most importantly, I am deeply thankful to my parents for their supporting and understanding. I would like to thank my husband Yanwei for his unyielding love, support and devotion all these years. My special and warmly appreciation to my little boy Steven, his coming and accompany makes me strong and happy.

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TEMPERATURE AND GENOTYPE INFLUENCE SWEET CHERRY

POLLINATION BIOLOGY

Abstract

By Lu Zhang, Ph.D. Washington State University December 2014

Chair: Matthew D. Whiting

In the Pacific Northwest of the U.S., the commercial productivity of several sweet cherry (Prunus avium L.) varieties with outstanding fruit attributes is poor, such as ‘Tieton’, ‘Regina’ and ‘Benton’.

Study of the reproductive characteristics (e.g. flower number/spur and fruit set) of these cultivars has revealed that low fertilization rate (i.e., fruit set), specifically related to maternal factors, is the main cause of low yields. This research project therefore studied the role of temperature on floral organ development and, stigma receptivity and ovule viability in four model sweet cherry cultivars:

‘Benton’ (self-fertile, low productivity), ‘Rainier’ (self-sterile, high productivity), ‘Tieton’ (self- sterile, low productivity), and ‘Sweetheart’ (self-fertile, high productivity). Stigma receptivity was assessed by evaluating stigma surface development, pollen hydration level, germination rate and pollen tube growth in vivo. Controlled-climate chambers were programmed to mimic cold, average, or warm spring flowering conditions. In addition field trials were conducted to develop practical strategies to improve fruit set.

The lengths of styles and filaments were most sensitive to temperature, being about 11% and 25% shorter in the low temperature environment compared to high temperature, respectively. Generally, pollen hydration and germination were poor under cool temperature and ovules were apt to lost

viabilities by warm temperatures. Two to three days post flowering, the pollen hydration level, germination rate and fruit set reached the optimal value. Compared with productive cultivars, the primary ovules of ‘Tieton’ and ‘Benton’ lost viability at a faster rate (e.g., 13% viable ovules in

‘Tieton’ vs. 77% in ‘Rainier’ seven days post pollination). Lastly, field trials with aminoethoxyvinylglycine (AVG) (commercial product: ReTain® ) applied at prior to and during flowering revealed the potential to prolong the ovule lifespan and improve fruit set. The percent of inactive ovules of ‘Tieton’ and ‘Regina’ decreased by ca. 180% and 50% while fruit set increased by ca. 120% and 60% by with 499 g/acre ReTain®, respectively. Combined, these results reveal a cause for poor productivity in several sweet cherry cultivars along with a potential commercial solution to overcome the problem.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... iii

Abstract ...... iv

TABLE OF CONTENTS ...... vi

LIST OF TABLES ...... x

LIST OF FIGURES ...... xi

CHAPTER ONE FLOWER DEVELOPMENT, POLLINATION BIOLOGY AND

MANAGEMENT IN SWEET CHERRY PRODUCTION ...... 1

1.1 Flower bud development and management ...... 4

1.1.1 Flower induction, initiation and their management ...... 5

1.1.2 Flower differentiation, dormancy and their management ...... 8

1.1.3 Final bud development and management ...... 10

1.2 Flowering, pollination and their management ...... 12

1.2.1 Flower structure, density and quality ...... 12

1.2.2 Pollinizer, pollinator and outcross ...... 14

1.2.3 Pistil role and effective pollination period (EPP) ...... 15

References ...... 21

CHAPTER TWO SWEET CHERRY FLORAL ORGAN SIZE VARIES WITH GENOTYPE AND

TEMPERATURE ...... 33

Abstract ...... 33

2.1 Introduction ...... 34

2.2 Materials and Methods ...... 37

2.2.1 Plant Material ...... 37

2.2.2 Temperature ...... 38

2.2.3 Experimental Design ...... 39

2.2.4 Statistical Analysis ...... 40

2.3 Results ...... 40

2.3.1 Floral Organ Size ...... 40

2.3.2 Correlation among Floral Organs ...... 43

2.4 Discussion ...... 43

2.5 Conclusion ...... 49

Acknowledgement ...... 50

References ...... 51

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CHAPTER THREE ASSESSING THE ROLE OF PISTIL IN SWEET CHERRY FRUIT SET

WITH CONTROLED ENVIRONMENT ...... 72

Abstract ...... 72

3.1 Introduction ...... 73

3.2 Material and Methods ...... 76

3.2.1 In Lab-Pistil Role Evaluation ...... 76

3.2.2 In-field Fruit Set Evaluation ...... 80

3.3 Results ...... 81

3.3.1 Stigma Development ...... 81

3.3.2 Pollen Hydration ...... 82

3.3.3 Pollen Germination ...... 84

3.3.4 Pollen Tube Growth ...... 85

3.3.5 Ovule Viability ...... 86

3.3.6 Fruit Set in Field ...... 88

3.4 Discussion ...... 88

3.5 Conclusion ...... 94

Acknowledgement ...... 95

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References ...... 96

CHAPTER FOUR THE EFFECT OF RETAIN® APPLICATION ON OVULE VIABILITY, FRUIT

SET AND QUALITY OF SWEET CHERRY IN PACIFIC NORTHWEST ...... 114

Abstract ...... 114

4.1 Introduction ...... 115

4.2 Material and Methods ...... 117

4.2.1 Orchard locations and plant materials ...... 117

4.2.2 Experimental Design ...... 119

4.2.3 Sample Treatments ...... 121

4.2.4 Statistical analyses ...... 122

4.3 Results and Discussion ...... 123

4.3.1 Fruit set ...... 123

4.3.2 Ovule viability ...... 125

4.3.3 Fruit quality ...... 127

4.4 Conclusion ...... 130

References ...... 131

LIST OF TABLES

Table 2-1. Correlation matrix among floral parameters of various sweet cherry cultivars: (a) Benton;

(b) Rainier; (c) Tieton; (d) Sweetheart.a, b ...... 57

Table 3-1. Pollen tube growth in ‘Benton’, ‘Rainier’, ‘Tieton’ and ‘Sweetheart’ sweet cherries flowers cultivated under high, moderate and low temperatures and sampled 8, 24 and 48 hours after the procedure of hand-pollination...... 101

Table 3-2. Fruit set of emasculated sweet cherry flowers receiving manual pollination in the field at daily intervals after anthesis in ‘Benton’, ‘Rainier’, ‘Tieton’ and ‘Sweetheart’...... 102

Table 4-1. Fruit qualities of sweet cherry cv. ‘Tieton’ by AVG treatment at 166,333 and 499g/acre rates and the control at 10% bloom in both commercial and experimental orchard located in

Washington State...... 136

Table 4-2. Fruit qualities and yield of sweet cherry cv. ‘Regina’ by AVG treatment at 166,333 and

499g/acre rates and the control at 10% bloom in commercial and experimental orchards located in

Washington and Oregon States...... 137

LIST OF FIGURES

Figure 2-1. Mean daily temperature during late winter and early spring at the WSU-Roza experimental orchard at 2 m in 2011 and 2012( Sampling date)...... 60

Figure 2-2. Diurnal variation in air temperature in controlled environment chambers...... 61

Figure 2-3. Flower sampling stages for organ evaluation. (A). Tight cluster stage, a. pedicel not yet extended; b. pedicel extended. (B). Sampling stages, a. Stage 1, tight cluster; b. Stage 2, first white; c. Stage 3, half white; d. Stage 4, first open; e. Stage 5, full open...... 62

Figure 2-4. Floral parameters measured on harvested sweet cherry flowers. PA, petal area; SA, sepal area; FL, filament length; SL, style length; OL, ovary length; PL, pedicel length; OD, Ovary diameter; and PD, pedicel diameter...... 63

Figure 2-5. A. Original digital image of petals, sepals and a reference square attached to colored paper; B. the same picture after image processing with the MATLAB algorithm...... 64

Figure 2-6. The parameters of floral organs at ‘tight cluster’ and ‘full open’ flower stages of four sweet cherry cultivars under high (H), moderate (M) and low (L) temperatures separately in year

2011 and 2012...... 67

Figure 2-7. Floral parameters ( ) of flowers at the ‘full open’ stage. (a), Petal areas of the four cultivars in 2011 and 2012 seasons (not influenced by temperatures); (b), Filament length of the four cultivars under high (H), moderate (M) and low (L) temperatures separately (not influenced by year); (c), Style length under high (H), moderate (M) and low (L) temperatures separately (not influenced by genotypes or year)...... 69

Figure 2-8. Development rate of floral organs from stage 2 to 5 compared to stage 1. (Flower stages:

Stage 1, tight cluster; Stage 2, first white; Stage 3, half white; Stage 4, first open; Stage 5, full open.

Floral organs: PA, petal area; SA, sepal area; FL, filament length; SL, style length; OL, ovary length;

PL, pedicel length; OD, Ovary diameter; and PD, pedicel diameter.) ...... 70

Figure 2-9. Main-effect sensitivity indices based on four-factorial design applied to the parameters of floral organs in four sweet cherry cultivars...... 71

Figure 3-1. Diurnal variation in air temperature in controlled environment chambers mimicking field environment of the past 10 years...... 103

Figure 3-2. Stigma development of sweet cherry flowers at different days post-anthesis observed from both surface and longitudinal sections under moderate temperature. (A) surface of stigma observed by SEM. (B) longitudinal sections observed by LM. (C, D) The day of anthesis. (E, F) 1 day post-anthesis. (G, H) 2 days post-anthesis. (I, J) 3 days post-anthesis. (K, L) 4 days post-anthesis.

(M, N) 5 days post-anthesis...... 104

Figure 3-3. (A) Stages 0 of Pollen hydration by observing pollens collected from stigmas 20 mins post-pollination. (B) Stages 1 of Pollen hydration. (C) Stages 2 of Pollen hydration. (D) Stages 3 of

Pollen hydration. (E) Stages 4 of Pollen hydration. (F, G) Pollen germination on stigma observed by fluorescence microscopy. (H) Pollen tube growth along style of flowers...... 105

Figure 3-4. (A) Callose accumulation in ovules where callose appeared fluoresces reaction, ovule without callose. (B, C) Callose appeared in 25% of ovules. (D) Callose appeared in 50% of ovules.

(E) Callose appeared in 75% of ovules. (F) Callose appeared in 100% of ovules. (G) The size of one ovule is larger than the size of another one within single ovary. (H) The two ovules within single ovary are of the same size...... 106

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Figure 3-5. Pollen hydration level assessed on pollen collected from the stigmatic surfaces of four sweet cherry cultivars 20 minutes after manual pollination at 24 hr intervals post-anthesis. Limbs were cultivated under high (A), moderate (B) and low (C) temperatures environment separately.

...... 108

Figure 3-6. Pollen germination (%) on the stigmatic surface of sweet cherry flowers there were manually pollinated at 24 hr intervals post-anthesis and cultivated under high, moderate and low temperature separately. Pistils were sampled 8, 24 and 48 hours after hand-pollination in ‘Benton’

(A), ‘Rainier’ (B), ‘Tieton’ (C) and ‘Sweetheart’ (D) sweet cherries...... 110

Figure 3-7. The frequency of primary ovules of four sweet cherry cultivars (A) ‘Benton’, (B)

‘Rainier’, (C) ‘Tieton’, and (D) ‘Sweetheart’, and secondary ovules (combining all cultivars) (E) that exhibited fluorescence across 0%, 25%, 50%, 75% or 100% of the ovules at different days post- anthesis cultivated under high, moderate or low temperature...... 113

Figure 4-1. Flower stages and ovule viabilities. Flower stages at ‘first white’(a) and ‘first open’(b) stages. (c) Callose accumulation in ovules where callose appeared fluoresces reaction, ovule without callose. (d,e) Callose appeared in 25% of ovules. (f) Callose appeared in 50% of ovules. (g) Callose appeared in 75% of ovules. (h) Callose appeared in 100% of ovules...... 138

Figure 4-2. Fruit set of sweet cherries cv. ‘Tieton’ and ‘Regina’ by AVG treatment at 166,333 and

499g/acre rates and the control at 10% bloom in both commercial and experimental orchards located in Washington State. (Multiple comparison within cultivars; Values within the same column group followed by the same letter do not significantly differ according to Duncan’s test (P=0.05))139

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located in Washington State. (Values followed by the same letter do not significantly differ according to Duncan’s test (P=0.05)) ...... 140

Figure 4-4. Fruit set observed from branches in both west and east directions of sweet cherries cv.

‘Regina’ by AVG treatment at 166, 333 and 499g/acre rates and the control at 10% bloom in a commercial orchard located in Oregon State. (Multiple comparison within branch directions; Values within the same column group followed by the same letter do not significantly differ according to

Duncan’s test (P=0.05)) ...... 141

Figure 4-5. Fruit set of sweet cherry cv. ‘Regina’ by 333g/acre AVG treatment and the control applied in ‘popcorn’, ‘10% bloom’, ‘50% bloom’ and ‘100% bloom’ period in a commercial orchard located in Oregon State. (Values followed by the same letter do not significantly differ according to

Duncan’s test (P=0.05)) ...... 142

Figure 4-6. Average Area Percentages of individual ovules lost viabilities of both primary and secondary ovules in sweet cherry cv. ‘Tieton’ by AVG treatment at 166, 333 and 499g/acre rates and the control when flowers were at ‘half white’ and ‘full open’ stages. (Multiple comparison within ovule types and flower stages; no significant differences between hours sampled post-flowering (i.e.

48h and 96h) and also no differences between experimental and commercial orchards; Values within the same column group followed by the same letter do not significantly differ according to Duncan’s test (P=0.05))...... 143

Figure 4-7. Percentages of ovules lost viabilities of both primary and secondary ovules in sweet cherry cv. ‘Tieton’ by AVG treatment at 166, 333 and 499g/acre rates and the control when flowers were at ‘half white’ and ‘full open’ stages...... 144

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Figure 4-8. Average Area Percentages of individual ovules lost viabilities of both primary and secondary ovules in sweet cherry cv. ‘Regina’ by AVG treatment at 166, 333 and 499g/acre rates and the control when flowers were at ‘half white’ and ‘full open’ stages. (Multiple comparison within ovule types and flower stages; no significant differences between hours sampled post-flowering (i.e.

Figure 4-9. Percentages of ovules lost viabilities of both primary and secondary ovules in sweet cherry cv. ‘Regina’ by AVG treatment at 166, 333 and 499g/acre rates and the control when flowers were at ‘half white’ and ‘full open’ stages...... 146

Figure 4-10. Fruit qualities and yield of sweet cherry cv. ‘Regina’ by 333g/acre AVG treatment and the control applied in ‘popcorn’, ‘10% bloom’, ‘50% bloom’ and ‘100% bloom’ period in commercial orchards located in Washington (WA) and Oregon States (OR). (a) Fruit Brix, OR;

(b)Fruit Brix-WA; (c) Fruit firmness, OR; (d) Fruit firmness, WA; (e) Fruit color, OR; (f) Fruit color,

WA; (g) Pedicel-fruit retention force (PFRF), OR; (h) PFRF, WA; (i) Fruit size, OR; (j) Fruit diameter, WA; (K) Fruit weight, WA.( Values within the same histogram followed by the same letter do not significantly differ according to Duncan’s test (P=0.05).) ...... 152

CHAPTER ONE

FLOWER DEVELOPMENT, POLLINATION BIOLOGY AND MANAGEMENT IN

SWEET CHERRY PRODUCTION

Sweet cherry (Prunus avium L.), also called wild cherry, bird cherry or gean, belongs to the family

Rosaceae. The sweet cherry is thought to have originated from the temperate areas somewhere close to the Caspian and Black Seas and was first used as food source from 5000-4000 BC (Kolesnikova,

1975). Worldwide sweet cherry production in 2012 was 2,256,519 tonnes and more than 60 nations are significant producers of sweet cherry. The top ten nations worldwide are Turkey, United States of

America, Iran, Italy, Spain, Chile, Uzbekistan, Syrian Arab Republic, Ukraine and Russian

Federation (http://faostat.fao.org/ ).

Sweet cherry is drupe fruit, which develops from a single carpel with a stone seed inside. Fruit flesh

(mesocarp) and skin (exocarp) of sweet cherry is originally from the outer and middle layer of a superior ovary. Most authorities usually split sweet cherry into three groups: the Mazzards, the

Hearts, Geans or Guignes, and the Biggarreaux. The Mazzards cherry is often small and inferior with various shapes and colors, and used as rootstocks mainly in South Africa, Canada and USA. The

Heart cherries are dark in color, heart-shaped and half-tender in flesh texture. The Geans cherry is obtuse, heart-shaped with tender and melting flesh, which is divided into two groups (Black Geans and Amber Geans) according to juice colors. The Bigarreaux cherry is light-colored, hard, heart- shaped and hard in flesh. (Webster and Looney, 1996) Fruit size of sweet cherry also varied with cultivars from large-sized greater than 22mm, medium 18-22 mm and small less than 18 mm

(Webster and Looney, 1996). The fruit diameters of new cultivars in USA, (e.g. Regina, Skeena), can reach as large as 32 mm (Long et al., 2008).

In the USA, Washington, Oregon, and California, are the main cherry production areas, and more than 50% of the nation’s sweet cherries are grown in Washington. Bing is the most famous and the classic cherry cultivar and, combined with, Lapins and Rainier, account for more than 90% of

Northwest cherry production (http://cahnrscms.wsu.edu/). However, poor financial returns of these classic cherry varieties is driving the interest of local growers in planting new cherry cultivars in the

Pacific Northwest, USA (Seavert, 2004; Long et al., 2008). The most promising cultivars include

Attika, Lapins, Skeena, Regina, Sweetheart and Staccato (Long et al., 2008). In Washington and

Oregon States, new sweet cherry varieties have advanced characteristic in resisting cherry Powdery

Mildew (Calabro et al., 2009) disease and fruit rain cracking (e.g. Regina), and in beautiful glossy skin and fruit size (e.g. Tieton) (Long et al., 2008). However, those cherry varieties with compelling fruit qualities usually beset in poor production (Lang, 2001). Mazzard (Prunus avium L.), Mahaleb

(P. mahaleb) and sour cherry (P. ceraus) were the most popular and traditional rootstocks in sweet cherry throughout the world (Webster and Looney, 1996). However, currently new rootstock group, such as Gisela 5 and Edabriz, are very productive, promoting increased spur formation on scions

(Lang, 2001). Therefore, the selection for matched rootstock/scion is impending for the extension of new cherry varieties (Lang, 2001).

Tree production is also affected by canopy architecture for the influence of light interception, light

distribution, and net photosynthesis and transpiration, which has been reported in apples (Malus domestica L.) (Hampson et al., 2002; Robinson et al., 1991). In sweet cherry, vase-shaped and central leader-shaped are the most traditional tree training and pruning system. Non-traditional training systems are developing to adapt to dwarf tree training, increase tree density and improve cropping precocity (Webster and Looney, 1996). Researchers and growers around the world are trying new systems. Spanish Bush was developed in the Ero Vally of Spain, the Tatura trellis system was first designed in Victoria Australia, Zahn Spindle was from northern Germany and Swiss was famous for its cherry four-wire system. In North America, Upright Fruiting Offshoots (UFO), Kym

Green Bush (KGB), Tall Spindle Axe (TSA) and Super Slender Axe (SSA) are the latest sweet cherry tree training and pruning systems. In addition to canopy management, planting orientation also was reported closely linked to tree productivity. North-south hedgerow orientation had advantages in flower bud density and high fruit set in pear (Khemira et al., 1993).

