Title: Self-and cross-incompatibility in black cherry (Prunus serotina)
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Title: Self-and cross-incompatibility in black cherry (Prunus serotina)
Physical Description: 92 leaves : ill. ; 28 cm.
Language: English
Creator: Forbes, Donovan Charles, 1933-
Copyright Date: 1969
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Subject: Prunus   ( lcsh )
Agronomy thesis Ph. D
Dissertations, Academic -- Agronomy -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1969.
Bibliography: Includes bibliographical references (leaves 84-91).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Donovan Charles Forbes.
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Bibliographic ID: UF00097757
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Resource Identifier: alephbibnum - 000416937
oclc - 37680337
notis - ACG4423

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SELF-AND CROSS-INCOMPATIBILITY IN

BLACK CHERRY (Prunus serotina)














By
DONOVAN C' ,*:, L














A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA
1969














ACKNOWLEDGMENTS


The author is grateful to his chairman, Dr. Earl S. Horner,

or the interest he took and aid he gave in the preparation of this

issertation. The kind assistance of the other members of the author

committee, Dr. Harry H. Griggs, Dr. Thomas E. Humphreys, Dr. Paul L.

fahler, and Dr. Stanley C. Schank, is also appreciated.

Special thanks is accorded to Dr. Ray E. Goddard, the author's

visor, for his guidance, encouragement, and help when it was most

needed; to Mr. Ray K. Strickland, who completed one phase of the

field work when the author was incapacitated; and to the University

f Tennessee Atomic Energy Commission Agricultural Laboratory and its

wo laboratory technicians, Carroll Shell and Louise Russell, for use

f facilities and aid in the microtechnique phase of the work.

Finally the author is indebted to the Tennessee Valley

ithority for allowing him time to do the research and for providing

id and equipment,











TABLE OF CONTENTS

PAGE

ACKNOWLEDGMENTS ...................... ii

LIST OF TABLES ...... . . . ..... vi

LIST OF FIGURES .................. . . .viii

INTRODUCTION .................. . . 1

Early Work .. . . . . . . . . ... . 4

Attempts at Interpretation ............... 4

Systems of Incompatibility ... . . . . . 5

Heteromorphic Plants ...... . . . . .. 6

Homomorphic Plants ...... . . . . . .. 7

Effect of Polyploidy on Incompatibility . . . . . 8

Variations of Incompatibility . . . . . . . .. 11

Unilateral Incompatibility .. . . . . . 11

Somatoplastic Sterility ... . . . . . . 11

Biochemistry of Incompatibility . . . . . . 12

Incompatibility in Plums and Cherries . . . . ... 14

Immature Fruit Drop in Cultivated Cherries . . . . . 16

Techniques ............. .... ...... 17

Pollen Collection, Germination, and Storage . . ... 17

Artificial Pollination .... . . . . . . 18

Time of Flower Removal .... . . . . . . 19

Flower Killing and Fixation .. . . . . . 20

Slide Preparation .... . . . . . . . 20







PAGE

MATERIALS AND METHODS . . . . . . . . . 21

Plant Materials . . . . . . . . . . 21

Principal Studies ...................... 21

Pollen Tube Length Study--Gainesville. . . . . 24

Fruit Set Studies . . . . . . . . . . 27

Techniques and Procedures . . . . . . . 28

Pollen collection ................ .. 28

Pollination techniques . . . . . . . . 30

Tagging for identification . . . . . . ... 32

Bagging for isolation ................... 32

Flower killing and fixation . . . . . . .. 33

Slide preparation ........ ....... .. 33

RESULTS AND DISCUSSION . . . . . . . . . . 35

Preliminary Trials ..................... 35

Date and Duration of Flowering . . . . . .. 35

Flower Location on the Tree . . . . . . . .. 36

Characteristics of Flowers and Racemes . . . . ... 36

Natural Crossing . . . . . . . . . . 36

Natural Selfing ................. ..... 37

Pollen Germination Tests . . . . . . . ... 38

Bagging . . . . . . . . . . . . . 38

Chromosome Count . . . .. 39

Pollen Tube Length Study . . . . . . . 39

Pollen Germination ..... ........... ... 39

Summary of results ..................... 47







PAGE

Pollen Tube Extension ................ ...... 47

Summary of results ..... .. ........ ..... 56

Fruit Set Studies . . . . . . . ... . . 57

Prevention of Insect Pollination without Bagging--

Gainesville . . . . . . . . . . . 57

Selfing and Crossing--Gainesville . . . . . 57

Selfing and Crossing--Norris . . . . . . . . 59

SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS . . . .... 64

Summary and Conclusions .................. 64

Recommendations for Breeding and Tube Length Studies . .. 68

Recommendations for Future Work . . . . . . .. 71

APPENDIX . . . . . . . . . . . . . 73

BIBLIOGRAPHY . . . . . . . . . .. . 83

BIOGRAPHICAL SKETCH . . . . . . . .. . . 92














LIST OF TABLES

TABLE PAGE

1. Effects of Polyploidy on Incompatibility in Several Auto-

tetraploids Arising under Controlled Conditions, by Species

and Author (from Lewis, 1949) ......... . .. 10

2. Pollination Treatments, Gainesville Studies, by Female

Parent . . . . . . . . . . . . 25

3. Pollination Treatments, Norris Fruit Set Study, by Female

Parent and Pollen ................. .. 29

4. Comparison of Pollen Treatments with Respect to Pollen

Germination, Tree Group A . . . . . . . 43

5. Comparison of Pollen Treatments with Respect to Pollen

Germination, Tree Group B . . . . . . . 44

6. Rank of Removal Intervals in Tree Groups A and B with

Respect to Pollen Germination .. . 46

7. Percent of Pollen Tubes in the Upper Third of the Style that

Reached the Middle Third, Over All Trees and Removal

Intervals, by Pollen Treatment and Tree . . . . .. 49

8. Percent of Pollen Tubes in the Upper Third of the Style that

Reached the Lower Third, Over All Trees and Removal

Intervals, by Pollen Treatment and Tree . . . . 50








TABLE PAGE

9. Percent of Pollen Tubes in the Upper Third of the Style that

Reached the Middle Third, Over All Trees and Pollen

Treatments, by Removal Interval and Tree ... . . 54

10. Percent of Pollen Tubes in the Upper Third of the Style that

Reached the Lover Third, Over All Trees and Pollen

Treatments, by Removal Interval and Tree . . ..... 55

11. Percent Fruit Set among Cross-Pollinated Flowers, by Pollen

and Mother Tree, Gainesville . . ....... 58

12. Percent Fruit Set among Self- and Cross-Pollinated Flowers,

by Pollen and Mother Tree, Norris . . . . . 60














LIST OF FIGURES

FIGURE PAGE

1. Location of Gainesville, Florida, test trees . . . 22

2. Location of Norris, Tennessee, test trees .. . . ... 23

3. Black cherry chromosomes at metaphase, not well spread . 40

4. Well spread black cherry metaphase chromosomes . .. 40

5. Abnormal pollen tube, upper one-third style, Tree 1 normal

self, 7 days . . . . . . . . . . . 52

6. Abnormal pollen tube, middle one-third style, Tree 6 x

Tree 8, 5 days (lower tube) ... ............ 52

7. Embryo from selfed fruit, Norris . . . . . . . 62

8. Fence row that included study trees, Norris . . . . 74

9. Heavily fruiting tree, Norris .. . . . . . . .. 75

10. Typical tree, Norris ... ................. 75

11. Fruit set from a cross, Norris ... . ....... 76

12. Fruit set from a self, Norris . . .. . ..... 76

13. Fruit set from a self, Norris . . . . . . . 77

14. Typical bag, Gainesville .... . . . . . 78

15. Petal removal prior to pollination, Gainesville ....... o 79

16. Pollen tubes, upper one-third style, Tree 4 no pollen,

3 days . . . . . . . . . . . . 80

17. Pollen tubes, stigmatic surface, Tree 1 no pollen, 5 days . 80


viii










18. Pollen tubes, stigmatic surface, Tree 4 no pollen, 5 days

19. Pollen tubes, lower one-third style, Tree 6 normal self,


5 days .

20. Pollen tubes,

days .

21. Pollen tubes,

5 days .


lo.er one-third style, Tree 6 x Tree 2,


lower one-third style, Tree 6 x Tree 2,

lower one-third style, Tree 6 x Tree 4,

. . . . . . . . . . . ..


22. Pollen tubes, lower one-third style, Tree 6 x Tree 5,

5 days . . . . . . . . . . ...

23. Pollen tubes, lower one-third style, Tree 6 x Tree 5,

7 days . . . . . . . . . . .

24. Pollen tubes, lower one-third style, Tree 6 x Tree 7,

5 days . . . . . . . . . ..


25. Pollen tubes,

5 days .

26. Pollen tubes,

days .

27. Pollen tubes,

5 days .


lower one-third style, Tree 6 x Tree 7,



junction ovary and style, Tree 7 x Tree 6,



upper one-third style, Tree 7 x Tree 8,













INTRODUCTION


Black cherry (Prunus serotina, Ehrh.) is the largest native

American cherry species in height and diameter and the only one that

provides large amounts of material for woodworking. On better sites it

may reach over 100 feet in height and upwards of five feet in diameter.

From earliest colonial times cherry has been a favorite furni-

ture wood. Its dark reddish-brown heartwood finishes to a beautiful

and distinctive luster giving it a quality look. Famous English and

American furniture craftsmen such as Duncan Phyfe, Hepplewhite, and

Chippendale used cherry for many of their creations. Those which re-

main today still retain their beauty and utility.

Cherry has been used in many ways from agricultural implements

to wartime tank construction, but in recent years its use has become

more and more restricted to high-value products because of its popu-

larity and diminishing supply. As late as 1940 the two outstanding

uses were furniture and backing blocks for use in printing. Today

lumber and veneer for furniture manufacture lead the list.

It is not a slow-growing species but makes good growth only on

the best of sites. On the Allegheny Plateau where sites are best,

dominant and codominant trees average about 2.5 inches in diameter

growth per decade between the ages of 13 and 33 (Defler, 1937, cited by

Hough, 1960). Since these better sites are limited in number and the

demand for cherry has been high, the best stands have long since been

depleted in all but this small section of the natural range, To make








matters worse, regeneration has come from low-quality trees, leaving

less desirable crop trees for future use,

The present outlook is not bright. Demand for high-quality

material is rising despite competition from synthetics, while the sup-

ply is diminishing. Furniture manufacturers realize this and are pro-

moting regeneration with better planting stock. It is a late start,

but progress is being made.

If a sufficient supply of good-quality material is to be assured,

future crop trees must be descendants of superior-quality trees now

bearing seed. High grading has removed almost all of these except

those in a small area in the Allegheny Mountains of New York, Pennsyl-

vania, and West Virginia known as the commercial range. But a few iso-

lated individuals of high quality can still be located in relatively

inaccessible and protected areas in most of the range. Along with

better trees in the commercial range, these are the trees that tree

improvement workers are using as parents in the first phases of black

cherry improvement,

Any breeding program must be adjusted to fit the needs of the

species studies. It appears that special consideration must be given

self- and cross-incompatibility in the case of black cherry. Prelimi-

nary evidence indicates that in the Gainesville, Florida, area the

species is probably completely self-incompatible under natural condi-

tions, but also that under certain artificial conditions, it might be

selfed. Near relatives of black cherry, the sweet and sour cherries

and the plums (Prunus avium, Prunus cerasus, and Prunus domestic,

respectively) all have a similar incompatibility system--one that


1Removal of only higher quality trees during harvest operations,








prevents nearly all selfing and also some crossing in certain species

but that allows for selfing in others.

