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Breeding and Physiological Studies

of a Bitter Compound in Carrots
presented by

Bruce Charles Carlton

has been accepted towards fulfillment
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BREEDIm AND PHYSIOLOGICAL STUDIES OF A BITTER

 

COMPOUND IN CARROTS

By

BRUCE CHARLES CARLTON

AN ABSTRACT

Submitted to the School for Advanced Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Horticulture

1960
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ABSTRACT

  

Carrot roots were selected on the basis of the amount of a bitter com—
pound (3-methy1-6-methoxy-8-hydroxy-3, 4-dihydroisocoumarin) developed
during storage in the presence of ethylene gas or apple emanations. Analyses
of individual roots from inbred lines and segregating populations demonstrated
an extremely wide variability in the quantity of the bitter compound developed
in roots within some lines. These extreme ranges and the differences obser-
ved between progeny means suggest a heritability for the trait and the possibi-
lity for elimination of this type of bitterness by selection and breeding.

Atypical ultra—violet spectra observed in roots of some breeding lines
indicated the presence of more than one compound, and necessitated modifications
in the calculations of the levels of bitterness from the spectrophotometric data.
There is evidence that the interfering compound or‘ compounds may be closely
related to the bitter principle named above, and may result from degradation of
the bitter principle in the root during storage. The nature of the interfering
substances and their fluorescent and taste properties remain to be elucidated.

An experiment utilizing carbonl4—1abelled ethylene gas for induction of
the bitter compound in a carrot breeding line showed that ethylene was not
incorporated into the carrot tissue although a quantity of the bitter principle
was synthesized by the roots. These results suggest that ethylene does not

act as a substrate in the biosynthetic reactions, but probably functions as a

 

 

A

 

 

 

 

 

    

BRUCE Cl-IARLES CARLTON ABSTRACT-2

catalyst. Synthesis of the bitter compound was found to depend on the simul-
taneous presence of both oxygen and ethylene. The biosynthesis of the bitter
principle appears to depend either on the availability of aerobically—produced
high-energy phosphates or on the presence of oxygen as an electron acceptor

in a terminal oxidation reaction.

  

 

 

 

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{h

at

 

a

 

BREEDING AND PHYSIOLOGICAL STUDIES OF A BITTER

COMPOUND IN CARROTS

By

BRUCE CHARLES CARLTON

 

A THESIS

Submitted to the School for Advanced Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

l 960

 

 

 

     

ACKNOWLEDGEMENTS

The author expresses his sincere appreciation to those who
have in any way assisted in this work. To the members of the guidance
committee, Drs. C. E. Peterson, S. K. Ries, F. C. Elliott, H. M. Sell,
and A. S. Fox, appreciation is expressed for their many suggestions and
guidance throughout the course of the work. Special thanks are due Dr.
N. E. Tolbert, for assistance in the radioisotope work, Drs. H. M.
Sell and A. L. Kenworthy for laboratory facilities, Dr. D. H. Dewey for
storage facilities, and to Mr. Roger Ritzert and Mr. Harold Schmalfeld
for their technical assistance.

The author is especially indebted to his wife Nancy for her
many hours of assistance with the manuscript and for her continued en-

couragement throughout the course of these investigations.

 

 

 

TABLE OF CONTENTS

INTRODUCTION 0 n c o 0 a n c c a l e o e 1 o c o c e o 0

REVIEW OF LITERATURE . ..... . . . . . . . . . . .
METHODS AND MATERIALS ..... . . . . . . . . . . .
A. Breeding Experiments. . . . . . . . . . . .....
B. Physiological Studies ................
1. Analytical Procedures .............
2. Radioisotope Experiment ....... . . . . .
3. Oxygen-Bitter Compound Relationships ......
RESULTS ..................... . . . .
A. Breeding Experiments ......... . . . . .
B. Physiological Studies ...... . . . . ......
1. Radioisotope Experiment ....... . . .

2. Oxygen-Bitter Compound Relationships . . . . . .

DISCUSSION . . . . . . ............... . . .
A. Breeding Experiments ............ . . . .
B. Physiological Studies ........... . . . . .

l. Radioisotope Experiment . . . . . . . . . . . .
2. Oxygen-Bitter Compound Relationships ......
SUMMARY AND RECOMMENDATIONS ....... . . . . .
LITERATURECITED.................. ..

APPENDIX. .................

Page

19
22
25
25
30
34
36
39
39
44
47
50
52
55

61

 

    

INTRODUCTION

The problem of bitter flavor in stored carrots was first noted in Cali-
fornia (Yamaguchi e_t 31; , 1955) in 1950. A similar problem was reported in
New York (Atkin, 1956). The situation has since become rather acute, espec—
ially in carrots to be used for processing, and may be one reason for the recent
decline in the carrot industry in Michigan (Agric. Market Serv. , U. S. D. A. , 1958).
Investigations have subsequently been initiated in California, New York and
Michigan to determine the causes and possible control of bitterness. During
the past five years these efforts have resulted in two major discoveries regard-
ing bitterness development in carrots. First, the isolation and identification
of 3-methy1-6-methoxy—8-hydroxy-3, 4-dihydroisocoumarin, one of possibly
many compounds contributing to bitterness, and second, the linking of ethylene
1/

gas to induction of this isocoumarin" type of bitterness in storage. No other
factors have been directly correlated with bitterness development and no satis-
factory and permanent means of control has been found, other than isolation of
carrots from ethylene emanations.

The present investigations were based on earlier results with bitter com-
pounds in other crops, notably the cucurbitacins in cucumber and coumarin in

sweetclover, for which inheritance has been determined and breeding programs

1/The term "isocoumarin" will be used to refer to the bitter compound under
study in this report. Although technically incorrect, this name has become
common in the literature and to change it would only lead to more confusion.

 

 

  

 

 

I" ._. . If a... .. "fa.
I _ .. . _ I

  

dual—root analyses, which ultimately would permit an estimate of genetic herita—
bility for the trait. The main objectives of this study were to determine if there
were inherent differences among carrots in their tendency to develop isocoumarin
and if there was any basis for a selection program. The discovery of some
breeding material with atypical spectral characteristics led to investigations into
some of the physiological aspects of bitterness.

It should be emphasized that the isocoumarin derivative under investi-
gation is not the only compound which contributes to the bitterness problem. In
fact, it has been thought by some (notably Sondheimer, 1957a) to be of rather
minor importance in the overall determination of bitter taste in carrots. Never-
theless, it is the substance about which the most information has been obtained and
seems a logical starting point from which to work on the problem. The ultimate
solution would seem to depend on the determination of the genetic basis for this
type of bitterness, elucidation of the biosynthetic pathways, and selection and

utilization of low-isocoumarin germplasm in carrot improvement programs.

 

 

 

 

 

   

 

  

REVIEW OF LITERATURE

Bitterness and off—flavors in various food products have long been re-
ported in the literature. Higby (1938) isolated and identified a bitter principle
from navel and valencia oranges called isolimonin, and cited the occurrence
of similar compounds in citrus such as limonin and citrolimonin. Kesterson
and Hendrickson (1953) described investigations on the occurrence and utili-
zation of naringin, a bitter gluco side found in grapefruit. In vegetable crops
Borchers and Nevin (1954) described a procedure for the quantitative estima-
tion of an unidentified bitter alkaloid in tomato. Bitterness in cucumbers, a
problem for many years, has been ascribed to a number of compounds by
Truscott and Gullet (1958), and by South African workers, Rehm e_t a_l. (1957a,
1957b, 1958) and Enslin e_t _a_l. (1954, 1956a, 1956b, 1957). The inheritance
of one type of bitterness was shown by Barham (1953) to involve a simple
dominant factor. Andeweg (1959) substantiated these results and found a single
non-bitter plant in 15, 000 tested, which indicated a very high prevalence of
the dominant bitter allele in commercial cucumbers and plant introductions.
Off-flavors in carrots were first mentioned by Brown e_t a_l. (1944) in
studies on the effects of fertility on color, flavor, and carotene content of the
roots. Hervey and Schroeder (1949) noted that carrots infected with aster
yellows virus developed astringent bitter—like flavors. In a comparison of

muck and upland soils as they influenced quality of reconstituted dehydrated

   

 

 

   

 

 

LBJ-{"6” . _; ',‘-_n'.

 

, .
r’ - ‘xand off-flavors in muck— grown carrots than in carrots grown on mineral soil.

