The genetics, nature and occurrence of self-and cross-incompatibility in four annual species of Coreopsis L. by Jagan Nath Sharma A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in GENETICS Montana State University © Copyright by Jagan Nath Sharma (1971) Abstract: Four annual species of Coreopsis L. (Compositae: Heliantheae: Coreop-sidinae), C. bigelovii. (A. Gray) H. M. Hall, C. calliopsidea (DC.) A. Gray, C. califomica (Nutt.) Sharsmith, and C. tinctoria Nutt., were studied to determine the genetics of their self-incompatibility mechanisms. Diallel -cross, backcross, and F2 studies revealed that these species have a sporo-phytic, multiple allelic, monogenic system of self-incompatibility. C. tinctoria had 7 multiple alleles, while C. bigelovii and C. califomica had 5 multiple alleles each. The number of multiple alleles could not be assigned to C. calliopsidea. Cytological studies' revealed a strong correlation between the sporophytic system of self-incompatibility and the stigma as the site of pollen inhibition. Meiotic chromosome numbers for all four species were determined as n=12. Secondary associations between different bivalents were found in all four species studied; these point toward some form of polyploidy associated with the genus. Significant heterosis for horticultural traits was detected and a method of producing F1 hybrid cultivars in Coreopsis tinctoria, using incompatibility as a technique, has been suggested. THE GENETICS, NATURE AND OCCURRENCE OF SELF- AND CROSS-INCOMPATIBILITY IN FOUR ANNUAL SPECIES OF COREOPSIS L„ ^ /

by

Jagan Nath Sharma

A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree

of

DOCTOR OF PHILOSOPHY

in

GENETICS

APPROVED:

Major Department

Co-chairman, Examining^Committee

Co-chairman, Examining Qqmmittee

^ 7 ) ______Deqn, College of Grg^uate Studies

MONTANA STATE UNIVERSITY Bozeman, Montana

December,..1971 - iii -

ACKNOWLEDGMENTS

The author would like to express his deepest appreciation to Professor H. N.

■ Metcalf and Dr. S. R. Chapman for their help and guidance during the course of this investigation.

He would also like to express his appreciation to the staff of the Plant and,

Soil Science, Genetics Institote, for their help and encouragement, and to

Montana State University for the use of facilities and financial assistance during the major part of the studies .

A special thanks to my wife for her long patience and understanding dur­ ing the past 3 1/2 years while caring for two children all alone, often in difficult circumstances.

) -iv -

TABLE OF CONTENTS

Page

V IT A ...... ii

ACKNOWLEDGEMENTS...... !Tii

TABLE OF CONTENTS ...... iv

LIST OF TABLES...... vi

LIST OF FIG U R ES...... x

ABSTRACT...... ' ...... xiii

INTRODUCTION...... I

REVIEW OF LITERATURE ...... 3

Incompatibility ...... 3 The genus Coreopsis L ...... 17

RESEARCH PROCEDURES, METHODS, RESULTS AND DISCUSSIONS ...... 22

Plant materials (culture) ...... 22 Confirming species identification ...... 22 Morphological variations in four annual species of the genus Coreopsis L , ...... 24 Materials and Methods ...... 24 R e s u lts ...... 26 Discussion...... 43 Genetics and incompatibility mechanism operating in four annual species of Coreopsis L ...... '. . 45 Materials and Methods ...... 45 R e su lts ...... 48 D isc u ssio n ...... 75 Factors associated with "S" gene action ...... 87 Materials and methods ...... 87 -V-

Table of Contents

(Continued)

Page

Results ...... 88 D isc u ssio n ...... 96 Hybrid vigor studies in Coreopsis tinctoria Nutt, using self-incompatibility as a tool for producing Fj h y b rid s ...... :...... 98 Materials and methods ...... 98 R e su lts ...... * ...... 99 Production of Fi hybrid seed on a commercial scale . . . 102 Discussion 104

SUMMARY AND CONCLUSIONS ...... HO

LITERATURE CITED ...... 112 -VL-

LIST OF TABLES

Context Tables

Number Page

I Chromosome number reported for the genus Coreopsis L. 19

2 Coreopsis species and cultivars used in these investi­ gations with seed sources and notes on growth habits. . . 23

3 Variability in inflorescence morphology and fertil­ ity ratings of ligulate and disc flowers of four species of Coreopsis L ...... 28

4 Variation in quantitative traits: Summary of the height and spread in four species of Coreopsis L ...... 29

5 Inter-group crossability behavior of the plants com­ prising the three compatibility groups studied in Coreopsis tin c to ria ...... 50

6 Crossability behavior of the 4 plants of Coreopsis tinctoria falling into incompatibility Group I ...... 51

7 Crossability behavior of the 2 plants of Coreopsis tinctoria falling into compatibility Group II...... 52

8 Crossability behavior of the 3 plants of Coreopsis tinctoria falling into compatibility Group III ...... 53

9 Inter group crossability behavior of the plants com­ prising three intra self cross-incompatible but cross-compatible groups studied in Coreopsis californica as measured by seed set success ...... 55

10 Crossability behavior of the four plants of Coreopsis californica falling into incompatibility . Group I ...... 56 -v ii-

Llst of Tables

Context Tables (Continued)

Number Page

11 Crossability behavior of the three plants of Coreopsis califomica falling into incompati­ bility Group II...... 57

12 Crossability behavior of the three plants of Coreopsis califomica falling into incompati­ bility Group III...... o ...... 58

13 Intergroup crossability behavior of the plants com­ prising the three incompatibility groups studied in Coreopsis bigelovii as measured by seed set success ...... 60

14 Crossability behavior of the two plants of Coreopsis bigelovii falling into incompati­ bility Group I ...... 61

15 Crossability behavior of the two plants of Coreopsis bigelovii falling into incompati­ bility Group H...... 62

16 Crossability behavior of the three plants of Coreopsis bigelovii falling into incompati­ bility Group III...... 63

17 Intergroup crossability behavior of the plants comprising two, intra self and cross-incompatible but inter cross-compatible groups studied in Coreopsis calliopsidea as measured by seed success ...... 65

18 Crossability behavior of the six plants of Coreopsis callopsidea falling into incompatibility Group I. . . . 66 -v iii-

List of Tables

Context Tables (Continued)

Number Page

19 Crossability behavior of four plants of Co calliopsidea falling into incompatibility Group n ...... 67

Results of the backcross in C. tinctoria plants showing reciprocal differences in a diallel cross. Reciprocal cross I, plants 5 and 9oo*.e..ooeoooooo.o* 68

21 Results of the backcrosses in C. tinctoria using plants showing reciprocal differences in a diallel cross. Plants chosen here are Nos. I and 4 ...... 69

22 Results of the backcross in C. califomica using plants showing reciprocal differences in a diallel cross. Plants 9 and I. . . . „ . 70

23 Results of the backcross in C. bigelovii using plants showing reciprocal differences in a diallel c ro ss. Plants 5 and 6 ...... 72

24 Segregation in Fg generation of a cross between self-incompatible and. self- compatible mutant of C. tinctoria...... 73

25 Genotypes assigned to the 10 plants (G. tinctoria) chosen for diallel cross...... 77

26 Genotypes assigned to the 10 plants chosen for diallel cross in C . califom ica...... 78

27 Genotypes assigned to 10 plants chosen for diallel cross in C. bigelovii ...... 80 -ix -

List of Tables

Context Tables (Continued)

Number Page

' 28 Results of the study of site of inhibition of pollen germination in four self-incompati­ ble/compatible species of Coreopsis L...... 94

29 Comparison of plant height, plant spread, number of buds per plant, number of flowers per plant and flower diameter for four par­ ental lines of Coreopsis tinctoria with their resultant Fj hybrids, Bozeman, Montana, 1968 ...... 100 -X-

LIST OF FIGURES

Figure Page

la Forms of ray petals. C. tinctoria upper row from left to right flat, semi-cactus lower row from left to right cactus, flat, tubular ..... 27

lb Forms of ray petals, left to right C. californica semi-cactus, C. bigelovii flat, C. calliopsidea f l a t ...... ' 27

2a Plant height. C. tinctoria Tall plant class 60-90 cms...... 31

2b Plant height C_. tinctoria Nana plant class 37-75 cms...... * . . . 31

Plant height C „ tinctoria Nana compacta class 15—22 cm s...... 000.0.06.0 32

4a Branching habit C. californica left erect branches, right drooping branches ...... 34

4b Branching habit C. bigelovii left erect branches, right drooping branches ...... 34

5 Branching habit C_. calliopsidea left erect branches, right drooping b ran ch es...... • 35

6 Achene morphology. Left C. tinctoria upper row smallest, middle row medium sized, lowest row largest. Right C. californica one morphological form dorsal and ventral view ...... 38

7a Achene morphology C. bigelovii left, dorsal and ventral view of ray achenes. Right, dorsal and ventral view of disc achenes 39 -x i-

Llst of Figures

(Continued)

Figure Page

7b Achene morphology C. calliopsidea left, dorsal and ventral view of ray achenes. Right, dorsal and ventral view of disc achenes...... 39

8a Leaf forms C. tinctoria. Most common form is the first group from le ft...... 40

8b Leaf forms C. californiea. Most common form is the first one from l e f t ...... 40

9a Leaf forms C. bigelovii. Most common form is the first one from l e f t ...... 42

9b Leaf forms C. calliopsidea. Most common form is the first one from left ...... 42

10 Coreopsis . Individual flowers in a head. Left only a few flowers of outer whorl have opened. Right, almost all the flowers are open ...... 46

Ila Meiotic studies C. tinctoria A P.M.C. Meta­ phase I with 12 bivalents ...... 89

lib Meiotic studies C. tinctoria A P.M.C. Dtafcin- esis with 12 bivalents grouped in a group of I four, I three and 2 twos...... 89

12a Meiotic studies C. califomica A P.M .C. Diafcin- esis with 12 bivalents. ■ One group of four biv­ alents ...... 90

12b Meiotic studies C. bigelovii A P.M.C. First anaphase M = 12...... 90 -x ii-

List of Figures

(Continued)

Figure Page

13 Meiotic studies C. calliopsidea A P.M.C. Metaphase I with 12 bivalents. 5 groups of t w o s ...... 91

14a Pollen studies C. tinctoria compatible pollination. Pollen grains germinating after 48 hours...... 93

14b Pollen studies C. tinctoria Incompatible pollina- tion. Pollen grains (a few in number) not germinating 48 h rs ...... 93

15 Hybrid Vigor in C. tinctoria. Fi hybrids Petite Purple (female) x^Self-compatible mutant' (male) 105 ,> / 16 Hybrid Vigor in C„ tinctoria.CultivariPetite 106

17 Hybrid Vigor in C. tinctoria „ Gultivart un- named dwarf ...... 107

18 Hybrid Vigor in C. tinctoria. Fi hybrid. Un- named dwarf (female)x'Self-compatible mutant* 108

19 Hybrid Vigor in C. tinctoria.Qultivarsself-corn- patible mutant ...... 109 -X iii-

ABSTRACT

Four annual species of Coreopsis L. (Compositae: Heliantheae: Coreop- sidinae), C. bigelovii. (A. Gray) H. M. Hall, C. calliopsidea (DC.) A. Gray, C. califomica (Nutt.) Sharsmith, and C. HnctoriaNutt., were studied to determine the genetics of their self-incompatibility mechanisms. Diallel - cross, backeross, and Fg studies revealed that these species have a sporo- phytic, multiple allelic, monogenic system of self-incompatibility. C. ■ tinctoria had 7 multiple alleles, while C. bigelovii and C_. califomica had 5 multiple alleles each. The number of multiple alleles could not be assigned to C. calliopsidea.

Cytological studies' revealed a strong correlation between the sporophytic system of self-incompatibility and the stigma as the site of pollen inhibition. Meiotic chromosome numbers for all four species were determined as vh=12. Secondary associations between different bivalents were found in all four species studied; these point toward some form of polyploidy associated with the genus.

Significant heterosis for horticultural traits was detected and a method of producing F^ hybrid cultivars in Coreopsis tinctoria, using incompati­ bility as a technique, has been suggested. INTRODUCTION

Four North American annual species distributed among three sections of the genus Coreopsis were chosen for study. Coreopsis tinctoria is a widely cultivated ornamental annual. Coreopsis californica, Coreopsis bigelovii and Coreopsis calliopsidea are showy and attractive and may have horticultural potential. The usefulness of these four species of Cor­ eopsis to breeders of ornamental plants could be better evaluated if their genetic characteristics were well-documented.

Self-incompatibility has been used as a means of producing hybrids which have, in many different crops , a yield advantage over open pollinated or pure line varieties (Allard, 1960). In general this in­ volves the production of inbreds which are homozygous for "S" alleles.

Homozygous lines can be produced with the sporophytic system, but their maintenance is a problem. Maintenance is possible in Brassica crops where incompatibility is overcome by bud pollination (Odland & Noll, 1950).