Since forest tree improvement programs usually include estab-

lishment of clonal seed orchards in which natural cross-pollination is

expected along with hand pollinations for production of progeny test

material, any incompatibility system in the species used must be un-

covered and cross-incompatible combinations pointed out. Also, such

a system must be known and overcome if any close inbreeding program is

to be successful.

The main purpose of this work is to determine if there is an in-

compatibility system in black cherry and if there is, how it compares

to the one in the other Prunus species. But since black cherry breed-

ing is in its early stages, new techniques will have to be adapted or

new ones developed to accomplish the primary goal. So as a secondary

goal several different studies aimed at adapting and developing tech-

niques have been incorporated. These include observations on flower

and flowering characteristics, natural and artificial pollination,

pollen collection, isolation techniques, and determination of presence

or absence of ploidy.













LITERATURE REVIEW


Early Work


Most people give Kolreuter (1964, cited by East and Park, 1917)

credit for the discovery of what he called self-sterility in plants.1

He found three Verbascum phoeniceum plants that failed to set seed with

their own viable pollen but seeded readily when pollinated by V. blat-

aria, V. n phlomoides, and V. lychnitis Darwin (1876) later

verified that V. phoeniceum was self-infertile along with V. nigrumbut

found two related species, V. thapsus and V. lychnitis, that were self-

fertile. Probably the next work on the subject was Herbert's (1837,

cited by East, 1929). He found self-infertility very common among

hybrids of the Amaryllidaceae and also that the non-hybrids Zephyranthes

carinata and Hippeastrum aulicum were self-infertile. A little later

Gartner (1844, cited by East and Park, 1917) found several additional

examples in other species including Dianthus aponicus, D, barbatus,

and Lobelia fulgens,

Several observers, including Mowbray (1830, cited by East, 1929)

and Munro (1868, cited by East, 1929) reported self-infertility in

Passiflora. Munro's work was especially important. He found self-

infertile plants that were sometimes cross-fertile and sometimes


1Hereafter I shall use the term incompatibility when pollen and
ovules were known to be functional and infertility when the reason for
non-fertilization was at the time unknown.

4







cross-infertile with others of the same species and even the same gen-

eration. Some plants of P. alata crossed easily with their own progeny.

In Oncidium, Scott (1865) showed that stylar tissue was pene-

trated freely by tubes from self pollen of self-infertile plants,

though fertilization did not occur. Muller (1868, cited by East, 1929)

corroborated Scott's findings and also showed each self-infertile

Oncidium flexuosum plant crossed with any other plant.


Attempts at Interpretation


Most work prior to the 20th century dealt with discovery of new

cases. Many were reported in addition to the ones already mentioned

including those by Bidwell, Bernet, Rawson (Darwin, 1876, cited by East,

1929); Lecoq (1862); Focke (1890, 1893, cited by East and Park, 1917);

and others. Work in the early 1900's uncovered many new examples of

incompatibility, including those reported by Backhouse (1911); Gardner

(1913); Raves (1921); Sutton (1918); Detjen (1916); Heribert-Nilsson

(1916, cited by East, 1929); Wada (1923, cited by East, 1929); Dahlgren

(1922, cited by East, 1929); but was more concerned with interpretation

of the phenomenon (East, 1929). Several noteworthy examples appeared.

Jost (1907) duplicated Hildebrand's (1866) earlier experiments

on Corydalis cava. Where Hildebrand had found absolute self-

infertility, Jost observed some self-fertility. Jost found that the

different behavior of these plants and of Secale cereal and Lilium

bulbiferum after self- and cross-pollination, was a difference in

pollen tube growth. In Secale, tubes penetrated the micropyle in eight

hours after cross-pollination but in 24 hours only reached the base of

the pistil after self-pollination.








Correns (1912) showed that pollen of self-incompatible Cardamine

pratensis germinated on stigmas of self-pollinated flowers but produced

only short tubes that did not penetrate the stigma. However, cross-

pollination produced pollen tubes that penetrated the upper ovary after

48 hours.

Correns (1912) and Compton (1912) suggested, as Jost (1907) had

previously, that diffusible substances which stimulated or retarded

pollen tube growth after cross- or self-pollination, respectively, were

present in the pistil. Compton felt that self-incompatibility might be

controlled by agents similar to those which govern immunity or suscep-

tibility in plants and animals.

Results of these and many earlier experiments were explained

when East and Mangelsdorf (1925) presented a new interpretation of self-

incompatibility in Nicotiana. They called it the oppositional factor

system. Filzer (1926) independently discovered the same system working

with Veronica syriaca. This work cleared up some puzzling aspects of

incompatibility and paved the way toward discovery of several additional

systems.


Systems of Incompatibility


There are six known systems of incompatibility. Two are found

in heteromorphic plants and four in homomorphic types.


Heteromorphic Plants

The first system (I) was first described by Darwin (1877, cited

by Dnyansagar, 1963) in the genus Primula and is known as distyly. Two

different flower types are borne on different plants. Thrum plants have

short styles and high anthers; pin plants have long styles and short







anthers. Compatible pollinations occur only between the two different

types. Genetic control is by one gene with two alleles, with sporo-

phytic determination of the pollen reaction. This type control has

been found in all distylic species so far analyzed (Dnyansagar, 1963)

including Primula sinensis (Bateson and Gregory, 1905), P. acaulis

(Gregory, 1915), P. officinalis (Dahlgren, 1916, cited by Lewis, 1949),

P. viscosa and P. hortensis (Ernst, 1936, cited by Lewis, 1949), P. ob-

conica (Lewis, 1949), Pulmonaria agustifolia (Darwin, 1876, cited by

Lewis, 1949), and Fagopyrum esculentum (Dahlgren, 1922; Garber and

Quisenberry, 1927).

The second system (II) is a more complex form of heterstyly char-

acterized by short-, medium-, and long-styled flowers. Each flower has

anthers at two different levels, each different from that of the stigma.

Darwin (1877) showed that compatible pollinations are between stigmas

and pollen from anthers at the same level. Fisher and Mather (1943) and

Fisher and Martin (1947) found that genetic control was by two loci,

each with two alleles which they called Ss and Mm. Long-styled plants

are ssmm; medium-styled, ssM ; and short-styled, S mm or S M (Lewis,

1949).


Homomorphic Plants

Under the homomorphic class are two subclasses, gametophytic and

sporophytic. Each includes two of the six incompatibility systems,

In the gametophytic subclass, mating type of pollen grains is

determined gametophytically and alleles of incompatibility genes act

individually in the style. Stylar pollen tube inhibition is the result

of incompatible matings (Crowe,1964).







In one system (III) genetic control is by multiple alleles at

one locus as in Oenothera (Crowe 1964). Prell (1921) first proposed

this type and East and Mangelsdorf (1925) used it to describe incompat-

ibility in Nicotiana sandarae. It was also used by Crane and Lawrence

(1931) to describe incompatibility in sweet cherry (Prunus avium),

The other gametophytic system (IV) is controlled by multiple

alleles at two loci as in Secale (Crowe, 1964). Present in several

grasses (Lundquist, 1956, 1961a, 1961b, 1964a, 1964b; Hayman, 1956), it

is also found in at least two members of the Solonaceae, Solanum pin-

natisectum and Physalis ixocarpa (Pandy, 1957, 1960). Pandy (1962)

concluded that the two-locus system is a derivative of the one-locus

system and suggested that the second locus is a duplication arising

from a structural chromosome change.

Under the sporophytic subclass, mating type of pollen grains is

determined sporophytically and alleles of incompatibility genes express

either dominance or individual action in both male and female parts.

Site of the incompatibility reaction differs in the two systems included

(Crowe,1964). In the first system (V) genetic control is by a single

locus with multiple alleles. Pollen tubes are inhibited in the style

as in Parthenium. In the second (VI) genetic control is by one locus

with multiple alleles and incompatibility is expressed between gametes

after fertilization as in Theobroma. Type V is common in dicotyledons,

but Type VI has been found only in Theobroma and Callistemon among the

dicotyledons (Crowe,1964).


Effect of Polyploidy on Incompatibility


An increase in chromosome number of an organism produces a

drastic upset in both the genic balance and the physiological processes





9

they control. In self-incompatible species studied, the effect of dou-

bling the chromosome number on incompatibility ranged from none to a

change to self-compatibility (Lewis, 1949). Effects on these species

are summarized in Table 1.

Lewis (1949) stated that the key to variable behavior of tetra-

ploids is found in tetraploid Oenothera organensis. In its style the

four S alleles, even when different, all operate without interaction to

inhibit haploid pollen carrying the same alleles. In diploid pollen

genic balance is unchanged if both alleles are alike; the pollen behaves

like haploid pollen. However, complex interactions occur between unlike

alleles, and diploid grains may show two different kinds of genic inter-

action depending upon the pair of alleles involved (Lewis, 1949).

With certain pairs of alleles, neither functions efficiently be-

cause of competitive interaction. In these cases there is a much re-

duced incompatibility reaction, and pollen tubes are only partially

inhibited even in styles carrying both alleles. The result is either

weak self-incompatibility or complete self-compatibility depending upon

the strength of the original reaction (Lewis, 1949).

With other pairs of alleles there is no competitive interaction,

but one allele is dominant to the other. Such pollen functions like it

is homogenic for the dominant allele. Plants producing only this type

pollen are completely self-incompatible (Lewis, 1949).

Competition and dominance in the diploid pollen grain explain

most effects of polyploidy found in other species. Effects in species

that show no change can be explained by dominance or lack of inter-

action, either dominant or competitive. Species that show a change to

self-compatibility must exhibit competitive interaction (Lewis, 1949).












Table 1

Effects of Polyploidy on Incompatibility in Several
Autotetraploids Arising under Controlled
Conditions, by Species and Author
(from Lewis, 1949)


Origin of
Species tetraploid Effect Author

Antirrhinum molle Colchicine Self-compatible Straub, 1941
Campanula ersicifolia Spontaneous Self-compatible Gairdner, 1926
Petunia xillaris?)* Colchicine Self-compatible Stout & Chandler
1941
Pyrus communis Spontaneous Self-compatible Crane & Lewis
1942
P. malus Progeny of Self-compatible Johansson, 1945
triploids
Solanum (3 species) Colchicine Self-compatible Livermore &
Johnstone, 1939
Trifolium reopen Colchicine Self-compatible Atwood, 1944
Ananas sativus Colchicine No effect Kerns & Collins
1947
Brassica campestris Colchicine No effect Howard, 1942
B rapa Colchicine No effect Howard, 1942
Oenothera organesis Colchicine Slight increase Lewis, 1943
in self-
compatibility
O rmbipetala Colchicine No effect Hecht, 1944
Raphanus sativus Colchicine No effect Howard, 1942
Taraxacum kok-saghyz Colchicine No effect Bannan,. 1946


* The species used was probably the garden form, P. violacea.