Truscott (1953—54) described two apparently different types of bitter flavors

in carrots. One, a "peppery hotness", could be eliminated by cooking and
was of no consequence in the processed product. The other was a bitter
effect which varied from root to root, persisted in the mouth, and could not
be eliminated by cooking. Yamaguchi it a_l. (1955) first observed bitterness in
California- grown carrots in 1950 and described the effects as "bitter, quinine—
like, soapy, alum—like and spicy". The bitterness also lingered in the mouth
and was concentrated in the phloem tissue. Further investigations revealed
that bitterness was not present in carrots at harvest but developed only after

6 to 12 weeks in 32°F storage. Experiments on cultural conditions showed no
Correlation between fertility levels and bitter flavor. Irrigation studies sug-
gested that maturity might affect the level of bitterness development. In New

York, Atkin (1956) also observed that bitterness was not present at harvest and

reported no correlation between spray programs, disease conditions, fumigants
or soil fertility and the development of bitterness in storage. The small varietal

differences were not considered significant. In his work carrots grown on muck

or sandy soils developed more bitterness than those grown in loam. No corre-
lation was evident between physiological age and bitterness, in fact immature

roots tended to become more bitter than those of large size.

  

Bessey (1957) in Michigan found no relationship between bitterness and
soil fertility, including major and minor elements, soil type (muck vs. upland)
or sugar content. Significant differences in bitterness development were ob-
served for variety, storage atmosphere and seeding date. Emanations from
apples and other respiring produce were found to accelerate bitterness devel—
opment. Ells (1958) showed that ethylene gas produced bitter taste, fluorescent
areas in the roots, and bitter principle development as measured by a spectro-
photometric method. Physiological age of the carrot was implicated as a factor
regulating development of bitterness although the evidence was not considered
conclusive.

Schmalfeld (1960a) indicated that the quantity of ethylene administered to
carrots influenced the ultimate level of bitterness. Pre-treatment of carrots
with ethylene and subsequent removal to ethylene-free atmospheres demonstrated
that exposure time influenced bitter compound development. Varieties of early—
maturing carrot types such as Nantes developed higher levels of isocoumarin
than did later types like Chantenay. It was concluded that physiological age
of the roots had a direct influence on bitterness formation. Three possible
roles of ethylene in inducing bitterness were postulated.

The effects of ethylene on biochemical and morphological characteristics
of plant tissues, especially in relation to ripening, have been reviewed by
Biale (1950), Pentzer and Heinze (1954), and Ulrich (1958). Minute quantities

of ethylene apparently cause changes in physiological activity which cannot

 

  

' The traced to known mechanisms of substrate degradation. Ever since Denny's
(1924) work on hastening of ripening in lemons through the use of ethylene
many possible roles of its action have been postulated. Michener (1935) and
Crocker e_t al_. (1935) suggested that ethylene’s effects may be produced in-
directly through one or more growth regulating hormones. Lynch (1936)
suggested that it acts as a prosthetic group or co-enzyme. Millere_ta_l. (1940)
related the coloring of mature oranges by ethylene to an accelerated decom—
position of chlorophyll without an effect on carotenoid synthesis.

In more recent work, Hall (1951) obtained evidence that ethylene arises
by enzymatic degradation of a number of active substances during respiration,
and that potential energy—yielding substrates are reduced to alcohols, which
appear to be the terminal substrates of ethylene synthesis. Porritt (1951)
showed that metabolic changes during ethylene—induced ripening such as chloro-
phyll destruction, sucrose inversion, and conversions of starch to sugar also
occurred under normal ripening processes. Ethylene merely accelerated these
changes. In an enzymatic study, Lee (1959) found that crude lipid extracts of
raw peas held at freezing temperatures developed much higher peroxide levels
than shelled peas of the same type. He postulated that ethylene production in—
side the pods might induce this effect and indicated that the lipid fraction of
ripening tissue could be intimately involved in the normal ripening process.
Lieberman and Spurr (1955) showed that ethylene production by broccoli de—

pended on oxygen content of the atmosphere, and that ethylene was the volatile

 

 

 

  

I ~ ,- . e produced in greatest quantity under aerobic conditions. Under
- - anaerobic conditions, ethylene production ceased and other volatiles, such

as ethyl acetate, acetaldehyde, and methyl mercaptan were produced. Burg
(1959) found that neither glycerol nor acetate was utilized as a precursor of
ethylene in apple tissue. The rate of incorporation was so low compared to
the labelled carbon dioxide evolved it was concluded that ethylene production
is not directly associated with the Krebs cycle organic acid, and that the
direct precursors of ethylene are quite distinct from this and other major
metabolic pathways. Ethylene evolution from apple tissue was later found

by Burg and Thimann (1960) to depend on oxygen and respiratory activity.
Studies involving the effects of osmotic and physical changes and the actions
of various inhibitors resulted in the hypothesis that ethylene production occurs
in the mitochondria.

Utilizing CM-labelled ethylene, Buhler e_t aL (1957) obtained incorpora-
tion with avocados and pears, while tomatoes, oranges, limes, papayas, apples
or grapes did not incorporate the material. In avocado and pear the rate of
incorporation was extremely low (0. 05 per cent) and the labelling appeared
almost exclusively in organic acids. Systematic degradations of succinic and
fumaric acids showed about three times as much activity in the carboxyl carbons
as in the second and third positions. These results suggested that ethylene is
incorporated into an unsymmetric intermediate in such a way that randomization

occurs preferentially in one of the carbon atoms of the ethylene skeleton. The

 

 

 

 

 

   

=__. , _rate of incorporation indicates that ethylene is a terminal production
. I of metabolism and is not metabolized any further in the plant. Hall (1959)
14
found that coleus and cotton leaves exposed to C -ethylene for 15 hours in-

4 throughout the entire plant. Of the 014 fixed,

corporated and transfused C1
fifty-five per cent was extractable by petroleum ether. Paper and column
chromatograms of water—soluble extracts revealed nine distinct compounds
with characteristic peaks. None of these was identifiable as known sugars,
sugar phosphates, amino acids or organic acids. The bulk of the C14 was
found in the leaf residues and very little in the roots.

An ultra—violet absorption method for the objective estimation of
bitterness in processed carrots has been described by Sondheimer _e__t_ aL (1955).
A correlation coefficient of -0. 82 was observed between flavor scores and
the peak heights in the ultra-violet spectrum. It was suggested that the dis-
crepancies between the two methods of evaluation might be caused by the
presence of other bitter compounds which did not absorb in this region,
since extracts from a few samples with greatly different taste scores showed
similar ultra—violet absorption spectra. Weak absorption in the ultra—violet
region could actually be due to carotenoids, which absorb primarily in the
380-520 millimicron region. Dodson_e_ta_l. (1956) and Dodson (1957) attempted

the isolation and identification of one of the bitter principles and found char-

acteristic Rf values on paper chromatograms and an ultra—violet spectrum

 

“>71

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and infrared maximum at 6. 01 microns. The compound was crystallized as

colorless platelets melting at 77°C and soluble in water and organic solvents.
A molecular weight of 268 was obtained and an empirical formula of C15H1505
suggested. However, no compound of these properties and molecular weight
had previously been described and no identification was made. Sondheimer
(1957a) isolated presumably the same bitter principle, since its physical

and chemical properties were identical to those of the compound described

by Dodson. Elemental analysis suggested an empirical formula of CHI-11204
however. The compound possessed an ultra—violet absorption spectrum similar
to those of hydrocarbon extracts of bitter carrots, and when added to steam-
distilled non—bitter carrots produced the characteristic bitter taste. It was
also shown that an oily steam—volatile substance present in some carrots

also produced a bitter taste. These observations indicated that bitterness

is caused by a number of factors of which the described compound is one,

and in many cases possibly a minor contributor to the bitter taste.

The bitter compound has been identified by Sondheimer (1957b) as 3-
methyl—6-methoxy-8—hydroxy—3, 4-dihydroisocoumarin and its chemical and
physical properties completely described. Isocoumarins have been described
in some detail by Sethna and Shah (1945) as a group of lactones derived from

o-hydroxy cinnamic acid, or as heterocyclic compounds with oxygen in the

 

 

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Isocoumarins Coumarins

A number of methods were given for the synthesis of isocoumarins from
B-napthoquinone and nitromethylene phthalide. No reference was made
to the synthesis of the 3-methyl-6-methoxy-8-hydroxy derivative or of
the biosynthesis of this compound or of other isocoumarins. However,

a number of possible biosynthetic pathways have been suggested. On
the basis of lower alpha—carotene levels in bitter than in non-bitter
carrots, the solubility of the bitter component in petroleum ether, and
its absorption on MgO columns, Yamaguchi gal; (1955) suggested that
bitterness might be a metabolic product connected with carotenoid syn—
thesis. Atkin (1956) observed that bitter carrots were paler in color
than non—bitter ones, but emphasized that the relationship between caro-
tene content and bitterness might be coincidental because of environmen-
tal influence. Schmalfeld (1960a) studied carrot types ranging from white
to dark orange in color and found no direct relationship between "degree

of pigmentation" and susceptibility to bitterness.