It may be achieved in crops which can be multiplied vegetatively. It may also be achieved in crops like alsike clover where the level of incompat­ ibility varies with the temperature (Townsend, 1968).

Hybrid seed is produced by sowing together in isolation two inbreds with different "S" alleles. Odland & Noll (1950) have, suggested the use of a double cross in cabbage, yrtiile Thompson (1964) proposed that a triple »2- cross be used for maximum efficiency in seed production. Hermsen (1969) has worked out a program of using "S" alleles for the production of hybrid seeds in several species and varieties of cruciferous crops (Brassica,

Raphanus). According to him a large number of "S" alleles may hamper the setting up of a practical program..

Self-incompatibility as a tool for hybrid seed production is useful only in the crops with high multiplication coefficients (Van Der Meer et al, 1968). In crops with low multiplication coefficients (ration, of Kg seed produced per acre/Kg seed sown per acre), like bean, some other methods like .the use of male sterility may be used with better results.

The following types of information must be obtained before effective plant breeding programs can be initiated in Coreopsis.

(1) The breeding systems of various species must be investigated..

This involves genetical and cytological investigations to determine the nature and the occurrence of self and cross-incompatibility because self-incompat­ ibility is an important and widely employed system which promotes out crossing and tends to maintain heterozygosity.

(2) The nature and extent of variation within each species (within cultivars of species where possible) must be evaluated for floristic traits. REVIEW OF LITERATURE

INCOMPATIBILITY

Self-incompatibility has been treated extensively in review papers by

Bateman (1952), Lewis (1954), and by Arasu (1968),• that by Arasu being

perhaps most comprehensive.

Self-incompatibility may be defined as "the inability of a plant pro­ ducing functional gametes to set seed when self-pollinated" (Brewbaker,

1957). It may be differentiated from the term "self-sterility” which is used ‘ in a wider sense to include sterility due to chromosomal abnormalities, the production of non-functional gametes and post-fertilization failure

(Crane and Brown, 1937; Crane and Lawrence, 1952 and Williams, 1964).

Self-incompatibility is widespread amongst flowering plants.. Every year several genera and species are added to the list of plants known to ■

exhibit self-incompatibility. Oryza, until recently cited as an example of a self-fertilized crop plant, has been added to the list of selfincompatible species. Oryza perennis ssp. barthii has been found to be self-incompatible

(Chu, et al, 1969). It is not intended here to list all the genera and species reported to be self-incompatible. Suffice it to say it occurs in every major phylogenetic line. It occurs not only in hermaphroditic species but also

in monoecious species with unisexual flowers on the same plant (Godley,

1955; Pandey, I960).

Systems of self-incompatibility: Self-incompatibility systems have —4 “ been classified by various authors (Arasu, 1968; Bateman, 1953; Crowe,

1964; Fisher and Mather, 1943; Tammisola et al, 1970; Lewis, 1944, 1954,

1949a; Linskens, 1965; Lundquist, 1965; Sears, 1937). Self-incompatibility in flowering plants may be divided in two major classes.

(1) Self-incompatibility of homomorphic angiosperms.

(2) Self-incompatibility of heteromorphic angiosperms.

Homomorphic Angiosperms: Self-incompatibility operating in homo­ morphic plants has always been classified according to the expression of an "Sji' allele in the male gametophyte. This is commonly referred to as the gametophytic system of s elf - incompatibility. In this system of self­ incompatibility "S" allele expression in pollen is not influenced by both

"S" alleles of the sporophyte. In the sporophytic form of self-incompat­ ibility "S" allele expression in a pollen grain is influenced by both "S" alleles of the sporophyte. Thus, until recently two. major systems of self­ incompatibility (gametophytic and sporophystic) were reported in homo­ morphic flowering plants.

This system of classification does not take into account the relation­ ship of the two "S" alleles in the diploid stigma, style and ovarian tissue and one "S" allele in the haploid female gametophyte. Recently, Tammisola and Ryynanen (1970) presented a new system of classification that takes into account both the male gametophyte, the interverihg diploid tissue

. (sporophyte) and the female gametophyte. A modified form of classification adapted from Tammisola et al (1970) is:

(A) Gametophytic-gam etophytic: self-incompatibility

(B) Gametophytic-sporophytic self-incompatibility

(C) Sporophytic-sporophytic. self-incompatibility

Gametophytic - gametophytic system of self-incompatibility: As the name suggests, the "S" allele in pollen grains is controlled gametophytical-

Iy and is not influenced by the "S" alleles of the sporophyte; also, either the male gametophyte does not come in contact with the female sporophyte, or it is not influenced by the female sporophyte.

At least two types of gametophytic - gametophytic incompatibility systems have been proposed. First is the type in which the pollen tube enters uninhibited into the ovule and sterilizes the egg cell if the two gametes are incompatible (Bateman, 1954). The second type is similar to that pro­ posed by Glenk (1964) for Oenothera. Certain pollen tubes, depending upon their genotypes,.fail to.reach certain ovules because of the lack ofchemo- tropic attraction. This system of self-incompatibility apparently operates in N arcissus, Hemerocallis, Lilium, G asteria, Ribes and Annona (Bateman,

1954; Arasu, 1968). - ( i -

/Gametophytlc - sporophyttc system of self-incompatibility: The

. self-incompatibility allele. ("S" allele) reaction in the pollen is controlled gametophytically. In the female organ "S" alleles do act independently.

However, a portion of the male gametophyte (the pollen tube) has to come into intimate contact with the female sporophyte; hence the name gameto- phytic-sporophytic system of self-incompatibility.

Genetically, both these forms of self-incompatibility (gametophytic- gametophytic and gametophytic- sporophytic) can be explained by what was first described by Prell (1921) as the hypothesis of oppositional factors and later (1925) by East and Mangelsdorf as the gametophytic system of self­ incompatibility in interpreting their data on Nicotiana.

The basic concept of this hypothesis, which has stood the test of time, is that self-incompatibility is governed by an allelomorphic series of alleles (SiSn) such that pollen tubes, having a particular "S" allele are in­ hibited in styles that carry the same allele and are not inhibited in those that do not. Thus, the progeny derived by crossing two self-incompatible plants (S 1S2 and S 3S4) would consist of four types of individuals (SiSg 1-SiS^

Sg^S’^S^.) in approximately equal numbers. All off spring would be self- x ■ . incompatible. ■ All crosses among individuals bearing the same pair of alleles would be incompatible. Crosses among individuals that have alleles ' x; -7 -

not in common would be compatible.

Prell, Lehman,. Filzer, East and Mangelsdorf (cited by Arasu, 1968)

assuming that the "S" allele of the male gametophyte is not influenced by

the sporophyte, termed this form of s elf - incompatibility gametophytic.

(C) Sporophytic - sporophytic system of self-incompatibility: The

self-incompatibility allele ("S" allele) in the pollen is influenced by the .

"S" alleles of the sporophyte. This leads to an expression of dominance

in pollen.

In the stigma and Style (diploid, maternal tissue) however, "S" alleles

may act independently or show dominance, hi some cases dominance in the stigma and style may be variable. This form of self-incompatibility was separated from the gametophytic form, as a result of work- in Brassica

oleracea var capitata (Kakizaki, 1930), and Cardamine pratensis (Beatus,

1934). Gerstel (1950) and Hughes and Babcock (1950),. working with Par-

thenium species and Crepis foetida respectively, propose dominance re­

lationships in pollen grains. Crowe (1954), working with Cosmos bipinnatus,

suggested variable dominance relationships between "S" alleles in the stigma

and style. The sporophytic influence of the "S" allele on the ovule was

demonstrated by Cope (1962) in Theobroma cacao. Studies by Imrie (1969)

in Carthamus flavescens and Martin (1965) in Ipomea helped in understanding -8 -

this complex system of self-incompatibility.

Heteromorphic Angiosperms: A variation of this system of self-in­

compatibility is found in some heteromorphic species. Incompatibility

is controlled by a diallelic "S" locus, acting in conjunction with a locus

conditioning flower structure (Brewbaker, 1964; Fisher and Mather, 1943).

Number of alleles at one lociis: Whereas the number of alleles at

a locus is generally very large with the gametophytic system, the number

identified in those species having a sporophytic system is generally less than

10 (Gerstel, 1960; Lewis, 1954; Pandey, 1960; Ramanamurthy, 1963; Sampson,

1967).

Mayo (1966), on the basis of computer simulation studies, estimated a high rate of loss of self-incompatibility alleles from a population and

a consequent high mutation rate to maintain the system. This suggested.up

■ to 40 alleles at one locus in the gametophytic system of self-incompatibility.

Wright (1964) estimated 7 alleles would be maintained in a population of

size 100 by a mutation rate I x 10 - 5 and in a population size of 55 by a mutation rate of I x 10 - 4. This model has a closer approximation to the

situation pertaining to the 4 species of Coreopsis studied in this research.

The maintenance of self-incompatibility despite the reproductive advantage of self-compatibility has been discussed by Crowe, (1964) and -9 -

Mayo, (1966). It was concluded that high levels of heterozygote advantage,

or heterosis, are a feature of those species in which the self-incompatibil­

ity system is maintained.

Anomalies in incompatibility systems: The genetic system in the

Gramineae differs from the commonly occurring form of the gametophytic

system in being controlled by two multi-allelic series of independent loci which act in a complementary way (Lundquist, 1965). In Physalis ixocarpa

(Solanaceae) the system is under the control of two independently segregating

multi-allelic loci, though the pollen reaction is gametophytically determined

(Pandey, 1957). Pandey (1962!)} !reported a two locus gametophytic system

of self-incompatibility operational in Solanum pinnatisectum.

' A broad spectrum of patterns of inheritance is reported in the spor-

ophytic system. In Iberis amara, eight alleles were identified including

an allele for self-compatibility. Crosses between plants with strongly

differentiated "S" alleles gave a normal seed set; whereas, crosses between

plants with similar "S" alleles resulted in a reduced seed set. The allele

Sf (for self-fertility) was dominant to one self-incompatibility allele and

recessive to the rest (Bateman, 1954). Inheritance may be further com­ plicated by the action of modifying genes . Compatibility levels have been

changed, apparently by selection of modifying genes, in Cichorium intybus -10-

(Pecauty 1962, cited by Imrie and Knowles, 1971) and-Helianthus annuus

(Luciano et al, 1965).

Distribution of types of self-incompatibility in Angiosperms: In those families where self-incompatibility is known, only one system has been found in any particular family. For example, the gametophytic system

is characteristic of the Solanaceae, and only the sporophytic system has been found in the Cruciferae and Composltae (Lewis, 1954; Pandey,, 1960).

Pandey (1960) suggests that the gametophytic and sporophytic systems of self-incompatibility may be found in the same family, depending on the nature of cytokinesis.

Factors associated with "S” gene reaction; Pandey (I960,- 1970) has.

reviewed in detail the time and site of the "S" gene action, breeding systems, pollen cytology and relationships in incompatibility. Genetic and cytological

studies have revealed that there is a correlation between floral morphology,

genetical control of self-incompatibility system, pollen cytology and site

of pollen inhibition.

Lewis (1949 and 1954) and Pandey (1957) reported a relationship be­ tween floral morphology and the genetics of self-incompatibility . All hetero- morphic plants had a diallelic control (with one or two loci), hi homo­

morphic angiosperms, self-incompatibility was controlled by a multi-

allelic system; the former had a sporophytic system; whereas, the latter -11-

had either gametophytic or a sporophytic system.

Lewis (1956) reported another relationship (in homomorphic plants

only) between site of pollen inhibition and system of self-incompatibility.

•In species with a gametophytic system, inhibition usually occurs in the style

or ovary; in those with a sporophytic system it occurs in the stigma.

Brewbaker (1957) discovered that the number of nuclei in the pollen

grain at anthesis is related to the type of self-in compatibility system of

the parent plant: species with a gametophytic system have binucleate pollen

while those with a sporophytic system have trinucleate pollen. Thus, four

features of incompatibility, the genetic system of control, floral character­

istics, pollen cytology and site of inhibition, were found to have interlock­

ing relationships.

It appears to be generally agreed that the; time of "S" gene action is

the critical factor in incompatibility relationships; however, a number of

exceptions have been cited (Cope, 1958, in Theobroma cacao; Hecht, 1964,

in Oenothera sp.). It is suggested that major exceptions are byproducts

of secondary evolution of incompatibility after the primary incompatibility

has been disturbed, or has broken down.

Physiology of "S" gene action: Studies after 1960 have devoted more

attention to resolving the phenomenon of the mode of "S" gene'action. -12-

East (1926, 1929) was the first to suggest the similarity of the in­ compatibility reaction to the immunity reaction in animals. This concept has been supported by various authors (Lewis, 1952, in Oenothera organensis cited by Arasu, 1968; Linskens, 1960, in Petuniacited by Arasu, I960. Strong support came from Nasrallah and Wallace (1967a, 1967b, 1970). They dem­ onstrated the presence of antigenic substances (proteins) that were specific to each "S" genotype in the tissue of unpollinated pistils of Brassica sp.