Variations of Incompatibility


Unilateral Incompatibility

This occurs when pollen tubes of a self-compatible species fail

to penetrate the style of a self-incompatible species, but the reverse

cross is compatible. Such results have been recorded by Mather (1943)

with Petunia, McGuire and Rick (1954) with Lycopersicon, Harrison and

Darby (1955) with Antirrhinum and Pandy (1962) with Solanum. Lewis and

Crowe 0958) believe this interspecific incompatibility may be con-

trolled by S alleles and that incompatibility substances in the pollen

are superimposed upon an essential pollen growth substance. Further,

these growth substances in pollen from self-compatible species are not

protected by superimposed groups and are inactivated by "antibodies" in

the style. Martin (1964, 1967) and Grun and Aubertin (1966) have

pointed out that other genes account for unilateral incompatibility in

some crosses. And Grun and Aubertin (1966) feel that mechanisms other

than pollen tube inhibition may cause the phenomenon.


Somatoplastic Sterility

This is found mainly in interspecific crosses and results in

decreased endosperm growth and eventual seed death in most cases.

Cooper and Brink (1940) found that in the cross Nicotiana rustic x N,

glutinosa death was caused by endosperm starvation due to an overgrowth

of the nucellus stopping the food supply. The nucellus in this case

blocked the gap adjacent to the chalazal pocket through which nutrients

reach the endosperm.

Cooper and Brink (1940) stated that Bradbury's (1929) selfed

sour cherries (Prunus cerasus) exhibited one or more of the








post-fertilization features of somatoplastic sterility. They felt that

this might have been the result of inbreeding,


Biochemistry of Incompatibility


Linskens (1963) lists two types of self-incompatible plants from

an experimental point of view:

1. Those in which the incompatible reaction occurs on the

stigmatic surface.

2. Those in which the reaction occurs during growth of pollen

tubes through the style.

In Type I the incompatibility barrier is probably the cuticle of

the stigma (Christ, 1959, cited by Linskens, 1963). In all cases inves-

tigated, pollen from plants with a stigma cuticular layer also had a

cutin-breaking enzyme system (Linskens, 1963). Kroh (1956, cited by

Linskens, 1963) and Tatebe (1959, cited by Linskens, 1963) obtained

normal seed set in incompatible pollinations of this type by bypassing

the cuticular layer. Kroh also showed that pollen from this type plant

placed on a cross-compatible plant for a short time then transferred to

a self penetrated the self stigma. Linskens (1963) stated that this is

evidence for a cutinase enzyme system linked to the incompatibility

barrier and that the cutinase can be activated irreversibly.

In Type II incompatibility, pollen tube growth is inhibited

within the style. Schlosser (1961, cited by Linskens, 1963) found

abnormal pollen tube behavior in incompatible crosses when compared

with compatible crosses. In about 50 percent of tubes studied the

generative nucleus did not divide, and the vegetative nucleus dis-

appeared just after germination without fulfilling its function,

There was also a disturbance of the normal carbohydrate metabolism







evidenced by a large deposit of fibrils on thickened walls, branched

tube tips, and an increased number of callose plugs (Linskens and

Esser, 1957; Schlosser, 1961; Tupy, 1961; all cited by Linskens, 1963).

Inhibited tubes also have a high respiration rate the first hours of

growth (Linskens, 1955, cited by Linskens, 196,3). And the fluorescence

patterns of style extracts taken after incompatibility reactions show

characteristic patterns after chromatographic separation; unidentified

substances that inhibit tube growth appear (Linskens, 1963).

Linskens (1963) believes pollen tube inhibition may be caused by

an antigen-antibody type reaction between a specific pollen protein and

a stylar protein with homologous specificity. Direct evidence to

support this conclusion comes from Lewis (1952) who found antigens that

were specific to different pollen genotypes in Oenothera and by Linskens

(1960) who found the same thing with protein in Petunia styles. Also

Linskens (1958, 1959, cited by Linskens, 1963) showed that a complex is

formed between pollen and stylar protein after an incompatibility

reaction. Selection of specific antibodies occurs during pollen devel-

opment as a result of intensive metabolic exchange between sporogen and

female tissue in the same flower. After pollination, antigen of the

pollen tube meets already selected antibodies in female tissue (Linskens,

1962, cited by Linskens, 1963).

Lewis (1960) showed that the S gene responsible for production

of the protein causing incompatibility has two cistrons. One controls

specific grouping of protein and the other controls the half-molecule

carrier responsible for protein activity in pollen and style.

According to Lewis mutations and recombinations are all changes in the

carrier cistron.







Linskens (1963) believes that S protein action occurs on the

pollen tube surface. Lewis' (1960) data support this hypothesis. He

found that S protein diffused out of both macerated and intact pollen.

This means that S protein either acts on the tube surface or outside in

stylar tissue. But since incompatible and compatible tubes in the same

style at the same time do not influence one another, it is likely that

S protein acts on the tube surface (Linskens, 1963).


Incompatibility in Plums and Cherries


M. B. Crane (1925) first reported self- and cross-incompatibility

in sweet cherry (Prunus avium). He showed that varieties involved could

be assigned to groups within which all but a very small percent of self-

and cross-pollinations failed. In one test over 18,000 selfed flowers

produced only 21 fruits. Other workers including Wellington (1926),

Tufts, Hendrickson, and Philp (1926), Sachoff (1931, cited by Crane

and Brown, 1937), Schanderl (1932, cited by Crane and Brown, 1937),

Gardner (1913), Schuster (1922), Florin (1924, cited by Crane and Brown,

1937), Einset (1932), Kobel and Steinegger (1933, cited by Crane and

Brown, 1937), Wenholz (1936), Sprenger (1908, cited by Crane, 1925),

and Hooper (1924) also showed that sweet cherry selfing is very rare.

Later Crane and Lawrence (1931) showed that incompatibility in

sweet cherry was comparatively simple with self-incompatibility the

rule and cross-incompatibility common, always reciprocally expressed.

But in the plum (Prunus domestic) incompatibility occurring in one way

of a cross and compatibility in the other was common. Darlington

(1930) pointed out that the degrees of fertility and complex results in

work with Prunus domestic were undoubtedly due to its hexaploid makeup







and manner of chromosome pairing. Bradbury (1929) found sour cherry

varieties both self- and cross-compatible, but selling produced a lover

percent set than did crossing.

Cytological studies by Evert (1922, cited by Crane and Brown,

1937) established the basic chromosome number in Prunus as 8 and that

all varieties of the sweet cherry were diploids (2x = 16). Kobel's

(1927, cited by Crane and Brown, 1937) work agreed with this. The sour

cherries, P. cerasus and Dukes, P. cerasus x P. avium are tetraploid

(Kobel, 1927; Darlington, 1928) as is P. cantabrigiensis (Faberge,

unpublished, cited by Crane and Brown, 1937). Black cherry (P.

serotina) is listed as tetraploid by Darlington and Ammal (1945).

Crane and Lawrence (1931) concluded that sweet cherry incompati-

bility was the oppositional factor type discovered in Nicotiana by East

and Mangelsdorf (1925) and discussed earlier in this paper under

incompatibility system III.

Roy (1938) compared (1) normal self-pollination, (2) self-

pollination in which styles were treated with growth promoting

phenylacetic acid before pollination, and (3) cross-pollination with a

compatible variety in sweet cherry. No significant differences were

observed between (1) and (2). Tubes which penetrated the style became

arrested in stylar tissue, and many appeared to swell up at their ends.

Tubes in (3) grew much more rapidly than in (1) and (2).

Earlier Afify (1933) studied pollen tube growth in cherries and

plums and distinguished between different pollen genotypes by behavior

of pollen upon germination and growth of tubes in the style. In sweet

cherries he found five types of pollen, those that: (1) failed to







germinate, (2) produced very short tubes which finally bent upwards and

ceased growth, (3) produced tubes that traveled about one-fourth the

stylar length, (4) produced tubes that traveled about one-half the

stylar length, and (5) traveled the length of the style and effected

fertilization.

So in Prunus there is a system of incompatibility which is

seemingly the same as that in several other genera. It is variable

according to chromosome number but is characterized by viable pollen

which germinates and produces tubes that penetrate the stigma, and by

slowed growth of tubes carrying the same alleles as those found in

stylar tissue,


Immature Fruit Drop in Cultivated Cherries


All cultivated varieties of cherry characteristically exhibit

three waves of fruit drop. The first occurs shortly after peak

flowering and is followed shortly by a second. The third drop is often

called the June drop since it usually occurs in that month.

Considerable work has been done to uncover the reason or reasons

for these drops including that of Detjen (1926) and Tukey (1933), but

Bradbury (1929) gives the best discussion of the problem. In her work

with sour cherry she made the following observations:

1o The first drop cannot be attributed to lack of pollination or

to failure of pollen tubes to reach the ovarian cavities. From 82 to 99

percent of first drop fruits had been pollinated and, in 76 to 98 per-

cent, pollen tubes had reached the ovarian cavities.






17

2. Degeneration probably begins in ovules of first drop fruits

before normal fertilization time. Both ovules were shriveled and

pollen tubes were growing at random in upper ovarian cavities,

30 Some unpollinated fruits were in the first drop, but the

large number of pollinated fruits in the first drop and unpollinated

fruits that were not in this drop indicate that pollination or lack of

it does not fully account for fruit development past the first drop.

4. A large part of the second drop is not due to lack of

fertilization. Embryos were present in 41 percent of dropped fruits

and 95 percent either contained embryos or pollen tubes in ovarian

cavities or ovules.

5. Partial embryo development has usually taken place in third

drop fruits of sour cherry.

Bradbury concluded that unfavorable nutritional conditions

probably play a major part in bringing about arrested development and

dropping of sour cherry fruits. Tukey (1933) agreed since he success-

fully cultured embryos from dropped fruits. Stage of development is

influenced not only by foods but also by pollination, fertilization,

and embryo development. Finally, Bradbury concluded that physio-

logical conditions in fruits leading to abscision layer formation

possibly are the same whether brought about by unfavorable nutritional

conditions or by lack of pollination or fertilization.


Techniques


Pollen Collection, Germination, and Storage

Schuster (1925) tried combing sweet cherry anthers off the

flower and was successful, but found brushing flowers over a screen an








even better method. Anthers snapped off and fell through the screen.

Schuster dried the collected anthers in open dishes until all pollen

was liberated and dry.

All work with cherry pollen germination testing has been with

sweet and sour horticultural varieties. Highest germination counts

were observed in various sugar solutions. Pfundt (1910) used 20-30

percent, Crane and Brown (1937) used 8-12, and Raptopoulas (1939),

10-25 percent sugar. These workers all got 50 percent or better

germination (Raptopoulas, 1939).

No reference was found that gave a method of pollen storage for

any species of Prunus, whether successful or not. Fresh pollen was

evidently used in all work.


Artificial Pollination

East and Mangelsdorf (1926) disagreed with the old idea that

stigmas are receptive only when they are secreting a substance assumed

to promote pollen germination. They pollinated as early as three days

before normal flower opening in Nicotiana without abnormal effect.

