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VHF—73 3 ‘

This is to certify that the

dissertation entitled

THE INHERITANCE OF BOLT RESISTANCE IN AN INTERSPECIFIC CROSS
SIBERIAN KALE (BRASSICA NAPUS) X CHINESE CABBAGE (B. CAMPES-
TRIS L. SSP. PEKINENSIS) AND AN INTRASPECIFIC CROSS CHINESE
CABBAGE x TURNIP (B. CAMPESTRIS L. SSP. RAPIFERA).

 

 

 

presented by
Carl E. Mero

has been accepted towards fulfillment
of the requirements for

Ph.D- degree in Horticulture

 

 

”’24 . MW“,

. / Major professor

Date 9% a {fig

MSU is an Affirmative Action/Equal Opportunity Institution 0-12771

 

THE INHERITANCE OF BOLT RESISTANCE IN AN INTERSPECIFIC
CROSS SIBERIAN KALE (BRASSICA NAPUS) X CHINESE CABBAGE
(B. CAMPESTRIS L. SSP. PEKINENSIS) AND AN INTRA-
SPECIFIC CROSS CHINESE CABBAGE X TURNIP

 

(B, CAMPESTRIS L. SSP. RAPIFERA)

 

By

Carl E. Nero

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Horticulture

1983

I ‘J \-<J\I /v

ABSTRACT

THE INHERITANCE 0F BOLT RESISTANCE IN AN INTERSPECIFIC
CROSS SIBERIAN KALE (BRASSICA NAPUS) X CHINESE CABBAGE
(B. CAMPESTRIS L. SSP. PEKINENSIS) AND AN INTRA-
SPECIFIC CROSS CHINESE CABBAGE X TURNIP
(B. CAMPESTRIS L. SPP. RAPIFERA)

 

 

 

By

Carl E. Hero

The inheritance of bolting in Chinese cabbage (Brassica

campestris L. ssp. pekinensis) was investigated by hybridizing

 

 

Chinese cabbage with Siberian kale (B, napus), Chikale (B,

campestris L. ssp. pekinensis x B, napus), and turnip (B, campestris

 

 

 

L. ssp. rapifera). The inheritance model was developed from ratios
observed in segregating populations from these crosses after various
durations of vernalizatiOn at 5°C and 16 hour daylength. Percent
of bolters was determined by the sum of visible bolters and longi-
tudinally cut plants with pointed apices, observed when the bolt-
resistant parent either visibly bolted or reached a marketable size.
Differences in bolting between Chinese cabbage, Chikale,
Siberaian kale, and an F2 population (Siberian kale x Chee Hoo)
under natural field vernalization suggested (1) differences in bolt-
ing habit observed after artificial vernalization at a constant
temperature (5°C) were similar to those under natural vernalization

with fluctuating temperatures, and (2) bolt resistance from

Carl E. Mero

Siberian kale was transferred to Chikale and the F2 p0pula-
tion.

Segregation for bolting response in the progeny from the
crosses Siberian kale x Chee Hoo, Siberian kale x Nozaki Early, and
Madarin x Siberian kale suggested that bolting response was condi-
tioned by a few major additive genes and that percent of bolters was
dependent on the Chinese cabbage cultivar used. Differences in
chromosome number between Chinese cabbage (n=10) and Siberian kale
(n=19) produced varying degrees of fertility in the progeny. The
observed segregations appear to have resulted from variable ploidy
levels, random assortment, and probable crossing over of chromosomes.
Segregation in F3 families from these crosses suggested one or more
major additive genes.

Based on these results and the observed segregations for
bolting response in the crosses Chinese cabbage x Chikale and Chinese
cabbage x turnip, it appears that 4 major genes with modifiers con-
ditioned bolting response. Genotypes for Chinese cabbage, Chikale,
Siberain kale. and turnip were proposed. Cytoplasmic effects on the

response to vernalization were also noted.

DEDICATION

To my Wife, Debbie,
and My Parents

ii

ACKNOWLEDGMENTS

The author wishes to express his special appreciation to
Dr. S. Honma for his guidance and advice during the preparation of
this thesis. Special thanks are also extended to Dr. H. Price,
Dr. J. Hancock, Dr. N. Adams, Dr. J. Hanover, and Dr. J. Nhalton
for their recommendations in the preparation of this manuscript.

Special thinks are also extended to Mr. Fred Richy, Mr.
Marshal "Lou" Pollack, and Mr. Bill Preist for their help and
cooperation in maintaining my field plots.