Fruit quality of sweet cherry is decided by many parameters including size, weight, firmness, color and brix (Drake and Elfving, 2002.). Bird and rain damages are the two main reasons in nature causing the economic losses of sweet cherry during harvest (Webster and Looney, 1996). Sweet cherry fruit ripening occurs in a double sigmoidal pattern with 25% of final fruit weight added in the last week before harvest where fruit color, flavor and firmness also changed considerably (Spayed et al., 1986). Sweet cherry is non-climacteric fruit and does not have starch reserves for respiration substrate after picked from trees, unlike apples or pears. Therefore the higher rate of respiration than apples relying primarily on sugars instead of starch was assumed as the reasons that result in short storage life-span (Webster and Looney, 1996). For sweet cherry, the period from packing to

marketing till consumption by customers should be managed within 4-6 weeks. Drake and Elfving

(2002) reported 14 days after storage, the appearance of fruit and pedicel reduced in Lapins sweet cherry.

1.1 Flower bud development and management

Flower buds of sweet cherry are lateral, fascicle and unmixed (simple) with usually one vegetative bud and 1-3 flower buds depending on varieties on each spur (Westwood, 1995). Within each floral bud, there are 2-5 flowers. In sweet cherry, flowers are born on spurs on 2-year and older wood and generally inclined to dispose laterally on short shoots or near to the base of longer shoots. In the

Columbia Basin of Washington State, the period of sweet cherry blooming is generally about two weeks for individual varieties and the date of the first flower opening is usually around the end of

March and early of April depending on climate and cultivars. According to our previous research

(Zhang and Whiting, unpublished), flower opening is asynchronous within the same tree, branch, shoot and even on a single spur.

According to Gasser (1991), flowering may be classified into three phases, first, induction and evocation, which decide whether or not to flower; second, organ initiation and specification at floral apex; third, differentiation and development of issues within organs. Buban and Faust (1982) considered flower bud formation in apple has three processes: induction of flower bud formation, histological transformation and morphological differentiation. In sweet cherry, though anthesis only appears within a couple of weeks, the cycle of flower development starting from induction to bloom

lasts 12 month. That is, flower bud induction occurs in May and June, flower organ development is from July to October, then bud growth stops and enters into dormancy phase from November to

February, and finally blooming happens in April after the final bud development from February to

March (Lang, 2001)

1.1.1 Flower induction, initiation and their management

Flower induction is not a change in histological or morphological levels but a stimulation from the environment to change the phase of vegetative buds to propagative status (Koutinas et al., 2010;

Wilkie et al., 2008). The precondition for a successful transition to floral bud is vegetative buds having being fully developed (Wilkie et al., 2008). In the model plant Arabidopsis thaliana L., four major pathways were considered stimulating flowering and these are photoperiodic, autonomous, gibberellins and vernalization. The identity genes of floral meristems were regulated by these pathways (Pineiro and Coupland, 1998). In apple, AFL (Apple Floricaula/ Leafy) gene was reported involved in floral induction to a great degree than other genes (Kotoda et al., 2000).

According to Raseira and Moore (1987), floral initiation begins with the first visible morphological alteration of a vegetative bud to a floral bud. In sweet cherry, flower bud induction and initiation take place right after fruit ripening and almost at the same time of the new shoot initial growth (Lang,

2001). The flower bud formation is considered controlled by the variation of endogenous plant hormones (Hoad, 1984). Of the five main types of plant hormones, (i.e. gibberellins, auxins, cytokinins, ethylene and growth inhibitors), gibberellins make the greatest contributions to flower

formation and development (Tromp, 1982; Bangerth, 2006). There was evidence that endogenous

GA coming from seeds of developing fruit had negative effect in fruit load of the next year and also that the applications of GA3 and GA4/7 inhibited flower formation (Bangerth, 2006; Lenahan et al.,

2006). This depressive influence of GA initially from seeds to flower formation causes a common phenomenon ‘alternate bearing’ in temperate tree fruit, e.g. apple and pear (Luckwill L.C. 1974;

1980). In ‘on year’, heavy crop loads produced extremely large amount of endogenous GA which cause sparse flower bud formation followed by an ‘off year’ bloom. In addition to GA, IAA was also informed inhibiting propagative organ formation (Bangerth, 2006) while cytokinin works for flower number promotion (Ramirez and Hoad, 1981; Chen, 1991).

In woody plants, there is a state called juvenility followed by a transition phase that tree starting to have flowers and fruits entering into the adult phase (Westwood, 1995). The length of juvenility phase is depending upon both environmental and genetic factors, e.g. approximately three years in

Mango (Mangifera indica L.), traditionally 6 years in apple (Wilkie et al., 2008), 3 years in pear

(Pyrus L.) (Koutinas et al., 2010) and 3 to 5 years in sweet cherry (Kotoda et al., 2000). During the juvenility phase, floral buds cannot be induced by seedlings (Goldschmidt and Samach, 2004). The initial time of first flower appearance in given genotypes can be advanced by different methods, such as nitrogen, dwarfing rootstock, irrigation and plant growth regulators (Raseira and Moore, 1987;

Oliviera and Browning, 1993). The most effective one is to accelerate the vegetative growth to assist seedlings reaching a large size and satisfying later phase transition (Koutinas et al., 2010). For example, by grafting on the dwarfing rootstock M9 in apple and Gisela 5 and 6 in sweet cherry, flower bud formation was accelerated in seedlings and precocity is improved compared to trees on

traditional seedling rootstocks (Koutinas et al., 2010, Whiting et al. 2005).

Several orchard management practices can be applied to regulate flower bud formation with the main principle ‘balance between vegetative and propagative growth’. Low vigor trees benefits flower number promotion, for example, proper summer pruning of young, especially current-season shoots on vigorously growing apple and cherry trees could leads to a well-accepted flower bud formation

(Miller, 1982; Elfving et al., 2005). Guimond (1998) suggested that early pruning in summer has advantages than late pruning in stimulating vegetative buds to flower buds. However, severe dormant pruning, on the contrary, will cause excessive vigorous growth and therefore reduce the initiation of flowers (Webster and Looney, 1996).

In addition, other practical management strategies such as defoliation, girdling, and branch bending to position limbs horizontally can also be applied to promote floral bud formation. Carbohydrates play a great function in floral initiation (King and Ben-Tal, 2001). Goldschmidt et al. (1985) confirmed that girding stops carbohydrates transported from canopy to shoots and therefore increased flowering intensity. On the other hand, light intensity can accelerate photosynthesis and increase the production of carbohydrates therefore it plays an important role in flower bud initiation.

Proper fertilization and irrigation may regulate vegetative growth in a better manner, e.g. improving the nodes in axillary buds, which will also promote flower growth in a better manner that is to imitate

more flower buds based on larger amount of shoot buds (Koutinas et al., 2010). Fertilization application to trees with a certain amount of phosphorus and nitrogen may stimulate flower bud formation (Hirst and Ferree, 1995; Williams and Renninson, 1965). Water stress can be applied as another orchard practical method to accelerate flower initiation. Stern (2003) found flower intensity increased considerably in spring by three low irrigation treatments, i.e. 0, 25% and 50% irrigation, where the control 0% trail only accepted 7mm supplementary irrigation. Water stress induces the amount of endogenous cytokinin which has a close relationship with flower initiation (Chen, 1991).

However, severe lack of irrigation during autumn can cause early leaf drop and consequence bud drop (Alburquerque, 2003).

In addition to gibberellic acid (i.e. GA3 and GA4/7) and cytokinins, other plant growth regulators have been applied to fruit trees to increase flower bud numbers. For example, the application of daminozide was found closely linked to the increasing concentration of GA and cytokinin and consequently promote flower formation (Ramirez and Hoad, 1981; Ryugo, 1986). In addition, ammonia, Apogee (Prohexadione calcium), Ethrel (Ethephon) have also shown efficacy as growth regulators that can increase flower bud initiation (Rohozinski et al., 1986; Lang, 2001; Elfving et al.,

2005).

1.1.2 Flower differentiation, dormancy and their management

Following flower bud initiation, the differentiation of floral primordium starts immediately after fruit harvest, following the differentiation orders: sepal, petal, stamen to pistil gradually (Beppu et al.,

2001; Engın and Ünal, 2007). Temperature the primary environmental factor that can cause abnormal flower differentiation and development in sweet cherry. Flower primordial is sensitive to high temperature especially from sepal differentiation to petal differentiation, during which period the occurrence of double pistil is induced at the highest frequency (Beppu et al, 2001). Whiting and

Martin (2008) suggested late July to August and field temperature > 35C is the critical time and temperature, separately, to cause pistil doublings. Though double pistil is more frequently happened to sweet cherries planed in warmer places, some cherry cultivars in colder area (i.e. Pacific

Northwest, USA) that are sensitive to warm temperatures, e.g. Tieton, are also susceptible to the occurrence of double pistils and in Tieton, double pistil percent can reach as high as 50%. Artificial shading is one of the most traditional methods to reduce the risk of double pistil occurrence. Beppu

(2000) reported 78% shading helped avoiding doubling but the treatment of 53% shading had no efficiency. Another method, over-tree evaporate cooling, was suggested by Whiting and Martin

(2008) as a more effective system to reduce canopy temperature compared to shading treatment.

In late autumn, flower bud will enter into a morphological stable phase which is called dormancy.

When the environmental temperature warm up in the spring, buds will break based on accepting a certain chills. Dormancy in deciduous fruit trees is a necessary phase that allows trees to survive cold temperatures (Lang et al, 1987). In warm winter region, the phenomenon of delayed bud break, flowering and flower organ malformation appeared in fruit trees due to unsatisfied chilling accumulation. Oukabli (2007) studied the anatomy of sweet cherry buds from trees grown under

Mediterranean climatic conditions and found abnormal differentiation of female gametophyte, anther and xylem vessel elements at the base of buds and flower primordial.

Dormancy-breaking agents (DBAs), such as gibberellins, cytokinins, mineral oils, and potassium are applied to plants to artificially accelerate bud break in deciduous fruit trees (Erez, 1987). Hydrogen cyanamide (HC) is suggested as the best DBAs for fruit trees and has been widely used in grape

(Vitis vinifera L.), apple, peach (Prunus persica), pear and sweet cherry (Jackson et al., 1995; Kuroki et al., 2009; Or et al., 2000; Siller-Cepeda et al., 1992; Weis et al., 1999). The commercial application of HC to fruit trees 2 - 5 weeks before nature bud break can increase flower numbers, hasten flowering and fruiting, improve fruit quality, and stimulate bud germination (Shuck et al.,

1995; Martinez et al., 1999). The concentration and application time of HC are varied with fruit species. In sweet cherry, generally 2-3% concentration is optimum and better be sprayed 4-8 weeks ahead of bud break. In addition to DBAs, shading and artificial defoliation can also help trees releasing from dormancy (Mohamed, 2008; Campoy et al, 2010). Field work combined with DBAs has more efficiency than single treatment. Mohamed (2008) used artificial defoliation (DEF) and hydrogen cyanamide (HC) on November 15, December 1 and December 15 separately. The results showed that the chilling requirement for bud breaking of the trail, DEF+ HC+ November 15, is lowest for both flower bud and vegetative bud. In addition, the selection of lower chilling requirement varieties of deciduous fruit trees in warm winter area is another effective method to avoid abnormal dormancy breaking.

1.1.3 Final bud development and management

According to Lang et al. (1987), bud dormancy may be classified into three stages: paradormancy, endormancy and ecodormancy. Paradormancy of buds is influenced by relative signals from organs elsewhere in the plant and can be eliminated once those tissues removed, (e.g. apical dominance).

Endormancy, also called rest period, is regulated by physiological factors inside the bud structure and requires a certain chilling accumulations to break this dormancy. Once the chilling requirement of buds has been satisfied, buds enter into ecodormancy period, which was caused by unfavorable environmental factors. In early spring, buds release from ecodormancy and start to develop when environmental temperature warms up and a certain heat units is accumulated. The requirement of heat or thermal time for the development of each bud/flower phonological period can be expressed by either growing degree-hours (GDH) or growing degree-days (GDD) (Zavalloni et al., 2006).

GDHs are hourly average temperatures (C) minus 4.5 C, which are accumulated daily (Richardson et al., 1974). GDDs are calculated similarly to GDH but with daily maximum and minimum temperatures. During this final flower part development, e.g. mature pollen grains and expanded petals and pistils, nitrogen and carbohydrate reserved from the last autumn are essential for flower development and the guarantee of fruit set (Lang, 2001).

The phonological development of sweet cherry floral buds is categorized into nine stages: ‘first swelling’, ‘side green’, ‘green tip’, ‘tight cluster’, ‘open cluster’, ‘first white’, ‘first bloom’, ‘full bloom’ and ‘post bloom’ (http://county.wsu.edu/chelan- douglas/agriculture/treefruit/Pages/Cherry.aspx). Alburquerque et al. (2008) reported GDH for sweet cherry flower blooming is around 7500-9500 varied with varieties. Compared with other Prunus species (e.g. almond), the heat requirement for flowering of cherry were high (Egea et al., 2003;

Alburquerque et al., 2008). In sour cherry, flower buds reach ‘green tip’ stage needs 24 GDDs, ‘tight cluster’ 44 GDDs, ‘first bloom’ 98 GDDs and ‘full bloom’ 123 GDDs, where GDD for different phonological stages were calculated starting from ‘side green’ stage (Zavalloni et al., 2006).

During flower bud and flower development, freeze damage is one of the most severe factors causing low productivities in sweet cherry. Freeze damage can occur in autumn before buds entering into dormancy, in winter because of deep super-cooling (hardiness) and also in early spring when buds have already release from dormancy (Webster and Looney, 1996). Frost injury during the development of cherry flowers ranged from 5-96% varied with cultivars and rootstocks (Choi et al.,

1998 3-55). In spring, the type of frost are important to determine the orchard management to minimize freeze damage. Radiation frost is more commonly appeared in dryer climate, which symptom usually closely related to fruit buds loss in the lower portion of the tree. There are many methods can be applied to reduce this frost, for example, orchard heating, wind machine to mix warm and cold air layers, water sprinkling during the frost by using the heat from ice fusion. Under moist climates, advective frost occurs at a high frequency and orchard heating is the only means that can relieve this freeze damage. In addition, installing an orchard in frost free area and selecting sweet cherry rootstocks with cold tolerance characteristic are the other methods that can be considered at the very beginning of tree planting.

1.2 Flowering, pollination and their management

1.2.1 Flower structure, density and quality

The structure of individual sweet cherry flowers is composed of five outer white sepals, five green petals, one pistil with a single stigma, style and ovary, and 20-30 stamens consisting of a filament and a pollen bearing anther. Pistils of Prunus species contain two ovules in a single carpel; one ovule

(i.e., secondary ovule) will lose viability prior to the other one (i.e., primary ovule) (Rodrigo and

Herrerro, 1998). The flower of sweet cherry is perigynous and the superior plane of the anthers is higher than the surface of stigma. The growth of filament occurs earlier than flower opening but the development of pistil completed till ‘popcorn’ stage of flowering (Zhang and Whiting, 2012).

Previous research has confirmed that the relative position of anthers and pistils impact the behavior of pollinators in apricot, peach and nectarine (Ruiz and Egea, 2008). Warm temperature hastened flower opening but not synchronous pistil development, and usually resulting in a reduced percent of fruit set (Rodrigo and Herrerro, 2002; Bepp et al., 1997). To determine the influence of pre-bloom temperature on flower development, a mobile green house has been adapted to trees in the orchard by horticultural researchers (Rodrigo and Herrerro, 2002). In addition, flower position and size were also reported influencing fruit set and fruit size in pomegranate trees (Punica granatum) (Wetzstein et al., 2013).

In Prunus species, increased flower density per branch generally is in accordance with increased fruit per branch (Iezzoni and Mullinix, 1992). Stöver (2000), Molina-Montenegro and Cavieres (2006) considered this positive relationship between the number of flower cluster and fruit due to pollinators’ behavior that they tent to visit high density bloom to save energy in a single food forage.

On the other hand, the compositions of nectar can also influence the behavior of honey bees (Apis mellifera L.) in avocado (Persea americana) (Afik et al., 2006). The nectar is usually consisted by aqueous solution of sugars, amino acids, organic acids, proteins, fats, vitamins, minerals and its compostion varied greatly depending on plant species and environment situations (Lenahan and

Whiting, 2006).

However, excessive flowering will produce excessive crops with an accompanying phenomenon of small fruits and alternate bearing in fruit species (Stover, 2000). Because of expensive hand thinning and un-practical fruit thinning in sweet cherry, chemical blossom thinner is economical and popular in the U.S. Pacific Northwest (Whiting et al., 2006). The application of naphthaleneacetic acid

(NAA), ammonium thiosulphate, ethephon, fish oil + lime sulphur and vegetable oil emulsion have been proved working efficiently in bloom thinning (Schmidt and Elfving, 2007; Lenahan and

Whiting, 2006; Whiting et al., 2006).

1.2.2 Pollinizer, pollinator and outcross

Most cherry varieties are self-incompatible, requiring pollination from other genotypes for successful fertilization. Interestingly, similar to self-incompatible cherries, fruit set of self-compatible cherries also can be increased by outcross. According to Stösser et al. (1996), around 50 pollen grains are transferred to stigma surface under nature situation in sour cherry. For this reason, it is essential to make sure during bloom, proper and adequate pollenizers being set in orchards. Stebbins (1963) suggested the optimum arrangement for maximum pollination is every other tree in every row a pollinizer and the minimum number of setting is every third row with a pollenizer. Growers use low pollenizer density mainly because of the poor commercial value of pollenizers. The number of pollinator hives depends on the space between commercial varieties and pollenizers. Bloom overlap and pollinizer compatibility are the two main factors for a successful pollination in sweet cherry.

Topwork grafting and inserting bouquets of compatible flowers can be conducted as emergency methods for orchard with poor performed pollenizer settings.

The European honey bee is the main insect visiting sweet cherries and its population counts up to around 97% of all sweet cherry pollinators. The bee’s behavior has a close relationship with the nectar, in particular the contents of sugars (Afik et al, 2008). It is recommended that 2-3 hives per ha is a sufficient density of hives though growers often consider higher density of bees according to the conditions of orchard. Since bees have low efficiency under hostile environment, (e.g. wind, rain, cool temperatures), hives should be set in a warm sunny area and separated locations if possible. In addition, it is necessary to put hives in a higher place and far away from the gate of fence in orchards to avoid the disruption from people and livestock. The application of pesticide spray should be considered carefully by using low-toxic pesticide to minimize the harm to bees and being conducted at night to keep away from pollinators’ daytime activities. In addition, competing flowers which are also attracting the forage of bees should taken away to save the energy of pollinators and bring maximum pollens to cherries. (Someriville, 1999) Researchers and breeders are also trying to use more dynamic bee cultivars to increase pollen load and fruit set, for example, Osmia Lignaria Say is under consideration because it shows a strong ability in collecting nectars and pollens and has strong endurance to low temperature conditions (Torchio, 1976).

1.2.3 Pistil role and effective pollination period (EPP)

The pistil is the female part of a flower and composed by stigma, style and ovary. Pollination is a process that starting from pollen landing on a stigma and hydrated, and then the pollen tube grow toward the ovary, exiting transmitting tissue and till one of the pollen tube reaching the ovule

(Kessler et al., 2010).