 

 

 

A possible relationship, or at least a similarity, between carotenoids

and fluorescent compounds in roots was indicated by Strain (1939). He
found large quantities of colorless fluorescing substances in carrot roots
which adsorbed immediately below alpha—carotene on magnesia chromato-
graphic columns. Fluorescing polyenes were investigated by Zeichmeister
e_t aL (1944, 1945), who found among a group of similar compounds a sub-
stance they termed phytofluene. It fluoresced under ultra-violet irradiation,
possessed an ultra-violet absorption spectrum with maxima at 367, 348 and
332 millimicrons in hexane solution, and was found in a number of plant
tissues such as carrot, tomato and orange. In Pyrocantha the biosynthesis
of phytofluene seemed to parallel the development of colored polyenes during
the ripening process. Phytofluene appeared to be involved in carotenoid syn-
thesis and did not accumulate in unripe fruit. None of the animal tissues
examined contained this compound. Porter and Zscheile (1946) isolated
two compounds from tomato which appeared to be polyenes. One had an ab-
sorption spectrum identical to that of phytofluene, the other exhibited maxima
at about 275, 285 and 295 millimicrons. It was suggested that these compounds
might be intermediates in carotene synthesis.

Goodwin and Kavanagh (1948, 1949, 1950, 1952) have described fluores—
cence studies of a whole series of compounds isolated from the roots of 135

species of vascular plants representing 69 plant families. All but six (which

  

  

3V ferns) exhibited fluorescent materials in the roots. In Avena (oats)

 

. .':' "ll-three distinct substances were found. The biosynthesis of these appeared

to begin the first few days after germination. One of these substances

was identified to be scopoletin (6-methoxy-7-hydroxy coumarin). Seed-

lings of carrot germinated in petri dishes also showed fluorescing substances

after a few days. A number of coumarin derivatives were examined, among

them umbelliferone, which is highly fluorescent and found in carrots. Clayton

and Larmour (1935) cite that coumarins often occur as glycosides in plant

tissue or associated with compounds of hydroxyphenyl propionic acid or

hydroxy cinnamic acid. Coumarins occur widespread in nature and have

been studied chiefly in sweetclover because of their hemorrhagic effects

on cattle (see review by Link, 1943-44). Investigations into the possible

‘ biosynthetic pathways of coumarins by Kosuge and Conn (1959) have shown
that cinnamic acid, phenylalanine, glucose, and acetate are all precursors
of coumarin, although even the most efficient (cinnamic acid) was diluted
7500 times. Their work showed that coumarin is rapidly metabolized to
melilotic acid and its glycosides and to o—coumaryl glucoside in Melilotus
Elia, sweet clover. Brown e_t a_l. (1960) fed suspect precursors of coumarin
to the perennial grass Hierochloe odorata, and found o—coumaric acid,
cinnamic acid, and shikimic acid to be the most efficient precursors. Acetic,
melilotic, salicylic, and ferulic acids were also incorporated to a lesser

degree. They concluded that the most efficient biosynthetic pathway probably

 

 

 

cinnamic acid - o-coumaryl glucoside was implicated as a possible important

 

side-route to the coumarins.

Breeding and inheritance studies in reference to coumarin synthesis in
SWeet clover have shown it to be under rather simple genetic control (Stevenson
and White, 1940; Homer and White, 1941; Smith, 1948). Goplen_e_t_a_l. (1957)
concluded that one major dominant gene controls the synthesis of coumarin
and a complementary locus controls the form (i. e. free or bound) of coumarin
in the plant. This evidence, along with the work cited earlier on breeding
for non—bitter characteristics of other crops, has suggested plant breeding
as a solution to the bitterness problem in carrots. The possibilities of breed-
ing for bitter-free carrots were suggested by a number of early workers,
among them, Yamaguchi e_t a1. (1955) who found that selections and mass pol—
linations of bitter and non-bitter roots showed a definite tendency for bitter-
ness to be inherited. Atkin (1956) suggested that the extreme differences
found between individual roots of the same lots might be due to genetic differ-
ences. He emphasized that there may be no statistical differences between
varieties or strains and still genetic differences may exist between individual
roots because of the extreme heterogeneity of carrot varieties. He concluded
that if root to root variation were due to genetic causes it should be possible
to breed bitter—resistant carrots.

Yeager (1957) employed breeding techniques to select for non—bitter

 

A drrots. Bitter—free roots were selected organoleptically from commer-

 

cial Chantenay varieties and allowed to mass-pollinate. The bulked seed

was grown and the composited progeny analyzed by a spectrophotometric

procedure-14 These composite samples showed bitterness of about the same

level as the parent variety. Some of the individual roots were not bitter to

taste, however, and selections of these were allowed to mass-pollinate.

The progenies of this second generation were only about one—fourth as ‘

bitter as the parent variety. :
Bessey (1957), in attempts to show that bitterness was transmitted

genetically, self—pollinated bitter and non—bitter plants which had been eval~

uated organoleptically and analyzed the progenies after four and one-half

months storage in a refrigerated root cellar. Although no bitter progenies

were found from any of the selections, it was emphasized that very little

bitterness in any material was found in that particular season and suggested

that environmental conditions may not have been conducive to bitterness devel-

opment. At this time ethylene gas had not been implicated in inducing bitter-

ness and controlled conditions for screening of breeding material were not

available. Progress in these directions has provided a basis for more inten-

sive investigations into the possibility of breeding non-bitter carrots.

1
—/Developed by W. F. Phillips, Beechnut Company, Canajoharie, New York

It

 

 

A

 

METHODS AND MATERIALS

A. Breeding Experiments

  

The materials used in these investigations were obtained primarily
from single-plant selections of commercial carrot varieties. One lot, mass-
selected for non-bitterness, was utilized in two crosses‘ and commercially—
stored carrots were used in one of the physiological studies. Two of the
original crosses were made in the winter of 1957—58 from single-plant selec-
tions grown in the field in 1957. The parent roots were stored for approxi-
mately ten weeks in the presence of apple emanations, selected by their
fluorescence ratings, and their bitterness content estimated by a spectro—
photometric method. The F1 progenies of these crosses were grown in the
field during the summer of 1958 along with some selected inbred lines, and
after harvest the roots were divided into three treatments for storage. In

one treatment the roots were stored in sealed respiration tanks at 36°F in a
continuous atmosphere of air containing 50 parts per million of ethylene gas.
In the second treatment they were packed in moist Sphagnum moss and kept

in a common storage at 38° F in the presence of apples, and in the third treat—
ment the roots were stored in respiration tanks at 36°F with a continuous flow
of ethylene-free air. After nine weeks all lines in each of the three treat—
ments were sampled by taking cross—sectional slices about one—third of the

i/Provided by A. F. Yeager, Department of Horticulture, University of New
Hampshire, Durham, N. H.

15.

 

 

 

    

y‘iip from the tip of each root. The slices were weighed accurately to
-.‘V'Il1thin 0. 1 gram, sealed individually in polyethylene film, and frozen at -10’ F
until analysis about two months later. Each root was examined under a 2537 A.
ultra-violet light and graded from 1 (no fluorescence) to 5 (extremely bright

fluorescence) for later comparisons with spectrophotometric data.

1.9.5.9

The remaining F1 crosses, backcrosses, and self pollinations were
made in the greenhouse during the winter of 1958—59 by both the emasculation
technique and in a few cases using genetic male sterility which appeared in
some plants. The crosses and resulting progenies are described in Tables 2,
3 and 4. Seed of these crosses and inbreds, as well as remnant seed from the
previous year, was sown in ten—foot rows on muck soil on May 21, 1959. The
roots were provided with as nearly uniform conditions of seeding, fertilization,
moisture, cultivation, and spray programs as were possible under field con-
ditions. The roots were harvested on September 11, transported in sealed
drums to storage, and placed in respiration tanks provided with continuous flow
of air containing 100 p. p. m. ethylene for a period of six weeks. Care was
taken to place all the progenies from a single cross in the same tank to mini—
mize any possible effects of differential flow rate from the main source. After
the six weeks' exposure period each line was sampled as previously described

and analyzed spectrophotometricaily within one month. Approximately 20 roots

 

 

    
   

 
 

 

 

P

a a?
“sampled of each inbred, F1, and backcross, and from 45 to 60 roots of

Two of the lines (Lots 11 and 12), both Sl's, were harvested on two
different dates three weeks apart. The roots were quite small at the first
harvest and of a marketable size at the second. Roots from both harvests
were exposed to ethylene for the same length of time under identical condi-
tions of temperature and humidity and analyzed for the bitter compound by a

method to be described.