Their work suggests that the inhibition is the result of an antigen antibody type of reaction. Using radioactive tracers, Linskens (1958, 1959, 1960, cited by Arasu, 1968) was able to show that the proteins of the pollen and style formed a complex in selfed pistils. The nature of these substances is not yet known.

Pandey (1967) worked on the electrophoresis of stylar tissues and suggested that each "S" allele is associated with a specific combination of peroxidase isozymes; these could cause the inhibition of pollen tubes by destroying the IAA present in them. Application of IAA in lanolin to lily styles and ovaries has resulted in improved seed set with reduced tendency for maternal inheritance. (Emsweller and Stuart, 1948).

Safonov et al (1969) reported that non-identity of protein systems in

Malus may be an important factor underlying physiological incompatibility. -13-

Different models have been proposed for "S" gene action. Lewis

(1965), in suggesting his dimer hypothesis of'gametophytic incompatibility,

stated "that (I) the "S" gene complex produces a polypeptide whose spec­

ificity, determined by the primary structure,is different Loru ,each allele.

Each allelic polypeptide is an identical molecule in pollen and style.

(2) The polypeptide polymerizes into a dimer in both pollen and style.

(3) the first step in the incompatible reaction is that the same dimers in

pollen and style and only the same, combine to form a tetramer with the

aid of an allosteric molecule which may be glucose, a protein of"one of many

small molecules. (4) The second step in the incompatible reaction is that

the tetramer acts on a genic regulator either to induce the synthesis of an

inhibitor or to repress the sythesis of an auxin of pollen tube growth".

Ascher (1966) suggested a similar model for "S" gene action: "The

. "S" alleles may be assumed to be regulators which produce monomers of

a dimer repressor of a high rate of growth operoh in the pollen tube.

"Similar monomers of pollen and style produce a functional dimer repressor

which inhibits this operon". Pandey (1967) suggested that the peroxidase isozymes, characteristics of each "S" genotype, were equivalent to Lewis's dimers. Ascher (1971) tried to correlate RNA synthesis in pollen and style with the self-incompatibility reaction; he suggested that RNA synthesis is required in the style for incompatibility reaction while it is the blockage I

—14- of RNA synthesis in pollen that probably causes the incompatibility reaction.

Breakdown of self-incompatibility system: Most of the studies con­ cerning the breakdown of self-incompatibility seem to have been done in flower­ ing plants having the gametophytic form. Review of the available literature reveals that four important mechanisms usually cause the breakdown. These are (a) polyploidy, (b) mutation, (c) temperature, and (d) mechanical methods.

Polyploidy: The effect of polyploidy on incompatibility has been studied by a number of workers (Lewis, 1943, 1947; Atwood and Brewbaker, 1950;

Brewbaker, 1954). It has been shown that diploid pollen, carrying two "S" alleles, can have one of three types of action:

(1) The two alleles function independently, so that an SjSg pollen grain would fail on all styles carrying either Sj or Sg.

(2) One of the alleles dominant over the other (e .,g .SjSg). In this case, SjSg pollen would function on all styles with SgSx genotype, but fail on styles carrying the S] gene.

(3) The two alleles are competitively interacting or mutually weak­ ening. .

Mutation: Arasu (1968) has summarized in detail the information available on mutation (induced and spontaneous) and its effect on incompatibility.

Recently, DeNettancourt■■11» UWvW1T-T V.IV. (1969) presented a brief review of the temporary and permanent effects which are usually observed after irradiation treatment -15-

of self- incompatible plants . Irradiation treatment has only negative effects

on the self-incompatibility systems (inactivation of the incompatible reaction and/or genetic losses at the S-locus). It is suggested that self-incompatible plants may be equipped with a switch system, or a mutagenic mechanism, which enables them to display a new specificity. This leads to an increase in the level of genetic polymorphism in the population to which the plant be­ longs .

Temperature; High temperature has been reported to result in break­ down of self-incompatibility, at least in the genus Lilium, (Ascher, 1970).

Townsend (1966, 1968, 1970), while studying the breakdown of self-incompati­ bility in Trifolium hybridum, found another "T" gene which seems to inactivate the "S" allele. It seems probable that this T locus is influenced by poly­ genes .

Mechanical Methods; hi Brassica oleracea incompatibility was over­ come by applying pollen to cut stigmas . Incompatibility has also been over­ come by bud pollination in different genera, e.g., Brassica (Thompson, 1957) and Guizotia (Naik and Panda, 1969). Higuchi (1968) reported induction of pseudo -fertility in Petunia by means of repeated pollinations. The under­ lying success of these methods has been attributed to the application of ripe pollen before the time of "S" gene action in the stigma (Lundquist, 1965). It - 16- is assumed that^in the cases where these methods succeed,the evolution of the incompatability system has resulted in the strongest barriers to fer­ tilization occurring at the same time that the stigma is most receptive. -17-

THE GENUS COREOPSIS L.

The genus coreopsis is a member of the plant family Compositae, in the tribe Heliantheae, sub-tribe Eoreopsidinae (Hoffman, 1894). The generic name is derived from the Greek Koris - a bug, and ops is - like; in allusion to the shape of the achene. The genus is known by the common name "tickseedV cultivated forms, notably in C. tinctoria Nutt., are called

"calliopsis".

Sherff (1936, 1955) considered Coreopsis to be comprised of 116 species in 11 sections. The genus is dispersed geographically in North America,

South America, and Africa. A few species have become established as adventives outside of their natural ranges.

■ Morphology: Bi terms of growth habit. Coreopsis includes a wide array of forms, including shrubs to three meters tall, other frutescent and suffrutescent forms, many perennial herbs of diverse form (including tuberculate-rooted and succulent ones), and a number of annual herbs, a few of which are specialized desert annuals (Sherff, 1936, 1955).

From various authorities (Sherff, 1936, 1955 and Munz and Keck,

1959), the majority of Coreopsis species can be characterized as having yellow ligulate florets; however, seven North American species have hi- or parti- colored ligulate florets. Among 55 species of the Americas, the ligulate florets in il are fertile and sterile in two. The fertility status of 47 is -IS1™ unknown. The fertility of but one African species is known. C. camporum has fertile ligulate florets (Sherff, 19361955). Fertility of the ligulate florets is an important taxonomic trait.

The disc florets are reported to be bisexual and usually fertile (Bailey,

1949).

Cytology; Cytologically, the genus Coreopsis is incompletely described.

Chromosome numbers have been reported for only 17 of 55 North American species. Reported species, chromosome numbers and authorities are given in Table I. The modal chromosome number appears to be x =. 12; however, there are conflicts in reported chromosome numbers for some species (for example, see Turner, Powell and King, 1962 or Crawford, 1970). hi addition, reported chromosome numbers include only six of ten North American sections.

> From an evolutionary point of view, cytological similarities among coreopsis and closely related genera are of interest. The sub-tribe Coreo- psidinae is polybasic (Melchert, 1968). The modal haploid number appears to be 12 and x ranges from 8 to 19. Table I

Chromosome Numbers Reported for the Genus Coreopsis L.

Section and species n 2n Authority

I. Electra (DC.) Blake

c. mutica DC. ca. 24, 26 Turner, Powell

c. mutica var. mutica ca. 112 Crawford, 1970 c. mutica var..Ieptomera Sherff 56 Crawford, 1970 c. mutica var. subvillosa DC. ca. 112 Crawford, 1970 c. mutica var. carnosifolia Crawford ca. 112 Crawford, 1970 c. mutica var. multiligulata Crawford 56 Crawford, 1970 c. mutica var. microcephala Crawford ca. 24, 26 56 Crawford, 1970

c. cuneifolia Greenman 28 Crawford, 1970'

2. Anathysana Blake

C_. cyclocarpa Blake 12 Powell & Turner, 1963

4. Pugiopappus (A. Gray) Blake

C. bigelovii (A. Gray) Voss 12 Sharma, hoc Ipc. C. calliopsidea (DC.) A. Gray 12 Sharma, hoc Ioc.

5. Euleptosyne (A. Gray) Blake

C. californica (Nutt.) Sharsmith 12 Sharma, hoc Ioc. Table I (continued)

Section and species n 2n Authority

6. Pseudo-Agarista A. Gray

C . petrophiloides Robins, et Greenm. • 52 Crawford, 1971

Coreopsis Sherff

C. lanceolate L. 24, 48 Bilquez, 1951 C. pubescens Ell. 28 Snoad, 1952 (cited by Darlington and Wylie) C . grand!flora Hogg ex Sweet 26: Gelin, 1937 C . auriculata L. 24 Bilquez, 1951 (cited by Darlington and Wylie) C . nuecensis Heller" 9 Turner, 1960 C. tripteris L. 26 Gelin, 1937

Calliopsis (Reichenb.) Nutt.

C . tinctoria Nutt. 12 Gelin, 1937; Turner, 1960 Sharma, hoc Ioc. C. basalis (Dietr.) Blake 12 26 Turner, 1960; Gelin, 1937 C. cafdaminefolia (DC.) T&C 12 26 Turner, 1960; Gelin, 1937

Eublepharis Nutt.

C. Iinifolia Nutt. 13 . Turner, 1960 - 21-

Horticultural aspects: Only a few of the 116 species of Coreopsis are commonly cultivated. Bailey (1949) listed 12 species which are cultivated in North America. Chittenden and Synge (1956) listed 23 species cultivated in Great Britain and Europe, where Coreopsis is more popular.

C. auriculata, C„ grandiflora, C. Ianceolata, and C. verticillata are encountered in perennial borders. The former two species are perhaps the most commonly encountered. Cultivated forms of these species are termed "coreopsis" by nurserymen.

Cultivated forms of annual species are classified as C. tinctoria; however, Everett (1960) implied that they descended from three species,

C. neucensis, C. basalis, and C. tinctoria. If this be the case, it rep­ resents an instance of interspecific hybridization among species with dif­ ferent base chromosome numbers, since for C. neucensis (x T 9), for C . basalis (x =. 13), and for C. tinctoria (x = 12). Cultivated annuals may be grouped into three classes by height: "Tall" plants vary from 60-90 cm,

"nana" plants range from 37-45 cm, and "nana compacta" types are 15-23 cm in height. The cultivated annuals are frequently misnamed "Calliopsis" in commercial seed catalogues. RESEARCH PROCEDURES, METHODS, RESULTS AND DISCUSSIONS

Plant Materials (Culture): Seeds of four species of Coreopsis were pro­ cured in 1967-68 from various sources for use in the present investigations.

Species and cultivar names, growth habit, and seed sources for these mater­ ials are presented in Table 2.

Seedlings were raised in vermiculite under mist in the greenhouse at

Bozeman, Montana.

Seedlings were transplanted into 2-inch Jiffy pots as soon as the first true leaves appeared. Jiffy pots were either transplanted directly into the field at Bozeman, or into 10-inch pots filled with a standard greenhouse soil mixture for greenhouse cultures. Where field culture was employed, the plants were spaced 18 inches apart, in rows 24 inches apart.

All species and cultivars were transplanted in the field and in the green­ house. Thus data reported have been collected both from field-grown and green house-grown plants during the period 1968-71.

Confirming species identification; Identity of the study material as to species was confirmed by comparison of random samples of 10 plants of each taxon (5 plants from field cultures, 5 from greenhouse culture) with keys and descriptions contained in the most recent monograph on the genus

Coreopsis (Sherff, 1955). Representative voucher specimens of each species have been deposited in the Montana State University Herbarium.. The Table 2. Coreopsis species and cultivars used in' these investigations with seed sources and motes on growth habits.. ' Growth Species Cultivar name if any habit Source class

C . tinctoria Nutt. 'Petite Purple'" nana compacta Geo. W. Park Seed Co. Greenwood, S.C. 29646 'Tiger Star" nana compacta 11 Tl' IT .

"Yellow Star" nana compacta " " " " ' -

"Tall tubular" tali' Indian Agricultural Research Institute, Kulu Valley, India "Compatible mutant" tall i to "Unnamed dwarf" nana Montana Agr. Exp.. Sta. ■■■ -' Bozeman, Mont. 59715 C. bigelovii (A. Gray) H.M. Hall — dwarf Rancho Santa Ana Botanic . . , Garden, Claremont, Calif 91711 C. calliopsidea (D.C.) A. Gray - dwarf Rancho Santa Ana Botanic " Garden, Claremont, Calif 91711 C. californica (Nutt.) Sharsmith — dwarf Rancho Santa Ana Botanic ■ Garden, Claremont, Calif 91711 and Botanic Garden, University of California at Los Angeles, Los Angeles, Calif. 90024 ■' - 21 - numbers alloted are: C. bigeloyit MONT 65881, C. calliopsidea MONT 65882,

C. callfomlca MONT 65883, and C . tinctorla MONT 65884.

■ Morphological variations in four annual species'of the genus Coreopsis L.