Appreciation and special thanks are also expressed to my
wife, Debbie, and to my parents and family for their immeasurable

encouragement and support.

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES

INTRODUCTION . . . . . .
LITERATURE REVIEW .

Vernalization .
Interspecific Hybridization .

MATERIALS AND METHODS .

Parent Material .
Chinese Cabbage
Kale . .
Chikale .
Turnip .
Hybridization
Vernalization .
Determination of Plant Age Effect on Vernalization
Response . . . . . .
Establishing Bolting Criteria
Vernalization Experiments . .
Experiment 1: Natural Vernalization of Siberian
Kale, Chinese Cabbage, an F2 Population (Siberian
x Chee H00) and Chikale . .
Experiment 11: Artificial Vernalization Studies
Siberian Kale x Chinese Cabbage . .
Experiment III. Artificial Vernalization Studies
of Siberian x Nozaki Early F3 Families . .
Experiment IV. Artificial Vernalization Studies of
Chinese Cabbage x Turnip .
Experiment V: Artificial Vernalization Studies of.
Chinese Cabbage x Turnip . . . .

iv

Page

vi

viii

42
43
44
44
45

RESULTS AND DISCUSSION

Experiment 1: Natural Vernalization Studies of
Siberian Kale, Chinese Cabbage, an F2 Population
(Siberian x Chee H00) and Chikale .

Experiment II: Artificial Vernalization Studies of.

Siberian Kale x Chinese Cabbage
Experiment III. Artificial Vernalization Studies
of Siberian x Nozaki Early F3 Families
Experiment IV: Artificial Vernalization of
Chikale . . . . . .
Chikale LB- 7 x Chikale 08- .2.
Chikale LB- 7 x Wong Bok
Wong Bok x Chikale 03- 2

Experiment V: Artificial Vernalization of Chinese .

Cabbage x Turnip Populations
SUMMARY AND CONCLUSIONS .
BIBLIOGRAPHY

Page

46

Table

10.

11.

LIST OF TABLES

Effect of plant size and duration of exposure to 5°C
on the percent bolting in Siberian kale .

Percentage of bolted plants at weekly intervals after
vernalization . . . . . . . .

Percentage of bolters under natural field conditions .

Bolting percentages after 5 durations of vernalization
for the various populations for the interspecific
cross Siberian kale x Chee Hoo . .

Bolting percentages after five duration of vernaliza-
tion for the various populations from the interspe-
cific cross Siberian kale x Nozaki Early .

Bolting percentages after five durations of vernaliza-
tion for the various p0pulations from the interspe-
cific cross Mandarin x Siberian kale

Percent bolters after five durations of vernalization
in two F3 families from the cross Siberian kale x
Chee Hoo . . . . . . . . .

Percent bolters after three durations of vernaliza-
tion of F3 families from the cross Siberian x Nozaki
Early . . . . . . . . . . . .

Chi-square test for goodness of fit to a single addi-
tive gene model for bolting after three durations of
vernalization of F3 families from the cross Siberian
x Nozaki Early . . . . . . . .

Bolting percentages after five durations of vernaliza-
tion for the various populations from the cross LB- 7
x 08- 2 . . . . . . . . . . .

Chi-square tests for goodness of fit to a two addi-

tive gene model for bolting after four durations of
vernalization for the cross LB-7 x QB-Z

vi

Page

36

42
47

52

54

56

63

65

66

69

Table Page

12. Percentage of bolters after five durations of
vernalization in the various populations from the
cross LB- 7 x Bong Bok. . . . . . . . . . 73

13. Chi-square tests for goodness of fit to a one addi-
tive gene model for bolting after 3 durations of
vernalization for the cross LB-7 X Wang Bok . . . 75

14. Percentage of bolters after five durations of vernali-
zation in the various populations from the cross Wong
Bok x 08-2 . . . . . . . . . . . . . . 76

15. Chi-square tests for goodness of fit to a one addi-
tive gene model for bolting after three durations
of vernalization for the cross Wong Bok x 08-2 . . 77

16. Proposed genotypes and phenotypes for bolting
response produced from the cross Siberian kale x
Chinese cabbage . . . . . . . . . . . . . 80

17. Percentage of bolters after four durations of
vernalization in the various populations from the
cross Mandarin x Milan White . . . . . . . . 81

18. Chi-square tests for goodness of fit to a two addi-

tive gene model for bolting after four durations of
vernalization for the cross Mandarin x Milan white . 84

vii

Figure

LIST OF FIGURES

Leaves of Chinese cabbage, hybrid, and Siberian kale;
Seedling of Siberian kale, hybrid, and Chinese
cabbage; Inflorescence of Chinese cabbage, hybrid,
and Siberian kale . . . . . . . .