Crane (1925) did the same with Prunus avium. Schuster (1925) normally

pollinated cherry as soon as stigmas showed drops of liquid on their

surfaces but pointed out that pollination was successful 48 hours or

more before usual flower opening. Stigmas were either receptive before

normal opening or some pollen remained on the stigma and germinated

later when stigmas were receptive, He found that a flower past matu-

rity showed a slight reddening of stylar tissue just above the ovary.

East (1919) showed that selfing could be successful in Nicotiana

either by pollinating buds or by pollinating late in the flowering

season. He concluded that an inhibitor was formed during the life of







the flower, but its production coincided with flower opening, Bud

pollination avoided the inhibiting effect and allowed tubes to grow

normally before being slowed by the inhibitor. The longer period for

tube growth allowed fertilization to be accomplished. Smith (1926)

showed that end-of-season fertility in Nicotiana is due primarily to the

last few flowers remaining on the stem longer than usual,

The perfect flowers in species of Prunus make emasculation a

consideration to remove any doubt about penetration of self-pollen

tubes, Schuster (1925) emasculated as does Sharp (1964) with Prunus

avium, Sharp simply pinches off stamens between his thumb and

forefinger. This is done mainly to avoid mixing pollens since Prunus

avium is highly self-incompatible. Schuster (1925) used a camel's-hair

brush to apply pollen. Sharp (1964) uses a pencil eraser.

Schuster (1925) reported that emasculating and leaving flowers

exposed was preferred to bagging. He found that when bagged, flower

pistils often came in contact with the bag surface and were either

heat- or frost-killed depending upon temperature. He also felt that

insects would not be attracted to flowers without petals and did some

work without bagging. But Schuster (1925) also showed that bagging

added to flower longevity provided no contact with the bag itself

occurred. So bagging9 by prolonging flower life, could aid in ob-

taining fertilization from incompatible matings.


Time of Flower Removal

Roy (1938), in his histological study of pollen tube growth in

Prunus, removed flowers four days after pollination. Whether in selfs

or compatible crosses more than half of all pollen tubes penetrating








the stigma failed to grow more than 0.5 mm. A few did reach 5.0 mm

in selfs and 6.5 mm in compatible crosses.


Flower Killing and Fixation

Roy (1938) used Flemming's and Karpechenko's fluids in a mixture

of equal parts of 95 percent alcohol, glacial acetic acid, and lactic

acid. For work where fine detail and lifelike appearance are not

required, Jensen (1962) suggests the use of FAA--formaldehyde, alcohol,

acetic acid--as a killing and fixing agent. Its action is fast and

material can be stored in it some time before use.


Slide Preparation

Standard techniques for slide preparation are found in any one

of several manuals or texts. Jensen's (1962), though much smaller than

Johansen's (1940) standard text, adequately covers the process and

gives more up-to-date information on new equipment. Jensen's suggested

variation of replacing tertiary butyl with normal butyl alcohol in the

dehydration phase is appropriate when temperatures may drop below 60

F, the freezing point of tertiary butyl.

Fine points of the process including use of new equipment were

reviewed with C. Shell (personal communication, 1967) and L. Russell

(personal communication, 1967) of the UT-AEC Laboratory in Oak Ridge,

Tennessee. They gave the author instruction in the entire process from

dehydration through staining and coverslipping.














MATERIALS AND METHODS


Plant Materials


A genetically controlled incompatibility system is more likely

to show up in crosses among close relatives than among non-relatives.

So a parent-offspring grouping was sought as test material. One of the

most likely arrangements of this type is an old tree with young flower-

ing trees under or very near it. A careful search around Gainesville,

Florida, located such a situation in an area uncut for some time which

had what appeared to be one or more offspring-parent groupings present,

Two older trees--possibly siblings themselves--and six younger ones

were selected as study trees. Locations of trees with respect to each

other and Gainesville, Florida, are found in Figure 1.

Six companion trees were selected in the Norris, Tennessee, area

to partially duplicate the Gainesville study. These were all young

trees found in a single fence row. Locations of these trees with re-

spect to each other and Norris, Tennessee, are found in Figure 2,


Principal Studies


One way that incompatibility has been shown in Prunus is to

observe lengths and rates of growth of pollen tubes after different

types of pollination. Another method is to make pollinations, then

check results by observing fruit set. Both methods were used.



















'dlI
C,!
C, Of~


























4- -m o ,m











--- c u------ ------ (



E 0 E -
to t 0 o to 0 ) to 0

HI H -
/~^ ^ ~^ V '<> ~'~~ ~*~*^ ^' *- -^- ^s &
C~ ~ (.cJ+ fE):A-^ St








Pollen Tube Length Study--Gainesville

Two groups were made of the eight selected trees because of

differences in flowering times. A breakdown of treatments for each

Gainesville tree group is given in Table 2. On group A the entire

scheme was reproduced three times to allow for removing flowers at 3-,

5-, and 7-day intervals after pollination. On group B two reproduc-

tions were used for 5- and 7-day removal intervals.

Pollination of young buds might be effective in avoiding an

incompatibility reaction. Such was the theory of East (1934), who be-

lieved that an inhibitor was formed within 24 hours of flower opening

in Nicotiana. Presumably, if germination occurred, tubes from such

crosses would be well down the style before an inhibitor was formed and

could possibly effect fertilization.

The no-pollen treatment, besides giving data on natural selfing,

was also meant to serve as a yardstick by which other pollinations

could be evaluated. This was required since flowers were not

emasculated. Hopefully, self tubes could be differentiated from non-

selfs in crosses based on their performance in natural-self checks.

This was not considered too difficult since a cross would be made in

an individual flower some time before pollen release in the same

flower.

Some question arose as to whether results of the tube length

portion of this work could be analyzed statistically. Unless most of

the applied pollen which would eventually germinate did germinate within

a short interval, tube length analysis would be confused. Tubes from

applied pollen would be indistinguishable from those of self pollen

from the same flower. Other factors which might affect an analysis












Table 2

Pollination Treatments, Gainesville Studies,
by Female Parent


Group A Group B
Female parents Female parents
Pollen treatment 1 3 4 5 6 7 8

No pollen + + + -

Early self + + + -

Normal self + + + + + + +

Pollen 1 + + + + + +

Pollen 2 + + + + + + +

Pollen 3 + + -

Pollen 4 + + -+ + + +

Pollen 5 + + +

Pollen 6 + + +

Pollen 7 -+ + +

Pollen 8 + + +


- No treatment

+ Treatment

Treatments:
1. No pollen: flowers bagged before opening, no pollen applied.
2. Early self: self pollen applied on flowers that would not
normally open for at least 24 hours.
3. Normal self: self pollen applied on flowers that would open
within one to two hours.
4. Crosses: cross-pollen applied to flowers that would open
within one to two hours.








include stigma receptivity and loss of an exceptional number of

observations. But the necessary elements of statistical design were

included so that the material could be analyzed if conditions were

favorable. Both tree groups were factorial experiments within a

completely randomized design. Each treatment was replicated twice.

After the different day intervals had passed, flowers were

removed, killed, and fixed in formalin-acetic-alcohol. Later, serial

slides were prepared from longitudinally cut pistils, and data were

taken on tube characteristics.

Since computer analyses would likely be used, all data were

taken on IBM data sheets. Each flower from each treatment unit was

identified as to its parent, pollen number, removal interval, location

(bag) number, and flower number. Actual counts of tube sections were

made on the upper one-third of the style and/or just above the stig-

matic surface, number within the upper one-third of the style (Group I),

number in the middle one-third of the style (Group II), number in lower

one-third of the style (Group III), and number in the ovary. Addition-

ally, numbers in Groups II and III were expressed as percentages of

Group I to exert a standardizing influence on the data. So, a total of

six variables appeared on each flower card.

Tube sections--pieces of tubes--were counted rather than actual

tubes, because longitudinal microtoming of styles produced sectional

pieces of the meandering tubes. These pieces were considered propor-

tional to actual pollen tube members and were the only means of

relating tube characteristics in one flower with those in other

flowers.







Data from the six categories mentioned previously were

transformed. The first four were transformed by adding 0.5 to each

value, then taking its square root. The last two categories were ex-

pressed as percent values so were converted to are sin values before

analysis.

Each of the six variables for each tree was subjected to a

separate analysis of variance using the Least Squares Analysis program

of the Virginia Polytechnic Institute Computing Center. This program

provides for unequal classes and will give unbiased estimates of main

effects with or without interaction.

Effects showing significant differences were then subjected to

Duncan's multiple range tests to pinpoint individual differences.

However, results of the analyses will not be presented. Reasons are

given in the results section; they pertain to unequal opportunity for

tube penetration and lack of time for penetration.


Fruit Set Studies

The Gainesville fruit set study served as a check on the tube

length study in that every pollen treatment in the latter was included

in the former. The procedures for both tube length and fruit set

studies were nearly identical except that some additions were made in

the latter. For Tree Group A each pollen treatment was placed on

flowers at three different locations rather than two. Also, both

bagged and unbagged non-hand-pollinated flowers were included. The

depetaled unbagged flowers served as a check on attraction of insects.

The divergence of the Gainesville fruit set and tube length

studies came when flowers were removed from the tube length study.








Fruit set flowers were left on the trees and collected and counted as

fruit. Analysis was to be based on success or failure of pollen treat-

ments to set fruit.

The Norris fruit set study was intended to strengthen its Florida

counterpart and possibly uncover intraspecies differences due to lati-

tude or other factors. A breakdown of treatments is given in Table 3.


Techniques and Procedures

Pollen collection.--To avoid the possibility of collecting

insect-contaminated pollen in the Florida studies, cuttings were

brought inside, flowers already opened removed, then pollen collected

from those flowers which opened afterward. Anthers were dehisced and

pollen dried in a refrigerated desiccator.

Trials proved that the best collection method involved gently

rolling the cherry flower raceme between two pieces of ordinary window

screen. With a little practice the collector can retain an almost pure

batch of anthers. Actual equipment included several prepared screens

made by mounting a piece of screen between two pieces of plywood with a

circular hole in the center of each. They looked like this:



plywood

screen




6 inches



Pressure was applied by hand with a piece of screen shaped like a prism

as follows:



















Table 3

Pollination Treatments, Norris Fruit Set Study,
by Female Parent and Pollen



Pollen
No pollen
Female self 1 2 3 4 5 6

1 + + + + + +

2 + + + + + +

3 + + + + + +

4 + + + + + +

5 + + + + + +

6 + + + + + +


- No treatment

+ Treatment












1 21 inches


I inches I 4 inches



The collection sequence was:

1. Flowering branches collected.

2. Smaller twigs cut off and immediately placed in water.

3. Opened flowers removed and discarded.

4, Other flowers allowed to open.

5. Racemes with most flowers opened removed and anthers

collected by the screen-on-screen pressure method.

6. Anthers placed in cotton-stoppered bottles in a refrigerated

desiccator.

Pollen was not tested prior to use, because the period of the

delay plus time required for pollinations might well have been longer

than the flowering period of some or all of the trees. But a later

check proved it viable,

In Norris whole racemes were bagged and flowers allowed to open.

When needed, flower-bearing limbs were removed, still bagged, and

placed in water. Racemes were then removed and used as applicators.