Stigma structure and receptivity

The stigma of sweet cherry flowers is classified into ‘wet’ style, which appeared stigmatic exdudate at the time of pollination. The stigmatic surface consists of numerous papillae cells, and those cells are very thin-walled and turgid at anthesis. In sweet cherry, those papillae shrivel within 1-2 day after anthesis and are collapsed completely 4-5 days post bloom (Stösser et al., 1996). According to

Swanson et al. (2004), stigmatic exduate is a medium that supporting pollen adhesion, hydration and tube germination. Wolters-Arts et al. (2002) have even observed a thin layer of water at the contact side between pollen and stigma during pollen swollen on the surface of stigma. The composition of stigma exudation varied widely depending on plant species and in rosaceous plants, and the exudation mainly contains lipids, carbohydrate and protein (Pusey, 2006). In sweet cherry, when those secretions no longer evident and the surface of stigma turn brown color, stigma will no longer appear receptive to pollens (Webster and Looney, 1996). Pollen germination and stigma receptivity declined significantly with stigma ages (Young and Gravitz, 2002).

The duration of stigma receptivity is highly vulnerable to environmental conditions, especially to temperatures. Generally, high temperature reduced stigma receptivity while low temperature enlarged this receptive period. In sweet cherry, the duration of stigma receptivity lasted up to 9 days under 10 C, whereas 5 and 2-3 days under 20 and 10 C, respectively (Hedhly et al., 2003).

Style and pollen tube kinetics

The stylar tissue connects the stigma and ovary and provides a pathway for pollen tube growth toward to ovary (Lush et al., 2000). In the center of the style, there is a special tissue with elongated cells and is called ‘transmitting tissue’. In Prunus species, gametophytic self-incompatibility usually occurred in styles and arrest pollen tube growth within the top third of style length (Yi et al., 2006).

Self incompatibility is genetically controlled by multiple S-alleles and SI alleles related glycoprotein has been tested out in sweet cherry styles (Tehrani and Brown, 1992; Mau et al., 1982).

In almond flowers, it took pollen tube growing along the whole style 4-5 days under favorable weather conditions (Griggs and Iwakiri, 1975). However, in sweet cherry, it only took out 2-3 days under field conditions of pollen tube reaching the basis of style, which was faster than apple and plum (Stösser et al., 1996). IAA plays role in pollen-pistil interaction and it reach the highest content in the stigma and was mainly distributed in the transmitting tissue when pollen is germinating (Chen and Zhao, 2008). In angiosperms, the synergids of ovary have been demonstrated controlling the final step of pollen tube guidance and reception (Kessler et al., 2010). Proper high temperature plays role in accelerating the rate of pollen tube growth, however, excessive warm temperature will reduce the pollen tube kinetics (Sanzol and Hererro, 2001). In sweet cherry, pollen tube growth and the population of microgametophyte reaching ovules performed well under 20 C, but microgametophyte population in ovules was reduced at 30 C or 10 C temperature condition depending on various genotypes (Hedhly et al., 2004). In addition to temperature, the number of pollen grains deposited on the stigma was also reported has a positive relationship with pollen tube growth rate in plum (Lee,

1980).

Foliar application of boron to fruit trees at bloom is a method to trigger pollen germination on the surface of stigma and improve pollen tube kinetics. Nyomora (2000) spray solubor (Na2B8O13•4H2O) which contain 20.5% B to flowers showed that moderate level (0.8 or 1.7 kg/ha) of B accelerated pollen germination and sped up pollen tube processing toward the ovary as well as improved the fruit set.

Ovule viability

Flowers of Prunus species have two anatropous ovules within a single carpel but usually one of the ovule will take part in fertilization with one seed produced (Rodrigo and Herrero, 1998). In our previous anatomical research, the development of sweet cherry ovule and embryo was classified into

8 stages and they are: ovule primordium Ⅰ- Ⅲ, mother cell of macrospore, macrospore, 2-nucleate embryo sac, 8-nucleate embryo sac and fertilization. Ovule viability and the embryo sac is a limiting factor for fertilization and fruit set (Stösser and Anvari, 1983). Pimienta and Polito (1982) have pointed out that the appearance of callose deposited in ovules revealed tissue degeneration. Ovule senescence starts with callose accumulation at the chalazal end of ovule and then extending through the inner integument till finally the whole ovule (Arbeloa and Herrero, 1985). In addition, the amount of starch in the ovular structures also has been reported positively linked to ovule degeneration

(Rodrigo and Herrero, 1998).

Ovule longevity in some tree fruit species (e.g. pear) is a specific genetic character, for example,

‘Doyenne du Comice’ pear is naturally short ovule life-span which results in a poor fruit set. Flower emasculation, which refers to petal, sepal and stamen removal and is a method usually been used for breeding and pollination research, has been confirmed hasten ovule degeneration (Hedhly et al.,

2009). Moreover, it is well accepted that in temperate tree fruit, high temperature accelerates the ageing of ovules (Sanzol and Herrero, 2001). The ovule longevity of plums showed an obvious decline under a constant temperature at 20 C compared to lower temperature 5, 10 and 15 C (Cerović et al., 2000). There are a couple of chemicals that influence synthesis can be applied at bloom to relieve ovule degeneration and therefore increase fruit set, for example, aminoethoxyvinylglycine

(AVG), putrescine, gibberellins and 6-benzylamino purine (Dussi et al., 2002; Stösser and Anvari,

1983; Edgerton, 1981)

Effective pollination period (EPP)

Effective pollination period was first described in apple by Williams (1965) and it is a period during which the flower is able to set a fruit irrespective of pollen source. The precise definition of the EPP is the longevity of ovules minus the time lag between pollination and fertilization, assuming this value does not exceed the receptive period of the stigmas. The EPP has been studied in many tree fruit species, e.g. apple, pear, sweet cherry, peach, plum and kiwifruit (Ortega et al., 2004). In sweet cherry, for individual flowers, the EPP was evaluated around 8-10 days and after 10 days, a decline in fruit set was observed (Ughini and Roversi, 1996).

Stigma receptivity, pollen tube kinetics and ovule longevity are the three main factors influencing the

EPP and those three factors are all highly variable depending on varieties within species, flower quality and environmental conditions (Egea et al., 1991; Young and Gravitz, 2002; Sanzol and

Herrero, 2001). Therefore relative management for improving stigma receptivity, pollen tube kinetics and ovule longevity, will also work for prolonging the EPP.

Pollination management plays a great role in determining fruit set and production in sweet cherry, and it is a complicate process which need healthy flowers starting from flower bud induction, initiation, differentiation, going through dormancy and finishing final floral organ development in the early spring. A successful pollination also depends on proper flower structure, density, nutritive conditions and the ultimate three key factors: stigma receptivity, pollen tube kinetics and ovule longevity that mutually influence the effective pollination period. In addition, the installment and regulation to supply adequate and proper pollinizers, pollinators are additional but indispensable factors contributing to prosperous pollination and fruit set.

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CHAPTER TWO

SWEET CHERRY FLORAL ORGAN SIZE VARIES WITH GENOTYPE AND

TEMPERATURE

Abstract

Floral organ attributes are important for pollinator attraction and pollination success in many plants.

As part of a broader effort investigating causes for variable fruit set among sweet cherry (Prunus avium L.) genotypes, we have studied the role of temperature on floral organs in cultivars exhibiting high (‘Sweetheart’ and ‘Rainier’) or low fruit set (‘Benton’ and ‘Tieton’) in field conditions. In 2010 and 2011, two-year old sweet cherry limbs collected at ‘tight cluster’ flower stage were distributed randomly among three controlled environment chambers programmed to mimic a warm, moderate, or cool blooming period. Entire flowers were sampled and dissected at ‘tight cluster’, ‘first white’,

‘half white’, ‘first open’ and ‘full open’ stages. There was high year-to-year variability in size of all organs except stylar and filament lengths. ‘Tieton’ floral organs were significantly larger than other cultivars with the exception of filament, style and pedicel lengths. Between the earliest and the latest developmental stages petal area enlarged ca. 4.5 times, filament length increased ca. 3 times and styles doubled in length, across all cultivars. Irrespective of cultivar, the lengths of the styles and filaments were the most sensitive to temperature, being about 11% and 25% shorter in the low temperature environment compared to high temperature, respectively. This suggests that temperatures after the ‘tight cluster’ stage play an important role in the growth of styles and filaments. In addition, interestingly, our current results revealed that low productivity cultivars, i.e.

‘Tieton’ and ‘Benton’, have relative short styles to its petal size compared to the other two high productivity cultivars.

2.1 Introduction

The commercial yield of sweet cherry (Prunus avium L.) varies among genotypes (Choi and

Anderson, 2012; Sanzol and Herrero, 2001). In Washington State, some cherry cultivars have compelling quality attributes but are beset with poor productivity (e.g., ‘Benton’, ‘Tieton’, and

‘Regina’) (Zhang and Whiting, 2012). Previous work has shown that genotypes do not perform similarly when considering key factors that determine final yield, such as effective pollination period

(EPP), sensitivity to freezing injury, fruit set, fruitlet drop, and floral organ traits (Burgos et al., 1991;

Browing and Miller, 1992; Ortega et al., 2004; Ruiz and Egea, 2008; Szpadzik et al., 2009).

Floral organ traits have been studied in fruit trees such as sweet cherry, sour cherry (P. cerasus) and apricot (P. armeniaca) (Rodrigo and Herrerro, 2002; Pérez-Sánchez et al., 2008). Pollinators tend to visit flowers with the traits (e.g., size, form and color) that imply maximum rewards (Klinkhamer and

Van der Lugt, 2004). Flower size is generally considered the most important attribute for pollinators and flowers with large perianth usually attract more pollinators, partially because of the positive correlation between the petal size and nectar rewards (Cochen and Shmida, 1993; Molina-

Montenegro and Cavieres, 2006). In fruit trees, such as peach (P. persica), pomegranate (Punica granatum) and rabbiteye blueberry (Vaccinium ashei), fruit set was significantly higher from larger flowers than smaller ones (Scorza et al., 1991; Johnson et al., 2011; Wetzstein et al., 2013). In

addition, pollinators generally minimize energy expenditure and tend to visit bloom where the distance between flowers is short (Molina-Montenegro and Cavieres, 2006). Therefore, flower densities influence the crop yield in most fruit species (Stover, 2000). The number of flower clusters per branch has a positive relationship with fruit numbers per branch in sour cherry (Iezzoni and

Mulinix, 1992). In addition to flower size, the relative position of stigmas and anthers can also influence pollination (Conner and Sterling, 1995). Ruiz and Egea (2008) reported a significant correlation (0.588, p<0.01) between height difference between the stigma and the superior plane of the anthers and decrease of fruit set in apricot. Due to poor pollen transfer, flowers of apricot with abnormally short styles exhibited significantly lower fruit set than normal flowers (Rodrigo and

Herrerro, 2002).

Characteristics of floral organs have evolved with their pollination habit and pollinator selections

(Conner and Sterling, 1995). Mating-related organs (e.g. stamens and pistils) vary less in size than attraction-related organs (e.g. petals) (Ushimaru et al., 2003). In an orchid species, the change of gynostemia length in response to evolution was smaller than the size of petals and sepals (Ushimaru and Nakata, 2001). Compared to outcrossing taxa, the corollas of self-fertile flowers are smaller based on the research of various species (Ornduff, 1969). Small corolla size in self-pollinating taxa is thought to benefit self-pollen deposition and limit the expenditures on pollinator attractions in

Clarkia xantiana (Runions and Geber, 2000). In addition, studies of cosexual flowers have exhibited that the variation of perianth size has greater influence on flowers of male functions (pollen donation) than male functions (seed production). The pollinator-mediated selection acts more strongly on the male fitness than female fitness in Campanula Americana and Wurmbea dioica (Johnson et al., 1995;

Vaughton and Ramsey, 1998). However, there are only few reports on the attributes of individual floral organs and the relationships among them in temperate fruit tree species.

Environmental conditions during anthesis influence the size of floral organs. The period of pre- anthesis temperatures examined varies in previous reports from, for example, dormancy to full bloom

(Alburquerque et al., 2008), from one month before anthesis to petal fall (Beppu et al., 1997), or from the separation of bud scale and initial protrusion of sepals to 50% full bloom (Rodrigo and Herrerro,

2002). During anthesis, high temperature can hasten flowering but also decrease flower numbers, reduce pistil weight and style length, and reduce corolla diameter (Adams et al., 1997; Whitman et al., 1997; Niu et al., 2001a). High pre-bloom temperature is also reported to cause underdevelopment of pistils in apricot (Ruzi and Egea, 2008). Low temperature also has negative effects on flowering, such as reducing flower perianth size in Ipomoea trichocarpa (Murcia, 1990). However, beneficial effectives of cold temperature to flower development and consequent fruit set were also reported.

The study of pear (Pyrus communis L.) yield and pre-bloom temperatures revealed a positive relationship between high yield and cool temperature (Browing and Miller 1992). In addition, the diameter of the perianth is reported more influenced by day temperatures compared with night temperature and this dimension has no relationship with temperature difference between day and night in ornamental plants (Pietsch, et al., 1995; Niu et al., 2001b).

Most reports of environmental effects on floral organ characteristics have focused on economically important ornamental species such as Campanula carpatica, Clarkia xantiana and Calopogon

tuberosus (Firmage and Cole, 1988; Niu et al., 2001a; Runions and Geber, 2000). There are few reports on similar relationships for temperate tree fruit crops, fewer still that relate to fruit set or yield. The aim of this work was to evaluate the effect of pre-anthesis air temperatures on sweet cherry floral organ development and characteristics. Two low-yield and two high-yield genotypes were studied and eight parameters of floral organ were measured including the area of sepals and petals, the length of filaments, styles, pedicels and ovaries, and the diameter of pedicels and ovaries.

2.2 Materials and Methods

2.2.1 Plant Material

This research was conducted at the Roza research orchard and laboratory facilities at the Irrigated

Agriculture Research and Extension Center of Washington State University, USA (N 46.2°, W

119.7°) during springs of 2011 and 2012. Four cherry cultivars were studied including two genotypes that exhibit high commercial productivity, ‘Sweetheart’ (self-fertile) and ‘Rainier’ (self-sterile), and two genotypes that exhibit low yield commercially, ‘Benton’ (self-fertile) and ‘Tieton’ (self-sterile).

Sections of un-branched 2 to 3 year old flowering wood were removed when most flowers had reached the ‘tight cluster’ stage (i.e., pedicels not yet extended) (Fig. 3A-a). Fifteen branches per cultivar were collected randomly from the outer canopy of 5- (2011) or 6-year-old (2012) trees at about 2 m. Branches were placed immediately in buckets with water and delivered within 30 mins to the lab for further treatment.

2.2.2 Temperature

Temperature before Sampling

Significant year-to-year variations of floral organ size were found in our research, so the early spring temperatures before our sampling periods were analyzed. All air temperature data were supplied by the Washington Agriculture Weather Net (http://weather.wsu.edu/awn.php) from the early January to the middle April (Fig.1).

Temperature Control

Three Adaptis A1000 growth chambers (Conviron, Winnipeg, MB, Canada) were utilized to examine the influence of temperature on flower development and organ attributes. Each chamber was programmed to mimic either a cool (low temp: 4°C, high temp: 12°C), average (low: 6°C, high:

18°C), or warm (low: 12°C, high: 24°C) spring temperature profile (Fig. 2). These three temperature regimes were developed from a study of the historical air temperatures in the Prosser region during the first two weeks of April (i.e., the weeks prior to anthesis). Yearly average minimum, medium and maximum temperatures of those prior-bloom days by individual hours were valued as low, moderate and high temperatures for growth chambers, respectively. The chambers environment had 70% relative humidity with a day/night light daily cycle at three levels, 0 (black, 12h, 19:00-6:00), 1 (dim,

2h, 7:00 and 18:00) and 2 (bright, 10h, 8:00-17:00).

2.2.3 Experimental Design

Any flowers not at the ‘tight cluster’ stage were removed from each branch. Branches were then randomly assigned to one of three chambers (i.e., temperature treatments) and cultivated in buckets with water, which was replenished daily. Flowers were sampled randomly from each chamber when they reached ‘tight cluster’ (stage 1, with obvious pedicels) (Fig. 3A-b), ‘first white’ (stage 2), ‘half white’ (stage 3), ‘first open’ (stage 4) and ‘full open’ (stage 5) stages (Fig.3B). In 2011 we collected

3 flowers randomly from each trail (5 branches) and in 2012 we sampled 5 flowers.

Harvested flowers were dissected manually within 30 minutes into 6 distinct organs: petals, sepals, filaments, styles, ovaries and pedicels. The dimensions (length and diameter) of filaments, styles, pedicels and ovaries, were assessed by digital caliper (Fig. 4).

Individual sepals and petals were flattened and fastened to A4 (210mm x 297mm) colored papers by transparent tape, and then digital images were collected of the sheets (Fig. 5). Image analysis was used to determine the total area of sepals and petals using a custom algorithm in MATLAB (The

MathWorks, Inc., Natwick, Mass.). The intensity image was converted to a binary image first and then the objects’ (individual sepals or petals) properties in pixels were measured. A 1 cm2 square was added to each paper (Fig. 5a) in order to compare it with the petals and sepals and to convert pixel data to area units.

2.2.4 Statistical Analysis

Comparisons of the traits of floral organs of the four cultivars under three temperature treatments, and main-effect sensitivity indices, were analyzed using the General Linear Models (GLM) program of the SAS statistical analysis. A correlation matrix among floral parameters was assessed by Pearson

Correlation program of the SAS statistical analysis. The floral organ development rates were determined by using the formula based on defined flower stages, as follows:

Floral Organ Development Rate= (Flower Stage N -Stage 1)/Stage 1

Where N was either 2, 3, 4 or 5; Flower stages, Stage 1, tight cluster; Stage 2, first white;

Stage 3, half white; Stage 4, first open; Stage 5, full open.

2.3 Results

2.3.1 Floral Organ Size a. Sepal and Petal Area

According to GLM analysis, petal area was not influenced by temperature (P=0.1822 >0.05), but was affected by year and cultivar at the final sampling stage (Fig. 6a). There were significant year-to-year variations in sepal and petal areas at the ‘full open’ stage of all the four cultivars. In 2011, petal area of ‘full open’ flowers was ca. 1.5 times greater than in 2012 depending on the cultivar (Fig. 6a). In both years, the sepal and petal areas of ‘Tieton’ were the largest among cultivars. In 2012, for example, the petal area of ‘Tieton’ was 1.6 times larger than that of ‘Sweetheart’ at the final sampling stage (Fig. 7a).

Petals exhibited the greatest rate of expansion among all the organs we measured. Across all cultivars, petal area increased ca. 4.5-fold between the first and final sampling times. In contrast, sepal area increased only moderately between the earliest and latest sampling dates, increasing by ca.

56% (Fig. 8). Compared with other floral organ traits, the sensitivity index ‘cultivar’ had the greatest influence on the expansion of sepals though this value reached only ca. 0.28 (Fig. 9). b. Filament and Style Length

Unlike sepal and petal areas, at the ‘full open’ stage there were no significant differences in filament or style lengths between years (Fig. 6b, c). At stage 5, style length was not affected by genotype, only temperature. Style length in flowers of all cultivars cultivated under cold temperature was obviously shorter than flowers under warmer conditions. Style length was 12.5 mm under cold temperature, and

13.9 mm and 14.0 mm when cultivated under high and moderate temperatures, respectively (Fig. 7c).

Similarly, the lengths of filaments were also shorter under the simulated cold spring compared to both warmer regimes, but this discrepancy was not significant in ‘Tieton’ cultivar (Fig. 7b).

Interestingly, though the organ important for pollinator attraction (i.e., petals) of ‘Tieton’ were largest among the four cultivars, the mating-related organs (i.e., filament and style) were not larger, or even shorter, compared with other cultivars. (Fig. 6a; Fig. 7b).