Metaphase [in Pollen Mother Cells of Siberian Kale,
Chinese Cabbage, the F1 Hybrid, Siberian Kale x Chee
H00 and Chikale . . . . . . .

Leaf and taproots of Chinese cabbage, hybrid, and
turnip . . . . . . . .

Stages in seedstalk development .

Minimum-Maximum air temperature chart for the period
May 4 to June 21, 1982 . . . . . . . . .

viii

Page

21

23

31
40

INTRODUCTION

Exposure for a period of low temperature is necessary for
flower initiation in many biennial species while it hastens flower-
ing in some annuals. This phenomenon, vernalization, is defined as
the change from vegetative growth to reproductive growth in response

to low temperatures. Chinese cabbage (Brassica campestris L. ssp.

 

pekinensis) develops flowering shoots prematurely (bolting) if

 

exposed to low temperatures, thereby reducing the market value. The
degree of bolting increases with duration of the cold period (tem-
peratures below 13°C) and flower stalk elongation is hastened by
subsequent warm temperatures and long days. Some species of crop
plants are responsive to vernalizing temperatures only after a
juvenile phase, however, the Chinese cabbage is sensitive with the
onset of germination.

Several cultural practices have been developed to reduce the
losses from bolting of spring grown crops. The use of protective
row coverings is a common practice in early season celery production,
but this is expensive and unsatisfactory during prolonged cold
periods. Adjusting planting dates and the use of larger transplants
can also reduce losses to bolting. However, a more satisfactory
solution to this problem is through the use of bolt resistant culti-

vars. In China, leafy nonheading Chinese cabbage cultivars which

have some bolt-resistance are grown in the spring season. Evaluation
of Chinese cabbage cultivars for bolting under natural field condi-
tions showed varying degrees of bolting but none of the nappa culti-
vars had the desired level of resistance.

The genus Brassica encompasses very diverse species which
vary in their response to vernalizing temperatures. Interspecific
crossing between Brassica spp. has been used to introgress genes
for disease resistance, incompatibility, as well as genes for quan-
titative traits such as yield. Therefore, it was hypothesized that
genes for bolt resistance could be transferred to Chinese cabbage
from species with greater vernalization requirments. An F3 popula-
tion from a cross between Siberian kale and Chinese cabbage was
screened at 10°C for 3 weeks and differences in susceptibility to
bolting were observed. Selections were made for quick and slow
bolting types for use in inheritance studies for bolting. This
material was also hybridized with Chinese cabbage. Additional
interspecific crosses were attempted between Chinese cabbage and
kale, as well as an intraspecific cross between Chinese cabbage and
turnip. The purpose of this study was to determine the inheritance
of vernalization response in crosses between (1) lines developed
from an interspecific cross beween Siberian kale and Chinese cabbage,
(2) Siberian kale and Chinese cabbage, (3) Chinese cabbage and turnip,
and to develop a breeding system for obtaining bolt resistance

Chinese cabbage cultivars.

LITERATURE REVIEW

Vernalization

 

Several reviews and books on the subject of vernalization and
ffiowerinduction have been published (Curtis and Clark, 1956; Chouard,
1960; Lang, 1965; Wellensiek, 1965; Purvis, 1966; Chailakhyan,

1968; Street and Opik, 1973). Vernalization is commonly defined

as the change from vegetative to reproductive growth in response to
low temperatures. This definition will be used in this study.
Gassner, in 1918, was the first to investigate the promotive effect
of low temperatures on flowering (Purvis, 1966). He observed a
definite relationship between duration of exposure to low tempera-
tures and flowering in certain winter cereals. Purvis and Gregory
(1937) determined that Petkus rye could be induced to flower by
exposing ripening seeds to low temperatures, however, crops differ
in the stage of development at which they become responsive to
vernalization. Boswell (1929) observed that cabbage plants were
responsive to low temperatures only after they had reached a cer-
tain minimum weight or size. According to Wellensiek (1965), many
biennials have a juvenile stage during which they are not responsive
to vernalizing temperatures. Chouard (1960) suggested that generally
sensitivity to low temperatures increased with age.