Pollination techniques.--Since black cherry has perfect flowers,

emasculation was considered. However, two problems made this imprac-

tical. One is the small size and delicacy of the flowers; pinching or

cutting off the anther-bearing portion is possible, but often the style

is broken or cut. The second is that success leaves a weak, unprotected







style. Breakage of the style is the usual result either during the

emasculation process or afterward due to jostling in a bag. This rules

out emasculation as a practical device except possibly in greenhouse

work, Here flowers would not be subjected to the frequent and some-

times extremely rough treatment common in unprotected trees.

In the Gainesville studies petals were removed with tweezers

when they showed a lifting or swelling. This is about one to two hours

before normal opening. At this time anthers are tucked under and

around the stigma and style with the stigma usually easily accessible.

Then it is a simple matter to touch the stigma with a pollen-laden

applicator. In this case the applicator was a small dowel covered with

sterile waxed paper. The matter of finding enough flowers at the same

stage of opening offered no problem. Once the process was fully under

way on a raceme, at least 8-10 flowers could be found at the same stage,

The pollination sequence in Gainesville was:

1. Opened flowers removed and discarded,

2. A treatment tag selected at random and attached to the limbo

3. Pollinator's hands and tools cleaned with ethyl alcohol,

4, Petals removed from flowers that would normally open within

one to two hours (evidence for this was swollen petals) 1

5. Appropriate pollen applied to the stigma of each flower.

6. Flowers bagged.

In Norris, pollinations were made on flowers that were previ-

ously bagged and allowed to open. Pollen was applied directly by

brushing flowers with an opened raceme of flowers from the male parent,


A slight variation used here for one treatment is explained
under principal studies.






32

Pollen was kept available by placing cut stems of bagged flowers in a

rack of milk bottles filled with water. The pollination sequence was

much the same as in Gainesville except that upon bag removal prior to

pollen application, immature and overmature flowers were removed.

Tagging for identification.-Tags were designed as follows,



S(pollen number)

(tree number) (days left on tree
after pollination)

S(replication number)


Pollen Tube Length Study Tag




(pollen number)
(tree number)
S(replication number)


Fruit Set Study Tag


All were prepared before work on a given tree began. Treatments were

assigned at random by drawing a tag from a bag after a flowering limb

was selected. In the pollen tube length study, different day intervals

were color coded with flags to permit easy identification and removal,

Bagging for isolation.--Crimped synthetic sausage casings were

used for bags. One was placed over all flowers covered by a single

treatment tag, Then cotton was placed around the limb near the base of

and inside the bag and a tying wire, known as a "Twist-em," used to tie

the bag around the cotton. A breathing quality of the bag material

along with the cotton allowed some air into the bag but not insects.








Limbs which were not strong enough to support the bag--many were in

this category--were supported by tying limbs to other limbs or in some

cases by attaching a small dowel to the bag and limb, then attaching

the dowel top and bottom to a higher limb. Theoretically, this second

method would keep the bag from being pulled off. However, several were

lost anyway.

Flower killing and fixation.--All flowers to be sectioned and

mounted on slides were killed, fixed, and stored in formalin-acetic-

alcohol (FAA) of the following formula:

70% ethyl alcohol -- - - - 90%

Formalin -------------- 5%

Glacial acetic acid - - - - 5%

Slide preparation.--Material was processed as follows:

1. Pistils removed from flowers.

2. Groups of pistils run through alcohol series to paraffin.

3. Material placed under vacuum in warm paraffin.

4. Material embedded in paraffin.

5. Blocks cooled, then cut longitudinally at 20 microns on

microtome.

6. Serial sections mounted.

7. Material dried thoroughly then run through xylene for

paraffin removal then through alcohol series to water.

8. Material stained with lacmoid and martius yellow,

9. Material destined slightly in alcohol, then placed in

xylene.

10, Slides coverslipped.








Under number 2 above, the alcohol series consisted of the following

mixtures:

1. 70% alcohol -- 150 ml H20, 250 ml 95% alc., 100 ml butyl

alcohol

2. 85% alcohol -- 75 ml H20, 250 ml 95% ale., 175 ml butyl

alcohol

3. 95% alcohol -- 225 ml 95% ale., 275 ml butyl alcohol

4. 100% alcohol -- 125 ml 200% alc., 375 ml butyl alcohol

5. 50-50 paraffin and butyl alcohol

6. Paraffin Paraplast mixture

Under 7, 8, and 9 of the process the series was the following:

1. xylene -- 3 minutes

2. xylene -- 2 minutes

3. absolute alcohol -- 5 minutes

4. 95% ethyl alcohol -- 5 minutes

5. 70% ethyl alcohol -- 5 minutes

6. water -- 5 minutes or more

7. lacmoid and martius yellow -- 10 minutes

8. water -- 15 seconds

9. absolute alcohol -- 30 seconds

10. xylene -- 3 minutes or more

The stain was a mixture of:

5 mg. martius yellow

5 mg. lacmoid

12 mg. water

enough NH4 to rise pH to 8















RESULTS AND DISCUSSION


Preliminary Trials


Since very little breeding work, if any, had been done with

black cherry prior to this study, especially with respect to controlled

pollinations, new techniques often had to be worked out. Methods used

by breeders of cultivated Prunus species were considered and used when-

ever possible. But familiarity with Prunus serotina came only after

development and trials of new techniques. All trials except the

chromosome count were carried out in the Gainesville, Florida, area,


Date and Duration of Flowering

Observations showed that both of these are largely dependent

upon weather conditions, mainly temperature. In the Gainesville,

Florida, area black cherry can be expected to flower sometime in late

February or early March. Cherries in the Norris, Tennessee, area

flower about one month later. Observations over a three-year period

showed much variation among individual trees with some flowering

several days or even one or two weeks before others in the same area.

Normally a tree flowered over a two-week period. This interval was

shortened to as little as five days when a hot period followed a cool-

to-cold first part of February in Gainesville.








Flower Location on the Tree

Flowers occurred with near equal frequency from top to bottom

and on all sides of a tree regardless of aspect. Careful observations

of naturally set fruit showed heavy fruiting all over an open-grown

tree. This indicated that the experimental design need not include

replication in blocks to offset variations in flowers due to location

on the tree.


Characteristics of Flowers and Racemes

Prunus serotina has perfect flowers which are about 1/4 inch in

diameter when fully opened. The 1/8- to 1/4-inch pistil is surrounded

by 15-18 stamens in a perigynous arrangement. Imminent flower opening

is evidenced by swollen or lifted petals. At this stage about one to

two hours in full sun remains to full opening.

Normally 50-70 flowers are borne on a raceme, but occasionally

very long racemes containing 80-90 flowers are encountered. Flowers

always begin to open at the base of the raceme, but some buds may be

bypassed by more rapidly opening flowers above. An individual raceme

may flower over a 2- to 5-day period.


Natural Crossing

Black cherry is insect pollinated. Although the principal

pollinator is the honeybee in Gainesville, other insects, including

other types of bees and several flies, were seen on flowers.

Temperature seems to highly influence insect activity, espe-

cially that of bees, One Florida cherry flowering season was cool, and

bee activity was limited to a short period each day. The resulting

crop was estimated at less than half that of years when temperatures








during flowering were in the 80 F to 900 F range during much of the

day. A cool season, then, is good for controlled pollination because

it spreads the flowering period out, but bad for natural pollination

because of limited bee activity.

Stigmas are exposed to cross-pollination before anther dehis-

cence in single flowers. Often flowers are open for one to two hours

before anthers show any sign of liberating pollen.


Natural Selfing

Nothing is known of what happens when a honeybee transfers self

pollen within a black cherry tree. What is known, however, is that

self pollen can easily be transferred from flower to flower or from

anther to stigma on the same flower by contact alone. If such trans-

fers produced compatible crosses, then black cherry crops would not

vary directly with honeybee activity as is the case in the Gainesville,

Florida, area.

The almost complete lack of selfing in black cherry in Gaines-

ville was first uncovered during trials of pollination techniques.

Unopened flowers, bagged and left, consistently failed to set fruit.

Artificially selfed flowers produced the same result. But crosses,

especially mixed-pollen crosses, set some fruit in almost every case.

An exception to the natural selfing rule in black cherry occurred on

one occasion during a second season of preliminary trials in Gaines-

ville; three flowers on one tree did set fruit when isolated. This is

the only instance of natural selling within a bag that occurred in

Gainesville studies over three years in thousands of flowers. Though a

highly suspicious result, there was no reason to doubt that natural

selfing had occurred.








Pollen Germination Tests

Tests on agar, agar with 10 percent sucrose, and distilled water

showed that black cherry pollen germinated well on all three media.

Germination on each one was 50 percent or better after three days of

incubation. Tube growth was regular with most tubes of similar length.

After three days, tubes of average length were long enough to have

reached the ovary in normal pollinations. This regularity of tube

growth and fairly short period of extension set the pattern for later

testing.


Bagging

Bagging is not only a means of isolation but also a way to keep

developing and mature fruits available and labeled, even though they

may become detached for one reason or another. It avoids the need for

constant attention over the ripening period to be sure that successes

are not lost before being counted.

Individual flower life varied within bags, with temperature an

important factor. Under cool conditions an individual flower would

last as long as nine days when pollinated and bagged. At the six- or

seven-day point pistils began to darken and wither. Very hot air

temperatures reduced these figures to as low as five- and three-day

periods, respectively, and prolonged contact with the bag usually meant

death to individual flowers.

Since Schuster (1925) in his work with sweet cherry concluded

that petal removal would deter insects, the possibility that isolation

was not needed was considered. However, the probability that unbagged

fruit would be eaten by birds or otherwise lost seemed high and bags

were used.








Chromosome Count

Ploidy influences incompatibility in Prunus. Darlington and

Ammal (1945) listed Prunus serotina as a tetraploid, and several tetra-

ploids in Prunus are self-compatible. But trees in the Gainesville

area, with the exception mentioned earlier, were all self-incompatible

in early trials. For this reason chromosome counts were made. Seed

for this study came from the Norris, Tennessee, area.

Root tip cells undergoing mitosis exhibited very long and fat

chromosomes--much of the cell was filled with them. By focusing up and

down on the microscope, each chromosome and/or broken parts were traced

Many squash preparations were examined, but in only five cells were

chromosomes dispersed enough to make counting practical (Figures 3 and

4). Counts ranged from a low of 22 to a high of 25 chromosomes plus

segments. One end of these short pieces was in almost every case very

near what appeared to be the point of breakage of a much longer

chromosome. Since there was no way to be sure of a count and continued

squashing produced no better separation, these trees cannot categori-

cally be classified as diploids. However, the arrangement of the short

segments indicates that they are diploid. They are definitely not

tetraploid.


Pollen Tube Length Study


Pollen Germination

The main purpose of this study was to supply a basis for compar-

ison of pollen treatments with respect to tube penetration. Lack of

germination was not expected for any pollen treatment including self,

since early trials had shown black cherry pollen would germinate well
























Figure 3. Black cherry chromosomes
at metaphase, not well spread.


Figure 4. Well spread black cherry
metaphase chromosomes.






41

on plain water, However, flowers on the same raceme showed highly dif-

ferent amounts of germination with the same pollen. This occurred even

though techniques used were meant to give individual flowers an equal

chance to respond to various pollen treatments. Some factors which

might have influenced this result are:

1. Differences in receptivity among flowers treated the same,

2. Interference with liberation of self pollen because of direct

damage caused by petal removal or indirect damage resulting from

exposure of immature flower parts.