Filaments grew faster than styles between the earliest and the latest stages. Filament length increased ca. 3 times whereas the styles roughly doubled in length by investigating all cultivars (Fig. 8).

Analyses of sensitivity revealed that the length of filaments and styles were more influenced by temperature than other organs (Fig. 9).

c. Pedicel and Ovary Characteristics

Compared with growth of other organs, the radial growth of pedicels occurred at the lowest rate, increasing by ca. 23% between the first and final stages. Across all cultivars, pedicel length, however, increased by about 1.4-fold during the same period (Fig. 8). Pedicel diameter was not influenced by temperature at the ‘full open’ stage. There were significant year-to-year variations in both the length and diameter of pedicels, with these two parameters being on average 30 and 15% higher in 2011 than 2012, respectively. Pedicel diameter of ‘Tieton’ was largest among four cultivars (‘Tieton’ averagely 1.66mm, ‘Benton’ 1.26mm, ‘Rainier’ 1.20mm, ‘Sweetheart’ 1.23mm) but the average pedicel length of ‘Tieton’ was the shortest (‘Tieton’ averagely 11.95mm, ‘Benton’

15.39mm, ‘Rainier’ 13.04mm, ‘Sweetheart’ 12.40mm).

The development rates of ovary length and diameter were both relatively low during anthesis compared with other floral organs; they increased by ca. 44% and 32%, respectively between the first and last sampling. By the final sampling at the ‘full open’ stage, there were no differences in ovary length or diameter across all temperatures but significant differences among varieties. The ovary diameters in 2011 were ca. 20-50% larger than those in 2012 depending on cultivars. In contrast, the ovary length in 2012 was larger than length in 2011 by investigating all the four cultivars and the maximum differences appeared in ‘sweetheart’ cherry that ovary length in 2012 was ca. 30% larger than in 2011.

2.3.2 Correlation among Floral Organs

Correlations among the size of sweet cherry floral organs we measured were generally very high and significant by investigating all samples (P < 0.01). Among all cultivars, the correlation between style length and filament length was the greatest, being as high as 0.87 for ‘Rainier’. Generally, the attraction-related organs (i.e., petal area) exhibited close relationships (CC>0.5) with mating-related organs (i.e. style and filament length). Correlation coefficients for these relationships were generally

0.75 or greater for ‘Benton’, ‘Rainier’ and ‘Sweetheart’ whereas in ‘Tieton’ they were slightly lower, ca. 0.6. Sepal area was the least related to other floral organs, and there were no relationships between sepal area and ovary length in ‘Benton’ and ‘Rainier’ cultivars where P-values were larger than 0.01.

2.4 Discussion

There were significant year-to-year variations in the size of floral organs of the four sweet cherry cultivars evaluated. Benedek et al. (1996) reported whole flower size and the relative position of stamens and styles of sweet cherry varied notably from year to year. In contrast, Pérez-Sánchez et al.

(2008) studied open flower diameter, petal length, petal width and pistil length and found minimal differences in sweet cherry floral parameters over three years. In our study, the dimensions of petals, pedicels and ovaries varied significantly between years for all cultivars at full bloom. In contrast, the lengths of styles and filaments were conserved between years in all cultivars (Fig. 6). This discrepancy among floral organs demonstrates the relative size of organs of single flowers in the two experimental seasons were not same. The relative size of floral organs is known to influence the

selection of pollinators and was conducive to successful pollination in Brassica napus (Cresswell,

2000). Alterations in the relative position of floral organs, such as flowers with abnormally short styles, was closely associated with reduced fruit set (i.e., fertilization rate) in apricot (Rodrigo and

Herrero, 2002). Insect-pollinated flowers such as sweet cherry are generally composed of attraction- related organs, such as petals, and mating-related organs, such as the stamens and pistils across years and temperatures. Researchers have proposed and proved an evolution theory that the physical fit between a flower and its animal pollinator determines successful pollination and this match is more important for organs involved in mating than for organs involved in pollinator attraction (Ushimaru et al., 2003; Wolf and Krstolic, 1999). In other words, this theory predicted that the size of mating- related organs were more stable than the size of attraction-related organs. In current research, we found that the size of mating-related organs varied less than the size of attraction-related organs across years. In Pogonia Japonica, gynostemium, a fusion of the stamens and pistils, also showed lower variation in size than petals (Ushimaru and Nakata, 2001).

In the current study, ‘Tieton’ flowers possessed the largest petals compared with other genotypes, but other floral organs were not similarly larger (Fig. 7). Consequently, stylar exertion in ‘Tieton’ was minimal. Because of less-exserted styles, pollinators often deposit more locally-collected self-pollen on the stigmas rather than pollen they carry from a pollenizer cultivar while they seek the footholds by pulling anthers (Thomson and Stratton, 1985). The low commercial productivity of ‘Tieton’

(Whiting and Zhang, unpublished), a self-sterile cultivar, might result from the relatively short styles disturbing the stigma to gain effective pollens causing low fruit set. Ruiz and Egea (2008) reported significant reductions in fruit set with decreasing height difference between the stigma and the

superior plane of the anthers. It is hypothesized that due to poor pollen transfer, flowers of apricot with abnormally short styles exhibited significantly lower fruit set than normal flowers (Rodrigo and

Herrerro, 2002). On the other hand, there was no evidence revealed that shorter styles shortened the days pollen tube passed through styles than longer ones. The growth of pollen tube along styles could be influenced by temperature, species, flower ages and self-pollination (Hedhly et al., 2003;

Sutyemez, 2011; Yi et al., 2006 and Ortega et al., 2004.) Therefore, the comparatively short styles of

‘Tieton’ may not affect EPP nor contribute to low fruit set in the field. Separate research in our lab has implicated short ovule longevity in ‘Tieton’ as the key factor in reducing fruit set (Zhang and

Whiting, unpublished).

Generally, self-fertile sweet cherry cultivars exhibit higher rates of fruit set compared with self- sterile cultivars under the same environmental conditions (Sutyemez, 2011). Compared to self-sterile flowers, self-fertile have a smaller corolla to reduce hekogamy and dichogamy by natural selection

(Runions and Geber, 2000). In the current study, individual petal areas of the self-sterile cultivar

‘Tieton’ were up to ca. 2.2 cm2 while petal area of the self-fertile cultivar ‘Sweetheart’ was only 1.2 cm2 at the ‘full open’ stage under high temperature conditions (Fig. 6a). However, the self-fertile cultivars studied herein, ‘Benton’ and ‘Sweetheart’, also exhibited different petal sizes, with those of

‘Benton’ being ca. 1.3 times larger than ‘Sweetheart’ at ‘full open’ stage (Fig. 7a). Compared to

‘Benton’, the flowers of ‘Sweetheart’ were smaller and their structures were more compact for self- pollination according to the ‘small corollas’ evolution theory of self-fertile flowers (Ornduff, R.,

1969; Runions and Geber, 2000). Though larger floral perianth was more attractive to bees, smaller and more compact flowers were more apt to guarantee successful pollination to self-fertile species under the situation with continuous rainy and windy days or lacking pollinizers. (Cochen and

Shmida, 1993; Sutyemez, 2011)

Flowers with long pistils tend to receive more pollen from outcross sources, and this additional pollen may increase the density and diversity of pollen and increase fruit set of these self-fertile cultivars (Thomson and Stratton, 1985). Paradoxically, similar to self-sterile genotypes, self-fertile cultivars with long styles may also exhibit high fruit set (Ruiz and Egea, 2008). Our results support this observation – in the current study, average style length of ‘Benton’ (low productivity) was 13.9 mm compared to 15.8 for ‘Sweetheart’ (high productivity), under moderate temperatures (Fig. 6b).

Combined, the large petals and short styles of ‘Benton’ flowers appear to be disadvantageous for fruit set despite being a self-fertile genotype. Based on the above discussions of flower structures of both self-fertile and self-sterile cherries, we suggest the selection of genotypes with long styles and, for self-fertile cultivars, small corollas of flowers to increase the probability of successful self- pollination for further selective hybridization.

Flower bud production, flower bud drop, flowering time, and fruit production of deciduous fruit trees have obvious year-to-year variations and researchers have attributed much of this variation to the effects of temperature (Ruzi and Egea, 2008). In previous work, temperature was also an important factor in determining floral organ dimensions (Rodrigo and Herrerro, 2002). Warm pre-bloom temperatures are known to accelerate anthesis but not the development of corolla size (Adams et al.,

1997). Prior to our earliest sampling (i.e., at the ‘tight cluster’ stage), air temperatures of early spring in 2012 were higher than those in 2011 (Fig. 1). Despite this, there were no differences between years in petal area measured in the growth chambers by ca. 2 days after sampling at ‘tight cluster’

(Fig. 6a). This suggests that warm spring temperatures did not affect corolla size before the ‘tight cluster’ stage. Previous reports have also shown that average daily temperature had no influence on

flower size prior to the ‘visible flower bud’ stage in Campanula carpatica (Niu et al., 2001a). With the process of flowering, however, corolla diameter decreased about 1 mm with every 1 ℃ decrease in average daily air temperatures in Campanula carpatica (Niu et al., 2001b). Murcia (1990) also reported that cold temperature delayed the time of anthesis and reduced the size of floral organs such as corolla length in late reproductive season. Interestingly, in the current study, temperature did not affect petal area at ‘full open’ stage for any cultivar. In contrast, the lengths of the styles and filaments at the ‘full open’ stage were about 11% and 25% shorter (except ‘Tieton’) in the low temperature environment compared to high temperature, respectively (Fig. 7b, c). This suggests that temperatures after ‘tight cluster’ play an important role in the final growth of styles and, to a greater extent, filaments. Zhang and Whiting (2012) reported reduced petal size of sweet cherry flowers cultivated under cold temperature conditions with no effect on the length of styles. Discrepancies among different reports on environmental effects on organ growth might be due to the differences in the timing of sampling and the onset of temperature treatments. Zhang and Whiting (2012) initiated their experiment at the ‘popcorn’ stage whereas we began the current experiments at the earlier ‘tight cluster’ stage. Therefore, it appears that the temperature between ‘tight cluster’ and ‘popcorn’, typically ca. 20 days in March, are important for the determination of the lengths of styles and filaments of sweet cherries in Washington State.

Pedicel and ovary diameters were not influenced by temperature when flowers were measured at the onset of ‘full open’ stage. Across both years and every genotype, the development rates of the diameters of ovary and pedicel were lowest of all the floral parameters evaluated in this study (Fig.

8). This demonstrates that the radial expansion of floral organs was not significant during anthesis.

This is an economically important trait since there is a positive relationship between fruit value and diameter (i.e., mesocarp). This result supports findings of Ushimaru and Nakata (2001) who concluded that there was no evidence that the widths of floral organs affected pollination success.

Our results that development rates of both ovary length and diameter were only around 30-40% and much lower compared with other organs (e.g. petal area 450%, filament length 300%) between flowers of ‘tight cluster’ and ‘full open’ revealed that our sampling period is not the essential period of ovaries growth in size. The report of ovary growth in sweet cherry was few, however according to previous research in stone fruit, the cell division for fruits in tart cherry (Prunus cerasus) ceases about 2 weeks after anthesis and 4 weeks in plum and peach (Westwood, 1995).

Floral organs have evolved to work together to perform important functions. To be receptive to pollen, promote out-crossing, and extend the effective pollination period, the relationships among floral organs have adapted to the environment (Thomson and Stratton, 1985; Ushimaru and Nakata,

2001). Nothaman et al. (1983) found positive correlations between stylar length and flower size in eggplant (Solanum melongena). Some correlations between organs were higher than the correlation among other traits in Arabidopsis thaliana (Brock and Weinig, 2007). Conner and Via (1993) reported the highest correlation between the length of filaments and corolla tubes of all the pairs of floral morphological traits while the relationship between the length of pistils and corolla tubes was less than other pairs by studying four species from Brassicaceae and one from Polemoniaceae. Male organs were more closely linked with floral corolla size than female organs in Arabidopsis thaliana

(Hill and Lord, 1989). In the current study, the correlations between petal area and both male

(filament length) and female organs (style length) were high, generally close to 0.75 in ‘Benton’,

‘Rainier’ and ‘Sweetheart’ cultivars. However, these values in ‘Tieton’, a genotype with low productivity, cultivar were lower, 0.62 (petal area-filament) and 0.56 (petal area-style length).

Interestingly, the correlation coefficient between filament and style lengths was the highest among all possible pairs of floral morphological traits in all genotypes we investigated in this study (Table 1).

Ushimaru (2002) studied the evolution of flower allometry and showed the similar results with

Mazus and Hosta species. According to that study, the correlation between female-male organs was much stronger than female-flower size or male-flower size correlations in selfing species. This close style-stamen relationship might be the result of pollen removal and pollen deposition selections by pollinators (Conner and Sterling, 1995).

2.5 Conclusion

Significant year-by-year variation was found in the size of attraction-related organs while mating- related organs were relatively stable. ‘Tieton’ flowers had a larger perianth but similar, or shorter styles and filaments compared with the flowers of other cultivars. Comparative short styles of both self-sterile cultivar ‘Tieton’ and self-fertile cultivar ‘Benton’ and large petals of ‘Benton’ could contribute to low pollination/fertilization success in these cultivars.

Between the earliest and the latest stages, filament length increased ca. 3 times and the styles doubled in length. The style and filament grew slowly under low temperature and they were more sensitive to the temperature after ‘tight cluster’ stage. In addition, we suggested that temperatures after ‘tight cluster’ play an important role in the final growth of styles and, to a greater extent, filaments.

The development of female and male floral organs was highly correlated compared to other organs; this may be the result of pollen removal and pollen deposition selections by pollinators. From the value of correlation coefficient, the floral parameters of ‘Tieton’ cherry were consistently lower than other cultivars which might suggest a slow evolution of flower structures in this low productive cherry cultivar.

Acknowledgement

We gratefully acknowledge financial support of this work from the Washington Tree Fruit Research

Commission and Washington State University’s Agricultural Research Center. We also thank Ms.

Laura Wells for valuable technical assistance.

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Table 2-1. Correlation matrix among floral parameters of various sweet cherry cultivars: (a) Benton;

(b) Rainier; (c) Tieton; (d) Sweetheart.a, b

(a)

Benton SA PA FL SL PL PD OL OD

SA 1.000

PA 0.500** 1.000

FL 0.360** 0.705** 1.000

SL 0.387** 0.724** 0.835** 1.000

PL 0.467** 0.806** 0.737** 0.723** 1.000

PD 0.390** 0.440** 0.425** 0.408** 0.554** 1.000

OL 0.122 0.27** 0.381** 0.482** 0.213* -0.011 1.000

OD 0.384** 0.734** 0.509** 0.554** 0.749** 0.564** 0.202* 1.000

(b)

Rainier SA PA FL SL PL PD OL OD

SA 1.000

PA 0.692** 1.000

FL 0.555** 0.738** 1.000

SL 0.564** 0.773** 0.856** 1.000

PL 0.463** 0.671** 0.756** 0.832** 1.000

PD 0.305** 0.414** 0.347** 0.449** 0.370** 1.000

OL 0.138 0.235* 0.392** 0.445** 0.476** 0.138 1.000

OD 0.376** 0.565** 0.515** 0.663** 0.576** 0.588** 0.152 1.000

(c)

Tieton SA PA FL SL PL PD OL OD

SA 1.000

PA 0.739** 1.000

FL 0.403** 0.618** 1.000

SL 0.454** 0.561** 0.750** 1.000

PL 0.59** 0.743** 0.572** 0.607** 1.000

PD 0.562** 0.646** 0.480** 0.566** 0.459** 1.000

OL 0.307** 0.393** 0.387** 0.360** 0.482** 0.193* 1.000

OD 0.616** 0.680** 0.374** 0.468** 0.574** 0.702** 0.374** 1.000

(d)

Sweetheart SA PA FL SL PL PD OL OD

SA 1.000

PA 0.525** 1.000

FL 0.424** 0.713** 1.000

SL 0.537** 0.795** 0.795** 1.000

PL 0.447** 0.732** 0.761** 0.774** 1.000

PD 0.341** 0.462** 0.277** 0.447** 0.377** 1.000

OL 0.352** 0.287** 0.548** 0.457** 0.343** 0.125 1.000

OD 0.350** 0.695** 0.509** 0.679** 0.603** 0.554** 0.152 1.000

a Correlation significant was at *p <0.05 and **p<0.01, respectively. Correlation coefficient (CC)<

0.5 means values were loose correlated and CC>0.75 means values were closely correlated. b PA, petal area; SA, sepal area; FL, filament length; SL, style length; OL, ovary length; PL, pedicel length; OD, Ovary diameter; and PD, pedicel diameter.

12 10

)

℃ 6 4 2011 2 2012

0 Temperature ( Temperature -2 -4 -6 Date

Figure 2-1. Mean daily temperature during late winter and early spring at the WSU-Roza experimental orchard at 2 m in 2011 and 2012( Sampling date).

High temperature 30.0 Moderate temperature

25.0 Low temperature

)

℃ 20.0 15.0 10.0

Temperature( 5.0

0.0

0:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00 8:00 9:00

10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00 18:00 19:00 20:00 21:00 22:00 23:00

Time

Figure 2-2. Diurnal variation in air temperature in controlled environment chambers.

(A)

a. b.

(B)

a. b. c. d. e.

(a) (b)

Figure 2-5. A. Original digital image of petals, sepals and a reference square attached to colored paper; B. the same picture after image processing with the MATLAB algorithm.

(a)

2.5 'Rainier' 2.5 'Sweetheart' 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5

0.0 0.0 Petal Area (cm2) Area Petal Petal Area (cm2) Area Petal 2011 2012 2011 2012 2011 2012 2011 2012 2011 2012 2011 2012 High Moderate Low High Moderate Low Chamber Temperatures in Year 2011 and Chamber Temperatures in Year 2011 and 2012 2012

2.5 'Benton' 2.5 'Tieton' 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5

(b)

20.0 'Sweetheart' 20.0 'Rainier' 15.0 15.0

10.0 10.0 5.0 5.0

0.0 Style Length(mm) Style Style Length(mm) Style 2011 2012 2011 2012 2011 2012 0.0 2011 2012 2011 2012 2011 2012 High Moderate Low Chamber Temperatures in Year 2011 and High Moderate Low 2012 Chamber Temperatures in Year 2011 and 2012

20.0 'Benton' 20.0 'Tieton' 15.0 15.0 10.0 10.0 5.0 5.0

0.0 0.0 Style Length(mm) Style Style Length(mm) Style 2011 2012 2011 2012 2011 2012 2011 2012 2011 2012 2011 2012 High Moderate Low High Moderate Low Chamber Temperatures in Year 2011 and Chamber Temperatures in Year 2011 and 2012 2012

(c)

'Sweetheart' 2.0 'Rainier' 2.0 1.6 1.6 1.2 1.2 0.8 0.8 0.4 0.4

0.0 0.0 Pedicel Diameter(mm) Pedicel 2011 2012 2011 2012 2011 2012 Diameter(mm) Pedicel 2011 2012 2011 2012 2011 2012 High Moderate Low High Moderate Low Chamber Temperatures in Year 2011 and 2012 Chamber Temperatures in Year 2011 and 2012

'Benton' 'Tieton'

2.0 2.0 1.6 1.6 1.2 1.2 0.8 0.8 0.4 0.4 0.0 0.0 2011 2012 2011 2012 2011 2012

Pedicel Diameter(mm) Pedicel 2011 2012 2011 2012 2011 2012 Pedicel Diameter(mm) Pedicel High Moderate Low High Moderate Low Chamber Temperatures in Year 2011 and 2012 Chamber Temperatures in Year 2011 and 2012

Figure 2-6. The parameters of floral organs at ‘tight cluster’ ( ) and ‘full open’ ( ) flower stages of four sweet cherry cultivars under high (H), moderate (M) and low (L) temperatures separately in year 2011 and 2012.