In most cold-requiring plants the receptive site for vernali-

zation is the apical meristem. Curtis and Chang (1930) demonstrated

3

4

that providing warm temperatures to the shoot tips of young celery
plants during vernalization inhibited flowering. Purvis (1940)
excised the shoot region of Petkus winter rye embryos and observed
that if proper nutrients were supplied for growth, these excised
apices could be vernalized. Other portions of the plant (leaves,
endosperm, etc.) are important as a source of nutrients needed in
the vernalization process (Lang, 1965).

1 Vernalization is unusual among biological processes in that
it is favored by low temperatures. The low temperature effect on
flower formation in cold requiring plants is quantitative in that
longer duration and lower temperature act to accelerate the flower-
ing response. Purvis and Gregory (1937) observed that in winter rye
the days to flowering were shortened by increasing the number of
days at the low temperature and by lowering the treatment temperature.
A similar situation has been observed in table beets (Chroboczek,
1931), and in all other plants that have been investigated in this
respect (Lang, 1965). Thus the concept of a temperature optimum for
vernalization appears to be relative to duration, degree, and plant
age. Lang (1965) suggested that as long as the vernalization process
is progressing, it progresses hithe same manner only at different
rates, resulting in the same maximal level of induction if enough
time is allowed.

The effect of low temperature on flowering is reversible by
high temperatures under certain circumstances. This phenomenon has

been called “devernalization” and has been observed in many cold

requiring plants including sugarbeets (Stout, 1946), table beets
(Chroboczek, 1931), celery (Thompson, 1034) and others (Lang, 1965).
The range of inductive and inhibitory temperatures can be very close
to one another. Temperatures at which neither induction or inhibi-
tion occurs are called "neutral" temperatures. Stout (1946)
observed that temperatures in the range of 17° e 18°C were neutral
while temperatures above 23°C were devernalizing in sugarbeets.
Weibe (1973) reported that temperatures above 18°C were inhibitory
to bolting while temperatures below 13°C can induce bolting in table
beets. The devernalizing temperatures are effective if given during
the vernalization period or immediately following. If a period at
"neutral" temperatures was given prior to exposure to high tempera-
tures, the high temperatures were no longer inhibitory to bolting.
This has been called stabilization of the vernalized state (Lang,
1965). In most plants examined, if thermo-induction has reached

its maximal level, devernalization is no longer possible (Lang,
1965).

In some plants a combination of low temperatures and long
days favor flowering. Chroboczek (1934) found that low temperature
treatment followed by warmer temperatures and long days resulted in
rapid flower stalk development in table beets. In spinach, flowering
is Optimum if long days are provided during or following the low
temperature treatment, although prolonged exposure to low tempera-
tures under short days or exposure to long days at intermediate tem-

peratures were also effective in promoting bolting and flowering

(Curtis and Clark, 1956). Purvis (1966) suggest that the vernaliza-
tion treatment itself does not induce flowering, but rather ensures
that the plant is able to respond to optimal conditions of light and
temperature.

Varietal differences in vernalization response have been
noted in many crop species, however, the number of genetic investi-
gations isy limited (Boswell, 1929; Hester and Magruder, 1938;
Lorenz, 1946; Moore, 1958; Thomas, 1980). Hall (1928) reported
that bolting was not simply inherited and that nonbolting was domi-
nant in sugarbeets, mangolds, and leeks. Van Heel (1927) observed
that in sugarbeets bolting was recessive and controlled by a single
gene. Sutton (1932) found that bolting in cabbage was conditioned
by recessive factors. Several reports on the inheritance of winter
and spring types of cereals have also been made (Cooper, 1927; Gaines,
1917; Purvis, 1939; Catch, 1979). These reports can be generalized
by stating that winter habit is recessive to spring habit and the
number of genes segregating, although generally low, depended on the
varieties. Gotoh (1980) suggested that differences in degree of
cold requirements in winter wheat varieties was conditioned by 2
genes. In celery bolting resistance was quantitative and recessive
(Elmsweller, 1934). Honma (1959) developed an artificial cold induc-
tion method to establisha cold units system that could be used to
separate bolt susceptible and bolt resistant celery lines. Using
this method, Bouwkamp and Honma (1970) determined that easy bolting

in celery was conditioned by a single dominant gene and that degree

of resistance was determined by modifiers. A similar cold unit sys-
tem was used by Dickson et al. (1961) to evaluate bolting resistance
in carrots, however, no genetic study was reported.