3. Interference due to a reaction between applied and self

pollen.

4. Failure to distribute pollen uniformly.

Although there is no way to be sure, a combination of Factor 1 and

Factor 2 seems the most likely reason for results obtained for the

following reasons:

1. Flower sections showed no evidence of physical damage to the

stigmatic surface; any damage must have been elsewhere.

2. Early trials using cross-pollens on unemasculated flowers

with petals removed showed that crosses consistently produced fruit,

3. Some early-pollinated flowers failed to extend anthers,

4, Pollen applicators were always kept covered with fresh

pollen and were checked regularly to be sure that pollen was being

left on the treated flower,

If some treated flowers on each raceme were a day or more away from

opening, applied pollen may not have germinated readily and self pollen

would have been liberated later. Self pollen would not have had an

opportunity to penetrate deeply if at all. Disregarding Factor 1, all








flower group means with the possible exception of early selfs should

have been at least equal to no-pollen-self means since no flowers were

emasculated. However, where the no-pollen treatment was included, it

was either first or second among treatments in number of tubes present,

There are only two possible explanations for this behavior (1) the

pollen application process interfered with release and/or growth of

self pollen, or (2) flowers that received the no-pollen treatment were

closer to the pollen-release stage as a group than were flowers in

other treatments. The second alternative is a weak explanation except

in the case of early self flowers which were selected on the basis of

immaturity. However, there is the direct evidence for the first alter-

native listed under reason 3 above. All early self flowers extended

anthers later than more mature flowers as expected, but some failed to

extend anthers after seven days. Any flower selected should have

opened within that period. The consistent last position of the early

self treatment is probably the result of both non-receptivity and

flower damage directly or indirectly (premature exposure) caused by

the pollination process,

Ranks of pollen treatments are shown in Table 4 and Table 5

Statistical comparisons were run but are not considered valid since

amount of pollen applied could not be controlled.

The fact that the no-pollen-self ranked high when used indicates

that not only was pollen transferred within individual flowers success-

fully, but that it germinated well.

Cross and normal self treatment results are confused because of

the likelihood that pollen from within the flower was also included in

counts. But the rankings did produce some patterns that give




















Table 4

Comparison of Pollen Treatments with Respect to Pollen
Germination, Tree Group A



Rank*
Tree 1 2 3 4 5 6

1 PO P2 Ph P3 NS ES

3 NS PO P2 P1 Ph ES

4 PO P2 P1 P3 NS ES


* Rank 1 had highest germination.

ES Early self

NS Normal self

PO No pollen


P1 Pollen 1

P2 Pollen 2

P3 Pollen 3

Ph Pollen 4


















Table 5

Comparison of Pollen Treatments with Respect to Pollen
Germination, Tree Group B



Rank*
Tree 1 2 3 4 5 6 7

5 P7 NS P2 PI P6 P8 Ph

6 P7 PI NS P2 P8 P5 Ph

7 P6 P8 P2 NS P1 P5 -

8 NS P7 P2 P6 P5 P1 Ph


- No treatment

* Rank 1 had most germination.

NS Normal self

P1 Pollen 1

P2 Pollen 2


Ph Pollen 4

P5 Pollen 5

P6 Pollen 6

P7 Pollen 7

P8 Pollen 8






45

indications of performance by some treatments. In tree group A, pollen

2 ranked second or third, and on group B it was third three times and

fourth once. Pollen 3 was only better than the applied selfs, and

pollen h was last in three of five cases. Pollen 7 always ranked high

and pollen 5 low.

Removal intervals were intended to give pollen tube growth rate

data, but the receptivity problem voided this possibility. Comparative

rankings are found in Table 6. The longer interval should have allowed

maximum pollen germination. It should also have included more germi-

nated pollen grains from flowers that became receptive later than

expected. But five out of seven times the 5-day interval produced more

germination. The 3-day interval was always last when included. Indi-

vidual flowers were again unpredictable. Quite often single flowers

from the 3-day interval showed more germination than others from longer

intervals. No definite reason can be given at this time for these

results. The following possibilities could well be responsible:

1. By chance, more flowers included in the 7-day interval were

pollinated too early and failed to promote germination as did most of

the early self.

2. A high percentage of flowers from the 5-day interval were

close to or had reached peak receptivity, with the result that more

pollen grains germinated.

3. Many flowers from the 3-day interval became receptive on

the second or third day and had no chance to reach a high level of

germination.




















Table 6

Rank* of Removal Intervals in Tree Groups A and B
vith Respect to Pollen Germination



Removal intervals (days)
Tree 3 5 7

1 3 1 2

3 3 1 2

h 3 1 2

5 1 2

6 2 1

7 1 2

8 2 1


- No treatment

* Rank 1 had most germination.








The consistent last position of the 3-day interval is easy to under-

stand and accept. But there seems to be no logical way to explain the

average second position of the 7-day interval.

On tree 3, pollen 2 from the 3-day interval ranked second com-

pared to seventeenth for pollen 2 from the 7-day interval, although the

3-day interval was the worst overall. On tree 6, pollen 8 from the 5-

day interval was superior to the same pollen from the 7-day interval.

And on tree 8, the normal self from the 5-day interval was superior to

the same treatment from the 7-day interval. Clearly, some factor

unaccounted for influenced the results.

Summary of results.--Among pollen treatments the no-pollen

treatment ranked first twice and second once in the three cases it was

used. Among removal intervals the 5-day period produced more germina-

tion than did the 3- or 7-day intervals, although the expected order

was 7, 5, 3 (highest to lowest). Other noteworthy results included:

1. Early selfing proved unsuccessful. It ranked last whenever

used.

2. Self pollen applied just prior to flower opening showed no

consistent pattern. It ranked first on two trees, next to last on two

others, and in varied positions on the other three trees.

3. Some cross pollens showed definite patterns. Pollens 2 and

7 always ranked high and pollens 4 and 5 always low.

4. Pollens varied in ability to germinate, but there was no

barrier to germination.


Pollen Tube Extension

In this phase the style was divided roughly into three equal

parts, and counts were made of tube sections in each part. Since no








control was possible over amount of pollen deposited on each stigma--

and, consequently, the number of tubes possible--an equalizing effect

was used; number of tubes in the middle and lower thirds of the style

was expressed as a percent of the number in the upper third. These

percentages were converted to arc sin values and analyzed statistically.

However, these findings are not considered a fair estimate of pollen

performance. Reasons are presented later.

Treatments for groups A and B are compared within and over all

trees with respect to pollen tube penetration into the middle third of

the style in Table 7. It is highly probable that self-pollen tubes

were included in counts of cross-pollen treatments. Therefore, no-

pollen treatment results could not be used to distinguish between self-

and cross-pollen tubes. Results did indicate that no-pollen-self tubes

failed to penetrate the lower third of the style. Because of these

findings, data for middle-third penetration by cross pollens are not

considered reliable, while the following lower-third data are thought

to be more accurate.

Treatments for tree groups A and B are compared within and over

all trees with respect to pollen tube penetration into the lower third

of the style in Table 8, Tube penetration into the lower third of the

style was much reduced over penetration into the middle third. Only

1.4 percent of the tubes reached this level. The most probable reason

for this lack of tube extension is insufficient time for tube travel.

Seven days was the maximum time allowed before flower removal and

fixation. This interval was selected for three reasons: (1) tubes

extended fully within three days in incubated pollen germination tests,

(2) pollen tubes had extended into the ovary by this time in Prunus














Table 7

Percent of Pollen Tubes in the Upper Third of the Style that
Reached the Middle Third, over All Trees and Removal
Intervals, by Pollen Treatment and Tree



Pollen treatment
Tree PO P1 P2 P3 P4 P5 P6 P7 P8 ES NS Av*

1 9.1 32.8 7.1 0 0 0 10.6

3 0 12.8 5.3 8.0 2.3 3.5 5.3

4 0 1.8 3.0 8.5 0 0 2.2

5 4.9 5.5 2.2 0 3.7 37.0 4.2 6.4

6 53.5 9.8 46.1 31.1 28.4 47.8 27.0 35.9

7 15.4 0 0 0 12.3 4.6 0 7.1

8 18.7 0 0 0 50.0 0.5 0 10.2


Av.* 3.6 33.1 7.5 8.3 29.4 30.1 12.0 21.3 36.0 1.3 20.1 20.7


- No treatment

* Weighted average

ES Early self

NS Normal self

PO No pollen


Pollen

Pollen

Pollen

Pollen


P5 Pollen 5

P6 Pollen 6

P7 Pollen 7

P8 Pollen 8















Table 8

Percent of Pollen Tubes in the Upper Third of the Style that
Reached the Lower Third, over All Trees and Removal
Intervals, by Pollen Treatment and Tree



Pollen treatment
Tree PO P1 P2 P3 P4 P5 P6 P7 P8 ES NS Av,*

1 0 5.2 0 0 0 0 0.5

3 0 0 1.1 0 0 0 0.3

4 0 0 0 0-0 0 0

5 3.3 0 0 0 0 0 0 0.9

6 2.7 0.4 1.1 3.7 1.8 0.6 1.2 1.8

7 0 0 0 0 9.1 0 0 3.5

8 .7 0 0 0 50.0 0 0 3.6


Avo* 0 2.6 0.6 0 0.6 3.6 9.6 1.3 0.4 0 0,8 1.4


- No treatment

* Weighted average

ES Early self

NS Normal self

PO No pollen


Pollen

Pollen

Pollen

Pollen


Pollen 5

Pollen 6

Pollen 7

Pollen 8








avium studies (Roy, 1938), and (3) flowers begin to deteriorate quickly

after seven days. However, tube travel continued past this stage and

probably was not completed until the style was well into decay.

Support for this view comes from the fruit set phase of this work.

Flowers in that phase treated exactly as those in the tube length phase

produced some fruit from nearly every cross. So tube penetration must

have continued in those flowers after flower removal in the tube length

study. Because of the lack of time for tube extension, no statistical

analysis is presented here.

The first visual evidence of possible incompatibility in black

cherry was found in this phase. One pollen tube from the normal self

on tree 1 produced a swollen tip or head (Figure 5). In studies of

Prunus avium (Roy, 1938), this condition was considered good evidence

for incompatibility. Another tube from the cross of tree 8 with tree 6

turned around and began to grow back up the style (Figure 6). This is

also evidence of incompatibility in P. avium (Roy, 1938). No other

examples of this type were found.

The consistently good overall performance of tree 6 as both male

and female is worth discussion. Some tubes from every pollen used on

this tree reached the lower style, even the normal self. No self on

any other tree reached this depth. Good performance by all pollens on

it indicates that tree 6 was highly receptive. It might even have a

genetic advantage in this respect over the other trees. But the most

important feature here is the penetration of self pollen. On P.

avium, Roy (1938) found that self-pollen tubes usually became arrested

in the style, and most of them appeared to swell up at their ends.

Afify (1933) found that self tubes were short and often bent upward














f *vy C M.


?*'' / ^


Figure 5. Abnormal pollen tube,
upper one-third style, Tree 1
normal self, 7 days.


Figure 6. Abnormal pollen tube,
middle one-third style, Tree
6 x Tree 8, 5 days (lover
tube).


'~i~j~liNM.