B. Physiological Studies

 

1. Analytical Procedures

The procedures for the quantitative estimation of the bitter principle
in carrot roots were based on an original method developed by Sondheimer e_t a_1.
(1955). Since the present investigations necessitated single-root analyses con—
sisting of the extract of a 3- to 4-gram sample, it was necessary to modify the
original procedure. The initial modification utilized in 1957 and 1958 employed
microblendor containers into which a weighed sample was placed with 40 ml of
the solventl{ blended for 45 seconds, and the extract decanted off into a small
bottle which was capped until analysis. This method proved quite satisfactory,
although settling out of particles in suspension caused a fluctuation in optical
density. Careful checking showed that this effect did not significantly change

the relative peak heights. Nevertheless, this method of analysis was time-

1
-/Skellysolve B, obtained from the Skelly Oil Co. , Chicago, Ill. , and purified
by the method of Graff e_t aL (1944).

 

 

 

   
  

inning and a faster and more suitable procedure was sought.
In 1959, a procedure was devised in which a previously weighed
sample was transferred while still frozen into a two-ounce sample bottle,
50 ml of the solvent added with an automatic pipetting machine, and the bottle
capped and allowed to stand at room temperature until extraction was com-
plete. A series of preliminary trials utilizing carrot slices of different thick-
nesses and varying time periods showed that a 24-hour extraction period gave
optimum results.

All samples were analyzed on a Beckman Model DK-2 Ratio Record-
ing spectrophotometer at wavelengths of 240, 265 and 290 millimicrons as
suggested in Sondheimer's (1955) procedure. In some cases where the back-
ground absorption was too high to obtain a reading, the sample was diluted to
a given volume with pure solvent and the bitter principle estimated on the
basis of the diluted sample. For each breeding line two or three samples
were selected at random and the complete ultra-violet spectra were traced
to test for any differences between lines.

The data from the breeding lines was tabulated and the levels of
bitterness are reported here as the bitterness index, in which only the 265 mp
peak was considered. For each breeding line the mean, standard deviation,
standard error, and coefficient of variation were computed. Histographs show-
ing the distribution of roots for bitterness index were also prepared for each

line to give a graphic representation of progeny behavior (see Appendix Figures

 

 

  
 

 

i .'
, g :ipetric data and the fluorescence ratings of each root.

J“.
i,
(.1 '
,

 

.-j;-f;{.~: n-“"'-"=. . __ 1'9.

 

2. Radioisotope Experiment

 

An experiment was designed to determine whether ethylene becomes
incorporated into the isocoumarin molecule. A quantity of doubly- labelled C14-
ethylene was obtainedl/with a specific activity of 11. 2 millicuries per milli- l
mole. The volume obtained was 0. 5 millimoles, which amounted to approxi— ‘
j
mately 11 ml of ethylene and a total amount of radioactivity of 5. 6 millicuries, ‘
or the equivalent of 20. 7 X 107 disintegrations per second.
The experiment was initiated on September 16, 1959. From a carrot
breeding line (Lot 15) which was thought to be genetically predisposed to de-
velop bitterness, six roots weighing a total of 411 grams were selected for
treatment. These were placed in a large dessicator having a volume of approx-
imately ten liters and a hole in the top. A tight—fitting rubber stopper was fitted
with a slim brass rod and two glass inlet tubes equipped with stopcocks. The
vial of labelled ethylene was also placed in the container and the lid heavily
greased. The stopper was fitted in place, all joints sealed with a plastic coat—
ing, and the seal of the ethylene vial was broken to release the gas by forcing
the metal rod through the stopper. The entire system was stored at 34°F and
was kept completely sealed. The next step was to determine how long the
oxygen in the container would last before anaerobic conditions would set in.

y

Purchased from Volk Radiochemical Company, Chicago, Illinois.

 

 

 

 

of oxygen present in the system initially would last about twenty days. Allow-

ing a margin of safety it was decided to re- gas the system after fourteen days
and sample the tissue for the incorporation of ethylene before allowing further
exposure. On October 1, fourteen days after the experiment was begun, the
CM-ethylene was scrubbed out of the system, following a procedure developed
by Young e_t a_1. (1951, 1952). The air flow through the system was regulated
as close to 100 ml per minute as possible and the scrubbing was continued for
tWenty-four hours. This rate provided an estimated ten to twelve complete air
changes in the system, which on the basis of published results, should have
completely removed the ethylene. About two hours after the scrubbing was
begun the cover of the container loosened and popped momentarily, probably
due to a combination of the pressure inside and the warming effect on the
grease seal. Possibly some of the labelled ethylene was lost during this inci-
dent. However, aliquots of the scrubbing solution containing the trapped ethy-
lene in the mercury complex were counted and showed about 5000-6000 counts
per second activity remaining in the solution. Lacking a liquid scintillation
counter it was impossible to determine accurately the counting efficiency.
However, on the basis of these approximate measurements, there appeared to

be enough activity remaining to continue the experiment.

After scrubbing was completed, the system was opened and three

I
— Unpublished report by R. C. Wright and T. M. Whiteman, cited in U. S.D. A.
Handbook No. 66, 1954.

 

 

 

 

 

 

 

  

4’5”" 'were sampled for the presence of radioactivity and isocoumarin, by a
- I. procedure to be presently described. The top (remaining green portion of
the petiole), tip (lower one inch of the root), and crown (upper one-half inch
of the root) were sampled. After sampling the system was resealed and the
ethylene regenerated by allowing HCl to run into the ethylene-containing
scrubbing solution. The system was placed in the coldroom to create a par-
tial vacuum in the chamber and after fifteen minutes the regenerating vessels
Were sealed off from the system with a stopcock.
At this time recent data on whole—carrot respiration rates were ob-
tained at the same temperature as used in these investigations (Schmalfeld,
1 960b). This data indicated a higher rate of oxygen uptake than had been pre-
viously calculated, and that only about seven days' oxygen supply could be contained
in the system. Therefore, some means of providing oxygen to the system was
sought which would circumvent the undesirable procedure of scrubbing out the
ethylene.

On October 1, seventeen days after the first sampling of the system,
approximately one and three-fourths liters of oxygen were added to the system.
A large Erlenmeyer flask was filled with oxygen, connected to the ethylene sys-
tem, and water allowed to run into the flask by gravity, thereby forcing oxygen
into the ethylene container. Carbon dioxide was then scrubbed out by means of

a reciprocating pump connected between a KOH scrubbing tower and the ethylene

 

 

 

 

 

 

    
  

_ eggfialner to prevent pressure build-up in the container from the increased

, t A Imetabolic rate after addition of oxygen.

I The addition of oxygen was repeated one week later and the experiment
terminated the following week when one of the exhaust tubes was accidently
broken and the ethylene lost. On November 6, after six weeks' exposure to
ethylene, the carrots were removed from the chamber, placed in a plastic bag,
and frozen at -10° F. Four days later they were removed and thawed. At this
time 387 grams of carrots remained. The whole roots were cut into fine

, 3 pieces, 100 ml water added, and the mass blended in a Waring blendor. To

r this mixture 500 ml purified Skellysolve B were added and the mixture shaken
for five minutes. The solvent was decanted off and 0. 5 ml aliquots were plated
out and counted. The residue was heated to boiling with 500 ml of water for
tWenty minutes, cooled and filtered, and O. 5 ml aliquots of the water extract
plated for counting. Samples of the insoluble residue were also smeared on
planchets for counting. The remainder of the Skellysolve extract was saved

for spectrophotometric analysis.