Familiarity with the range of variation for various traits' is essential to under­ standing the nature of research materials. Preliminary studies to determine patterns of quantitative variation for several traits were carried out from

1968-1971.

Materials and Methods: Unless otherwise noted, studies were made on

10 unrelated plants of each species and/or cultivar Which were cultured as described above. Five characters were measured on each plant. Plants were grown at random both under field conditions and in the greenhouse.

Measurements of variation in inflorescence morphology included determining the relative fertility of disc and ray florets by microscopic examination to determine the presence of functional pistils and by scoring seed set in mature flowers. Mean number of ray flowers per head was based on counts from two heads on each plant. The type of ray petal was scored as flat or tubular vs star shaped (cactus type). Mean number of involucral bracts was counted on the same flowers for which number of ray flowers was scored. Similarly the number of whorls of bracts was scored. Mean head diameter in cm was based on two heads/plant using three plants/species and/or cultivar. Receptacle chaffiness, an important taxonomic trait, was -25-

scored as chaffy or non-chaffy based on microscopic observation of two

r eceptacles/plant for 10 plants.

Height and spread of plants were recorded in all the cultivars available

in C. Jdnctoria. Height in cm was measured from the soil surface to the tallest point of the mature plant. Spread in cm was measured as the greatest diameter of the mature plant. In the other three species no named cultivars were available, hence the characters under study were scored on ten unre­ lated randomly chosen plants in each species.

Variation in color range of ray flowers was. studied with the help of the color chart published by the Royal Horticultural Society - London. Color matching was done in all the cultivars and species on a sunny clear day at noon time. Ten plants within each species and variety were scored.-

The range of variation in leaf form was studied by collecting the first true leaves from more than forty plants within each species. Variation was described in terms of deviation in leaf form from the form most common to the cultivar and/or species. Representative samples of common and deviant forms were photographed.

Following the procedures used in describing variation in leaf form,

morphological features of achenes from four species were compared with the

standard description given by Munz and Keck (1959) . Important forms were photographed. -26-

Results; The results of the study of quantitative variation in five traits are summarized in Tables 3 and 4. In C. tinctoria maximum var­ iability in inflorescence morphology was observed in the shape of the ray petals . The most common shape was flat. Two more forms, one with thin- pointed ligulate florets (termed "cactus" type), and another with tubular (Fig. la) ligulate florets were observed. In C. califomica no unusual variants were

■noted. The same was true of C. bigelovii and_C. caUiopsidea. (Fig. lb).

C. calliopsidea was extremely variable with regard to the fertility of the ligulate florets. Some of the plants had only 2-3 (out of 8) female fertile ligulate florets, while in some others all 8 florets were fertile. On the other hand, in some plants all 8 ligulate florets were sterile; One plant had no

■ray petals; only male-fertile disc florets were present. -

Habitwise, C. tinctoria can be separated into three classes. It stands . distinct from the three other species studied in not having basal leaves.

It also has distinct branches. Variation in height and width within each class of this species and the three other species studied is summarized in Table 4.

Class A. Tall. Height, 60-90 cm. This group consists of plants that are tall and need support to stand erect if planted alone. Branches, shown distinctly, are either dichotomous or trichotomous. .The branches are not hidden in the leaves. (Figure 2a) -27-

.14 3 6 7 8

Fig. la. Forms of ray petals. C. tinctoria upper row from left to right flat, semi cactus,Lower row from left to right cactus, flat, tubular

Fig. lb. Forms of ray petals. Jgft to right C. californica semi cactus, C. bigelovii flat, C. calliopsidea flat. Table 3

Variability in Inforescence Morphology and Fertility Ratings of Ligulate and Disc Flowers of Four Species of Coreopsis L.

Species or Floret fertility Mean no. Ray Whorls Mean no. Mean Nature cultivar Ray Disc of ray petal of in- of in- head of re- florets florets petals volucral volucral diam. cepta- bracts bracts em. ble

C. tinctoria

'Petite Purple1’ sterile fertile 7.6+0.43 flat 2 6.8+.62 3.8+.41 chaffy

'Tiger Star' sterile fertile 7.6±0.40 cactus 2 6.5+.32 3.8+.43 chaffy

"MSU selection" sterile fertile 7.4+0.15 flat 2 6.5+.87 3.9t.l2 chaffy

"Katrain self­ compatible sterile fertile 7.3+0.14 cactus 2 6.0+.68 3.3+.13 chaffy mutant"

"Tall tubular" sterile fertile 7.6+0.52 tubular 2 6.8+.85 3.6+.47 chaffy

C. bigelovii female fertile 8.6+1.3 flat 2 6.0+.51 4.07+.36 chaffy fertile

C. calliopsidea highly fertile 8.0+3.3 flat 2 3.5+.89 3.2+.47 chaffy variable

C. californica female fertile 8.8±0.78 flat & 2 4.0+.11 2.8+5.7 chaffy fertile semi- cactus Table 4

Variation in Quantitative Traits :■ Summary of the Height and Spread in Four Species of Coreopsis L.

Species Cultivar Plant Pedicel Plant height height spread cm. cm. cm.

C. tinctoria "Tall tubular" 77.66*2.26 Not applicable 21.16+1.69

I! IT 'Petite Purple' 19.45tl.02 Not applicable 22.02+1.29

Il Il '’Tiger star' 26.55+1.67 Not applicable 21.77+2.19 I N3 VOI Tl Il "Unnamed dwarf" 21.25+2.04 Not applicable 21.30+1.10

Tl IT "Compatible mutant" 32.69+1.28 Not applicable 29.35+1.28

C . californica Not applicable 11.91+1.44 34.58+1.37 14.58+1.16

C. bigelovii Not applicable 10.41+1.37 34.66+0.49 15.08+1.67

C. calliopsidea Not applicable 19.91*2.15 . 27.75+1.91 22.08+1.78 - 30-

Class B. Nana. Height, 37-45 cm. This group consists of plants that are intermediate in height. These plants do not need any support to stand erect if planted alone. Branching is dichotomous or trichotomous. Leaves are not basal, but the inflorescence and the leaves do not expose the branches.

(Figure 2b).

Class C. Nana Compacta. Height, 15-22 cm. This class consists of true dwarf plants. Branches are hidden in the leaves and the inflorescence tops the plants. (Figure 3).

Study of growth habit variation in the available plants of C . cal ifomica revealed that they have bas ically one form. The plant height ranged between

10 cm. and 14 cm. while spread ranged between 12 cm. and 16 cm. All the plants had basal leaves and floral branches topped the plants (Figure 4a).

Two main types of branches were observed:

Type A. Erect floral branches and support not required.

Type B. Drooping floral branches and support needed.

As with C. californica, the plants of C. bigelovii had a common form.

(Figure 4b).

Variability for height and spread has been summarized in Table 3.

Height ranged between 7 cm. and 12 cm ., while the range in spread was between 12 cm . and 18 cm . Fig. 2a. Plant height. C. tinctoria. Fig. 2b. Plant height. C. tinctoria. Tall plant class 60-90 cms. Nana plant class 37-75 cms. -32- -33-

All the plants had basal leaves and floral branches topped the plants

(Figure 4).

Once again two main types of branches were observed.

Like C. califomica, type A.

Like C. califomica, type B.

As with C. bigelovii, the plants of CL calliopsidea had one basic form

(Figure 5).

Variability for height and spread has been summarized in Table 3.

Plant height ranged between 12 cm. and 23 cm., while spread ranged between 18 cm. and 23 cm „ All the plants had basal leaves and floral branches topped the plant (Figure 5).

Once again, two main types of branches as described for_C. califomica were detected. In summary C. califomica, C. bigelovii and C. calliopsidea had one basic form. Leaves were almost basal and flowering branches topped the plant growth habit variation within species is recorded in the table.

C . tinctoria differed entirely from these species in growth habit, having dichotomous branches with leaves.

Only one morphological form of achePie type was found in C . tinctoria .

This matched the accepted description of achenes (Munz 1959). hi terms of variability (not reported earlier) three size forms were found (Figure 6).

One unnamed cultivar selected at the Montana Agricultural Experiment -34-

Fig. 4a. Branching habit C. californicaf left erect branches, right drooping branches.

Fig. 4b. Branching habit C . bigelovii left erect branches, right drooping branches. -35-

Fig. 5. Branching habit C. Calliopsideaa left erect branches, right drooping branches. -36-

Station. always had large seeds. No such relationship was noticed in other varieties.

Seed oil composition: A sample of the seed of a tubular-floreted mutant form of C. tinctoria was submitted to the Industrial Crops Laboratory,

Northern Utilization Research & Development Division, ARS, USDA (Peoria,

111.) for determinations of seed oil content and composition. This seed sample had been raised in the Netherlands by the Dutch seedhouse of Sluis en Groot.

In letters dated 19 June 1970 and 28 August 1970 respectively, F. R. Earle,

Head, New Crops Screening Investigations, NURDD, reported the following:

"The Coreopsis samples were received last week, and three portions are being analyzed now. There is no difference in the oil (24.3%) or protein

(24.5%) (as is). Esters have been made of these three (samples), but the analysis of them is not complete. A previous sample had essentially the same composition (26% oil and 24% protein) and its oil was much like safflower oil. It had a little less linoleic acid (73% vs. 80%) and a little more palmitic

(12% vs. 5%)."

"We analyzed the three portions mentioned in my June 19 letter and this unintentional checkup speaks well for our analysis. The iodine values were 148.8, 148.6, and 149.3. The amount of linoleic acid (as methyl ester in mixed methyl esters) was 79.0, 79.2, and 79.9 percent." -37-

Thus, should agronomic comparisons prove favorable, coreopsis

might yield an oilseed competitive with safflower for end-uses.

Only one morphological form was found in C= califomica species.

These matched the description of achenes by Munz (1959). No major varia­

bility was observed in this species (Figure 6).

Seed dimorphism was observed in C_. bigelovii. The ray achenes were morpholgically different from the disc achenes. No variability within ray

or disc achenes were observed (Figure 7a). Ray and disc achenes in this

species matched the accepted description of achenes (Munz 1959).

Two distinct forms, like those reported in C. bigelovii; were observed

in C. calliopsidea . AU the heads , depending upon, the fertility of the ray florets, had ray achenes distinctly different from the disc achenes (Figure 7b).

Description of the variation in leaf form foHows Munz (1959). The first true leaves were scored for variation. In C. tinctoria the commonest form was found to be bipinnate with three linear divisions. The variations

observed were bipinnate with zero, one or two linear divisions (Figure 8a).

In C. .califomica the most common leaf form was linear, the variations

observed were with one or two linear divisions present on ,the main linear leaf blade (Figure 8b). Bi C. bigelovii the commonest form observed was

bipinnate with three linear divisions. It was ovate in outline. The • 11 U » 38-

- T

Achene morphologyw Fig. 6. Left C. tinctorla upper row smallest, middle row medium sized, lowest row largest. Right C. califomica One morphological form dorsal and ventral view. IllllllllllIiniIIlTTT

Fig. 7a. Achene morphologic, bigelovii. Fig. 7b. Achene morphology,C. calliopsidea. Left, dorsal and ventral view of Left, dorsal and ventral view of ray ray achenes. Right, dorsal and achenes. Right dorsal and ventral ventral view of disc achenes. view of disc achenes. -40-

Fig. 8a. Leaf formSyC. tlnctoria. Most common form is the first group from left.

Fig. 8b. Leaf form^C. californica. Most common form is the first one from left. -41- variations observed were bipinnate with four linear-divisions (Figure 9a),.

In C„ calliopsidea the commonest form of leaf was bipinnate with two linear divisions. It was ovate in outline, the variations observed were simple linear leaf and leaves with one division (Figure 9b).

A color range study done on cultivars of C. tinctoria revealed that it has two basic colors - purple and yellow. Within purple, the follow­ ing variations were seen:

Dahlia Purple 931

Pansy Purple 928

Purple Madder 1028

Within yellow, the following colors were encountered:

Buttercup Yellow 5

Indian Yellow 6

Saffron Yellow 7

In the yellow group some varieties had ligulate florets with a dot of purple. This dot could be as broad as 2.5 cm. In at least one cultivar,

Tiger Starv, both yellow and purple were present in blotches.

C. califomica, C_. bigelovii and C_. calliopsidea fall in one color range.

Their basic color was yellow with the following range:

Buttercup Yellow 5 Indian Yellow 6 Saffron Yellow 7 -42-

Fig. 9a. Leaf forms, C. bigelovii , Most common form is the first one from left.