(a)

2.5

) 2 2

1.5 2011

1 2012 Petal Area Petal (cm 0.5

0 Benton Rainier Tieton Sweetheart Cultivars

(b) 14

8 H 6 M

4 L Filament Length(mm) Filament 2

0 Benton Rainier Tieton Sweetheart Cultivars

13.5 13 12.5

12 Style Length(mm) Style 11.5 11 H M L Temperature

4.5

3.5 SA

3 PA FL 2.5 SL 2 PL

1.5 PD OL 1

DevelopemntRateof Organs floral OD 0.5

0 Stage2 Stage3 Stage4 Stage5 Flower Stage

Figure 2-8. Development rate of floral organs from stage 2 to 5 compared to stage 1. (Flower stages:

Stage 1, tight cluster; Stage 2, first white; Stage 3, half white; Stage 4, first open; Stage 5, full open.

Floral organs: PA, petal area; SA, sepal area; FL, filament length; SL, style length; OL, ovary length;

PL, pedicel length; OD, Ovary diameter; and PD, pedicel diameter.)

PL Year

SL Cultivar

Floral Organs Floral Temperature FL Stage PA

0.0 0.2 0.4 0.6 0.8 1.0 Sensitive Indices

Figure 2-9. Main-effect sensitivity indices based on four-factorial design applied to the parameters of floral organs in four sweet cherry cultivars.

CHAPTER THREE

ASSESSING THE ROLE OF PISTIL IN SWEET CHERRY FRUIT SET WITH

CONTROLED ENVIRONMENT

Abstract

Several sweet cherry (Prunus avium L.) cultivars have compelling quality attributes but are beset with poor productivity (e.g., ‘Benton’, ‘Tieton’, and ‘Regina’). Our research investigated the role of temperature on stigma receptivity and ovule viability in four model sweet cherry cultivars:

‘Sweetheart’ (self-fertile, high productivity), ‘Benton’ (self-fertile, low productivity), ‘Rainier’ (self- sterile, high productivity), and ‘Tieton’ (self-sterile, low productivity). The development of the stigma surface, pollen hydration in vivo, pollen germination in vivo and tube growth in vivo were all observed and counted as proxy for stigma receptivity. In addition, the senescence of primary and secondary ovules was analyzed with fluorescence microscopy. The stigmatic papillae began deteriorating by the second day after anthesis and had collapsed by the sixth day post anthesis, across cultivars. We characterized pollen hydration into 5 stages and found that maximum pollen hydration and pollen germination occurred on the stigmas of flowers that had been open for 2-3 days, depending on cultivar and air temperature. Pollen hydration and germination, and ovule senescence were accelerated under warm temperatures. Under warm conditions there were ca. 80% fully viable ovules in ‘Rainier’ compared to ca 30% viable ovules in other cultivars by 7 days after flowers opened. The results reveal that low commercial productivity of sweet cherry cultivars in the Pacific

Northwest of U.S. is likely due to rapid ovule senescence and this is accelerated in warm conditions.

3.1 Introduction

In the Pacific Northwest of the U.S. the anthesis of sweet cherry occurs in early April and the first day of anthesis varies by ca. two weeks (Zhang and Whiting, 2012). Successful pollination provides the premises for fertilization and fruit set, and the latter one was reported to be accelerated by endogenous hormone produced (i.e. GA, auxin) from well-developed embryo, which is the masterpiece of fertilization (Rodrigo and Herrero, 1996; Sanchez et al., 2004). Most commercial sweet cherry genotypes exhibit gametophytic self-incompatibility, requiring out pollen from other compatible genotypes for successful fertilization (Choi and Andersen, 2001). Therefore, the pollinator factor including the behavior of honey bees (Apis mellifera L.) and the bloom overlap of pollinizers are important, but these two elements can be promoted by installers’ perseverance (Afik et al., 2008; Someriville, 1999; Imani, 2013). It is interesting that compared with self-incompatible cultivars, fruit set of self-fertile sweet cherry genotypes is generally higher (Sutyemez, 2011).

Previous reports of sweet cherry fruit set have demonstrated a significant range, from 0% to 70%

(Tosun and Koyuncu, 2007; Beyhan and Karakaş, 2009). In the PNW, the commercial productivity of several sweet cherry cultivars with outstanding fruit attributes (e.g. ‘Tieton’, ‘Regina’ and

‘Benton’) is poor, generally lower than 10% (Whiting and Zhang, unpublished), the value classified as the threshold of low fruit set (Bekefi, 2004).

Effective pollination period (EPP) is a concept that reflects the influence of genotype on pollination and therefore fruit set. EPP was first developed by Williams in 1965 and the definition is the longevity of ovule minus the time lag between pollination and fertilization, assuming that this value does not exceed the receptive period of the stigmas (Williams, 1965; Ortega et al., 2004; Tonutti et

al., 1991). Therefore, stigma receptivity, pollen tube kinetic and ovule longevity jointly determines

EPP. In cherry, the period for pollen tube germinated from the stigma to reach ovules is usually around 2-5 days depending on cultivars and temperatures (Sutyemez, 2011). Ughini and Roversi

(1996) reported the duration of EPP in sweet cherry covers a period from 2/3 to 3/4 of the entire duration of the flowering period.

Three keys steps in the fertilization process are related to the stigma receptivity: pollen grain adhesion, pollen germination, and pollen tube penetration into the transmitting tissues (Hedhly et al.,

2003). Stigmas lose the capacity to support pollen tube penetration first, pollen germination second and, finally for pollen adhesion (Sanzol et al., 2003). Stigma receptivity is associated with the exfoliation of cuticles, production of stigma secretion and integrity of papillae structure (Harrison,

2000; González, 1995). In Prunus fruit crops, stigma receptivity becomes greatest after flowering and this value decreased with time (Hedhly et al., 2003; Ortega et al., 2004; Yi et al., 2006). In addition, stigma receptivity is very susceptible to ambient unstable environment and pathogens

(Nicholson and Hammerschmidt, 1992).

The EPP of the species that the embryo sac matured after flowering is greatly determined by ovule longevity (Pimienta and Polito, 1982). Pistils of Prunus species contain two ovules in a single carpel; one ovule (i.e., secondary ovule) will lose viability prior to the other one (i.e., primary ovule)

(Rodrigo and Herrerro, 1998). The primary ovule is generally considered as the one that receives pollen tubes to complete fertilization (Bradbury, 1929; Arbeloa and Herrero, 1991). Ovule

degeneration was reported positively linked to callose amount appeared within ovules but negatively related to the amount of starch because fewer starch was observed in degenerated ovules (Pimienta and polito, 1982; Rodrigo and Herrero, 1998).

The environmental conditions during anthesis, pollinator activity, and genotype are the three main factors that affect the success of pollination (Hedhly et al., 2003; Mohamed, 2008). Warm pre-bloom temperatures can accelerate sweet cherry anthesis, but not floral organ development, resulting in reduced weight of pistils (Rodrigo and Herrerro, 2002). The rate of pollen germination on the stigma surface is increased under warm temperature, and the time for pollen tubes to grow the length of the styles is shortened by 6 days at 30ºC compared to 10ºC in sweet cherry (Hedhly et al., 2003). Stigma and ovule senescence are accelerated under warm temperatures and EPP cannot be prolonged by single fast pollen tube growth (Ortega et al., 2004). EPP of sweet cherry is also at a conflict state between pollen growth and pistil senescence under cold temperature (Sanzol et al., 2003b).

This research examined the role of genotype and temperature on the pistil components of EPP, including stigma receptivity and ovule longevity.

3.2 Material and Methods

3.2.1 In Lab-Pistil Role Evaluation a. Plant Material

This research was conducted in 2011 at two locations: Washington State University’s Roza research orchards in Prosser, USA and a commercial orchard in The Dalles, Oregon, USA. Four sweet cherry cultivars were selected including two genotypes that exhibit high commercial productivity,

‘Sweetheart’ (self-fertile) and ‘Rainier’ (self-sterile), and two genotypes that exhibit low yield commercially, ‘Benton’ (self-fertile) and ‘Tieton’ (self-sterile). At ca. 10% bloom throughout trees, thirty section of two-year-old flowering branches per cultivar were collected randomly from outer canopy of fifteen trees, placed in water and delivered to lab immediately. Flowers that were older or younger than ‘popcorn’ stage were removed from the branches. Subsequently, stamens and perianths were manually removed (emasculation) of the flowers left in the branches to avoid self-pollination.

In addition, 2-3 branches with intact flowers were set aside for controls and isolated from treated flowers in the growth chambers (i.e., inadvertent pollen transfer) by mesh veils.

Thirty branches were randomly divided into three groups, placed in buckets with water and cultivated in three controlled environment chambers (Adaptis, A1000, Canada). Each chamber was programmed to mimic either a cool (lowest:4°C, highest: 12°C), average (lowest:6°C, highest:

18°C), or warm (lowest:12°C, highest: 24°C) spring (Fig. 1). These three temperature regimes were developed from a study of the historical air temperatures in the Prosser region during the first two weeks of April (i.e., the weeks prior to anthesis). Yearly average minimum, medium and maximum

temperatures of those prior-bloom days by individual hours were valued as low, moderate and high temperatures for growth chambers, respectively. The chambers environment had 70% relative humidity with a day/night light daily cycle at three levels, 0 (black, 12h, 19:00-6:00), 1 (dim, 2h,

7:00 and 18:00) and 2 (bright, 10h, 8:00-17:00).

b. Observation of Stigma Surface

Flowers without emasculation treatment cultivated in moderate temperature set chamber were started to be sampled out at 1 day intervals for stigma surface observation since the first day of anthesis and this sampling continued six days. The development of stigmas was observed by scanning electron microscopy and light microscopy of longitudinal sections.

Scanning Electron Microscope – Styles with stigmas were cut off at the junction between style and ovary in the pistil and five fresh flowers were sampled as a trial of each cultivar. The surface of fresh stigma was observed directly under Hitachi S-570 scanning electron microscope (Hitachi Ltd.,

Tokyo, Japan). Images were digitally captured using Quartz PCI 4.2 (Quartz Imaging Corporation,

Vancouver, B.C. Canada).

Light Microscope- Pistils of ten flowers were collected and dissected on continued six days after flowering. Samples were fixed in 2% glutaraldehyde in 50mM phosphate buffer overnight at 4ºC and dehydrated through ethanol starting with 30%, 50%, 60%, 70%, 80% and 95% for 10 minutes each,

ending with 2 rinses in 100% ethanol 10 minutes each. Then at room temperature, samples were rinsed through 50% (with ethanol) and 100% propylene oxide and infiltrated with propylene oxide/

SPURRS at 2:1, 1:1, 1:2 orderly till embedded in 100% SPURRS overnight in hood. 500-800nm stigma longitudinal sections were cut by ultra microtome (Reichert-Jung, Cambridge Instruments

GmbH, West Germany), stained with 0.05% toluidine blue O. The stigma longitudinal sections were then examined using a Leitz Aristoplan Florescence Microscope (LEP Ltd., Wetzlar, Germany).

c. Pollen Hydration Test

Previously collected, compatible pollen from NY 54, a small-fruited wild cherry selection used commercially as a seed source for seedling P. avium rootstock, was delivered to the surface of stigma manually using a soft brush. Hand pollination was conducted to fifteen randomly selected emasculated flowers from each temperature-controlled environment daily from the beginning of flowering. Twenty minutes after pollination, entire flowers were harvested at the pedicel-spur junction. Stigmas of 3 groups of five flowers were dipped into mineral oil to rinse the pollen from the stigmas onto glass slides. Pollen was observed Olympus BX51 Fluorescence Microscope with DP70

Digital Camera System at 4X and randomly three visions were captured of each slide by Spot

“Advanced” Camera Software. According to the degree of the shape, hydrated pollens were classified into five stages (i.e. stage 0, 1, 2, 3 and 4) (Fig. 3A-E). Hydration level ( ) was counted by the following formula:  fc X  N

Where is the hydration level; N is the total number of pollen; f is pollen amount of different stages; and c is the hydration stage (1 – 5).

The hydration level of collected pollen was estimated using the above formula, then the data were analyzed using the General Linear Models (GLM) program of the SAS statistical analysis.

d. Pollen Germination, Tube Growth and Ovule Viability Evaluation in vivo

On the first day of anthesis in control flowers, hand pollination using previously collected compatible pollen were given to stigma surface of flowers. Emasculated flowers of each trial were randomly divided into six groups with 10 in each. Each group was randomly assigned to receive pollination at one of 6 different intervals (at 24 hr), with the final pollination taking place on the 6th day after anthesis. Pistils were collected at 3 intervals (8hr, 24hr, 48hr) post pollination for microscopic assessment of pollen germination, pollen tube growth, and ovule viability.

Pistils were fixed in FAA solution comprised of 95% ethanol: glacial acetic acid: 37% formalin

10:1:2 (v/v/v). Flower tissues were rinsed with distilled water three times for 30 mins each and then transferred to 5N sodium hydroxide solution. Four to six days later, when tissue was softened, samples were transferred to a 0.1% Aniline Blue solution and left until the pistils became transparent, ca. 10-14 days. Whole pistils were then placed on a slide with 1-2 drops of 50% glycerol and then squashed with a cover lip. Slides were examined by using Olympus BX51 fluorescence microscope

equipped with a DP70 Digital Camera System.

Pollen germination rate was determined by counting pollen grains that possessed a tube that was at least the length of the pollen grain (Fig. 3F, G). The length of the longest pollen tube was recorded as a percentage of the total length of the style was evaluated as pollen tube length (Fig. 3H). Ovule viability was determined by assessing the fluorescence from both ovules in each ovary. The fluorescent (i.e., non-functional) areas of both primary and secondary ovules were classified into five stages (i.e. full viability, 25%, 50%, 75% and 100% lost viabilities) (Fig. 4A-F).

The data of trials from the two locations (i.e. Prosser and The Dalles) was combined and therefore the percent of pollen germination of each treatment were calculated based on 20 flowers. Pollen tube length was analyzed using the General Linear Models (GLM) program of the SAS statistical analysis followed by Duncan’s multiple range test at P=0.05. Based on no significant differences between two locations of the percentage of callose of individual ovules, each data were counted within cultivars, temperatures and flower stages based on 20 ovules.

3.2.2 In-field Fruit Set Evaluation

Ten-year old sweet cherry trees of four cultivars, ‘Sweetheart’, ‘Rainier’, ‘Tieton’ and ‘Benton’ all grafted on ‘Gisela 5’ rootstock were selected in Roza research orchard. Branches were selected in the early spring of both 2011 and 2012 before flowering; flowers older and younger than ‘popcorn’ stages were manually removed at the pedicel-spur junction and the remaining flowers were manually emasculated. The branch sections were then covered by rectangular iron wire frames measuring 80

cm in length. These frames were wrapped around with plastic mesh cloth to prevent bee pollination.

Thirty-six spurs in 2011 and sixty spurs in 2012 on 3 and 10 branches of each cultivar, separately, with emasculated flowers were randomly divided into six groups and one group was randomly selected to accept hand pollination every day. Fruit set was determined six weeks post pollination by counting fruit on these branch sections. Fruit set data were analyzed using the General Linear Models

(GLM) program of the SAS statistical analysis followed by Duncan’s multiple range test at P=0.05.

3.3 Results

3.3.1 Stigma Development

Viewed from above, the surface area of sweet cherry stigma is nearly circular to slightly oval with a concave center. The stigmatic diameter of the sweet cherry genotypes we assessed varied between

850-1000 μm and its surface was fully covered by ca. 1800-2200 papillae cells of various sizes, the diameter of which ranged around 10-30 μm at the surface. (Fig. 1A) Longitudinal sections through the stigma reveal that the papillae cells are elongated along the axis of the style, 5-15 times greater in areas by longitudinal section than the glandular cells below them. There were typically 1-3 layers of papillae cells on the stigma (Fig. 1B,D) irrespective of cultivar.

Under moderate temperature, the papillae structures of the stigma began to degenerate by the second day after flowering (Fig. 1E, F). This degeneration of papillae cells was characterized by a cellular collapse that left a withered layer of cuticular pellicle on the stigma surface. With the progression of flowering, there were increasingly more collapsed papillae and, among the papillae, a fluid was

exuded that filled gaps between papillae and covered the stigmatic surface. This exudate accumulated first on the perimeter of the stigma before filling in toward the center of the surface. On the sixth day after anthesis, there were almost no integrated papillae cells and the whole stigma surface was covered with exudate (Fig. 1M, N).

3.3.2 Pollen Hydration

Upon deposition to the stigmatic surface, pollen is hydrated and swells prior to germinating. The extent of pollen hydration was assessed 20 minutes after manual pollinations. We observed differences in the extent of hydration after deposition on the stigmatic surface and we classified the process of hydration into five stages based upon pollen grain shape (Fig. 3 A-E). Pollen hydration stages of A-D were characterized as having elliptic pollen with varying length-width ratios. Stage A pollen was long and narrow with ca. 2.5:1 length-width ratio; stage B pollen was rounder than A with

2:1 length-width ratio; in stage C, the two ends of pollen was more round and full than pollen B and with 1.7:1 length-width ratio, and stage D pollen had a 1.5:1 length-width ratio. Stage E pollen was very nearly spherical with three obvious exines, a state just prior to germination.

Pollen hydration level varied with flower age (i.e., days after opening) and both pistil genotype and temperature were influential. Twenty minutes after manual pollination, pollen hydration was low on flowers that were within 1 day after anthesis irrespective of genotype. For example, in ‘Benton’ cherry under warm temperature, the pollen hydration level was less than 1.5 on the first two opening days (i.e. 0 and 1 day) while this value is ca. 3.0 on 2 days after flowering (Fig. 5). The degree of

pollen hydration reached its peak on flowers that had been open for 2 – 3 days, across cultivars at the moderate temperature. Pollen hydration level declined when pollen was applied to stigmas on flowers that had been open for 3+ days (Fig. 5). Under moderate temperature, pollen hydration on

‘Sweetheart’ stigmas was greatest on flowers that were open for two days. In contrast, the most rapid pollen hydration on ‘Tieton’ flowers took place on flowers that had been open for 4 days (Fig. 5B).

Moreover, the comparison of the maximum hydration level among those four cherry cultivars showed that this value was all ca. 2.6 of ‘Rainier’, ‘Tieton’ and ‘Benton’ cherries, which was ca.

30% higher than of ‘Sweetheart’ cherry under moderate temperature.