% t







before ceasing growth. But Roy (1938) also very rarely obtained one

or two fruits after self pollination. On tree 6 with self pollen,

tubes did not have swollen ends nor were any turned upward. However,

both of these characteristics might have appeared had tubes been

allowed more time to grow. On the other hand, one or more might also

have penetrated the ovary. But fruit set study results indicate that

fertilization probably would not have occurred.

Flower removal intervals for groups A and B are compared within

trees and over all trees and pollen treatments with respect to pollen

tube penetration into the middle third of the style in Table 9.

In tree group A, the pattern of tube penetration with respect to

removal intervals was the same as that in pollen germination: the 3-

day interval was low; the 5-day, high; and the 7-day, intermediate.

But tubes penetrated the middle third on every tree in the 7-day

interval, whereas tree 4 had no penetration in the 5-day interval, and

trees 3 and 4 had none in the 3-day interval. The pattern here as in

germination seems to reflect the stage of receptivity in the trees,

although there is no explanation for the 7-day period being inter-

mediate. Tree group B showed an overall advantage for the 7-day

interval though it ranked first on only one tree. Over all trees in

both groups the 7-day interval produced the highest percentage of tubes

reaching the middle third of the style. This was caused by the very

large number of penetrations observed on tree 6 in the 7-day interval.

Intervals for groups A and B are compared within trees and over

all trees and pollen treatments with respect to pollen tube penetration

into the lower third of the style in Table 10. Only the 5-day interval

produced tubes at this depth in tree group A, and these were at very
















Table 9

Percent of Pollen Tubes in the Upper Third of the Style
that Reached the Middle Third, over All Trees and
Pollen Treatments, by Removal interval and Tree



Removal interval (days)
Tree 3 5 7 Av.*

1 3.6 14.0 5.4 10.4

3 0 10.8 6.1 5.1

h 0 0 3.5 2.1

5 6.3 5.0 6.1

6 30.6 38.2 34.5

7 7.1 6.5 6.9

8 15.4 4.1 9.8


Av., 0.4 19.9 24,8 20.7


No treatment

Weighted average















Table 10

Percent of Pollen Tubes in the Upper Third of the Style
that Reached the Lover Third, over All Trees and
Pollen Treatments, by Removal Interval and Tree



Removal interval (days)
Tree 3 5 7 Av.*

1 0 0.8 0 0.5

3 0 1.0 0 0.3

4 0 0 0 0

5 0.9 0 0.8

6 3.1 0.4 1.7

7 3.9 1.9 3.4

8 4.0 3.0 3.5


Av.* 0 2.4 0.5 1.4


No treatment

Weighted average






56

low percentages. In group B, the trend set in the middle third of the

style was reversed, and the 5-day interval ranked first. Percent pene-

tration was much lower, but some penetration occurred in every tree-

interval combination except tree 5, interval 7.

Summary of results.--The several noteworthy results included:

1. Pollen tubes from the no-pollen-self treatment, so highly

ranked in pollen germination, penetrated to the middle third of the

style only on tree 1, with no tubes reaching the lower third.

2. Early self tubes reached the middle third only on tree 3,

with no further penetration.

3. Normal-self tubes reached the middle third on trees 3, 5,

and 6, and the lower third on tree 6. Tree 6 pollen showed exceptional

ability to penetrate tree 6 stylar tissue.

4. Cross-pollen results were clouded by the strong possibility

that self pollen tubes were included in the counts. Based on no-pollen-

self results it is highly probable that they were included in tube

counts from the middle third of the style but unlikely in lower third

counts.

5. The pattern of tube penetration with respect to time of

flower removal after pollination showed less penetration in the

shortest interval as expected, but also less extension in the longest

than in the intermediate interval.

6. Two instances of abnormal pollen tube development, evidence

for an incompatibility system similar to that in Prunus avium, were

observed.








Fruit Set Studies


Prevention of Insect Pollination without Bagging--Gainesville

On each tree in group A, 60 flowers were depetaled. Half were

bagged; half were not. No fruit set occurred among bagged flowers, but

one unbagged flower did set on tree 3. If this low set could be dupli-

cated over a large number of flowers, the results might be acceptable

for most breeding work. However, the one fruit that did set causes

some doubt as to the possible success of the procedure.


Selfing and Crossing--Gainesville

Both early and normal selfs were included on tree group A. Only

normal selfs were included on group B. Not a single flower of any self

set fruit. This paralleled findings in preliminary trials. After ob-

serving results of the pollen tube length phase of this work, lack of

fruit set in the early self is understandable. Very little germination

occurred, and in all but one instance tubes traveled no farther than

mid-style. Since normal selfs produced many tubes with some penetrat-

ing into the lower style, the only reason for lack of fruit set is the

incompatibility mechanism.

The overall fruit set among cross-pollinated flowers was 5.9

percent (Table 11). Pollens varied from 1.7 to 12.0 percent. Mother

(female) trees varied from 1.7 to 14.2 percent. Some results followed

trends set in the tube length study, but others did not. Tree 6 was a

good pollen parent on each tree where it was used and a good mother

tree for all pollens except 5 and 7, which failed on all trees. No

tree crossed successfully with every other tree. Tree 1 crossed with

every tree except tree 8. Tree 8 crossed only with tree 6.
















Table 11

Percent Fruit Set among Cross-Pollinated Flowers,
by Pollen and Mother Tree, Gainesville


Mother Pollen parent (tree)
tree 1 2 3 4 5 6 7 8 Av.*

1 6.7 3.3 13.3 7.8

3 0 0 10.0 3.3

4 6.7 0 0 2.2

5 10.0 0 5.0 10.0 0 0 4.0

6 4o0. 10.0 25.0 0 0 10.0 14.2

7 10.0 15.0 0 0 20.0 0 7.0

8 0 0 0 0 10.0 0 1.7


Av.* 10.0 4.4 1.7 9.3 0 12.0 0 3.3 5.9


- No treatment

* Weighted average









With these data alone, only compatible crosses can be distin-

guished with accuracy. But there are some indications that tree 8

might be incompatible, or partially so, with one or more trees. It

crossed successfully with tree 6 both as a male and female but with no

other tree. However, it is not possible under the diploid system of

incompatibility found in other species of Prunus for tree 8 to be com-

pletely cross-incompatible with all but one of the study trees. The

reasoning here is that if tree 8 is completely incompatible with all of

the others, then all must have the same alleles for incompatibility.

If this were true, the others would also be completely incompatible

among themselves. The logical deduction is that poor pollen germina-

tion or some other influence prevented fertilization or fruit set.


Selfing and Crossing--Norris

Percent set by male and female parent for selfs and crosses is

found in Table 12, Of the 17,921 flowers allowed to self, 3,859 pro-

duced fruit for a 21.5 percent set. Every study tree produced selfed

fruit, Individual trees ranged from a low of 9.9 to a high of 42.3

percent. Out of 1,181 cross-pollinated flowers, 516 or 43.7 percent

set fruit, Male-female combinations varied from 0 to 80.0 percent set.

Tree 1 produced a lower set from crosses than from selfs, the only tree

to do so. It was pollinated just as the other trees began to flower.

Racemes from other trees used to cross pollinate may not have liberated

much pollen with the result that fewer flowers were pollinated.

However, this does not explain why these flowers did not self.

Percentages in Table 12 are based on counts that were probably

made just before what is called the third or June drop in cultivated
















Table 12

Percent Fruit Set among Self- and Cross-pollinated Flowers,
by Pollen and Mother Tree, Norris



Mother Pollen (tree) Av. for
tree Self 1 2 3 4 5 6 Crosses*

1 9.8 17.4 0 8.0 2.5 0 5.0

2 11.5 40.0 13.5 20.5 36.7 65.6 34.3

3 42.3 63.2 63.4 22.5 80.0 63.6 58.8

4 28.5 48.4 55.4 54.3 70.0 55.9 57.0

5 29.8 37.9 56.4 47.6 67.7 36.0 50.0

6 16.8 74.4 14.7 30.2 38.5 32.3 38.7


Av.* 21.5 54.1 45.6 33.9 32.0 46.8 46.9 43.7


- No treatment

* Weighted average





61

cherry varieties. At that time--two weeks after peak flowering--fruit

was well developed. Ten selfed fruits--two from trees 1, 3, 4, and 5;

one from trees 2 and 6--were removed, opened, and inspected. Three of

the ten--two from tree 4, one from tree 6--appeared normal. The others

were either empty or had shriveled seed coats; five of these showed

insect larvae damage. Normal appearing seeds were sectioned by hand

and observed under the microscope. A typical picture of a developing

embryo from this type seed is shown in Figure 7.

Of ten crosses examined, all were filled, but seven showed

insect damage. Developing embryos were similar to that in Figure 7.

Five weeks after peak flowering only ten selfed fruits remained

of the 3,859 that set, but 117 of the 516 crosses that set remained on

the tree. There is little doubt that the insect infestation caused a

large part of the fruit to drop, but the wide difference between self

and crosses is unexplained. These results are similar in one respect

to Bradbury's (1929) with sour cherry and Cooper's and Brink's (1940)

with Nicotiana hybrids. In both cases selfs and interspecific hybrids

showed restricted embryo development when compared to intraspecific

crosses. What is important is that black cherry did self and that

selfed fruit remained on the tree past the usual drop periods of other

species of Prunus.

With regard to selfing, the Norris study produced completely

opposite results from those in Gainesville. In Gainesville black

cherry exhibited an incompatibility system similar to the one in sweet

cherry. In Norris it was self-fertile, a characteristic also exhibited

by the sour cherry, Prunus cerasus, The latter is a tetraploid.

















































Figure 7. Embryo from selfed fruit, Norris.






63

Chromosome counts will be made on Norris area cherry again and

on those from Gainesville to accurately determine their ploidy, but

there could be another reason for successful selfing in Norris cherry.

It is possible that there are one or more self-compatible alleles in

the Norris population. If this is the case, then there are at least

two races of black cherry based on self-compatibility. The small

amount of evidence now available supports this alternative.

Presence of selfing coupled with lack of emasculation of cross-

pollinated flowers makes impossible the classification of crosses in

the Norris study. The higher percent retention of crossed fruits does

indicate that crossing was successful, however.














SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS


Summary and Conclusions


Black cherry is one of our most valuable timber species and is

now included in several tree improvement programs. Its breeding

habits, however, had not been studied closely prior to this work,

Consequently, this study is intended to supply black cherry breeders

with not only a description of incompatibility in the species but also

a working knowledge of breeding habits and techniques used in making

controlled pollinations,

Lack of literature on breeding black cherry and techniques asso-

ciated with the process made necessary preliminary trials. These were

conducted prior to the major work and covered flowering habits, natural

selfing and crossing, pollen germination tests, isolation of flowers,

and a chromosome count. Techniques established for other species were

adapted for black cherry when possible and new ones developed when

necessary.

The major work was subdivided into studies of pollen tube

length, or penetration into the style, and fruit set. Pollen tube

characteristics, including amount of germination and depth of penetra-

tion,were observed for selfs and crosses among trees selected because

of likelihood of close relationship. The objective was to obtain

microscopic evidence for self- and/or cross-incompatibility among study

trees based on behavior of pollen tubes in the style. Fruit setting








was used as a check on microscopic evidence and to uncover possible

regional differences between the Norris, Tennessee, and Gainesville,

Florida, areas. Pollinations that readily produced fruit were con-

sidered compatible.