Fig. 9b. Leaf forms, C. calliopsidea, Most common form is the first one from left. -43-

Diseussion: It is clear that the four species of the genus Coreopsis

studied are quite variable. Coreopsis tinctoria was found to have consider­

able variability with regard to the major characters studied. This was

expected because of a wide collection of cultivars under study in this species „

Variability within each variety of C. tinctoria, as reflected by standard deviation^as considerably less than other species (Tables 3 and 4). This may be due to constant selection practised by plant breeders to maintain so-called purity within each cultivar. Achene size, as seen by gross morph­ ology, proved to be interesting in this species; only one cultivar (unnamed dwarf selection made at the Montana Agr. Exp. Sta.) had large-sized seeds; the others had small, medium and large seeds. Color range and shape of ray floret was highly variable in this species. Coreopsis tinctoria was found to be highly stable with regard to its having sterile ray petals; however, one plant with female fertile ray petals was found. It may represent a mutant form that could not be maintained.

C. californica, C. bigelovii and C. calliopsidea all had a common pattern of plant form, plant height and width. One interesting variability

observed in all the three species was two forms of branching which bear flowers. Horticulturally, flowers and branches that drqpp down have a distinct disadvantage over the erect ones. -44-

Color and form of ray petals did not' show a great range of variation within and between these three species. The reason for this low variability in form of ray petals may be the limited samples studied for these species .

Selection done by plant breeders does not seem to be a major factor as these species are not widely cultivated in gardens.

With regard to size of the capitulum and fertility status of the disc and ray florets, Coreopsis calliopsidea showed maximum variability. Two plants without any ray petals were scored. Fertility status of ray petals was very variable in this species.

Dimorphic achenes were found in C . bigelovii and in C. calliop­ sidea.

These studies exposed variation in important characters which were previously not reported in these four species of Coreopsis .

A larger collection covering diverse habitats is recommended in C. californica, C. bigelovii and C. calliopsidea for unearthing further var­ iability in these interesting annual species. - 45 -

Genetics and Incompatibility Mechanisms Operating in Four Annual Species of Coreopsis L.

The phenomenon of self and cross - incompatibility is widely distributed

throughout higher plants. The inheritance of this phenomenon is of basic,

genetical interest and may be of practical significance to horticulturalists

in developing and maintaining hybrid seed and stock. The inheritance of self-

and cross-incompatibility was studied in four annual species of Coreopsis L.

Materials and Methods: Ten random plants of each species investigated

in the previous study were crossed in a diallel pattern in the field.

Each plant was enclosed in a muslin bag that was supported by a two unit metal fram e. Bagging was done as soon as the buds were about to open.

In C. tinctoria five cultivars were available, so two plants of each cultivar were bagged; in the other three species all ten plants-were chosen at random.

Flowers of Coreopsis, as with other Compositae, are borne in what

is termed a capitulum. Two photographs (Figure 10) show clearly the pattern

in which the individual florets of the capitulum open. The best time for pol­

lination of the head is when nearly all the florets start showing stigmas,

or when the capitulum appears to be "fuzzy". Pollinations before this stage

may lead to erroneous conclusions because the seed set may be consider­

ably reduced and the plants assumed incompatible. Compatible pollina­

tions at this stage yield 20-55 ripe seeds/head 11 to 20 days after pollina­

tion, depending upon the species. - 46-

Fig. 10. Coreopsis*Individual flowers in a head. Left, only a few flowers of outer whorl have opened. Right, almost all the flowers are open. •47-

Plants were first verified for self- incompatibility; three open capitula were chosen in each self-incompatible plant for pollinations. Pollinations were carried out by rubbing a pollen-filled head from the male plant across the capitulum. This was best accomplished about 10 A.M. Three hundred crosses were made in each species during the first week of July, 1969.

Scores on self-incompatibility and cross-compatibility were recorded by counting the number of seeds produced per capitulum.

At the end of 1969 all parental plants in 3 species which showed recipro­ cal differences in their crossibility behavior were transplanted to the green­ house. Selected crosses were made and F1 plants were raised and back- crossed to both the parents. AU F1 plants were scored for their cross­ incompatibility and cross-compatibility behavior with the parents. A

test of goodness of fit was applied to the data grouped in these two classes

(cross-compatible vs. cross-incompatible) to compare them with a theor­ etical ratio based on the hypothesis of a sporophytic type of incompatibility.

Plants of C. calliopsidea did not survive transplanting (they were truly annual in habit); therefore, this species was not included in the back-cross studies. Crosses involving self-incompatible plants of C tinctoria

"Petite Purple’ as a female parent and a self-compatible mutant as a male parent, were made in the winter of 1969. The self-compatible mutant -48-

was not used as a female parent because of the complexity of emasculating

the hi-sexual disc florets .in the capitulum. Fifteen F1 plants were raised

in the greenhouse in 25 cm . pots . Three heads in each plant were bagged for studying the expression of the "S" gene in F1 plants . The remaining flowers were allowed to bloom in isolation for mixed pollination, hi the fall

of 1969, mix-pollinated seeds were collected. Thirty-six F^ plants were raised and studied for segregation of the "S” gene. These plants were kept

in isolation and not allowed to intercross. Each plant was studied for self­

incompatible or self-compatible status. This type of study was possible only

in C. tinctoria. No self-compatible plants were available in the other 3

species of Coreopsis under study, so that appropriate generations could not be developed. Any cross which gave 20 or.more seeds was regarded as cross-compatible. In plants which were recorded as cross-incompatible, seed set was never more than 3/head. All materials for seed counts were O hand-thrashed . The data collected were analyzed statistically , using X techniques.

Results: Three mating groups were identified among crosses within

C . tinctoria. These groups are referred to as groups I, II and II res­ pectively. Groups of plants in C. tinctoria are summarized in Table 4.

Grouping was done on the following basis:

Plants belonging to one group were always self-incompatible. - 49-

These were also cross - incompatible, barring some reciprocal

differences.

Plants belonging to 2 different groups were, barring some anomalous

conditions, cross-compatible in both directions.

Plants of Group I were 100% cross-compatible with the plants of Group II

(both ways). In crosses involving Groups I and HI, plant number 4 of Group I, when used as a female parent, was found to be cross-incompatible with the plants 8 and 9 of Group HI, but for this exception, Group I was cross-compatible with Group III both ways. Groups II and III were cross-compatible both ways

(Table 5).

Group I consisted of four plants (I, 2, 4 and 6). Barring reciprocal dif­ ferences listed in the table, these plants formed a separate self and cross- incompatible group. Detailed intra-class crossibility behavior along with reciprocal differences in these four plants are given in Table 6.

Group II consisted of .two plants (3 and 7). No reciprocal differences were found among these plants. Crossability behavior of this Group or class is presented in Table 7.

Group HI consisted of three plants (5, 9 and 8). Barring one reciprocal difference (and a cross -compatible combination between plants 5 and 8) . plants of this group formed a separate self and cross-incompatible group

(Table 8). -50-

TaBle 5

Inter-group Crossability Behavior of the Plants Comprising the Three Compatibility Groups- Studied in Coreopsis tinctoria

Group. I Group T I . Group III (Plants (Plants (Plants 1,2,4,61 3,7) 5,8,9)

Group I (0) Reciprocal + + Plants differences 1,2,4,6 (see text)

Group II + (0) Plants 3,7

Group III + with excep- (0) with excep- Plants 5,8,9 tions of recipro­ + tions of reci­ cals (see text) procals (see text

Cross-compatible. = +

Cross-incompatible ^ 0.

Self-incompatible 0 -51-

Table 6

Crossability Behavior of the Four Flants of Coreopsis tinctorla Falling Into Incompatibility Group X

Cross Seed set observed: Cross is therefore:

'None.. Full . Cross Cross:in=, Self Self in= compatible compatible compatible compati­ ble

1 x2

2 x1

1 x 4 4 x 1 2 x 4

4x2 x

2x6 x

6 x2

I x l x

2 x2 X 4 x 4

6 x6 x

1 x6 x

6x1 % 4 x 6 % 6 x 4

Reciprocal differences have been boxed - 52 -

TiELble;?

Crossablllty Behavior of the Two Plants of Coreopsis tlnetbria Palling Into Compatibility Group II. ---- • f

Cross Seed set observed: Cross is. therefore:

None Full Cross, Cross in­ Self Self in­ compatible compatible compatible compati­ ' j ble

■3x7 x x

7 x 3 x x

3x3 x x

7x7 x x - 5 3 -

Table 8

Crqssahility Behavior of the Three Plants of Coreojp^is ' tinctoria Falling into pompability Group T H

Cross Seed set observed: Cross is therefore:’

None' Full Cross- Cross in­ Self Self in- compatible ,compatible compatible compati? ble

• ; 5. x„ 9 x X

9 x 5 X X

5 x 8 X X

8 x 5 X X

8 x 9 X x ■

8 x 8 X X

5 x 5 X X

8 x 8 X X

9 x 9 X X

Reciprocal differences have been boxed

Plants No.,-5 and No , 8, though, hot cprss—incompatible,, had to be in­ cluded in this group, being common with plant number 9. - 5 4 -

Three mating groups were identified among crosses with C. califomica.

These groups are referred to as Groups I, II, and III respectively. Group­

ing was based on the same criteria as for C. tinctoria.

Plants of group I were 100% cross-compatible with the plants of Group II and III in all directions. Thus, there were no anomalies between the three groups, an ideal situation not met in other species (Table 9).

Four plants, 1,2,9 and 10, formed Group I. These plants formed a

separate self and cross-incompatible group. Reciprocal differences were observed: When plant number 9 was used with plants I, 2 or 10 it proved to be cross-compatible. Details of intra-group :cr.o,ssability behavior are given

in Table 10.

Group H consisted of three plants, 3, 4, and 6 . No reciprocal differ­ ences were found among these plants. Crossability behavior of this group is presented in Table 11.

Three plants, 5, 7 and 8 formed Group HI. No reciprocal differences were found among these plants. Cross ability behavior of this group is present­ ed in Table 12.

Three mating groups were identified among the C. bigelovii crosses.

These groups are referred to as groups I, II and III respectively. Grouping was based on the criteria used for C. tinctoria. - 5 5 -

Table 9

Intergroup Crossability Behavior of the Plants Comprising Three lntra^self, Cross-incompatible but Cross­ compatible Groups Studied in Coreopsis californica as Measured by Seed Set Success

Group I Group II Group III Plants 1,2,9,10 Plants 3,4,6 Plants 5,7,8

Group I 0 + + Plants 1,2,9,10 Except recipro­ cal differences

Group II 4* 0 + Plants 3,4,6

Group III + + 0 Plants 5,7,8

CrossT'Compgtlble = +

Cross^incoiapatihle ' — Q

Self ^incompatible. = 0 5 6 -

Table 10

Crossability Behavior of the Four Plants of Coreopsis californica Falling Into Incompatibility Group I

Cross Seed set observed: Cross is therefore:

None Full Cross Cross in­ Self Self in­ compatible compatible compatible compati­ ble

I x l x x

2 x 2 x x

9 x 9 x x

10 x 10 x x

1 x 2 x X

2 x 1 x X

1 x 9 X X

9 x 1 x X

I x 10 x X

10 x I x X

2 x 9 x X

9 x 2 X X

2 x 10 x X

10 x 2 x • X

9 x 10 X X

10' x 9 x X

Reciprocal differences have been boxed -57-

Table 11

Crqssability Behavior of the Three Plants of Coreopsis californica Falling Into Incompatibility Group II

Cross Seed set observed: Cross is therefore:

None Full Cross Cross in­ Self Self in­ compatible compatible compatible compati­ ble

3 x 3 x x

4 x 4 x I x

6 x 6 x x

3 x .4 x x

4 x 3 x X

3 x 6 x X

6 x 3 x X

4 x 6 x X

6 x 4 x x - 5 8 -

Table 12

Crossability Behavior of the Three Plants of Coreopsis californica Falling Into Incompatibility Group III

Cross Seed set.observed: Cross is therefore:

None Full Cross Cross in­ Self Self in­ compatible compatible compatible compati­ ble

5 x 5 x x

7 x 7 x x

8 x 8 x x

5 x 7 x x

7 x 5 x x

5 x 8 x x

8 x 5 x x

7 x 8 x x

8 x 7 x x

All plants in this group are 100% self- and cross-^incompatible - 5 9 -

Plants of Group I were 100% cross-compatible with the plants of Group II in any direction. Plants of Group I were also 100% cross-compatible with the plants of Group III in both directions. In crosses involving Groups II and

III, plant number 5 of Group E, when used as a female parent, was cross- incompatible with plant numbers 6, 7, 8 and 10 of Group HI, used as pollen parents; but for these exceptions, the other plant of Group II was cross­ compatible with plants of Group III both ways (Table 13).

Two plants (I and 4) formed Group I (Table 14). These plants formed a separate self and cross-incompatible group. No reciprocal differences in mating were observed. Intra-group crossability behavior is given in Table

14.

Two plants (3 and 5) formed cross and self-incompatible group II.

No reciprocal differences were found. Crossability behavior of this class is presented in Table 15.

Three plants ( 6, .7 and 8 ) formed cross and self-incompatible Group HI.

No reciprocal differences were found. Crossability behavior of this group is presented in Table 16.