Under these three temperatures (i.e. warm, moderate and cold), there were no obvious differences of the highest pollen hydration level across cultivars, however, the duration of high pollen hydration levels did vary with the temperature regimes. For example, the highest pollen hydration level of

‘Rainier’ was ca. 2.5, irrespective of temperature. However, this high value was maintained across 4 days (1-4 days after flowering) under high temperature, whereas this level only lasted 2 days (2, 3 days after flowering) under moderate and low temperatures. In addition, the first day starting with the highest pollen hydration level was delayed by colder temperatures in ‘Benton’, ‘Sweetheart’ and

‘Rainier’ cherries. For example, the highest hydration level in ‘Sweetheart’ occurred 1 day post flowering under both high and moderate temperature, but this highest value occurred 3 days after flowering under cold temperature. (Fig. 5)

3.3.3 Pollen Germination

We recorded germination rates of pollens hand-pollinated to flowers of different ages (i.e. days post- flowering) and sampled these flowers at three time intervals following each manual-pollination. The extent of pollen germination after deposition on sweet cherry stigmas varied with the age of the flower and the incubation temperature (Fig. 6). Generally, pollen germination was greater under warm temperatures compared to moderate temperatures which had higher pollen germination than low temperatures. Under cool conditions we recorded 30 – 40% pollen germination. In contrast, pollen germination was 80 – 100% under the warmest temperature regime across cultivars and comparing similar flower ages. Pollen germination was greatest for pollen applied to flowers that had been open for 2 or 3 days. Germination declined when pollen was applied to flowers that had been open for 4+ days except for ‘Benton’ and ‘Rainier’ under cool temperatures.

Pollen germination was also varied with genotype. Pollen germination rates on ‘Tieton’ were nearly

100% under both warm and moderate temperatures, compared to a maximum of ca. 80% and 60% on

‘Sweetheart’, 90% and 85% in ‘Rainier’ and 100% and 80% in ‘Benton’ under the two temperatures, respectively. Under moderate temperatures, the highest rate of pollen germination was achieved for

‘Tieton’ by 2 days after flowering, whereas maximum pollen germination in ‘Benton’ and

‘Sweetheart’ did not occur until 4 days after anthesis. Under warm conditions pollen germination on

‘Tieton’ was ca. 50% on the first day post-anthesis, from samples collected 48 hr after pollination. In contrast germination on ‘Benton’ and ‘Sweetheart’ was ca. 20% and 10%, respectively (Fig. 6).

The percent of pollen germination in samples collected 8 or 24 hr post-pollination was lower than in samples collected 48 hr after pollination. According to our result, generally ca. 60% trials of pollen germination at 8 hr were lower than germination after 48 hr by 10-30% in value. However, compared to later sampled stigmas, the pollen germination ratio were still at a high value in earlier samples, for example, on the fourth day of flowering of ‘Tieton’ under moderate temperature, pollen germination radio was around 80% 8 hours followed by 100% 48 hours after pollen delivered to the surface of stigma. This means pollen tube germinated quickly once there was a proper environment. (Fig. 6)

3.3.4 Pollen Tube Growth

Pollen tube growth was not influenced by flower ages (i.e., 0-5 days post flowering) by investigating all the cultivars according to ANOVA analysis. Under low temperature conditions there was very little pollen tube growth, particularly in the low-yielding cultivars ‘Tieton’ and ‘Benton’ (Table 1).

The maximum tube length was 20% of the total length of styles in low temperature which appeared in ‘Tieton’, ‘Sweetheart’ and ‘Rainier’ cherries while this maximum value reached 100% in high temperature in ‘sweetheart’ and 60% in moderate temperature in both ‘Tieton’ and ‘Sweetheart’ after

48 hours of pollination. Generally, our results revealed that the higher the temperature the better the growth of pollen tubes except in ‘Tieton’ where styles reached ca. 21% the average length of the styles by 48 hr post pollination under the high temperatures and was significantly lower than the value under moderate temperature. However, even under high temperature, the average pollen tube length was less than one-third of the whole length of styles irrespective of cultivar.

3.3.5 Ovule Viability

In sweet cherry, there are two ovules in each ovary. The fluorescent areas of the ovules revealed the appearance of callose and, therefore, the degeneration of the tissue and loss of viability. We observed that when neither ovule exhibited fluorescence, usually one of the ovules was ca. 20%-50% larger than the other depending on flowers’ age (Fig. 4G). The ovule exhibiting no or less fluorescence or the larger ovule was considered as the primary ovule and the other ovule was considered secondary. The viabilities of both the primary and secondary ovules were classified into five levels (Fig. 4A-F). We categorized the ovules without fluorescence as ‘full viability’ (Fig. 4A).

At 25% fluorescence, color can appeared in either micropylar end or chalazal end of ovules (Fig. 4B,

C). Except these two ends, the degeneration of nucellus appeared earlier than of integument where the ovules with 50% and 75% fluorescence involved the degeneration of the whole integument (Fig.

4D, E).

Our assessment of ovule viability addresses both the primary and secondary ovules within each ovary. Both temperature and genotype affected viability of the primary ovule. In contrast, there was no significant effect of genotype in the viability of the secondary ovules of individual flowers.

Overall, the apparent lifespan of ovules was extended under cool temperatures and shortened under warm conditions. For example, there was never less than of 70% viability of primary ovules in

‘Tieton’ under the low temperature regime, compared to ca. 13% viability under high temperature

(Fig. 7C). In ‘Sweetheart’, ‘Benton’, and ‘Rainier’, all primary ovules maintained full viability up to four days post-flowering, under both moderate and low temperatures. In contrast, the percent of primary ovules at full viability was ca. 85% one day post-flowering and only ca. 55% by four days

after flowering under warm conditions.

Ovule viability varied among cultivars. Viability of the productive, self-sterile cultivar ‘Rainier’, for example, was ca. 80% after 6 days at the warmest temperature when viability of all other cultivars was 25 – 36%. Under moderate temperature conditions, primary ovules exhibiting with full viability in ‘Rainier’ did not fall below ca. 90%. In contrast, the lowest percent of primary ovule viability was ca. 50% in ‘Benton’ and ‘Sweetheart’, and 60% in ‘Tieton’, all exhibiting these lows on the final sampling, seven days post flowering. Ovules of ‘Rainier’ appear to be less-influenced by temperature and senesce at a lower rate compared with other cultivars. Interestingly, ovule viability of the two high productivity cherry cultivars (i.e., ‘Rainier’ and ‘Sweetheart’) was higher than the two low productivity cultivars (i.e., ‘Tieton’ and ‘Benton’) under moderate temperatures which mimic the average temperatures during flowering in Washington State. Specifically, primary ovules of ‘Rainier’ and ‘Sweetheart’ maintained full viability for 3 and 4 days post-anthesis, respectively. In contrast, full ovule viability in ‘Benton’ and ‘Tieton’ was maintained for only 1 day from anthesis.

High temperature also accelerated senescence of secondary ovules. For secondary ovules, the percent of ovules with full fluorescence color reached as high as ca. 20% under high temperature 4 days post flower opening while under moderate and low temperature condition, the number of ovules with full fluorescence were almost zero even at 7 days post flower opening (Fig. 7E). In addition, about 10% of the secondary ovules exhibited lost viability on the day of opening, irrespective of cultivar.

3.3.6 Fruit Set in Field

There was little variability among cultivars in fruit set from the field trial in which flowers, on 2-3 year old branches were hand pollinated. The differences of mean fruit set among these four cultivars were tiny across the days of pollination, where the greatest mean fruit set is 25.4% in ‘Sweetheart’ cherry and the lowest is 20.8% in ‘Benton’ cherry.

For all cultivars, fruit set was low when pollen was applied on the day of anthesis. Fruit set either doubled (‘Tieton’) or tripled (‘Benton’, Rainier’, and ‘Sweetheart’) when flowers were pollinated 1 day after anthesis compared to the previous day (Table 2). The highest fruit set occurred for flowers that were on their 2 days after anthesis, irrespective of cultivar. Fruit set decreased with older flowers and there were almost no fruit from flowers pollinated on the 5th and 6th days from anthesis.

On the third day after opening, fruit set of flowers in ‘Rainier’ cherry reached as high as 67% while this value was only 43% in ‘Benton’ cherries. Interestingly, ‘Sweetheart’ exhibited high fruit set (i.e., ca. 50%) for continuous two days, exhibiting 10% more in fruit set than other cultivars on the fourth day after flowering.

3.4 Discussion

Natural fruit set of sweet cherry cultivars grown under commercial conditions in Washington State varies considerably. The current study has revealed differences in stigma receptivity and ovule

viability among productive and unproductive cultivars as well as a role of air temperature during bloom. During anthesis, papillae on the surface of stigma of flowers gradually lose their turgidity

(Sanchez et al., 2004). There was a severe degeneration of papillae structures by 5 days after anthesis and this timing coincided with a decrease in the percentage of pollen that germinated (Fig. 2 M,N;

Fig. 6). Stigma receptivity relies on papillae integrity (González et al., 1995.) In the current study, stigmatic exudation was not present in flowers when they first opened, becoming apparent generally on the second day after opening (Fig. 2 E, F). Similar results have been reported in Silky Oak

(Grevillea robusta A. Cunn.) (Kalinganier et al., 2000) in which flowers lacked exudate upon opening. In almond, exudate was not observed on in peripheral but only the central region of stigmas till petal unfurling (Yi et al., 2006). Exudate secretion started 1-2 days post-anthesis in sweet cherry, this timing coincided with increased stigma receptivity and fruit set of flowers in field conditions

(Fig. 2 E-H; Fig. 5; Fig. 6; Table 2). The production of exudate has previously been linked to stigma receptivity in fruit tree crops such as sweet cherry and peach (Herrero and Arbeloa, 1989; Uwate and

Lin, 1981). Yi et al. (2006) also reported that stigmatic secretions could hasten the process of pollen hydration, germination and tube growth. Proteins, sugars and lipids have been identified in the secretions from wet type stigmas (e.g. sweet cherry) in Arabidopsis (Arabidopsis thaliana) and sunflowers (Helianthus annuus) (Sanchez et al., 2004; Shakya and Bhatla, 2010). It is unknown whether the composition of stigmatic exudate may vary among sweet cherry cultivars nor whether this could play a role in pollen hydration and, ultimately, fruit set.

Stigma maturity, considered as the first day that stigma can accept pollen, may occur either before or after flowering, depending on species. In almond (P. amygdalus), stigma receptivity was optimal

until flowers were past the fully open stage (Yi et al., 2006). Kalinganier et al. (2000) reported a lack of pollen germination on stigmas at 2 days or 1 day prior to opening, and a peak in stigma receptivity was reported to be 2 days post anthesis, in Silky Oak. In our research, the sweet cherry stigma was able to accept pollen on the first day of opening, though the pollen hydration level and the extent of pollen germination were low compared to results on older flowers (Figs. 5, 6). Generally, stigma receptivity of sweet cherries increases post-anthesis, reaching the maximum level after 2-3 days, and subsequently declines across cultivars and temperatures. Similar to our observations, the study of pollen germination in vivo of almond cultivars also revealed a decrease of stigma receptivity with time from flower opening after the peak of pollen tube numbers 0 or 2 days post blooming (Ortega et al., 2004).

There is much research on pollen tube growth in drupe fruits, e.g., sweet cherry, apricot (Prunus armeniaca), almond and peach (Prunus persica). It has been demonstrated that pollen tubes require 4

– 5 days to pass the entire length of the style in almond (Ortega et al., 2004), 6 – 7 days in apricot

(Rodrigo and Herrerro, 1998), ca. 7 days in peach (Arbeloa and Herrero 1987), 4 days in pear (Pyrus communis) (Sanzol et al., 2003a) and 2 – 4 days in sweet cherry (Sutyemez, 2011). In the current study, it appears unlikely that pollen tubes are able to pass the length of the styles within 2 days post- pollination since very few pollen tubes achieved sufficient length to reach the length of the style by

48 hr after pollination (Table 1), irrespective of cultivar and temperature. Indeed, the average pollen tube length was less than one-third of the length of the whole style in all the trials in our research.

This phenomenon may be explained (Yi et al., 2006; Sutyemez, 2011) that growth of pollen tubes was discontinued in the styles and it was slowly for tubes to transmitting through the one-third top of

stigmas. In addition, pollen tubes grew faster along the styles of self-fertile cultivars than self- incompatible ones in sweet cherry (Sutyemez, 2011). In current research, we observed that under high temperature – pollen tubes grew through 33% and 28% of the stylar length for the self fertile cultivars ‘Benton’ and ‘Sweetheart’, respectively. In contrast pollen tubes had grown through 21% and 24% the length of the styles in the self-sterile cultivars ‘Tieton’ and ‘Rainier’, respectively

(Table 1). Interestingly, according to the ANOVA analysis we found that pollen tube growth was not influenced by the flower ages. Once the pollen germinates, pollen tubes will grow through the styles at the same speed, irrespective of the date on which flowers were pollinated. On the other hand, our current research has shown that the percent of pollen germination varied with flower ages. Therefore, this discrepancy revealed that the degenerations of styles were later than the stigmas where the former one supplied pollen tube grows and the latter one for pollen germination.

Fluorescence microscopy reveals callose tissue when stained with aniline blue, and this fluorescence is indicative of ovule tissue that is degenerating and losing viability (Stösser and Anvari, 1982). It was hypothesized that the appearance of callose in ovules was due to the production of ethylene

(Clayton et al., 2000), the plant hormone chiefly responsible for degradative changes in all floral organs (Zhang and O’Neill, 1993). In a sweet cherry ovary, there are two ovules with the primary ovule being fertilized while the secondary one usually aborts (Bradbury, 1929). Therefore, for our analyses we labeled the secondary ovule as the one that accumulated callose (i.e., exhibited fluorescence) prior to, and/or to a greater extent, than the primary ovule (Pimienta and Polito, 1982).

Our results for show that senescence of the secondary ovules did not vary among genotypes, and that fluorescence, and loss of viability of secondary ovules was much higher than of that of primary

ovules, irrespective of temperature (Fig. 7). Stösser (1982) and Arbeloa (1991) reported that callose accumulation began at the chalazal end of the ovule and expanded through the inner integument, finally spreading throughout the entire ovule. However, in our research, we found callose may also first appear at the micropylar end and, in some cases, the integument close to the micropylar end

(Fig. 4B, C). It remains to be seen whether this has any bearing on fruit set.

In Prunus species, the stages of flowering, starting from mature ovules, varied depending on genotype (Egea and Burgo, 2000). From the current research, we found that the primary ovule of

‘Tieton’ under high temperature and ‘Benton’ under both high and moderate temperature (both low fruit set) degenerated on the day of flower (Fig. 7A, C). In addition, about 10% of the secondary ovules lost viability on the first day of opening by both high and moderate temperatures irrespective of cultivars (Fig. 7E). This result clearly demonstrates that sweet cherry ovules, especially ‘Tieton’ and ‘Benton’, have already degenerated before anthesis. Similarly, ovule degenerations due to high temperature were different among cultivars in almond (Ortega et al., 2004). In the current study, the percentage of fully viable primary ovules was as high as 80% in ‘Rainier’ compared to 25 – 36% in the other cultivars by seven days after anthesis and under high temperature (Fig. 7). This suggests that one mechanism for high productivity of ‘Rainier’ in field conditions is the relative vigor of its ovules, extending the effective pollination period (EPP). Similarly, early senescence of ovules in

‘Tieton’ and ‘Benton’ likely reduced the effective pollination period and contributed to their low productivity under field conditions. These traits would be accentuated in years during which warm temperatures are experienced during bloom. EPP is highly variable among cultivars within the same species and temperature plays an important role (Sanzol et al., 2003b). We observed that stigma

receptivity (i.e. pollen hydration, germination) was low on the day of first opening. Furthermore, under high temperatures, ca. 90% of ‘Tieton’ primary ovules were exhibited fluorescence by seven days post anthesis while only ca. 30% of ‘Rainier’ lost partial viabilities (Fig. 7). In addition,

‘Tieton’ and ‘Rainier’ are both self- sterile cultivars and cannot start pollination until flower opening.

Therefore, we can estimate that the most efficiency EPP of ‘Tieton’ sweet cherry is between 2 and 7 days after flowering and the most efficiency EPP of ‘Rainer’ cherry is longer. The shortened EPP value can be a reason that resulted in the low commercial production of ‘Tieton’ sweet cherry.

In current study, temperature played a great role in pistil’s function, i.e. stigma receptivity and ovule longevity. Generally, high air temperature expanded the duration of high pollen hydration level, facilitated the pollen germination rate, and accelerated pollen tube growth in sweet cherry pistils, but also hastened ovule degeneration according to our results. This temperature response has been observed in previous research (Kliewer, 1977; Pirlak, 2002; Sanzol et al., 2003b; Ortega et al.,

2004). Poor fruit set and smaller fruits were caused by temperatures of 33 – 40 C during flowering in several Vitis vinifera L. cultivars (Kliewer, 1977). Under cool conditions, pollen germination and growth were reduced and ovule longevity was extended. However, under low temperature, the extension to ovule longevity was not sufficient to offset the low pollen germination rate and reduced pollen tube growth in pear (Sanzol et al., 2003a) and fruit set was reduced. According to our research, the role of temperature on ovule viability was cultivar-dependent. ‘Rainier’ and

‘Sweetheart’ (high productive) maintained a comparably longer ovule life under high temperature than other cultivars. We speculate that the rapid senescence of ovules of unproductive cherry genotypes may be the main reason resulting in poor production.

We recorded the highest fruit set from flowers that were pollinated 2 days after opening, irrespective of cultivar (Table 2). This stage coincides with the flowers having relatively high stigma receptivity and significant exudate covering the surface. Griggs and Iwakiri (1964) reported greatest fruit set with flowers pollinated 3 days after emasculation, compared to flowers pollinated earlier. Combined current research on fruits set by hand pollination and the same location and year study on fruit set under natural open bee-mediated pollination, average fruit set in natural and by hand pollination of

‘Benton’ was 18.0% and 20.8% separately, of ‘Tieton’ was 26.7% and 22.1%, of ‘Rainier’ was

42.3% and 22.4%, and of ‘Sweetheart’ was 55.7% and 25.4% (Table 2). There were almost no differences in fruit set between natural pollination and hand pollination in the low productive cultivars ‘Tieton’ and ‘Benton’. On the other hand, those cultivars, i.e. ‘Rainier’ and ‘Sweetheart’, with high productivity in a commercial setting showed declined fruit set by hand pollination. This phenomenon can be explained by the reports of Guerra et al. (2010) that progress of emasculation could result in ovule senescence and very low fruit set, depending on the cultivar.

3.5 Conclusion

Results from this series of experiments showed that the optimal pollen hydration level, germination rate and fruit set by manual pollination appeared in the samples 2-3 days post-flowering but stigmatic papillae started to degenerate one day after the flower opening. These results may be useful to breeding programs that rely upon manual pollinations to set seed. Pollen tube growth was not affected by flower age but was accelerated by warm temperatures. In addition, according to current research, it was hard to find pollen tube passed through the whole styles within 2 days in sweet cherry. Generally, pollen hydration and germination were poor under cool temperature and ovules

were apt to lost viabilities by warm temperatures. However, exceptions were found in our results exhibiting the ovule viabilities in high productive cherry cultivar ‘Rainier’ and ‘Sweetheart’ exhibited tolerance to warmer temperatures compared to low productive cherries. Therefore, we suggested that warm temperatures accelerating ovule senescence could be the reason resulting in low production of some sweet cherry cultivars in Pacific Northwest of U.S. Our current research is investigating the ability of plant growth regulators to delay the senescence of ovules and improve fruit set.

Acknowledgement

We gratefully acknowledge financial support of this work from the Washington Tree Fruit Research

Commission and Washington State University’s Agricultural Research Center.