Conclusions on the pollen germination phase of the tube length

study accompany the following noteworthy results:

1. Pollen moved from anthers to pistil of the same flower

without insect visitation and germinated well. Transfer occurred

either by direct contact or through air currents. Both are possible.

The former must have accounted for most of the transfer because of the

close proximity of anther and stigma and lack of air movement in bags.

2. There was considerable variation in results among flowers

receiving the same treatment. The best explanation for this is that

flowers exhibiting external indications of opening (swollen petals)

were not receptive to pollen of any type until shortly after the time

of normal flower opening.

3. Selfing of young buds was unsuccessful. This failure re-

sulted because of "2" above and also because petal removal had a

detrimental effect on the maturation sequence as evidenced by the fact

that anthers occasionally failed to extend.

4o Selfing just prior to flower opening varied from good to

poor among mother trees. This, too, was caused by "2" above.

5. Cross-matings also produced variable results; some males

were always good, some always poor, others intermediate in amount of

pollen germinated. Part of this variation can be attributed to "2"

above, but there was evidence that some pollens germinated better than

others in general. Since all pollens were treated alike, some must








have had a genetic advantage; they probably had less stringent

germination requirements.

6. There was no barrier to germination of any pollen. When a

flower was receptive, any pollen including its own would germinate on

the stigmatic surface.

Pollen tube extension results gave limited support to the case

for self-incompatibility and some cross-incompatibility but did not

exclude self-compatibility completely. Most interesting findings and

conclusions based on them include:

1. Pollen tubes from self pollen distributed within individual

flowers penetrated the middle third of the style on only one tree even

though much pollen germinated. In trees of this phase of the overall

study, self-pollen tubes got a late start and also grew more slowly

than did those from cross pollen. These are the reasons for lack of

natural selfing in the Gainesville trees. However, self tubes did

penetrate more deeply than do self tubes in other Prunus species.

2. Pollen tubes from self pollen applied on young buds also

reached the middle third of the style on one tree only. Selfing the

young bud offers no advantage in obtaining longer tubes that might

effect fertilization.

3. Pollen tubes from self pollen applied just prior to flower

opening reached the middle third on three trees and the lower third on

one. Self pollen, though not as fast growing as cross pollen, can

penetrate the lower style and possibly farther on self-incompatible

trees, Flowers were removed before these and tubes from crosses could

penetrate the ovarian cavity. Styles were beginning to decay after








seven days, but tubes were still growing. This is supported by fruit

set results.

4. Two instances of abnormal pollen tube development were

observed; one was a self pollination and the other a cross. This lack

of physical evidence of incompatibility plus the deeper penetration of

self tubes in Prunus serotina than in other Prunus species are evidence

that the incompatibility reaction is not as strong in Prunus serotina.

5. Natural self pollination between anthers and adjacent

stigmas of individual flowers reduced the effectiveness of this work.

In future pollen tube length studies, either emasculation must be

practiced or a radioactive marker used to label applied pollen.

Fruit set study results varied greatly between the Gainesville,

Florida, and Norris, Tennessee, test areas. The most important result

in this phase and probably in the entire study is that all trees in the

Norris phase selfed while no selling occurred in Florida. Further,

embryo development was normal in the three selfed fruits observed.

Since chromosome counts ruled out the Norris trees being tetraploid,

there might be one or more self-compatible alleles in the Norris

population. If so, the Norris and Gainesville population could be

different races. However, environmental effects must be studied be-

fore a firm conclusion is reached.

The overall fruit set percent among crosses in Gainesville was

5.9 compared to 43.7 in Norris. In Gainesville no tree crossed with

every other tree; in Norris all crosses were successful at least one

way. Selfing is a definite factor in these results, but since the

percent of successful crosses was much higher than that of selfs (43.7

compared to 21.5) in Norris, either technique was better in Norris or

Norris trees have a reproductive advantage over those in Gainesville.






68

One depetaled flower exposed to possible insect visitation set

a fruit, Although this is not enough evidence to eliminate depetaling

as a method of avoiding insect pollination, it causes some doubt as to

its value.


Recommendations for Breeding and Tube Length Studies


1. Determine whether the particular trees to be worked are

self-compatible or not. The easiest procedure is to bag several

racemes on each tree prior to flower opening and observe them for fruit

set. Self-fertile individuals should not be used as females in a cross-

breeding program at this time; no satisfactory and, at the same time,

practical emasculation method is available. Further, use of self-

fertile trees as males will introduce self-compatibility genes into a

self-incompatible breeding group.

2. If trees of a breeding group originated in several geographic

regions, expect a wide range of flowering times. Even local trees will

vary up to a week or ten days in initiation of flowering. Satisfactory

long-storage procedures will be available soon from Robert Farmer,1 but

until that time do not count on keeping pollen more than one month, It

can be successfully stored for that period in a refrigerated desiccator.

This should be long enough to cross early trees with late ones,

3. Leafing out is an excellent indicator of the approach of

flowering in black cherry; the two are closely correlated. Flowers

will not open until full leafout. Therefore, no close examination is

needed until a tree is in full leaf.


1Physiologist, TVA Forestry and Fisheries Laboratory, Norris,
Tennessee.








4. Temperature is closely correlated with both initiation and

period of flowering, In 850 F and over weather, expect only five to

seven working days per tree. Daytime temperatures under 650 F will

hold up flowering; between 650 and 850 F, the flowering period will

last at least two weeks on each tree.

5. Use fresh pollen when parents are close together and are

flowering at the same time. Small branches containing bagged flowers

can be kept in water and the pollen used up to three days. Pollen

collection and short storage will be required in other situations.

6. Select stout upright limbs of at least 3/16-inch diameter

for bagging. Often a larger limb can be trimmed so that several racemes

close to the base will fit into a bag. The synthetic sausage casings

used in this study are cheap and easy to use, but a better type is made

of a special paper. It is large, light weight, and has a plastic view-

ing window. Forestry Suppliers, Jackson, Mississippi, handles it.

7. Use a camel's hair brush to apply collected pollen and also

fresh pollen, if desired. The brush will hold more pollen than stiff

applicators and offers little chance for flower damage. Have plenty of

them available since each pollen treatment on each tree will require

one. Also place only small amounts of pollen to be applied in an appli-

cation bottle because of the transfer of pollen from unemasculated

flowers. Discard this pollen after use on a single tree or clone.

8. Pollinate only those flowers that have recently opened. On

a raceme there are usually two or three distinct classes of flowers.

Early during flowering only freshly opened flowers and buds are present.

Within two or three days there will be overmature, freshly opened, and

buds. Five or six days after initial flowering, only overmature and a






70

few freshly opened flowers remain. Pollination is best when the three

classes are present; more flowers are at peak receptivity. Strip buds

and overmature flowers from the raceme; 15 to 25 flowers should be left.

Overmature flowers have brownish-green sepals compared to the bright

green receptive flowers. Also, look for shriveled petals on overmature

flowers. Beginning discoloration--reddish brown spot--on the style is

a sure sign of overmaturity.

9. Remove synthetic sausage casing bags as soon as pollinated

flowers show signs of overmaturity. Slit the bag longitudinally and

carefully open it to avoid dislodging flowers. Some flowers will fall

at this time and afterward on all racemes, even though pollinated.

Paper bags can be left on the tree without heat buildup and will pro-

tect developing fruit from birds.

10. Fruit will turn a bright red, then dark red, then black.

Begin harvesting at the dark red stage.

11. In tube length studies labelled pollen or some form of emas-

culation must be used because of presence of self pollen. Complete re-

moval of calyx and corolla is unsatisfactory because it leaves an

unprotected style. For a small-scale project anthers can be clipped

off with small scissors. This is a very tedious and time-consuming

job, but it will work. Labelling applied pollen with a radioactive

tracer will allow rapid pollination procedures. The more detailed

exposure to film and subsequent processing to get tube tracings can be

done in the laboratory when convenient. Satisfactory procedures are

listed in Jensen's (1962) Botanical Histochemistry under autoradiog-

raphy.






71

12. Plan to remove flowers for killing and fixation after sev-

eral periods with the longest at least 10 days. Styles will begin to

dry up and wither six to seven days after opening and possibly sooner

in very hot weather, but tubes will not penetrate the ovary until seven

or more days after pollination. Flower removal after different inter-

vals will allow study of fresh tissue at all stages of tube penetration.


Recommendations for Future Work


Future work can be subdivided into two classes: work with

(1) self-compatible, and (2) self-incompatible types.

Since no seedlings known to be the result of self pollination

are available, there is no way to estimate the extent of inbreeding

depression likely to be encountered, This should be studied first

under class io If depression is moderate to low or negligible, produc-

tion of pure lines should be relatively easy for two reasons: (1) only

isolation is necessary to obtain selfs and (2) the interval from seed

to flowering tree is about five years, a short period for a forest tree.

Once lines are developed that exhibit desired characteristics, they

could be crossed to combine traits and hopefully obtain hybrid vigor.

A large crossing program dealing with self-compatibles would

require either a new and acceptable emasculation method or development

of male-sterile lines. Both of these approaches should be investigated.

For self-incompatible trees the most needed work is in the area

of cross pollination techniques. Such innovations as controlled insect

pollination, adaptation of normally insect carried pollen to a type

dispensed by air currents, and better means of isolation all should be

studied.






72

Work on pollen and seed storage is being done now, but accepta-

ble methods are not yet available. There is room for more of this

work.



































APPENDIX
















































Figure Fence row that included study trees, Norris.
Figure 8. Fence row that included study trees, Norris.










I


Figure 9. Heavily fruiting
tree, Norris.


Figure 10. Typical tree,
Norris.
























Figure 11. Fruit set from
a cross, Norris.


Figure 12. Fruit set from
a self, Norris.










6 v r -.F
W- F r
'7\


Figure 13. Fruit set from a self, Norris.





















































Figure 14. Typical bag, Gainesville.























































Figure 15. Petal removal prior to pollination, Gainesville.


































Figure 16. Pollen tubes, upper Figure 17. Pollen tubes, stig-
one-third style, Tree U no matic surface, Tree 1 no
pollen, 3 days. pollen, 5 days.


Figure 18. Pollen tubes, stig- Figure 19. Pollen tubes, lower
matic surface, Tree 4 no one-third style, Tree 6
pollen, 5 days. normal self, 5 days.






























Figure 20. Pollen tubes, lower
one-third style, Tree 6 x
Tree 2, 5 days.


d i
















Figure 21. Pollen tubes, lower
one-third style, Tree 6 x
Tree 4, 5 days.


Figure 22 Pollen tubes, lower Figure 23. Pollen tubes, lower
one-third style, Tree 6 x one-third style, Tree 6 x
Tree 5, 5 days. Tree 5, 7 days.
































Figure 24. Pollen tubes, lower Figure 25. Pollen tubes, lower
one-third style, Tree 6 x one-third style, Tree 6 x
Tree 7, 5 days. Tree 7, 5 days.


Figure 26. Pollen tubes, junction Figure 27. Pollen tubes, upper
ovary and style, Tree 7 x one-third style, Tree 7 x
Tree 6, 7 days. Tree 8, 5 days.



































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