Two mating groups were identified among the C. calliopsidea crosses.

These groups are referred to as Group I and Group H respectively. Group­ ing was done as previously described. - 6 0 -

Table _ 13

Intergroup Crossability Behavior of the Plants Comprising the Three Incompatibility Groups Studied in Coreopsis bigelovii as Measured by Seed Set Success

Group I Group II Group III Plants 1,2,9,10 Plants 3,4,6 Plants 5,7,8

Group I Plants 0 + • ' + I, 2, 9, 10 Except recipro­ cal differences

Group II Plants 3,4,6 + 0 +

Group III Plants 5,7,8 + ' + 0

Cross—compatible = +

Cross—incompatible = 0

Self—incompatible 0 - 61-

■ Table 14

Crossability Behavior of the Two Plants of Coreopsis higelovii Falling Into Incompatibility Group I

Cross Seed set observed: ' Cross is therefore:

None Full ■ Cross Cross in­ Self Self in­ compatible r_ compatible compatible compati­ ble

I x l x x

4 x 4 x x

1 x 4 x x

4 x 1 x x

All plants 100% self^ and cross^incompatible -62-

Table 15

Crossability Behavior of the Two Plants of Coreopsis bigelovii Falling Into Incompatibility Group II

Cross Seed set observed: Cross is therefore:

None Full Cross Cross in­ Self Self in­ compatible compatible- compatible compati­ ble •

3 x 3 x x

5 x 5 x x

3 x 5 x x

5 x 3 x x

All plants .within this group 100% crossr and self^incompatible. - 6 3 -

Table 16

Crossabiiity Behavior of the Three - Plants of Coreopsis bigglovii •,Falling Into Incompatibility Group. Ill

Cross Seed set observed: Cross is therefore:

None Full Corss Cross in­ Self - Self in­ compatible compatible compatible compati­ ble

6 x. 6 X X

7 x 7 X X

8 x 8 X X

6 x 7 X X

7 x 6 X X

6x8. X X •

8 x 6 X -X

7x8 X X

8x7 X X

All plants in this-group are 100%'s e l f a n d cross-incompatible; - 64“

Plants of Group I were 100% cross-compatible with the plants of Group II

in both directions. No reciprocal differences were observed (Table 17).

Six plants, I, 3, 4, 7, 9 and 10, formed Group I. These plants formed a separate self-and cross - incompatible group. No reciprocal differences were found among plants in this group. Crossability behavior of this group is presented in Table 18.

Four plants, 2, 5, 6 and 8 , formed Group II. These plants formed a separate self-and cross-incompatible group. No reciprocal differences were found among plants in this group. . Crossability behavior of this group is presented in Table 19.

Seed set data for F j's and reciprocal backcrosses are summarized in

Tables 20 and 21 for C. tinctoria, Table 22 for C_. californica and Table 23 for C. bigelovii. hi C. tinctoria reciprocal differences between Fj's of the same parent were detected. Similarly, backcross results.depended on the recurrent parent. A similar pattern was detected for C. californica and for

C. bigelovii.

Data from the C. tinctoriaF? of a cross, self-incompatible (female) x self-compatible (male) are summarized in Table 24. Of 36 Fg plants

scored, 7 were self-compatible and 29 were self-incompatible. Table 17

Tntergro'up Crossability Behavior of the Plants Comprising Two, Intra-shlf and Cross-incompatible but. Ititer-cross-r compatible"Groups Studied in Coreopsis calliopsidea as.Measured by Seed Set Success

Group I Group.II Plants Plants I, 3, 4, 7, 9, 10 2, 5, 6, 8

Group I Plants I, 3, 4, 7, 9, 10 0 +

■ 7

Group II Plants 2, 5, 6, 8 + 0

Cross-compatible = +

Cross—incompatible = 0

Self-incompatible 0 - 6 6 -

Table 18

iCrossability Behavior of the Six Plants in Coreopsis calliopsidea Falling Into Incompatibility Group I

Cross Seed set observed: Cross is therefore:

None Full Cross Cross .in­ Self Self, in­ compatible compatible compatible compati­ ble

X 3 x 3 X 4 x 4 X 7 x 7 X 9 x 9 X 10 x 10 X I x 3 x 3 x 1 X .1x4 X 4 x I 1x7 7x1 .1 x 9 9-x I I X 1 0 10 x I +j CU 3x4 CO

4 x 3 H j- CU 3 x 7 OJ 7 x 3 CO 3 x 9 § 9 x 3 3 x 10 10 x 3 4 x 7 7x4 4x5 9x4 4 x 10 10 x 4 7 x 9 9x7 7 x 10 10 x 7 9 x 10 ■ 67-

Table 19 ,

Crossability Behavior of Four Plants of C_. calliopsidea Falling Into-Incompatibility Group II

Cross Seed set observed: Cross i 3•therefore:

None Full Cross Cross in­ Self Self in­ compatible compatible compatible compati­ ble

2 x 2 X •- X

5 x 5 X X

6 x 6 X X

8 x( 8 X X

2 x 5 X X

5 x 2 X X

2 x 6 X X

6x2. X X

2x8 X ■ X

8 x 2 X X •

5x6 X. X

6x5 X X

5x8 X X . '

8x5 X X

6x8 X X •

8x6 x X Table 20

Results of the Backcross Using, Plants Showing Reciprocal Differences in a Diallel Cross in Ch tinctoria Reciprocal Cross I . Plants 5 and 9

.....

Crosses Genotypes Crossability behavior Prpb > level Actual ratios Hypothetical ratios

Cross comp. iCross incomp. Cross comp. I Cross incomp..

Plant 5 x ' 60 0 N/a' N/a ' N/a S4S4 x S3S4 N/a Plant 9

Plant 9.x S5S4 X "0 60 N/a N/a N/a S4S4 N/a Plant 5

Plant 5 x 27 33 0.6 (30) I : 1(30) S4S4 X S3S4 .50- 250. h S4S4

Plant 9 x S3S4 -X 2 58 o.o ( 0) 0 S3S4 : I(SO) X (see Text) F1 S4S4 Table 21

Results of the Back Crosses Using Plants Showing Reciprocal Differences in a Diallel Cross in C. tinctoria Plants Chosen here are Nos. I' and 4

Crosses Genotypes Crossability behavior Prob. level Actual ratios X2 Hypothetical ratios

Cross comp. Cross . incomp. Cross comp. Cross incomp.

PI x P U SlS, x 80 0 N/a N/a N/a N/a S1S3 I 4

P U x pi Si:? =V 2 0 . 80 N/a N/a N/a N/a

PI x F . (80) I 38 42 0,2 1(40) : 1(40) Sl^Z =sIsI 0.75- 0.50 X sIsS

X S1S2

X S2S3 Table 22

Results of the backcross Using Plants Showing Reciprocal Differences In a Diallel Cross in C . califprnica Plants 9 and I

Crosses Genotypes Crossability behavior Prob. level Actual ratios ,X2 Hypothetical ratios

Cross comp. Cross incomp. Cross comp. Cross incomp.

9 x 1 80 S2S3 x SlS2 0 N/a N/a N/a

1 x 9 0 80 N/a N/a S1S2 x S2S3 N/a

Female parent 43 37 0*45 (AO) I : 1(40) S2S3 x S1S2 .750 - 0.500 9 x F1 SiS3

S2S2

S2S3 Male . 0 80 ( 0) 0 : 1(80) parent S1S2 X S1S2 No deviation in.observed I x F1 SiS3 and expected ratios 5252

5253 Table 23

Results of the Backcross Using Plants Showing Reciprocal Differences In a Diallel cross in U- bigelovii Plants 5 and 6

Crosses Genotypes Crossability behavior Prob. • level Actual ratios X2 Hypothetical ratios

Cross comp. Cross incomp. Cross comp t Cross inComp.

6 x 5 S4S5 x S2^ S4 100 0 N/a N/a N/a N/a

5 x 6 . 0 100 N/a N/a S254 X S4 ) S5 N/a N/a i

I Male parent 5 x 82^4 x S2)S 4 0 100 0 0 Co). 1(100) No different ces in obser­ S2>S5 ved and exj pected s4 > s4 ratios'

s4 > s5 Female parent S4S5 x S2> S4 53 47 0.36 I (50) : I (50) 0.750 - 0.500 6 x F^ plants 32 >85 S4 S4

84 >85 - 7 3 -

Table ^ 24 •

Segregation in Fl Generation of. a ■Cross Between Self-incom­ patible and self-compatible mutant.of C. tinctofia

No. of plants Total Prob. level s.r ? S.C. 3 : I

29 7 36 . 0.50 .500-.250

/ -75-

Discussion: Results of the diallel cross, reciprocal differences, as

well as the anomalies in Groupfs';I found in inter-class compatibility and

and intra- class - incompatibility can be explained by a scheme proposed by

L . K. Crowe(1954) for Cosmos bipinnatus. This scheme is a modified form

of the scheme proposed earlier by Gerstel (1950) and by Hughes and Babcock

(1950), which was known as the Compositae scheme, but now is referred to

as the sporophytic form of self-incompatibility. According to this scheme the following assumptions are made:

1. A single locus controlling self-incompatibility

2. Multiple alleles for the locus

3. Dominance relationship in the pollen grains (allelomorphic genes)

4. Dominant or independent relationship in the maternal tissue (i.e.,

stigma, style) between allelomorphic genes.

Following this scheme genotypes can be assigned to the 10 plants in­

volved in the diallel crosses of C. tinctoria. These genotypes (along with the

dominance relationship of "S" alleles) explain the three mating groups that

form intra-cross and self-incompatible groups with reciprocal differences.

Explanation of anomalous behavior of some plants that cause inter-class

incompatibility can also be given. The genotypes assigned to these plants,

along with the dominance relationship of "S" alleles (on male as well as on - 76- female side) is given in Table 25.

Results of the diallel crosses in C. californica could not be explained on the basis of Nicotiana scheme proposed by East et al (1925) and designated as the gametophytic system of self-incompatibility.

In order to explain the reciprocal differences involving plant number 5 of Group II with plant numbers 6, 7, 8 and 10 of Group HI (one directional inter-class cross - incompatibility), the scheme proposed for Coreopsis tinctoria is appropriate.

Following this scheme genotypes can be assigned to the 10 plants chosen for diallel crosses. These genotypes (along with the dominance relationship of "S" alleles) explain the presence of three mating groups that form intra­ class, cross and self-incompatible groups. These genotypes also explain the reciprocal differences involving plant numbers 6,7,8 and 10 of Group HI with plant number 5 of Group II. The genotypes assigned to these plants, along with the dominance relationship in "S" alleles (on male as weU as female side) have been presented in Table 26.

As in the case of G- tinctoria, results, of the diallel cross in C. bigelovii could not be explained on the basis of the Nicotiana scheme proposed by East et al (1925) and designated as the gametophytic system of self-incompatibility.

. In order to explain the reciprocal differences (involving plant number 9 - 7 7 -

Table 25

Genotypes Assigned tp -the Ten Plants of .CL tinctoria Chosen for Dialled. Cross'

Plant No. Genotype Dominance relationship

C? Pollen ^ Stigma & style

I SS ■s, - \ S S1 \ S0 I 2 I / 2 1 / 2

2 SlSi' N/a N/a-

3 S9S No dominance Z X S2 > Sx

4 S1 S0 S0 \ S S1 \ S0 I 3 3 Z I I / 3

5 N/a, N/a S4S4

6 S1 S 1 S1 \ S' S ^ S 1-4 1 / 4 I z 4

7 No dominance ' % ■ . L > Sx.

8 N/a ■ N/a S3S3-

9 No'dominance. S3S4. S3 > S4

10 Crossed wi th all other plants, 'ience it has a separate genotype S^S^ or SgS^ - 78-

Table ■ 26

Genotypes Assigned to Ten Plants.Chosen for Dialled. Cross in _C. calif6,mica

Plant No, Genotypes C f side dominance f side (stigma relationship in & style) domin­ S alleles ance in S alleles t

I No dominance S1S2 S 1 > S 2

2 : SiS2 No dominance S 1 > S 2

3 S 3 > s 4 S3S4 S 3 > S 4 .7

4 S3S4 S 3 > S 4 - 3 3 > S 4

5 S4S5 No dominance S 4 > S 5

6 J P . JP ; S3S4 S 3 > S 4 V

7 No dominance S4S5- S 4 > S 5

8 S 4 S 5 No dominance 3 4 > S 5

9 S2S3 • S 2 > S 3 S 2 > S 3

10 No dominance. s I s 2 S 1 > 3 2 >

dominance - 79-

when used as a female parent with plant numbers I, 2 and 10) in plants of

Group I, a scheme proposed in Coreopsis tinetoria seems to be appropriate.