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Pollen Tube Length (%) Temperature Intervals ‘Benton’ ‘Tieton’ ‘Sweetheart’ ‘Rainier’ Average Maxi Average Maxi Average Maxi Average Maxi H 8h 14c 40 18bc 50 18b 60 15ab 40 24h 21b 40 20b 50 22ab 80 19ab 60 48h 33a 90 21b 70 28a 100 24a 90 M 8h 12cd 50 14bcd 20 13b 30 10b 20 24h 14c 40 18bcd 40 19ab 40 24a 30 48h 14c 40 26a 60 21ab 60 25a 40 L 8h 10d 10 10d 10 10b 10 12ab 20 24h 10d 10 11cd 10 11b 20 13ab 20 48h 11d 10 10cd 20 15b 20 12ab 20

*Length of longest pollen tube as a percent of the total length of the style

Values within the same column followed by the same letter do not significantly differ according to

Duncan’s test (P=0.05).

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Table 3-2. Fruit set of emasculated sweet cherry flowers receiving manual pollination in the field at daily intervals after anthesis in ‘Benton’, ‘Rainier’, ‘Tieton’ and ‘Sweetheart’.

Time of Pollination Cultivar (Days after Full Open) Benton Rainier Tieton Sweetheart 0 10.7de 7.8de 16.1c 9.8cd 1 29.5bc 23.7c 30.5b 27.1b 2 43.0a 67.1a 46.5a 50.4a 3 38.0ab 39.7b 36.5ab 49.4a 4 20.0cd 13.8d 17.1c 26.4b 5 4.5e 4.5de 8.1cd 13.3c 6 0.0e 0.0e 0d 1.4cd

Values within the same column followed by the same letter do not significantly differ according to

Duncan’s test (P=0.05).

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High temperature 30.0 Moderate temperature

25.0 Low temperature

)

℃ 20.0 15.0 10.0

Temperature( 5.0

0.0

0:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00 8:00 9:00

10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00 18:00 19:00 20:00 21:00 22:00 23:00

Time

Figure 3-1. Diurnal variation in air temperature in controlled environment chambers mimicking field environment of the past 10 years.

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A. B.

C. D. I. J.

E. F. K. L.

G. H. M. N.

(M, N) 5 days post-anthesis.

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A. B. C. D. E.

F. G. H.

Figure 3-3. (A) Stages 0 of Pollen hydration by observing pollens collected from stigmas 20 mins post-pollination. (B) Stages 1 of Pollen hydration. (C) Stages 2 of Pollen hydration. (D) Stages 3 of

Pollen hydration. (E) Stages 4 of Pollen hydration. (F, G) Pollen germination on stigma observed by fluorescence microscopy. (H) Pollen tube growth along style of flowers.

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A. B. C.

D. E. F.

G. H.

Figure 3-4. (A) Callose accumulation in ovules where callose appeared fluoresces reaction, ovule without callose. (B, C) Callose appeared in 25% of ovules. (D) Callose appeared in 50% of ovules.

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4.00 B. Moderate Temperature

3.50

3.00

2.50 Rainier 2.00 Tieton

Hydration Level Hydration 1.50 Benton Sweetheart 1.00

0.50

0.00 0 1 2 3 4 5 Days after Flowering

4.00 C.Low Temperature

3.50

3.00

2.50 Rainier 2.00 Tieton

Hydration Level Hydration 1.50 Benton Sweetheart 1.00

0.50

0.00 0 1 2 3 4 5 Days after Flowering

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4.00 A. High Temperature

3.50

3.00

2.50 Rainier 2.00 Tieton

Hydration Level Hydration 1.50 Benton Sweetheart 1.00

0.50

0.00 0 1 2 3 4 5 Days after Flowering

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A. Benton 48h

100 90 24h 80 8h 70 60 50 40 30 20 10

0 Percentage of Pollen Germination(%) Germination(%) Pollen of Percentage 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 High Moderate Low Days after flowering under different temperatures

100 B. Rainier

90 48h 80 24h

70 8h 60 50 40 30 20

10 Percentage of Pollen Germination(%) Germination(%) Pollen of Percentage 0 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 High Moderate Low Days after flowering under different temperatures

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C. Tieton 100 48h 90 24h 80 8h 70 60 50 40 30 20 10

Percentage of Pollen Germination(%) Germination(%) Pollen of Percentage 0 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 High Moderate Low Days after flowering under different temperatures

D. Sweetheart

100 48h 90 24h 80 8h 70 60 50 40 30 20

10 Percentage of Pollen Germination(%) Germination(%) Pollen of Percentage 0 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 High Moderate Low Days after flowering under different temperatures

(A), ‘Rainier’ (B), ‘Tieton’ (C) and ‘Sweetheart’ (D) sweet cherries.

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A. Benton-Primary Ovule

100 90 0 80 1

70 2 60 50 3 40 4 Frequency(%) 30 5 20 10 6 0 7 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 High Moderate Low Ovule Senescence (%) under Different Temperatures

B. Rainier-Primary Ovule 100 90 0 80 1 70 60 2 50 3 40

30 4 Frequency(%) 20 10 5 0 6 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 7 High Moderate Low Ovule Senescence (%) under Different Temperatures

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C. Tieton-Primary Ovule 100 90 0 80 1

70 60 2 50 3 40 4 Frequency(%) 30 5 20 10 6 0 7 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 High Moderate Low Ovule Senescence (%) under Different Temperatures

D. Sweetheart-Primary Ovule 100 90 0 80 1

70 60 2 50 3 40 4 Frequency(%) 30 5 20 10 6 0 7 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 High Moderate Low Ovule Senescence (%) under Different Temperatures

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E. Secondary Ovule 100 90 0 80

1 70 60 2 50 3 40 4

Frequency(%) 30 20 5 10 6 0 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 7 High Moderate Low Ovule Senescence (%) under Different Temperatures

Figure 3-7. The frequency of primary ovules of four sweet cherry cultivars (A) ‘Benton’, (B)

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CHAPTER FOUR

THE EFFECT OF RETAIN® APPLICATION ON OVULE VIABILITY, FRUIT SET AND

QUALITY OF SWEET CHERRY IN PACIFIC NORTHWEST

Abstract

Our previous research revealed that low productivity of several sweet cherry (Prunus avium L.) cultivars is due to the premature senescence of ovules. ReTain®, is a plant growth regulator with the active ingredient aminoethoxyvinylglycine (AVG) that inhibits ethylene biosynthesis. This research assessed the potential to improve fruit set of sweet cherry cultivars with chronically low commercial productivity with applications of ReTain®. Application timing and rate studies were conducted with

‘Tieton’ and ‘Regina’ trees in experimental and commercial orchards in Washington State and

Oregon State. Three ReTain® rates were compared (166 g/acre, 333 g/acre and 499 g/acre) with water-treated control trees with applications made at ca. 10% bloom. The role of application timing was assessed with 333 g/acre of ReTain® applied at ‘popcorn’, 10% bloom, 50% bloom, and full bloom. Fruit set was determined on two limbs per replicate tree. In addition, ovule viability was assessed on flowers 72 hours after ReTain® applications in the field at the first white and fully open stages in the field. We recorded significant improvements of fruit set from treatment with ReTain®.

Usually, the higher the ReTain® dosage the higher the fruit set across all the trails. Fruit set was increased by 120% and 60% in ‘Tieton’ and ‘Regina’ sweet cherries by 499 g/acre ReTain®, respectively, compared to controls. However, the efficiency of ReTain® increasing fruit set was not influenced by application timings. On the other hand, ovule degeneration was reduced by ReTain®,

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but, this reduced extent was not significant among applied dosages. In addition, we conclude from current work that generally fruit qualities (i.e. color, size, weight, firmness and soluble solid) of sweet cherries were not affected by ReTain® applied during anthesis.

4.1 Introduction

Some cherry cultivars (e.g., ‘Regina’, ‘Benton’ and ‘Tieton’) are popular commercially due to excellent fruit quality attributes, storage life, and resistance to rain-induced splitting (particularly

‘Regina’) yet are routinely low-yielding (Lang, 2001). ‘Regina’ is large (30 mm diameter), firm, rain crack resistance and also resistant to foliar infections of powdery mildew infection (Calabro et al.

2009). ‘Tieton’ is an early-maturing cultivar prized for its very large fruit size (32 mm) and attractive fruit with a natural glossy appearance and thick pedicels (Olmstead et al., 2000). ‘Benton’

(‘Columbia’) cherry is a self-fertile cultivar with large fruit size, high firmness and natural character of fruit splitting tolerance (Simon, 2006).

Rapid ovule senescence is known to limit fruit set in several sweet cherry (Prunus avium L.) genotypes (Zhang and Whiting, unpublished; Postweiler et al., 1985; Stösser and Anvari, 1983).

Previous research has revealed that ovule degeneration has been linked to the appearance of callose which seems to act as a barrier to the translocation of metabolites into the nucellus (Rodrigo and

Herrero, 1998). With this callose-tracking test, there are huge differences between primary ovules and secondary ovules where the first one effectively accepting sperms from pollen tubes while the latter ones usually shrunk during bloom in Prunus (Bradbury, 1929; Pimienta and Polito, 1982).

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High temperature during flowering has been reported as the main cause for ovule degeneration and, consequently, reductions in fruit set in Pyrus and Prunus fruit (Sanzol et al., 2003; Cerović et al.,

2000; Sutyemez M. 2011). Hedhly et al. (2007) reported complete senescence of ‘Sunburst’ sweet cherry ovules by 3 days post-opening in response to warm treatment (ca. 6 C higher in maximum temperature than the control) compared to 4 days in the control. Our previous work evaluating the influence of temperature on pistil function in sweet cherry showed that after 6 days of cultivation under elevated temperature, 80% of the primary ovules maintained their viability with a naturally productive cultivar ‘Rainier’ whereas about 35% ovules were viable in the cultivar ‘Benton’ which exhibits low productivity (Zhang and Whiting, unpublished). We hypothesize therefore, that reducing ovule senescence in chronically under-productive cherry cultivars may increase fruit set and subsequent yield.

Aminoethoxyvinylglycine (AVG) is a naturally occurring non-protein L-amino acid that was discovered in the early 1970s. This compound temporarily inhibits the activity of the enzyme ACC synthase, delaying ethylene-mediated ripening and senescence processes (Jobling et al., 2003).

ReTain® is a plant growth regulator containing 150 g/kg AVG in the form of a water soluble powder

(Valent BioSciences Corporation, IL, USA). ReTain® has been registered for use in apple, pear, stone fruit, walnut, pecan and cucumber in several states in the USA. Generally, ReTain® is applied to fruit trees weeks before harvest to reduce fruit drop, delay fruit maturity, and increase fruit size and firmness (Dussi et al., 2002; Green and Schupp, 2004.). In ‘Kordia’ sweet cherry, ReTain® reduced the incidence of fruit rain cracking by ca. 45% in Tasmanian, Australia (Bound et al., 2014).

ReTain® application is also common to prolong fruits storage life during postharvest (Jobling and

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Pradhan, 2003; Palou and Crisosto, 2003.). ReTain® is also be applied to flowers to delay flowering in rabbiteye blueberry (Vaccinium ashei Reade) and therefore to reduce the risk of spring freeze injury and increase the yield (Dekazos, 1979; Wood, 2011). Previous reports have demonstrated that the application of ReTain® improved fruit set in pears, sweet cherry, apple and nut crops (Dussi et al., 2002; Bound et al., 2014; Edgerton, 1981; Wood, 2011).The rates of AVG application ranged from 50 to 1000 mg/L during bloom for fruit set, pre or post-harvest for fruit quality depending on plant species (McFadyen et al., 2012).

There are very few reports about the ReTain® application in sweet cherries and none that examine its effect on ovule viability (Bound et al., 2014). Our current research investigated the influence of

ReTain® application rate and timing on ovule viability, fruit set, and fruit quality of several low- yielding sweet cherry cultivars.

4.2 Material and Methods

4.2.1 Orchard locations and plant materials a. Experimental orchards in Washington State

Washington State University’s ‘Roza’ experimental orchard located north of Prosser at lat. N

46°12′long. W 119°46′at an elevation of 203 m. Twelve 10-year-old ‘Tieton’/‘Gisela®12’ cherry trees trained to a Y-trellised architecture and spaced 1.5 m × 4.2 m, in rows orientated south-north were utilized in this study. Twelve 12-year-old ‘Regina’ trees were also selected from an

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experimental block that was planted south-north rows spaced 2.4 m × 4.5 m grafted on ‘Mazzard’ rootstock . Trees were trained to a steep leader system with three main leaders maintained at a height of 4.5 m. European honey bees (Apis mellifera L.) were introduced to the orchard at first bloom as pollinators at a density of two hives per acre. Standard orchard management practices were carried out for disease and pest control according to WSU Crop Protection Guide for Fruit Trees (Layne et al., 2014).

b. Commercial ‘Tieton’ orchard in Washington State

This orchard was located north of Prosser, Washington. Similar to the Roza farm, twelve 12-year-old

‘Tieton’/‘Gisela®5’ trees, trained to ‘steep leader’ type and spaced 3 m × 4.2 m were randomly selected for this trial. ‘Bing’ trees were interplanted as the pollinizers as every third tree in every third row. Honeybees were introduced to this orchard just prior to flowering at a density of 2 hives per acre.

c. Commercial ‘Regina’ orchard in Washington State

This orchard was located near Zillah, Washington State at lat. N 46°24′long. W 120°10′and at an elevation of 249 m. Sixty trees of eight-year old ‘Regina’/‘Gisela®6’ trees were selected for this research. Trees were trained to ‘steep leader’ system and spaced 3 m × 4.9 m. The honeybee hives density was 2 hives per acre and set in the orchard before flowering.

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d. Commercial ‘Regina’ orchard in Oregon State

This commercial orchard was located near The Dalles, Oregon in the Mill Creek area with lat. N

47°51′long. W 122°12′at an elevation of 115 m. Sixty 5-year-old ‘Regina’/‘Gisela®12’ trees planted at 300 trees per acre were used in this trial. The trees were trained to a modified Spanish Bush system with four permanent leaders. The fruit is grown off renewable laterals. Honeybees were introduced to this orchard and the density is 2 hives per acre.

4.2.2 Experimental Design

Experiment 1. AVG rates application

In Washington State, three ReTain® rates, (i.e., 166 g/acre, 333 g/acre and 499 g/acre ReTain® ) and control (distilled water) were applied to both ‘Tieton’ and ‘Regina’ sweet cherry trees at 10% bloom period in both commercial (B, C) and experimental (A) orchards. Twelve trees in the orchards were selected and divided into four groups for ReTain® and control. Two to three year-old branches with estimated ca. 150 flowers on it from two directions of individual trees were labeled out for fruit set count and fruit quality test. Two branches were selected from each direction of single tree, therefore, totally twelve branches were used for each treatment. ReTain® was dissolved in distilled water and sprayed to trees by a pressurized portable sprayer (Rear Nifty Series, Rears Manufacturing Company

Inc., Oregon, USA).

In addition, ten branches other than above labeled ones of all the AVG rates application trials in

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Washington State, were removed from trees 24 hours after spraying. These flowering branches were placed in buckets with water in the field and delivered to the lab immediately. Flowers at ‘first white’

(Fig. 1a) and ‘first open’ (Fig. 1b) stages were left on the branches while flowers of other stages were manually removed. These branches were subsequently placed into controlled environment chambers set to mimic field temperature with a diurnal low of 6°C and high of 18°C. The chambers had 70% relative humidity with a day/night light daily cycle at three levels, 0 (dark, 12 h, 19:00-6:00), 1 (dim,

2 h, 7:00 and 8:00) and 2 (bright, 10 h, 8:00-17:00). Twenty flowers labeled at ‘first open’ stage of each trail were randomly removed from 10 branches 48 hours and 96 hours post first-opening, separately, for further ovule viability test. Flowers at the ‘first white’ stage were labeled by black marker on the pedicels and observed every 12 hours until they reached the ‘first open’ stage and then be sampled in the same way. Perianth and stamens were got rid of from the flowers and the pistils were stored in FAA solution comprised of 95% ethanol: glacial acetic acid: 37% formalin 10:1:2

(v/v/v).

In Oregon State, the same ReTain® rates and control (distilled water) were sprayed to ‘Regina’ cherry trees in the commercial orchard (D). Twenty-four trees selected and divided into four groups.

Branches were selected following the same method we used in the orchards in Washington State and marked for further fruit set count and fruit quality test. One branch was labeled out from each direction of single trees, and therefore, also totally twelve branches were used for each treatment.

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Experiment 2. Flower timing by AVG application

In commercial orchards in both Washington State (C) and Oregon State (D), forty-eight cherry trees of ‘Regina’ cultivar were divided into eight groups which were trials by the application of 333 g/acre

ReTain® dissolved in distilled water 100 gallon/acre or control (distilled water) at ‘popcorn’, ‘10% bloom’, ‘50% bloom’ and ‘100% bloom’, respectively. ReTain® dissolved in distilled water with the concentration 3.33g/gallon and sprayed to trees by using pressurized portable sprayer. One two-year- old fruiting branch was selected and labeled out from each side (west and east) of single tree, therefore totally 12 branches were selected for each trial, for further fruit set record and fruit quality test.

4.2.3 Sample Treatments

Fruit set Flower numbers (all stages) on labeled branches were counted around 50% bloom and then fruit numbers were counted six weeks after full bloom.

Fruit quality test Fruits were sampled on the day according to commercial harvest standard. Fruit colors were classified into seven degrees according to the color cards produced by CTIFL (Centre

Technique Interprofessionnel des Fruits et Legumes). The weight of fruits was determined by electronic scale Adventurer TM Pro AV2102c within minimum range at 0.01g (Ohaus Corporation,

Pine Brook, NJ, USA). Digital calipers were used to measuring the fruit largest width, i.e. diameter of individual fruits. Soluble solids were determined by using Pocket Refractometer PAL-1 (Atago

USA., Inc). Pedicel-fruit retention force (PFRF) of single fruits was measured within 8 hours after

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field harvesting by using Digital Force Gauges Model DS2 (Imada, InC. ®, Northbrook, IL, USA).

Fruit sample size for quality test was depending on fruit number yielded from individual branch, but the maximum fruit number of each trial was controlled within 50 fruits.

Ovule viability test Flower tissues previously collected in FAA were rinsed with distilled water three times for 30 mins each and then transferred to 5N sodium hydroxide. Four to six days later, when pistil tissues were softened, they were transferred to 0.1% Aniline Blue and left until pistils became transparent. Whole pistils were placed on a slide with 1-2 drops of 50% glycerol and then squashed with a cover lip. Slides were examined by using Olympus BX51 florescence microscope with DP70

Digital Camera System. Both primary and secondary ovules of individual flowers were observed, and ovule viabilities were classified into five stages (Fig. 1 c-h) according to our previous description

(Zhang and Whiting, under review).

4.2.4 Statistical analyses

Data of fruit set, average area percentages of individual ovules lost viabilities, fruit quality (i.e. color, diameter, weight, firmness and brix), and PFRF were statistically analyzed by the General Linear

Models (GLM) program of the SAS statistical analysis followed by Duncan’s multiple range test at P

= 0.05. Ovules that exhibited fluorescence were considered as having lost viability in the frequency analysis of ‘percentage of ovules lost viabilities’.

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4.3 Results and Discussion

4.3.1 Fruit set

There were no significant differences in fruit set of neither ‘Tieton’ nor ‘Regina’ among the three commercial and experimental orchards we studied in Washington State (data not shown). Compared between cherry species within control trials, fruit set of ‘Regina’ was 68% higher than that of

‘Tieton’ where the average fruit set of ‘Tieton’ was 11% and of ‘Regina’ was 17% (Fig. 2). Bekefi

(2004) reported that the value of fruit set in cherries depends on cultivars and above 30% were classified as extremely high, between 20 and 30% were high, between 10 and 20% were considered medium and below 10% were low. In the same experimental orchard in Prosser (A), the fruit set of sweet cherry ‘Bing’, dominant traditional PNW cherry cultivar, was close to 30% (Whiting et al.,

2006). Therefore, ‘Tieton’ and ‘Regina’ were both considered having low fruit set, which would directly influence final production.