3. Oxygen-Bitter Compound Relationships

On the basis of the high respiration rates observed for carrots and
Bessey's (1957) observations that carrots did not develop as much isocoumarin
when stored under lowered oxygen tensions, an experiment was designed to test

the dependence of isocoumarin development on the presence of oxygen. A bushel

 

 

 

   

   

. .5
-\
r

 

.455 ' 1/
~.__'ii:oumarin-free carrots was obtained' and divided randomly into five

 

. . 'II’I -. E. 1 ueaments’ as follows: ‘

A. Provided with a continuous flow of pure (ethylene—free) air.
B. Provided with a continuous flow of air containing 100 p. p. m.
ethylene.
C. and D. Sealed in an atmosphere of nitrogen (standard purity)
containing approximately 100 p. p. m. ethylene.
E. Sealed in an atmosphere of nitrogen. 1
The pure air and air plus ethylene treatments were provided by the methods ‘
used to evaluate the breeding material. In this instance a smaller number of
roots were used and were placed in respiration jars. The nitrogen and nitrogen
plus ethylene treatments were achieved by placing the carrots in large dessi-
cators provided with single sleeve-openings in the top. The dessicators were
evacuated by a water—pump vacuum and filled with nitrogen gas from a cylinder
which was connected to the system by heavy-walled tubing. The process was
repeated three times to flush as much oxygen as possible out of the system.
After the third flushing the system was again evacuated and ethylene was
metered in to a concentration of approximately 100 p.p. m. Nitrogen was then
introduced to complete the atmosphere until the internal pressure approximated
atmospheric and the sleeve turned to seal the system. All treatments were kept

in a 33° F storage until sampling.

1
—/Provided by Gerber Products Company, Fremont, Michigan.

 

 

 

”taken from each. The samples were prepared by the Sondheimer method and

 

analyzed for the presence of isocoumarin by tracing the complete ultra—violet.
spectra. Treatments A and B were resealed for later sampling. The remain-
ing roots from treatment C were placed in the pure air system to determine
whether pre-treatment with ethylene under anaerobic conditions would induce
isocoumarin synthesis if the roots were later placed in an ethylene-free
oxygen— containing atmosphere. Four weeks later (eight weeks total) treat-
ments A, B, D and E were sampled, as well as the remainder of treatment C
in the pure air system. At this time samples of the atmosphere from treat-
ments D and E were taken and analyzed for carbon dioxide and oxygen contents

With an Orsat gas analyzer, capacity 70 per cent.

 

 

   

RESULTS

  

A. Breeding Experiments

1 958

The relative amounts of bitter principle in the F1 of a cross between
two open-pollinated carrot roots and in the S1 progenies derived from these
roots are shown in Table 1.

TABLE 1

Bitterness Indices of Carrot Lines after Eight Weeks' Storage

 

Mean Bitterness Indexl/

 

Line Air + 100 p. p. m. C2H4 Apple Emanations Control
LC 1375 S1 0. 42 0. 15 0. 14
RCC 3M 81 0. 36 0.10 0. 14
(RCC 3M X LC 1375) F1 0. 41 0. 09 0.09

 

_1/ Treatments underlined are not significantly different.

The two parental roots were selected from the field in 1957 by their fluorescent
characteristics after storage in the presence of apples for ten weeks. The
parent root from RCC 3M fluoresced brightly under ultra-violet light, while
that from Long Chantenay did not. Spectrophotometric analyses indicated
bitterness values, as calculated by Sondheimer's (1955) formula of 7. 0 and 0. 0

for the two roots, respectively. Later calculations based on the revised method

 

 

 

 

   

 

_ in ,

, . only the 265 my reading yielded bitterness index values of 0. 34 and

 

- '
II In.
\

- .f' 0.29 for the two roots, which are more in accord with the mean values for

the progeniesas shown in Table 1. What appears by one method of calcula-
tion to be a cross of low and high bitterness roots is shown by another method
to be a cross involving two roots of probably intermediate bitterness. These
observations led to an examination into the causes of the fluctuations in rela—
tive peak heights and will be discussed in more detail later.

This first year's data showed no significant differences in mean bitter-
ness indices between material of identical genetic composition when stored
for eight weeks in an atmosphere of ethylene-free air and in a storage con-
taining apples. Roots of the same material stored in an atmosphere containing

100 p.p. m. ethylene gas developed higher levels of the bitter compound.

£9

The results of spectrophotometric analyses for the bitter compound in
thirty breeding lines are tabulated in Tables 2, 3 and 4. The histographs
showing frequency distributions of the bitterness indices of these lines are
available in Appendix Figures 1, 2 and 3. Most F1 means exceeded the highest
parent, suggesting that dominance and possibly heterosis may be present. The
ranges of bitterness indices in these breeding lines varied between 0. 20-0. 42
in the least variable line (Lot 15 82) and 0. 00—0. 61 in the most variable (Lot
20 F2). These represent variability ranges from two—fold to nearly twenty-

five fold between roots within lines.

 

 

 

                                                     

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. . L.’ '-
'-_ ,._ .5 ' i
I

1335.1 acients Between Bltterness Index1 and Fluorescence Rating
' for Thirty Carrot Breeding Lines

   
 

 

 

 

r Signif. Lot No. Gen. r Signif.
Cross No. 1 . Cross No. 2
P1 —0. 51 * 17 F1 +0. 11 N. 5.
F1 -o. 07 N. s. 18 BC1 -0. 37 N. s.
BC1 +0. 01 N. s. 19 F2 +0. 45 **
BCl +0. 45 * 20 F2 +0. 59 **
F2 -0. 16 N. s. 21 s2 +0. 05 N. 5.
F2 -0. 07 N. s. 22 s2 -0. 15 N. s._
F2 +0. 04 N. 5.
F2 +0. 01 N. s.
, Fl x F1 +0.35 *
1 F1 x Fl -0. 06 N. 5.
1121 S1 +0. 08 N. S. Other Crosses
' ‘ 11b s1 -0. 39 N. s. 23 F1 +0. 06 N. s.
12a s1 -0. 37 N. s. 24 F1 -0. 15 N. 5.
12b 51 -o. 43 N. s. 25 F1 +0. 12 N. s.
13 s2 +0.35 N. s. 26 F1 +0. 04 N. s.
14 s2 +0.06 N. s. 27 F1 -0. 26 N. s.
15 s2 0. 00 N. s. 28 s4 -0. 02 N. s.
16 s2 -0. 39 N. s

* Significant at the 5% level.
"’3‘ Significant at the 1% level.

 

/
‘ AS measured spectrophotometrically.

 

 

 

  

vi. _l.

 

I The mean bitterness indices for the two inbred lines harvested three
weeks apart showed higher values for the earlier harvest in both cases, as
shown in Table 2. Correlation coefficients for bitterness index and fluores—
cence as determined visually with an ultra-violet light are presented in Table
5. The values ranged from +0. 59 to —0. 51, with most of the coefficients ap—
proaching zero. No definite relationship between these correlation coeffi-
cients and the mean bitterness values or the type of ultra-Violet absorption

spectrum was evident.

B. Physiological Studies

The ultra—violet absorption spectra of random samples of roots from
each of the breeding lines exhibited extreme differences, although for a given
line the spectra were practically identical. As shown in Figure 1, some of
these atypical lines exhibited spectra with a number of peaks, instead of the
characteristic two peaks of pure isocoumarin (Figure 2). The breeding lines
showed varying degrees of ultra-violet spectra between two extremes. Exam-
inations of these spectra revealed that, in general, the atypical peaks occurred
in the regions of 243, 249, 254 and 261 my.

A solution of pure isocoumarin which had exhibited the characteristic
spectrum shown in Figure 2 when freshly prepared, was found to undergo
Changes when stored at 33°F over a period of time, as shown in Figure 3.

COmparisons of these spectra with those exhibited by some of the breeding

 

  

 
  

    

Optical Density

 

 

  

 

Optical Density

 

l l
240 265 278
Wavelength (mp)

  
  

Figure l. Atypical ultra—violet absorption spectra of Skellysolve B
extracts of carrot breeding lines.

     
   

 

   

265 278
Wovelength(m}1)

Figure 2. Ultra—violet absorption spectrum of a Skellysolve B solution
of crystalline bitter compound.

 

 

A
a;
U)
c
to
Q
E
.0 .
v:
Q.
0

 

 

 

 

 

l l l
240 265 278 300
Wavelength (mp)
Figure 3. Ultra—violet absorption spectra of a Skellysolve B solution
of crystalline bitter compound stored in the dark at 33°F.
.6 Carrot Extract
’/”Lor7,Roor/8

.5 —
i E
‘g .4
Q:
Q >—
3 .3 —
.2
Va —
Q '2 _

I —- . .
Crystal/me bitter Compound (IOmg/I)
l l
l .o l 1 I I I I 1 I 1

I
i 240 265 278 300
Wavelength (mp)

, Figure 4. Ultra—violet absorption spectra of a fresh carrot extract
and a solution of bitter compound stored at 33° F for 14 weeks.

 

 

 

  

, .' limes showed remarkable similarities, as shown in Figure 4. In an effort to

determine whether a definite relationship existed between the spectra of carrot

 

extracts and that of the pure compound, attempts were made to accelerate
breakdown of the pure compound by exposure to white light, heat, and addition
of water. These attempts were unsuccessful. The breakdown of the pure com-
pound in purified Skellysolve B solution appeared to be a spontaneous reaction
that occurred at a slow rate.