Following this scheme we can assign genotypes to the ten plants chosen for

diallel c ro ss. These genotypes (when keeping in view the dominance relation­

ship of "S" alleles) explain the presence of three mating groups that form

intra-class, cross and self-incompatible groups. ■ These genotypes also ex­ plain some of the anomalies discussed above. The genotypes assigned to these plants, along with dominance relationship in "S" alleles (on male as well as on female side) are presented in Table 27.

No backcrosses were possible because of cultural difficulties. This

species is strictly annual and Fj*s could not be maintained for attempting

■crosses with the parents .

Interpretation of the diallelic cross in Coreopsis calliopsidea was the most difficult task. Absence, of reciprocal differences, along with the inability to maintain parents for backcrosses was the main stumbling block.

Results of the diallelic cross could be, interpreted both for the game- tophytic or the sporophytic system of self-incompatibility; Both views

are being presented here. It is unlikely that this species has a gametophytic

system of self- incompatibility. Cytological studies pertaining to the site

of inhibition discussed later show that the sporophytic system of self-incom- . - 80-

Table 27 ; Genotype Assigned, to Ten Plants Chosen for Diallel Gross in Cv bigelo^ii'

Plant No. Genotype • Cf side dominance $ side dominance relation in S Cstigma &.style) alleles relationship in. S alleles

I S1S2 S1 > S2 5I > S2

3 S2S3 S2>V S2 > S3

4 Si > S, ^ S1S2 ' S1 > S2

5 . No dominance S2S4 S2 > S4

6 No dominance V5 S4 > S5

7 No dominance V 5 S4 > S5

8 No dominance S4S5 S4 > S5

10 No dominance S4S5 s4 > s5

2 and 9 - These plants were universally cross-compatible so could not be assigned a genotype in absence of.any reciprocal differences or otherwise. The only thing that can be said is that they had•genotypes different .f from each other and the rest of the plants., —81=

patibility is operative in this species as well. (There is a strong correlation between the incompatibility system, pollen morphology and germination as well as the site of inhibition.)

Ten plants used for diallelic cross could have come from parents with genotypes (I) S]S2 and ( 2) S2S3, with dominance in ”S" alleles operating in pollen as well as in stigma and style. Four genotypes of plants with 2 inter­ cross-compatible and intra-cross -incompatible groups will be present from such a cross.

81)82x82)83

S1>S2 81)83 Group Ei

S2^2 82)53 Group II

Plants of genotype 81)82 and S]) S 3 in Group I will form a single intra­ incompatible group with each other, provided there is a dominance in " 8 " alleles on both parents.

Plants of genotype 8^82 and 8^83 in Group II will form a single intra­ compatibility group with each other, provided there is a dominance in " 8 " alleles in both the parents.

Based on our assumptions (I and IQ plants of Group I will cross readily with those of group II in both directions. Alternatively, even if the game- tophytic system of self-incompatibility is operating there will be 2 genotypes and 2 groups. - 82-

S]S2 x S2Ss

SjSs Group I

S2S3 Group H

The crossability behavior will also be like the one we got from the diallelic cross.

However, the true picture could have been clear from backcrosses since dominance relationship would have changed the ratios of such a cross. A hypothetical model which could not be put to test because of the reasons stated earlier is being presented.

Sporophytic system: Let original parents,?! and PH,have the following genotype with dominance operating in "S" alleles both bn male and female sides.

P I - Si>S2 and P H S2)S3

A cross involving PIxPH wiH give plants of 4 genotypes:

S1S2, S1S3, S2S2, S2S3. Four genotypes will form two groups, each group will have self and cross - incompatible plants, with two genotypes,

SiS2 and S 1S3 - Group I; S2S2 and S2Ss - Group DL On crossing P 1 with Fi plants, 50% of the plants wiH be cross-compatible. The rest (%0%) will be cross -incompatible. Thus:l:l.rp.tio of cross-compatible to cross - incompatible will be obtained with Pi.

pI x Fi - 8 3 -

S])S2 x S])S2 cross-incompatible

Si)S3 cross-incompatible

82)82 cross-compatible

82)83 cross-compatible

On crossing PU with Fj plants 50% of the Fj plants will be cross-compatible; the rest will be cross-incompatible with PH. Thus, once again 1:1 ratio of cross-compatible to cross-incompatible will be obtained.

P E x Fj .

81)82 cross -compatible

81) 83 cross - compatible

82 ) 82 cross - incompatible

82)83 cross-incompatible

Gametophytic system: Let original parent plants have the same genotype, but let the poUen reaction of " 8 " allele be influenced by the male gametophyte itself; also let there be no dominance relationship in " 8 " alleles in stigma, and style.

PI - SjS2J HI S2Ss

A cross involving PI x P II will give plants of 2 genotypes only:

SjSs and S 2Ss. These plants wiU form two cross-compatible groups; however plants of same genotype will form a self and cross-incompatible - 84 “

group. Thus, purely on the basis of mating system, it will look like the first case; however backer os s data will be different from the one expected if the sporophytic system is functioning in the plants forming these two major groups.

On crossing PI with Fj plants, .100% of the plants will be cross-compatible; therefore, instead of I: I ratio of cross -compatible Fj's to cross - incompatible

Fi’s, there will be I : 0 ratio of cross-compatible Fj’s to cross-incompatible

F i’s.

PIxF1

S1S2 S1S3 cross -compatible

S2S3 cross-compatible

On crossing PU with F 1 plants the ratio of cross-compatible Ff plants to cross-incompatible Fi plants will remain I : I as in sporophytic system.

P II x F1

S2S3 x S1Sg cross-compatible

S2S3 cross - incompatible

This hypothetical model has tried to explain how backcross data (PI x Fi) would have confirmed the genetic system of self-incompatibility operating in this species.

However, based on cytological studies, it is proposed that a system similar to the other three species is operative in Coreopsis calliopsidea. -85-

The backcross data for C. tinctoria can be explained in terms of the genotypes assigned as a result of the diallel study. The backcross data sup­ port the diallel results. Expected backcross ratios are either 1:1 or 0:1 o r 1:0 for self-compatible ys . self-incompatible, on the assumptions pre­ viously discussed. These ratios were tested using methods and in all cases the fit of observed to expected was satisfactory (P ).10).

For the other two species for which backcross data are available, C. californica and C. bigelovii, similar conclusions are reached based on the same logic.

The F2 data for C_. tinctoria further support the assumptions previous­ ly discussed. A classical Mendelian ratio .75 (self-incompatible):.25 (self­ compatible). is expected. This ratio was tested using techniques and the fit was satisfactory (P ). 10).

The results indicate that Coreopsis tinctoria, Coreopsis californica and Coreopsis bigelovii have a sporophytic system of self-incompatibility.

In Coreopsis calliopsidea data do not clearly support the hypothesis of a sporophytic system of self-incompatibility.

In terms of classical genetics each of the three species has multiple alleles at a single locus. Dominance relationship is obligatory in pollen grains, while variable on the female side. With the presently known tools - 8 6 “

of genetical investigations, it was not possible to differentiate the dominance .. relationship within female gametophyte (ovules). Variable dominance relation­ ship could be expressed in stigma, style, ovary and ovules or could be operative only in diploid tissue.

In terms of number of alleles C. tinctoria had a minimum of seven alleles,

C. californica had five alleles and C_. bigelovii had five alleles: It was not possible to assign number of alleles in C. calliopsidea. The number of alleles is less than 10 as is expected in the sporophytic system of self-in­ compatibility (Gerstel,1950j Lewis,19545 Pandey,1960$ Ramanamurtfey 1963;

Sampson,1967). Fortunately, incompatibility alleles had high penetrance and expressivity. This was helpful in the analysis of data based on number of seeds set per head, a criterion used in differentiating self-incompatible and cross - incompatible crosses from compatible crosses.

Expression of the self-compatible allele (SC allele) in C. tinctoria was not clear cut. Fg progeny of the cross self-incompatible female X self-compatible male, kept at 70°F with a 12 hour light period in a growth chamber had self-compatible plants which did set seeds, but the seed set was reduced (7 to 15 seeds per head). Normal seed set, as recorded earlier in self-compatible plants, ranged from 20 to 30 per head. Out of 7 self­ compatible plants only 2 did show normal expected seed set." One possible explanation for the low seed set is the presence of modifying genes which alter the behavior of SC genes. - 87-

Factors Associated with "S" Gene Action

In many cases, sporophytic and gametophytic systems of incompat­ ibility can be separated on either a cytologicalbasis or by the site of in­ hibition of pollen growth. Generally, in the case of the sporophytic system, meiotic configurations are normal, and cytolqiBjiesis'occurs relatively late in microsporogenesis. Also, the stigma is the site of pollen inhibition, and pollen tubes fail to function normally. Cytological studies and stigma decapitation studies were carried out to verify genetic results supporting the hypothesis of a sporophytic system of iimcompatibility in the four species of Coreopsis studied.

■Materials and Methods: Young heads (buds) were fixed in 3:1 absolute alcohol and glacial acetic acid. Ten random plants per species were used in the study of the course of microsporogenesis. At least 10 pollen mother, cells per plant were scored. The aceto-orcein smear method (La1Gpur,-1941) was used for staining. A representative pollen mother cell was-photographed in each species.

Pollinated heads of 60 plants of each of the four species, two heads per plant were fixed in absolute alcohol overnight at. room temperature or in a refrigerator. Pistils were dissected, and following the method described by Boiler (1948 pp. 14-16) for pollen tube studies in sweet cherries, material was stained on microscope slides with Resorcin Blue (Lacmoid Blue) in 30% -88- alcohol. The tissue was covered with a standard cover slip and squashed by- hand (thumb pressure on the cover slip). After one minute excess stain was drawn off. Slides were examined under a compound microscope at 250X.

The callose plug of the pollen tube stains a brilliant deep blue.

Bud pollinations were carried out on 60 plants of each species, two heads per plant. Young heads were mechanically opened and pollinated with its own mature pollen from a plant known to be incompatible. Seed set was scored on each head so pollinated as an estimate of the effectiveness of bud pollination in overcoming self-incompatibility. In addition, stigmas and the distal portion of styles of the same number of plants were excised and mature pollen was applied to the remaining portion of the style . Pollen from incompat­ ible plants was used in this study. Seed set in compatible pollinations was compared with seed set from incompatible pollinations to estimate the effect of decapitation in overcoming incompatibility.

Results; All cytological studies indicated normal meiotic behavior during microsporogenesis in all species studied. In all four species, most notably in C. calliopsidea, secondary associations between different bivalents was regularly observed (Figures 11a, 11b, 12a, 12b andl-3). Cytokinesis occurred after telophase I.

In all cases pollen fertiflity was over 80%. A byproduct of this study was determination of chromosome numbers for three previously unreported - 89-

W 5

* • t » a ■ r

Fig. 11a. Meiotic studies C. tInctoria 4 A P.M .C. Metaphase I with 12 bivalents.

Fig. 11b. Meiotic studies C. tine tor ia * A P.M.C. Diakinesis with 12 bivalents grouped, in group of I fours, I threes, and 2 twos. Fig. 12a. Meiotic studies C. califom ica, A P.M.C. Diakinesis with 12 bivalents. One group of four bivalents.

I ' i A ■« e I

- :<

Fig. 12b. Meiotic studies C. Mgeloviia A P.M.C. First anaphase N s 12. - 91-

r

*

Fig. 13. Meiotic studies C. calliopsidea. A P.M.C. Metaphase I with 12 bivalents . 5 groups of twos. species (Figures 11a, 11b, 12a, 12b and 13). C. californica' (n =. 12), C. Mg-

elovii (n = 12) and C. call i ops idea (n = 12), (Sharma, Metcalf, Chapman and

Smith,in press).

Pollen germination was examined 4, 6, 8 , 12, 24, 48 and 72 hours after pollination. The methods of examination have already been discussed. In

3 out of the 4 species (C. californica was different) studied, germination of

a.few pollen grains started after 8 hours, but the maximum number of the. pollen grains around the ‘stigma were germinated only after 48 hours. Studies

of pollen tube growth were thus done only after 48 hours (Table 28).

Cros s - compatible pollinations could be easily differentiated from

the incompatible ones by the number of germinated and penetrated pollen grains

■ around the stigmatic area. There were considerable changes in the size of

the stigma and style in compatible pollinations, where the size was much

larger in comparison to the incompatible one (Figure 14a). Total number of pollen grains germinated (both those that penetrated and the ones that could not) and ungerminated, around the stigma were markedly large in compatible pollinations. In compatible pollinations, C. calliopsidea was the only species which had maximum number of pollen grains around the stigma.

These grains, however, failed to germinate as expected. In other species not many pollen grains were found around the stigmatic area.

Bud pollinations, were ineffective in three species, C. californica, - 93-

Fig. 14a. Pollen studies C. tinctoria compatible pollination. Pollen grains germinating after 48 hours.

Fig. 14b. Pollen studies C. tinctoria Incompatible pollination. Pollen grains (a few in number) not germinating after 48 hrs. Table 28

Results of Study of Site of Inhibition of Pollen Germination in Four Self-incompatible/compatible Species of Coreopsis L.