Generally, applications of ReTain® increased fruit set for both ‘Regina’ and ‘Tieton’ cherry cultivars.

This agrees with previous research that documented increases in fruit set in apple, pear and sweet cherry (‘Regina’ and ‘Kordia’) with application of ReTain® during bloom (Dussi et al., 2002;

Edgerton, 1981; Lombard and Richardson, 1982; Greene, 1980; Bound et al., 2014). In Oregon State, the maximum fruit set value of trials by ReTain® treatments was ca. 50% higher than controls, but not all trails were significantly improved in fruit set by ReTain®. In Washington State, fruit set of

‘Tieton’ increased by 120%, in ‘Regina’ cherry increased by 60% with 499 g/acre ReTain® application compared to control trails, which were also higher than other trials with lower ReTain® dosage treatment (Fig. 2). Therefore, current result revealed that high rates AVG had better

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efficiency on improving fruit set. Dussi et al. (2002) also concluded that the higher concentration of

ReTain® gave the higher fruit set and number of fruits per limb.

It is interesting that, fruit set of west-facing branches was generally higher than that of east-oriented limbs, for example fruit set was ca. 25% higher on west- than east-branches compared within controls in the orchard in Oregon State (Fig. 4). However, there were no remarkable differences of fruit set on branches between west- than east- orientations in Washington State (data wasn’t listed).

Previous research also suggested there were no differences of fruit set within north-south planted trees (i.e. east and west oriented branches), though fruit set of north-south rows were significantly higher than of east-west rows based on the consideration of light (Lombard and Westwood, 1977;

Khemira et al., 1993). Considering our introduction of honey bees in orchards, those insects behavior were negatively influenced by weather conditions (e.g. windy) (Southwick and Moritz, 1987).

Therefore, we speculated that the wind direction of the orchard we used in Oregon State can be the reason resulted in this fruit set differences between branch orientations.

In ‘Regina’ sweet cherry from both Washington and Oregon State orchards , there were no significant differences in fruit set among application timings(Fig. 3, Fig. 5). Therefore, we speculate that a broad period, from ‘popcorn’ to ‘full bloom’, may be effective for using ReTain® to increase fruit set in sweet cherry. However, compared with controls in the Oregon ‘Regina’ orchard, fruit set of the trials at 10% and 100% bloom exhibited significant lower fruit set within controls compared to other bloom period. This suggests that water sprays may decrease fruit set and supports previous research that spraying water during flowering could be a potential thinning agent to decrease fruit set

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in palm (Phoenix dactylifera L.) (Awad, 2006). Because there were no significant differences in fruit set among ReTain® treatments mixed with water at different blooming time (Fig. 3, Fig. 5), we speculate that any negative influence of water on fruit set was more than overcome by the efficiency of ReTain® to improve fruit set.

4.3.2 Ovule viability

Ovule viabilities can be assessed mainly by three methods and they are making serial tissue sections by microtome, watching and staining fresh tissues directly, and squashing pretreated softened and transparent ovules on object slides. Sarranine, hematoxylin and toludine blue are the three main dyestuffs that usually used in staining plant ovules and ovule sacs followed the process of making serial sections, i.e. plant tissue fixation, dehydration, embedding and sectioning (Cao and Russell,

1997; Caiola et al., 2000; Zhang et al., 2010). This method was considered, to a certain extent, lacking uniformity since it mostly depends on subjective description of the researchers (Webster and

Looney, 1996). Fluorescein diacetate (FDA), a nonpolar ester, which only stain living cells and is universally used in testing pollen viabilities, is also but rarely reported be applied in examining ovule degeneration by using fresh plant materials (Cao and Russell, 1997; Sutyemez, 2011). Using the aqueous solution, aniline blue, which stain callose with a clear and glistening blue color has been demonstrated as a more accurate and common method of testing ovule viabilities than other methods(Currier, 1957).

Across all trials, secondary ovules were more apt to lose viability than primary ovules when sampled

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2 days after opening, and ReTain® treatments can somewhat decrease ovule senescence. In control trials, the extent of senescence of secondary ovules was ca. 1.5 – 3 times higher than senescence of primary ovules, across cultivars and flower stages (Fig. 6, Fig. 8).

In sweet cherry, it is the primary ovule that exhibits extended viability and is fertilized. Primary ovule senescence was delayed by ReTain® applications except to flowers at the ‘full open’ stage in

‘Regina’. The percentage of individual ovules exhibiting lost viability decreased by 182% and 53% at 499 g/acre ReTain® compared to control at the ‘half white’ stage for ‘Tieton’ and ‘Regina’, respectively (Fig. 6, Fig. 8). Generally, the differences among ReTain® rates at reducing ovule degeneration were not significant except the trial of flowers at ‘half white’ in ‘Regina’. It was opposite to the current results on fruit set, which revealed the higher the ReTain® rates the better performance on fruit set. Callose appearance in ovules were considered as a barrier to the translocation of metabolites but no evidence proved the relationship between callose amount and the extent of ovule degeneration (Rodrigo and Herrerro, 1998). Combined, we inferred that ReTain® functioned in reducing callose of ovules but callose accumulation was not the only element deciding ovule viability. Starch was also reported linked to ovule degeneration as it played a significant role in embryo sac nutrition (Arbeloa and Herrero, 1991). On the other hand, we found that area percentage of individual ovules lost viabilities in ‘Regina’ was generally higher than that in ‘Tieton’ compared within controls. This value in ‘Tieton’ cherry was 14.1% and 24.1% while in ‘Regina’ cherry was

40.9% and 32.3% applied by AVG at ‘half white’ and ‘full open’ flower stages, respectively (Fig. 6,

Fig. 8). However, fruit set of ‘Regina’ cherry was 1.7 times higher than that of ‘Tieton’ cherry from the same orchard we collected samples for ovule viability test. In addition to ovule degeneration and

126

nutrition, the characters of cherry genotypes (e.g. stigma receptivity, effective pollination period) also played role in deciding successful fruit set (Ortega et al., 2004).

Interestingly, there was a reverse result of ovule degeneration in trials of the two flower stages (i.e.,

‘half white’ and ‘full open’) between these two cultivars. Simply compared within controls, ovules had higher viabilities of flowers sampled at ‘half white’ than at ‘full open’ stage in ‘Tieton’ cherry, however, in ‘Regina’ sweet cherry, ovules viabilities were lower of flowers at ‘half white’ than at

‘full open’ stage. In current study, flowers from trees at ‘half white’ were cultivated under moderate temperature longer than flowers sampled at ‘full open’ stage before ovule degeneration test.

Therefore, this result revealed that the moderate temperature setting which mimic the field environment of Pacific Northwest of U.S was more proper for ‘Tieton’ cherry than ‘Regina’ cherry.

Unsuitable high temperature hastened ovule degeneration in fruit crops (Kliewer, 1977; Ortega et al.,

2004). Furthermore, we speculated that during bloom from ‘half white’ stage to 2-day post-opening,

‘Regina’ cherry required a lower temperature environment than ‘Tieton’ cherry.

4.3.3 Fruit quality

Unlike sour cherry, most sweet cherries in commercial market were consumed freshly. Therefore, the fruit qualities in the aspects of fruit appearance (i.e., fruit size and skin color), taste (i.e., sweetness) and texture (i.e. firmness) are especially important that deciding the consumers’ selection and marketing sale (Chauvin et al., 2009; Ross et al., 2010). Most Harvest time of sweet cherry varies with varieties and local climates, generally from mid-June to early August in the Pacific Northwest

127

of the U.S. Sweet cherry fruit maturity is judged principally by the color of the exocarp and there are several color plates that are utilized commercially, including the CTIFL scale used herein. Fruit exocarp darkens with maturity in dark sweet cultivars such as those we studied. Generally, fruit color was not affected by ReTain® except with treatments to ‘Regina’ in the commercial orchard in

Washington and the highest rate in the WSU experimental ‘Tieton’ orchard. Compared to fruit from control trees, the application of ReTain® reduced fruit exocarp color by 13% and 5% from treatment with 166 g/acre and 499g/acre, respectively in the ‘Tieton’ commercial orchard, and by 3% and 9% in the ‘Regina’ experimental orchard. In addition fruit exocarp color was also reduced by 15% from

333 g/acre ReTain® in ‘Regina’ in the commercial orchard in Oregon.(Table 1; Table 2)

Fruit diameter of both ‘Tieton’ and ‘Regina’ was unaffected by ReTain® treatments in all orchards.

Similarly, fruit weight was also unaffected with the exception of average 1.2g/fruit significantly increase from ReTain® applications of all three dosages in the experimental ‘Regina’ cherry in

Washington. Generally, fruit firmness was not improved by ReTain® treatment in any trial except an increase of ca. 13% by 166g/acre ReTain® application in ‘Tieton’ cherry. Similarly, there was no significant effect of ReTain® treatments on fruit soluble solids (obrix).

Combined, ReTain® application at bloom in both ‘Tieton’ and ‘Regina’ cherries were not detrimental to fruit qualities including color, size, weight, firmness and brix. Those five traits are main factors that deciding fruit quality of sweet cherries (Kappel et al., 1996).

In the current study, the value of fruit color and firmness of cherries from experimental orchard was

128

ca. 35% higher and 25% lower, respectively, than cherries in the commercial ‘Tieton’ orchard by a simple comparison within controls (Table 1). This revealed that sweet cherries in this work harvested from commercial orchards were ‘seeming less mature’ than fruits from experimental orchards.

However, the soluble solids of fruits from these two orchards were almost the same. Besides, sweet cherry is non-climacteric fruit and does not have starch reserves, and therefore sugar contents of sweet cherry will not increase during storage and till final consumption (Webster and Looney, 1996).

For this reason, we confidently conclude that compared to experimental orchard, this ‘seeming less mature’ will not influence sweet cherries’ sweetness, on the contrary, higher firmness will guarantee a longer storage life.

Pedicel-fruit retention force (PFRF) was not affected by ReTain® treatments in commercial orchards

(Table 2). That is, ReTain®’s function as an ethylene inhibitor had an effect on fruit maturity (i.e., delaying exocarp color), but not on abscission zone developement. This may be due to the timing of application. Mean PFRF of fruits from WA was ca. 30% lower than that from OR where the average

PFRF of ‘Regina’ cherry in WA was 0.87kg and in OR was 1.27kg (Fig. 10). Zhao et al. (2013) also reported the value of PFRF in sweet cherry was fluctuated by various factors and there were significant differences in cherry PFRF among genotypes and years.

Compared among ReTain® timing trials, fruit qualities (e.g. Brix, firmness, color, PFRF and weight) generally were not significantly varied, apart from an exceptions that in the commercial orchard of

OR, brix and size of fruits treated by ReTain® at 100% bloom was higher than other timing trial (Fig.

10).

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4.4 Conclusion

We conclude that ReTain® improved fruit set and ovule activity in both ‘Tieton’ and ‘Regina’ sweet cherries and generally the higher the chemical dosage the higher the fruit set across all the trails.

However, there were not significant differences of fruit set among ReTain® spray timings. In the final fruit quality test, the parameters of color, size, weight, firmness and soluble solid were not declined by ReTain® used at blooming.

130

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Table 4-1. Fruit qualities of sweet cherry cv. ‘Tieton’ by AVG treatment at 166,333 and 499g/acre rates and the control at 10% bloom in both commercial and experimental orchard located in

Washington State.

Commercial Experimental Location Orchard Orchard AVG 166g/ 333g/ 499g/ 166g/ 333g/ 499g/ Treatment acre acre acre Control acre acre acre Control

Color 3.4c 4.0a 3.7b 3.9a 5.3b 5.5b 6.0a 5.3b Diameter (mm) 29.3a 29.2a 29.4a 29.2a 27.7a 27.7a 28.1a 27.9a

Weight (g) 11.3a 11.8a 11.9a 11.5a 10.4a 11.0a 10.8a 10.9a

Firmness 401.7a 361.7b 360.0b 355.9b 295.3a 276.3b 263.6b 262.4b

Brix 20.0b 21.5a 20.0b 20.3b 20.2b 18.2c 21.2a 19.8b

Values within the same column followed by the same letter do not significantly differ according to

Duncan’s test (P=0.05).

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Table 4-2. Fruit qualities and yield of sweet cherry cv. ‘Regina’ by AVG treatment at 166,333 and

499g/acre rates and the control at 10% bloom in commercial and experimental orchards located in

Washington and Oregon States.

Comme Experim Comme rcial ental rcial Orchard Orchard Orchard Location (WA) (WA) (OR) 166g/ 333g/ 499g/ 166g/ 333g/ 499g/ 166g/ 333g/ac 499g/ Treatment acre acre acre Control acre acre acre Control acre re acre Control

Color 5.0a 4.4b 4.2b 3.8c 5.6b 5.9a 5.3b 5.8a 5.1ab 4.6b 5.3ab 5.4a

Diameter (mm) 27.7a 27.3a 26.9b 27.5a 26.6a 25.3b 26.2a 25b 22.9a 22.1a 23.0a 23.8a

Weight (g) 11.3b 11.8ab 12.2a 11.7ab 10.1b 9.7c 10.5a 8.9d . . . .

Firmness 244.1b 249.7ab 242.5b 257.3a 261.6a 224.6b 241.5c 258.3a 272.4a 271.3a 249.3a 265.2a

Brix 16.4a 16.9a 14.9a 15.8a 18.9c 20.5a 18.6c 19.6b 16.6a 14.7a 16.2a 17.0a

PFRF *(Kg) 0.80bc 0.84ab 0.77c 0.87a . . . . 1.15ab 1.09b 1.05b 1.27a

*PFRF pedicel-fruit retention force

Values within the same column followed by the same letter do not significantly differ according to

Duncan’s test (P=0.05).

137

c d e f

a a g h c d e f b

g h

138

a 30 ab 25 a

20 b 166g/acre AVG b b 15 333g/acre AVG

Fruit Set Set (%)Fruit b 499g/acre AVG 10 Control 5

0 Tieton Regina Sweet Cherry Cultivars

Figure 4-2. Fruit set of sweet cherries cv. ‘Tieton’ and ‘Regina’ by AVG treatment at 166,333 and

139

50 a 45 333g/acre AVG 40 ab Control 35

ab 30 ab b 25 b b b 20 Fruit Set Set (%)Fruit 15 10 5 0 popcorn 10%bloom 50%bloom 100%bloom Treatment Timing

Figure 4-3. Fruit set of sweet cherry cv. ‘Regina’ by 333g/acre AVG treatment and the control applied in ‘popcorn’, ‘10% bloom’, ‘50% bloom’ and ‘100% bloom’ period in a commercial orchard located in Washington State. (Values followed by the same letter do not significantly differ according to Duncan’s test (P=0.05))

140

80 a 70 abc abc ab 60 abc abc

bc 50 c 166g/acre AVG 40 333g/acre AVG 30 Fruit Set Set (%)Fruit 499g/acre AVG 20 Control 10 0 West East Branch Direction

Figure 4-4. Fruit set observed from branches in both west and east directions of sweet cherries cv.

Duncan’s test (P=0.05))

141

70 a 60 a a ab a a 50

40 b

30 333g/acre AVG

Fruit Set Set (%)Fruit Control 20

0 Popcorn 10% Bloom 50% Bloom 100% Bloom AVG Treatment

Duncan’s test (P=0.05))

142

166g/acreAVG 70 333g/acreAVG 60 a 499g/acreAVG ab ab

50 b a Control 40 b b 30 a a b a

20 a a Lost Viabilities (%) Viabilities Lost b 10 b b

Primary Ovule Secondary Ovule Primary Ovule Secondary Ovule Average Area Percentages of Individual Ovules Individual of AreaPercentages Average Half White Full Open Ovule Types and Flower Stages

143

100.00 166g/acreAVG 90.00 333g/acreAVG 80.00 70.00 499g/acreAVG 60.00 Control 50.00 40.00 30.00 20.00

10.00 Percentage of Ovules lost viabilities viabilities (%) Ovules of lost Percentage 0.00 Primary Ovule Secondary Ovule Primary Ovule Secondary Ovule Half White Full Open Ovule Types and Flower Stages

144

166g/acreAVG

70 333g/acreAVG a a 499g/acreAVG a a ab 60 b b b Control 50 ab a 40 bc a 30 c b b

20 b Viabilities (%) Viabilities 10

0 Primary Ovule Secondary Ovule Primary Ovule Secondary Ovule

Average Area Percentages of Individual Ovules Individual of Lost AreaPercentages Average Half White Full Open Ovule Types and Flower Stages

145

100.00 166g/acreAVG

90.00 333g/acreAVG 80.00 499g/acreAVG 70.00 Control 60.00 50.00 40.00 30.00 20.00

(a)

(a) 146

19 a 18 ab ab 17 abc abc c 16

Brix c 15 c

12 popcorn 10%bloom 50%bloom 100%bloom Treatment Timing

(b)

17 a ab ab

16 ab b b b Brix 15 b

12 popcorn 10%bloom 50%bloom 100%bloom Treatment Timing

(c)

147

(c)

300 a a 290 a 280 a 270 a

a 260 a 250 a

Firmness 240 230 220 210 200 popcorn 10%bloom 50%bloom 100%bloom Treatment Timing

(d)

300 290 280 270

a 260 ab ab ab ab 250 ab ab

Firmness 240 b 230 220 210 200 popcorn 10%bloom 50%bloom 100%bloom Treatment Timing

(e)

148

(e)

a 5.5 a a a 5 a

a 4.5 a

Color b 4

3.5

3 popcorn 10%bloom 50%bloom 100%bloom Treatment Timing

(f) (f)

5.5

5 a

ab ab bc bc 4.5 bc

Color c

4 d

3.5

3 popcorn 10%bloom 50%bloom 100%bloom Treatment Timing

149

(g)

1.6 a a 1.5 1.4 ab ab 1.3

ab ab bc 1.2 b 1.1

PFRF (Kg) PFRF 1 0.9 0.8 0.7 0.6 popcorn 10%bloom 50%bloom 100%bloom Treatment Timing

(h)

1.6 1.5 1.4 1.3 1.2 1.1 a

PFRF (Kg) PFRF 1 ab 0.9 abc bc cd cd cd 0.8 d 0.7 0.6 popcorn 10%bloom 50%bloom 100%bloom Treatment Timing

(i)

150

(I)

29 28 27

a 26 ab 25 abc abc 24 abc 23 bc Diameter (mm) Diameter bc c 22 21 20 popcorn 10%bloom 50%bloom 100%bloom Treatment Timing

(J)

29 a a 28 b ab b b b

26 c 25 24

23 Diameter (mm) Diameter 22 21 20 popcorn 10%bloom 50%bloom 100%bloom Treatment Timing

(k)

151

(K)

12.5

a a a a

12 a a a

11.5 a

11 Weight (g) Weight

10.5

10 popcorn 10%bloom 50%bloom 100%bloom Treatment Timing

Figure 4-10. Fruit qualities and yield of sweet cherry cv. ‘Regina’ by 333g/acre AVG ( ) treatment and the control ( )applied in ‘popcorn’, ‘10% bloom’, ‘50% bloom’ and ‘100% bloom’ period in commercial orchards located in Washington (WA) and Oregon States (OR). (a) Fruit Brix, OR;

(b)Fruit Brix-WA; (c) Fruit firmness, OR; (d) Fruit firmness, WA; (e) Fruit color, OR; (f) Fruit color,

152