The observations on these atypical spectra resulted in a modification of
the calculations of bitterness values. The method of calculation proposed by
Sondheimer yields a bitterness level expressed as height of the 265 mp peak
above a base line passing through the 240 and 290 mp points. However, the
unusually high 240 mp readings observed in many of the breeding lines altered
the slope of this base line to such an extent as to bias the 265 mp peak height
downward. For this reason the 240 mp value was not used in the work reported
here.

Dilution studies on the pure crystalline compound dissolved in purified
Skellysolve B showed a direct proportionality between concentration and heights
of the 265 and 300 mp maxima and 240 and 278 mp minima. These data indi-
cate that the use of a base line is unnecessary in the calculation of bitter com-
pound concentration (see Appendix Table 1). On this basis the 265 mp value
alone was chosen for calculations of the bitterness indices reported in this

work. The 265 mp was preferred over the 290 reading because the latter value

 

 

 

J - :_.'. --.'
.‘I .-
.« 1..

'ys occurs on a side peak and results in significant error with small wave-

   

       

  

length fluctuation. In many of the atypical spectra the 300 mp peak is also de-
pressed. Admittedly in some cases the 265 mp measurement also occurs on
a side peak, but the resulting errors are not as great in proportion to the peak

height as with the 290 mp reading.

1. Radioisotope Experiment

 

The results of the 014—labeled ethylene experiment are shown in Table
6 below.
TABLE 6

Incorporation of Cl4—ethylene into Carrot Tissue, Expressed as Counts per
Second over Background

 

 

 

Time Skelly B Water Residue
2 Top 0 0 0
Tip 0 O 0. 2
Weeks Crown 0 0. l 0. 1
6 Whole
Weeks Roots 0. l 0 0

 

Although some slight elevation in activity above background was obser—

 

ved in a few cases, the differences were not significant and the degree of in-

corporation was essentially zero. The spectrophotometric analyses, although
inconclusive at two weeks, definitely showed the presence of the bitter com-
1

pound at six weeks, as shown in Figure 5.

 

 

 

 

  

Optical Density

 

 

  

 

  

l I
265 278 300
Wavelength (mp)

Figure 5. Ultra-violet absorption spectrum of a Skellysolve B
extract of 014—ethylene treated carrots.

 

   

 

 

  

..‘.'.-' 2'. mmtter Compound Relationships

 

  

The results from the carrots treated with ethylene under aerobic and

   

anaerobic conditions are tabulated in Table 7.

TABLE 7

Effects of Aerobic and Anaerobic Storage Conditions on Development of the
Bitter Principle in Carrots

 

 

Treatment 4 Weeksl—/ 8 Weeksi/
A Pure air 0. 0 0. 0
B Air+ 100 p.p.m. CZH4 1.7 2.3
C N2 + 100 p.p. m. C2H4 0. 0 ———
D N2+ 100 p.p. m. C2H4 "" 0.0
E N2 - - - O. O
2/
Pretreatment— - - - 0. 0

 

l /
— Mean of two samples calculated by Sondheimer's formula.

2/

— Pretreatment of C2H4+N2 for four weeks followed by pure air for four weeks.

The complete ultra-violet spectra are reproduced in Figures 6 and 7.
Examination of the roots at the four-week sampling showed them to be

in good condition in the anaerobic treatments but in fair condition in the aerobic

treatments. Those under the latter conditions had developed some mold growth

and there was evidence of the early stages of rot infection. No evidence of rat

 

 

  

Optical Density

Optical Density

[Tj

   

_ (Air +lOOp.p.m. C2H4

  

 

 

l l l
240 265 278 300
Wavelength (mp)

Figure 6. Ultra-violet absorption spectra of carrots exposed to
ethylene under aerobic and anaerobic conditions four weeks.

v(Air+/OOppm. C2H4

 

 

1

l
240 265 278 300
Wavelength (mp)

Figure 7. Ultra-violet absorption spectra of carrots exposed to
ethylene under aerobic and anaerobic conditions eight weeks.

 

 

 

 

 

 

Taste evaluations of the roots

.. observed in the anaerobic treatments.

 

showed the presence of bitterness only in the air plus ethylene treatment.
At the eight-week sampling the roots under anaerobic conditions were

in much better physical condition than those in the aerobic treatments, es-

pecially in regards to mold growth and rotting. There was a decided odor

of fermentation in the anaerobically-treated roots, but bitter taste was again
evident only in the air plus ethylene treatment.

Gas samples taken from the nitrogen plus ethylene and the nitrogen
treatments at eight weeks showed carbon dioxide contents of 54 and 80 per

cent, respectively. The oxygen contents of both treatments were too low

to m easure.

 

 

. _-_.. ‘

    
   
 

DISCUSSION

A. Breeding Experiments

The observations in 1958 on the breeding lines placed in three treatments
shOWed very low values and no significance between the controls (no ethylene)
and the roots stored in the presence of apple emanations. Those exposed to
100 p.p. m. of ethylene gas for nine weeks developed higher levels of the bitter
compound, as measured spectrophotometrically. These results indicate that
the storage of carrot roots from breeding lines in the presence of apples as a
means of inducing bitterness was not satisfactory. The concentration of ethy-
lene in the storage atmosphere undoubtedly depends upon variety and quantity
of apples, degree of maturity of the fruit, temperature, and ventilation. In
these experiments the ethylene content of the storage atmosphere was obviously
too low to provide an adequate screening of the material. As a consequence,
all breeding material was screened thereafter under controlled conditions in

respiration tanks fed from a common ethylene-air source.

E

The 1959 results of spectrophotometric determinations indicate that
dominance, and possibly heterosis, is acting in the F1 progenies. Although
the bitterness indices of progenies and their parental roots are not strictly

comparable because the two must be grown in separate growing seasons, the

 

 

 

 

  

2' .: “It's nevertheless indicate a definite trend toward hetero sis in the F 1's.
The extreme ranges in bitter content observed between roots within
some progenies are especially significant. For example, in F 2's, bitterness
indices of roots within one line (Lot 20) ranged from 0. 00 to 0. 61. In another
F2 (Lot 5) roots varied from 0. 03 to 0. 74, nearly a twenty-five fold range
within the one line. Most other Fz's varied over ranges of four- to ten-fold,
while the bitter compound levels in inbreds and backcrosses varied from two—
to four-fold. This extreme variability observed within lines is probably not
due to environmental effects, which under the conditions of these studies would
be expected to be low. This might be demonstrated by using some of the $2
inbreds as examples (see Table 2). If one assumes that the total variability
in these lines is environmental, which is extremely unlikely since these orig-
inally heterozygous lines were inbred for only two generations, then the maxi-
mum environmental effects would account for only a two— to four-fold varia-
bility range within lines. The only plausible explanation for the much greater
observed variability is that the differences between plants are primarily due
to genetic factors. These wide ranges suggest the possibility of selecting
carrot roots that produce the bitter compound in low concentrations after ex-
posure to ethylene. In fact, the occurrence of no bitter compound at all in
one root (in line 20 F2) suggests the possibility of isolating genotypes incapable

of synthesizing the compound at all. More work is necessary to isolate single

 

 

 

 

”t <_ ‘. cts lacking the bitter principle and inbreeding to fix the genotypes.. Selec-
tion in the breeding program must be based upon a standard ethylene expos-
ure and precise quantitative determinations of bitter compound in order to
obtain non-bitter roots for use in establishing inbred lines and in producing
hybrids.

As shown in Appendix Table 2, significant differences existed between
F2 means of progenies derived from Crosses 1 and 2. In both instances
the mean of one F2 is approximately double that of another from the same
F1 cross. This fact alone would suggest a degree of heritability, since if
a genetic difference did not exist between the F1 roots, no differences would
be expected between progenies grown under the uniform conditions provided
in these studies.

Comparisons of progeny means with bitterness indices of parent roots
shOWed a low degree of association. Under conditions of fairly high herita-
bility this correlation should be much higher. No clear explanation for
these results is at hand, although as pointed out earlier there may be inter-
actions of environment with genotype. Furthermore, since heritability de—
pends on the transmittance of genes which are additive for the trait in ques-
tion, a condition of heterosis such as is suspected in this case would result
in a high non—additive genetic variance and thus a low degree of heritability.