Species and Cross Time in hours Total pollen Mean number Mean no. Mean no. Mean no. form for pollen to grains around germinated penetra­ grains grains penetrate stigma pollen ted not pene­ not cvger- stigma fie ld grains grains trated 'minated

C. tinctoria selfed 48 11 3.87 2.76 1.83 7.80 (self-incomp.)

C. tinctoria selfed 48 198 195.67 192.00 3.67 2.56 (self-comp.)

C. tinctoria S i1XSi2 48 288 285.00 280.00 5.00 3.00 (self-incomp.)

C. tinctoria Si1XSC 48 285 281.37 279.00 3.00 4.00 (Self-incomp.) • Y

C. californica S i1XSi2 72 241 236.00 228.54 7.57 5.00 (Self-incomp.) Table 28 (continued)

Species and Cross Time in hours Total pollen Mean number Mean no. Mean no. Mean no. . form for pollen to grains around germinated penetra# grains grains penetrate stigma pollen ’ ted not pene­ not ger­ stigma field grains" grains trated minated

C. californica selfed 72 10 5.00 3.37 2.76 5.00 (Self-incomp.)

C. bigelovii Si^xSig 48 199 ' 197.00 190.87 6.67 2.00 (self-incomp.) -95-

C. bigelovii selfed 48 20 3.50 3.50 0.00 18.33 (self-incomp.)

C. callippsidea Si^xSig 48 213 200.00 196.57 4.00 13.00 (self-incomp.),

C. calliopsidea selfed 48 36 4.87 ' 2.87 2.00 32.21

Note: Studies done on 20 plants, 2 heads per plant, with 3 gynoecia per head examined - 96-

C. bigeloyii and C. calliopstdea. In C. tinctoria 10 to 15 seeds were set in three of the 60 plants examined.

In the decapitation studies the results are similar. Only three plants

of C. tinctoria set seed following pollination of the decapitated style. These were the same plants which set seed from bud pollinations.

Discussion: Cytological studies in four species of Coreopsis revealed that cytokinesis takes place as late as after Anaphase.II during microsporo- genesis; hence even a late "S" gene action will give rise to pollen,under spor- ophytic influence with all the attributes of a sporophytic system (like dom­ inance among "S" alleles), irrespective of the number of nuclei in the pollen grains at anthesis. The experimental results show the stigma as the site of inhibition. This^points towards two things: First, "S" gene products in the pistil are also distributed in stigma; and second, the pollen grains have the "S" gene products in them as soon as they land on the. stigmatic surface.

Bud pollination and stigma decapitation studies performed in all four species of Coreopsis point out that "S" gene products are distributed all

• over the pistil,, and time of "S" gene action is very early in the development of the female sex organ. How early cannot be answered from these studies.

Bud pollinations did succeed in,3 plants of C. tinctoria due to some unknown physiological reasons their "S" gene products accumulated only in the stigma, or there were some modifying genes which altered the general trend of "S" - 97- gene action in this regard. Townsend (1970) has selected some incompatible lines with modifying genes in alsike clover. These genes altered the "S" gene action, making them temperature sensitive. Three plants of g . tinctoria thus offered a good opportunity to study "S" gene action in C. tinctoria. - 98-

Hybrid vigor studies In Coreopsis tinctorta Nutt., using self-incompati­ bility as a tool for producing Fj hybrids.

Heterosis,, or hybrid vigor, is a well-known phenomenon in many plant

species „ A study was initiated to determine if significant heterosis for

important horticultural traits was expressed in C. tinctoria .

Materials and methods: Three Fj lines were developed by crossing

three open-pollinated cultivars with a. self-compatible male parent, The

Fj lines were compared with their respective parents and with each other with respect to five morphological traits.

Parental materials were:

1. 'Petite Purple* (open-pollinated cultivar)

2. 'Tiger Star' (open-pollinated cultivar)

3. Un-named dwarf (open-pollinated Mont. Agr. Exp. Sta. selection)

4. Self-compatible mutant (inbred for 2 generations only)

Fp hybrids developed were:

!. 'Petite Purple' x self-compatible mutant

2. 'Tiger Star' x self-compatible mutant

3. MAES selection x self-compatible mutant

Use of the self-compatible mutant as common male parent facilitated. Fj .

seed production. >

hi 1968, 12 plants of each parent and of the resultant Fj hybrids were - 99- arranged at random in each of three blocks in the field at Bozeman, Montana.

The following traits were measured on all plants: (I) plant height in cm., measured from the soil surface to the tip of the tallest portion of the mature plant; ( 2) plant spread or width in cm., measured at the widest portion of . the plant; (3) number of buds per plant; (4) number of flowers per plant;

(5) diameter of flower heads in cm., measured across the spread of the fully open flower head. Data were analyzed by standard analysis of variance (ANOVA) techniques.

Results: Mean values and standard deviations for all crosses and parents for all traits measured are summarized in Table 29.

The major difference between the Fj hybrids and their parents was found to be greatly increased flower production by the Fj hybrids, as indicated by the bud and flower counts. Plant dimensions were modified, in the F^ hybrids, more in terms of spread than in height. This is indicated by the height/spread ratios, which were all less than 1.0 for the Fj hybrids, but clearly less than 1.0 for but one of the four parental lines. Only small differences in flower size were found among the Fj hybrids and their parents.

The Fj hybrid 'Tiger Star' x self-compatible mutant was significantly shorter than the two other Fj hybrids. The Fj hybrid 'Petite Purple' x self-compatible mutant was also of desirable height. These two hybrids were Table 29

Comparison of Plant Height, Plant Spread, Number of Buds Per Plant,. Number, of Flowers Per Plant, and Flower Diameter for Four Parental Lines of Coreopsis fin eto ri a with their resultant F^ hybrids, Bozeman, Montana, 1968

Line Line Plant Plant H/S Buds per Flowers/ Flower no. ■ id en tity height spread ratio plant plant diameter cm. cm. no. no. cm.

Parental . lin e s :

I. ilP etite Purple" 19.4+1.0 22.0±1.3 0.88 58.0±5.4 60,4+4.8 3.810.4 -OOT-

,2. "Tiger Star" 26.5±1.7 21.8±2.2 1.22 60.5±7.8 61.4±7.0 4.0±0.4

"3. Unnamed MAES 21.2±2.0 21.3+1.1 0.99 62.8±2.5 63.312.3 3.910.1 dwarf selection

4. Self-compatible 32.6+1.3 29.8±5.8 1.09 43.5±1.4 38.615.9 3.210.1 mutant Table 29 (continued)

Line Line Plant Plant H/ S Buds per Flowers/ Flower no. identity- height spread ratio plant plant diameter cm. cm. no. no. cm.

F-l hybrid lines:

I. "Petite Purpler 27.8±2.2 32.7±2.3 0.85 257.6+6.1 258.9+2.7 4.1+0,3 x self-comp.

2. "Tiger Starrt 25.6±2.8 5I . 6±2.2 0.50 271.0±16.8 273.9±18.9 4.6+ 0.I x self-comp.

3. Unnamed, dwarf 29.7±0.4 39.8±0.8 0.75 252.2±8.4 257.9±9.5 4.4+0.4 x self-comp. - 102-

similar in height to 'Tiger Star', but differed significantly in height from the

other parental lines-. They were judged to be of desirable height.

All three Fj hybrids produced plants with greater spread than their parents. The Fj hybrid 'Tiger Star' x self-compatible mutant had maximum

spread and was significantly different from the other Fjs in this regard.

AU the Fj hybrids were significantly superior to the-parents in numbers of buds and flowers produced. Among the F 1 hybrids, 'Tiger Star' x self­

compatible mutant was superior to 'Petite Purple* x self-compatible mutant

and to ' un-named dwarf x self-compatible mutant* in flower and bud production.

The Fi hybrids did not differ among themselves or from 'Tiger Star' v. in size of bloom, but produced larger flowers than 'Petite Purple*, the un­

named MAES dwarf selection, and the self-compatible mutant.

The Fi hybrids all had the star ray floret pattern. The self-compatible

mutant parent, common to all, had star ray florets . It is apparent that, in these hybrids, star ray floret pattern was dominant over flat ray floret pattern.

The Fi hybrids could be segregated in the field from the parental lines with­

out the aid of precise measurements. Apparently, the F 1 hybrids are much better, hort!culturally speaking, than their parents, and, if put into production,

could readily displace current commercial lines.

Production of Fi hybrid seed on a commercial scale: A simple mechanism

for production of Fi hybrid seeds of annual species of Coreopsis cvvfv . A - 103-

could be of significant value to flower breeders. In essence, such a mech­

anism calls for two homozygous self-incompatible but cross-compatible lines.

Such lines would be grown side-by-side in alternate rows, cross-pollination

being achieved via insect activity.. The seed harvested from the self-incompat­ ible parent would be Fj hybrid in nature; that from the individual row's could be bulked or segregrated depending on the nature of the progeny from various parental lines and/or the intended commercial end-use of the seed.

There are two problems that must be solved before such a program could be successful. First, production of homozygous self-incompatible but

cross -compatible lines that are good combiners is quite a task;, secondly, maintenance of.these lines is possible only if "S" gene action occurs some­ time after floral morphogenesis (only then are bud pollination and other techniques effective).

The technique to be followed is a simple one; the only requirements are that the pollen parent must be self-compatible and that the incompatible plant should be capable of being multiplied vegetatively. We have discover­

ed these requirements to be satisfied in Coreopsis tinctoria, in which diverse

genetic material, lines with good combining ability, and at least one self­

compatible mutant are available as a result of our research and from other

sources. Herbaceous cuttings of C. tinctoria strike root with 90% success, - 1 0 4 - which is significant since the female self-incompatible parent must be multi­ plied vegetatively from an initial single plant. In order to have adequate stocks of this type, the increase program can be started in autumn in the greenhouse. The stock plant will remain vegetative at night temperatures of 55-60°F and will yield a large number of cuttings which, when suitably manipulated, will afford a further increase of stock. Reserve stock for use in succeeding years should be maintained.

The plants raised from cuttings should then be planted out with the seedlings of self-compatible plants (raised from seeds' and maintained in isolation) in alternate rows. Seed harvested from the self-incompatible plants will be hybrid for commercial sale.

Discussion: Fp hybrids were superior, as ornamental plants, to the best parent. What constitutes , a better variety in an ornamental plant and how FI. hybrids may be better can be described in terms of a medium tall plant with many flowers which persist over a long period.

Heterosis is apparent for important horticultural traits. A simple mechanism to produce Fl seeds could be of significant value to flower breeders. Fig. 15. Hybrid Vigor in C. tinctoria. z/ Fj hybrid: Petite Purple'(female) x Self- Compatible mutant" (male). -106-

Fig. 16. Hybrid Vigor in C. tinctoria. Cultivar: 'Petite Purple'. Fig. 17. Hybrid Vigor in C. tlnctorta. CultivanUnnamed dwarf? - 108-

Fig. 18. Hybrid Vigor in C. tinctoria. ^ Fj hybrid? Unnamed dwarf' 1[female) x self-compatible mutant (male). - 109-

Fig. 19. Hybrid Vigor in C. tinctoria , Cultivan self -compatible mutant. SUMMARY AND CONCLUSIONS

Studies concerning variation in four annual species of the genus

Coreopsis revealed that there is considerable variation within and among species in important horticultural traits, such as plant height, plant width, branching habit and size of the capitulum . Such variations may be of consider­ able importance to plant breeders.

Diallel backcross and Fg studies revealed that three of the four species

(C. tinctoria, C. califorriica and C. bigelovii)have a multiallelic, monogenic system of self- incompatibility. Pollen reaction was influenced by the sporo- phyte, which is expressed as obligatory dominance of "S" alleles in the pollen grains. Dominance relationship between "S" alleles in the pistil was variable.

Cytological, bud pollination and stigma and style decapitation studies have revealed normal meiosis with secondary associations between different bivalents present in all the four species. This phenomenon points out a probable role of polyploidy associated with this genus. The stigma as the site of pollen inhibition, is, exhibited in all four species. Distribution of "S" gene products all through the pistil does not allow breakdown of self-incompati­ bility barrier by stigma and style decapitation. Chromosome numbers for three perviously unreported species have been determined.

Genetical and cytological studies show a strong correlation between the - 111-

stigma as the site of pollen inhibition,associated with the sporophytic system

of self-incompatibility. Based on these observations it is suggested that.C.

calliopsidea has also a sporophytic system of self-incompatibility.

Self-incompatibility could be used with advantage in producing hybrid

seeds. In C. tinctoria heterosis expresses itself in form of more (greater number) buds and flowers in hybrids than in parents. Fj hybrid seed production on a commercial scale is feasible. LITERATURE CITED

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Sharma, Jagan If Sh23 The genetics, nature cop. 2 a n d occurrence of self- and cross incompatibility in four annual species of Coreonsis L______%AMg AND AOPRKSa