Under these conditions the solution to the inheritance depends on the fixation

    

l

 

 

 

  
 

 

_; .2551” the additive elements in inbred lines. In an improvement program,

however, care must be taken not to overlook other characteristics of

the material, such as yielding ability, color, sugars, and other desirable
traits. A breeding program considering all these characteristics as well
as selection for non-bitterness might effectively utilize a mass pollination
or family breeding technique. The application of this type of mild inbreed-
ing to fix desirable characteristics would at the same time allow for fur—
ther selection by retaining some heterozygosity in the population. This
technique is preferred to selfing, which fixes genotypes so rapidly that
many of the desirable features may be lost in the early generations and
further selection would be useless.

The open-pollinated material from which these crosses were derived
was extremely heterozygous, which undoubtedly accounts for the wide seg-
regation observed in the F1 progenies and to a lesser extent in the inbreds.
At the present time extremely high and low segregants are being crossed
in the greenhouse in an attempt to obtain more conclusive genetic evidence
through the use of parent-progeny regression techniques. Of course, if
the additive F2 genetic variance is low due to high interaction variance,
then the F2 heritability will also be low. Further selections and parent-
progeny tests in F3, F4 and possibly in F5 may be necessary to conclusively
evaluate heritability.

Examinations of the correlation coefficients for bitterness index and

 

 

 

"These results did not agree with the high correlations obtained by

 

Schmalfeld (1960a). However, it was pointed out by Schmalfeld that
these correlations were high only when the bitter compound was present
at low levels, indicating that the visual range does not approach the
spectrophotometric range in sensitivity. This could explain the lack of
correlation between the fluorescence ratings and the quantity of bitter
compound present in the roots, where the bitterness values were generally
high. It should also be emphasized that with an open—pollinated and highly
heterozygous crop, such as carrot, that all lines and all genotypes do not
respond alike. Depression of vigor related to inbreeding can vary widely
between lines and pleiotropic effects may occur to greatly influence response.
A third explanation might be that the presence of breakdown products
in considerable quantities alters the degree of fluorescence, since only
specific chemical bonds capable of resonance can bring about fluorescence.
According to Glasstone (1946) fluorescence is the emission of radiant energy
which has been absorbed by a molecule, either at the same or at another
frequency. When radiation is absorbed the electronic energy is raised to
higher energy levels or "excited". If this additional energy is not removed
by collisions with other molecules, the electronic energy can return to its
stable or ground state only by the release of the excess energy as fluores-

cence. The situation in the case of the bitter compound may be that, as

 

 

  

. radiant energy is absorbed, some of it is utilized in a molecular decomposi—
.tion. If this occurs, the excess energy does not accumulate, and thus the
breakdown product in the carrot may not fluoresce. Whether or not this
occurs is speculative. At any rate the low degree of association between
fluorescence and the quantity of bitter compound present in carrot roots
points out the inadequacy of selection of breeding material by fluorescence
observations. For critical work selection should be based on a more objec-

tive procedure.

B. Physiological Studies

During the course of spectrophotometric analyses of roots from carrot
breeding lines, atypical ultra-violet absorption spectra were noted, and these
spectra appeared to be identical with those of crystalline bitter compound
which had been stored in purified Skellysolve B solution at 33° F for a period
of time. These observations strongly suggest that isocoumarin is present in
more than one form in carrots roots, and roots (and hence their progenies)
Vary in the degree to which isocoumarin is present in the "pure" form. The
fact that the spectra of some carrot extracts are identical with those of a pure
Solution in which decomposition has occurred suggests that the unusual peaks
in the ultra—violet spectrum are due to rearrangements or degradation of the
molecule. The most likely type of chemical rearrangement of a lactone-ring

Structure would be the opening of the lactone ring. Two types of reaction are

 

 

 

 

 

    

fi’tprobable. Hydrolysis of a ring lactone can _occur as discussed by
, ' -,~1d (1953), and shown in the following equation:

OH I? OB

0001-1

H300 1101-13 H3C0 CHZCHOH CH3
H2
This type of reaction might occur in the carrot root, but would not be ex-
pected in a purified Skellysolve solution of the crystalline compound. The
possibility exists, however, that a sufficient quantity of water was retained

in the solvent after purification to initiate a hydrolysis reaction.

.19;

A more likely type of rearrangement for this compound is a ring-chain
tautomeric shift. As discussed in Newman (1956), this type of reaction can
occur in any system in which an equilibrium can be established between an
open-chain compound and its cyclic isomer. Five and six-member lactone
rings, of which the bitter compound is one, are especially susceptible to
this type of tautomeric shift. The reaction involves a rupture of the ring and
a subsequent enolization to the enoic acid form- Such a shift could occur in
the bitter compound as follows:

0H 0 0H
II

COOH

H3C0 HCH3 H300 CH = CH—CI—l3

H2

 

 

 

 

..'. tion spectra, both in the ultra-violet and infra-red, and can occur in both

--,- aqueous and non—aqueous solutions as well as in the gaseous phase. The
stability of the cyclic isomer appears to depend primarily on the pres-

ence of substituents attached to the lactone ring. In this case the presence

 

of the 3-methyl group may favor ring closure resulting in an equilibrium
which tends more toward the cyclic form.

Other possibilities of degradation could involve cleavages of the
functional methyl, methoxyl, and hydroxyl groups from the aromatic ring,
or possibly rearrangements within the lactone ring such as a shift in posi-
tion of the oxygen to produce a coumarin derivative. These might occur
in the fresh carrot roots by enzymatic catalysis, but appear to be less
likely general explanations since they would not be expected in solutions
of the pure compound.

The possibility that the interfering compound or compounds are
precursors of the isocoumarin molecule should not be ruled out. Although
no evidence has yet been obtained to support this possibility, there are un-
doubtedly precursors formed during the biosynthetic process and it is entire-
ly possible that some of these may be identical to breakdown products of the
pure compound mentioned previously. The products of the crystalline com-
pound could possibly be identified by comparing the absorption spectrum with

that of the undecomposed compound at lower energy levels, such as the infra-

 

 

 

 

  

 

   

:i 5
region. Comparisons of these results with the absorption spectra of

_I-"abarrot extracts should provide more conclusive evidence as to whether the

two are the same.

The occurrence of breakdown products in carrot extracts would bring
up an important point in regard to the solution of the bitterness problem,
which, of course, is an important ultimate objective of this work. Although
the criteria of bitter flavors are not well known, it is entirely possible that
the breakdown product may not be bitter to taste. The answer to this problem
rests in the fractionation and separation of the two or more compounds which
occur in the carrot, and subsequent flavor evaluations of the isolated com—
pounds. If the breakdown product(s) should be found to be non-bitter, and is
produced by degradation of the isocoumarin molecule, a solution to the bitter—
ness problem could conceivably be achieved by selecting for the presence of
the breakdown compound. This line of approach is suggested by the work with
the bitter cucurbitacins in cucumber (see Rehm and Wessels, 1957). Bitter—
ness in cucumber fruits is controlled by a genetic mechanism which suppresses
the occurrence of cucurbitacins in non—bitter types, probably by degradations

or diversions of the bitter compounds to other metabolic pathways.

1. Radioisotope Experiment

 

Although it has been shown rather conclusively that ethylene gas will

initiate the development of isocoumarin (Ells, 1958), there has been little

 

 

 

 

three mechanisms of action have been proposed. Ethylene could be acting as

a catalyst, as a "triggering" or initiating substance, or it could enter directly

into the bitter compound as a substrate or co-substrate. With available tech-
niques it should be possible to distinguish at least between the first two possi-
bilities and the third by employing radioactive "labelled" ethylene to initiate
the development of isocoumarin. An experiment utilizing this approach has
been described.

Based on the original amount of radioactivity, calculations assuming a

ten per cent efficiency of the counting equipment indicated that as low as O. 01

 

per cent incorporation of the labelled ethylene into isocoumarin would give a
total of about eighteen counts per second, an activity well within the range of
significance. A rate of incorporation this low would actually be of little signi—
ficance since it is entirely possible that this concentration of ethylene could
be trapped "per se" in the carrot tissues without ever being metabolized.

After exposure of an inbred line (Lot 15 $2) to C14-labelled ethylene
for periods of two weeks and six weeks, no incorporation of the labelled mater-
ial was found in either instance. These observations parallel those obtained

4—ethylene into fruit tissue

by Buhler e_ta_l_. (1957), in which incorporation of C1
Was obtained with only two species out of seven tested, and in these two at a

rate of only 0. 05 per cent. Their conclusions that ethylene is a terminal

 

 

 

 

 

 

 

 

  

. ct of metabolism would appear to be strengthened by the work reported

there. However, their work involved fruits which are laiown producers of

ethylene. The present experiments may not be exactly parallel, since the
rate, if any, of ethylene evolution by carrots has not been reported.

As shown in Figure 5, a quantity of the bitter compound was synthe-
sized in the carrots corresponding to a bitterness index of 0. 15 during the
course of the experiment. Simultaneous exposure of the same breeding line
to unlabelled ethylene produced somewhat higher levels of the bitter compound,
as shown in Table 2 (Lot 15). Since no labelled ethylene was incorporated .
into the compound, it can be concluded that ethylene probably does not act
as a substrate or co—substrate in the synthesis of the bitter compound.
Although there was some question as to the availability of oxygen in this ex-
periment, other results obtained in the studies on oxygen requirements indi-
cate that the oxygen supply was adequate, although not optimum. The lower
level of bitter compound development can probably be attributed partly to a
somewhat lowered oxygen tension and partly to a shorter time of ethylene ex—
posure in the radioisotope experiment as compared to the breeding experiments.
Conclusive statements as to the role of ethylene will ultimately depend on
further and more extensive work. Investigations utilizing suspected isocou-
1'I‘Iarin "precursors" in conjunction with labelled ethylene should provide
evidence as to the actual metabolic intermediates involved, and to more directly

link the role of ethylene to isocoumarin biosynthesis.

 

 

  

 

-'l:’
$3.3

__ fidygen-Bitter Compound Relationships

The investigations into the oxygen requirements of bitter compound
development showed the simultaneous requirement of both e thylene and mole-
cular oxygen for synthesis of isocoumarin to be initiated. No bitter principle
was detected under conditions of ethylene without oxygen or oxygen without
ethylene. It was also found that four weeks' pre-treatment of ethylene under
anaerobic conditions would not initiate isocoumarin development even when
the roots were later placed in an aerobic situation.

These results would appear to have implications in the biosynthetic
pathways involved in isocoumarin synthesis. The results obtained by Burg
and Thimann (1960) showed the dependence of ethylene evolution on the presence
of oxygen and respiratory activity and it has been shown here that development
of isocoumarin depends both on the presence of ethylene and oxygen. These
results suggest that the biosynthesis of the bitter principle in the stored root
depends either upon the availability of aerobically—produced high-energy phos-
phates or on the presence of oxygen as a terminal electron acceptor in the
biosynthetic process.

As demonstrated by Schmalfeld (1960a) the ultimate level of the bitter
compound in carrot roots depends on the length of time of exposure to and

concentration of ethylene during pre—treatment. These results and the re-

sults described in the present investigations suggest that ethylene does indeed

 

 

 

 

i—L.

 

., -=. ction as a heterogeneous catalyst and not as a substrate in the biosynthems

 

of the bitter compound. Future work regarding these aspects would probably
be mast fruitful if directed toward determining whether ethylene is produced
by the carrot root and whether the effect of anaerobic conditions is to lower
the availability of high—energy compounds necessary for isocoumarin synthesis,
or to prevent a terminal oxidation reaction which is dependent on oxygen as
the electron acceptor. Studies should show whether specific inhibitors have
the same effects on isocoumarin synthesis as on ethylene evolution, and
whether other electron acceptors than oxygen may be able to carry on the

biosynthesis under anaerobic conditions.

 

 

 

SUMMARY AND RECOMMENDATIONS

 

The objectives of this work were to determine whether inherent dif-
ferences existed between carrot roots in their abilities to synthesize 3-methyl-
6-methoxy-8—hydroxy-3, 4-dihydroisocoumarin, a bitter compound commonly
called isocoumarin, and to determine some of the physiological properties
of this compound. Quantitative analyses of the bitter compound in individual
carrot roots from thirty breeding lines involving seven crosses showed ex-
treme variability between roots within lines. Variability ranges as great as
twenty-five fold were observed in an F2 of one cross, while ranges as low
as two-fold were observed in some inbreds. Evidence of dominance or heter-
osis was observed in F1 progenies, and significant differences were found
between separate F2 progenies derived from the same original cross. These
factors suggest a degree of heritability for the trait, although it was probably
low in these early generations due to a high proportion of non—additive com—
ponents. Further work involving parent—progeny regression techniques in F3,
F4, and later generations should give a more definite estimate of heritability.
The observations in this work suggest that selection and breeding would be a
possible means of eliminating this type of bitterness from carrots.

The atypical ultra-violet absorption spectra observed for many of the
breeding lines indicated that more than one compound was present in these

roots. Subsequent investigations showed some of these atypical spectra to be

 

 

 

'1' u: - cal to those of the pure crystalline compound after storage in the sol-

 

. vent at 33°F for varying periods of time. These atypical spectra may result
from enzymatic or non-enzymatic degradations or molecular arrangements,
or from the presence of precursors of the bitter compound in the stored root.
Such reactions might involve hydrolysis, functional group cleavages, or more
probably, ring—chain tautomeric shifts. Further work is necessary in frac-
tionation and identification of the interfering compounds utilizing such tech-
niques as column chromatography and infra-red spectroscopy. The bitterness
and fluorescent properties of these compounds should also be investigated,
since one solution to this type of bitterness could involve selection for the de-
gradation products if they should be found to be non-bitter.

Studies aimed at determining the role of ethylene in inducing the devel-
opment of the bitter compound in storage demonstrated that ethylene was not
incorporated and metabolized by carrot roots from one breeding line, although
the bitter compound was synthesized by the roots. These results suggest that
ethylene does not act as a substrate or co-substrate to become part of the
molecular structure of the bitter principle, but probably functions as a heter-
ogeneous catalyst by accelerating the biosynthetic reaction rates. Further
studies involving feeding experiments with radioactive—labelled "suspect" pre—
cursors should prove valuable in elucidating the biosynthetic pathways and in

more specifically linking the role of ethylene to the process.

 

    
  
  
  
   
  
  
   
  
  
  
  

cular oxygen and ethylene simultaneously did synthesis of isocoumarin take
place. These results seem to be related to the findings of Burg (1959) and
Burg and Thimann (1960), who found that ethylene evolution by apple tissue
appears to depend on the availability of high-energy compounds produced by
aerobic respiration in the mitochondria. It appears that such high-energy
compounds might also be required to drive the biosynthetic reactions of iso-
coumarin formation, or alternatively, that oxygen functions as a direct elec-
tron acceptor in a terminal oxidase reaction. More intensive investigations
into the interrelationships of aerobic and anaerobic conditions with bitterness
formation may bring about valuable means of controlling the problem as well
as providing data on the metabolic processes which take place in storage

organs, SUCh as carrot I'OOtS.

 

   

 

     

LITERATURE CITED

 

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Barham, W. S. 1953. The inheritance of a bitter principle in cucumbers.
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Bessey, P. M. 1957. Studies on the occurrence, measurement, and control
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Borchers, E. A., and C. S. Nevin. 1954. Quantitative estimation of a bitter
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Brown, H. D., M. K. Miller, K. Alban, R. Short, R. Schulkers, and C. Murname.
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Buhler, D. R., E. Hansen, and C. H. Wang. 1957. Incorporation of ethylene
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l s - I
'. '-' .-
_- .

   

 

   

 
 

 

., "Clayton. J. S. , and R. K. Larmour. 1935. A comparative color test for
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. 1950. Fluorescence of coumarin derivatives
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. 1952. The fluorescence of coumarin deri-
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sin. . t

 

 

 

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APPENDIX

 

    

TABLE 1

Compound in Skellysolve B Solution];

 

- -Relation of Concentration to Ultra-violet Absorption Spectrum for Pure Bitter

 

Wavelength (mp)

 

 

 

 

 

 

 

 

Conc.
236 240 265 278 290 300
3 mg/l --- .0033 .055 .0017 .010 .018
6 mg/l . 014 . 017 . 068 . 013 . 023 . 029
12 mg/l .014 .017 .067 . 012 .021 . 028
18 mg/l .014 .016 .067 .012 .021 .029
24 mg/l .014 . 016 .070 . 012 . 020 . 029
-1-/ Values expressed as ratio of optical density to concentration.
TABLE 2
Analyses of Variance of Bitterness Indices of F2 Progenies
Source D. F. S. S. M. .S. F
Cross No. l - RCC 3M X LC 1375
Total 179 7. 06 -- —
Between Lines 3 1. 84 0. 61 20. 3**
Within Lines 176 5. 22 0. 03
Cross No. 2 - Imp. 1381 X RCC 3M
Total 107 3. 69 - --
Between Lines 1 0. 77 0. 77 27. 5**
Within Lines 106 2. 92 O. 028

 

*"‘F value significant at the 1% level.

 

 

 

 

 

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