Although the vernalization process is generally similar in
cold requiring plants, there is variation between and within species
for many of the aspects of vernalization. This portion of the litera-
ture reveiw will focus on vernalization responses of the Brassica
species used in this study: B; oleracea, B, campestris, and B; 22225

a natural allotetraploid between oleracea and campestris. Brassica

 

oleracea includes many vegetable crops: vars. italica (broccoli);
gemmifera (Brussels sprouts); capitata (cabbage); botrytis (cauli-
flower); acephala (collards and kale); gongylodes (kohlrabi); as
does B, campestris ssp.: rapifera (turnips), pekinensis (Chinese
cabbage), and chinensis (pak choi). The majority of B, £32 5 forms
are used as fodder and oil crops with the exception of rutabaga,
which is grown as a vegetable crop.

Shinohara (1959) investigated the variation within and
between Brassica species for the phase of development in which
vernalization had a role in bolting and flowering. He observed that
members of the oleracea spp. were susceptible to vernalization only

during the "green plant stage" while species campestris could be

 

seed vernalized. Shinohara also reported that B, ggpgg had the
vernalization characteristics of B, campestris. He concluded that
Brassica species with the same genomic constitution have similar
requirements for vernalization, however, the degree of these

requirements varied within species.

Several reports on the response of cabbage to low tempera-
tures have been made (Boswell, 1929; Miller, 1929; Detjen and McCue,
1933; Ito and Saito, 1961; Heide, 1970; and Patil, 1981). Boswell
(1929) observed that if fall grown cabbage plants were too large
when exposed to low winter temperatures, the plants would form
flower stalks instead of heads the following spring. After inves-
tigating this phenomenon, Boswell (1929) and Miller (1929) concluded
that exposure to low temperatures is a requisite for flowering in
cabbage, but only after plants had obtained a certain size. Detjen
and McCue (1933) investigated the effect of nutrition, time, and
temperature at planting and various growth checks on bolting of
cabbage cultivars. They concluded that susceptibility to the vari-
ous environmental conditions was dependent on hereditary factors
which controlled bolting. The optimum plant size (5 to 6 weeks)
and temperature (4° - 9°C) for vernalization of cabbage varied with
cultivar (Ito and Saito, 1961); however, in general, longer dura-
tions (8 weeks) and lower temperatures (4°C) could induce bolting
and flowering in younger plants and hastened flowering (Patil, 1981).
Temperatures above 18°C could inhibit flowering in cabbage if plants
were exposed to these high temperatures during the cold period or
immediately afterwards. However, if a cold period of 8 weeks was
used, plants were no longer devernalized by exposure to high tem-
peratures (Heide, 1970).

Similar vernalization responses were reported in Brussels
Sprouts (Verkerk, 1954; Thomas, 1980). Verkerk (1954) observed that

Brussels sprouts seedlings were more responsive to low temperatures

9

after they were 8 weeks old; longer durations of cold exposure could
induce flowering in younger plants. The optimum temperature for
vernalization of Brussels sprouts appeared to be 4° to 7°C, depend-
ing on the cultivar, with longer durations at temperatures up to
14°C producing flowering. Thomas (1980) compared early season and
late season cultivars of Brussels sprouts and observed that the
early season cultivars bolted and flower more rapidly in response
to less low temperature than the late slow growing cultivars.

Information on bolting and flowering in kohlrabi is limited.
Marrewijk (1976) observed that kohlrabi plants will flower if exposed
to 8 to 10 weeks at 4°C followed by 2 weeks at 10°C.

Moore (1958) observed that collard varieties differed in
bolting tolerance to low temperatures under field conditions.
Parham and Moore (1959) exposed collard seelings, cultivar Georgia,
with 5 mm stem diameters to 5°C for 0, 2, 4, or 6 weeks. No bolt-
ing was observed in the O or 2 weeks treatments, 50% of the plants
bolted at 4 weeks and 100% bolted at 6 weeks. They suggested that
5 weeks at 5°C was the critical temperature and duration for floral
development for this cultivar of collards. Cheng and Moore (1968)
investigated the relationship between seeling size and length of
cold exposure to flowering in collards, cultivar Yates. Stem dia-
meters were used to group plants into classes then each class was
exposed to 5, 6, or 7 weeks at 5°C. Results of this study suggested
that plants of Vates must be at least 4 mm stem diameter to be

induced to bolt and flower by low temperatures and the period for

10

appearance of the first flower buds was shortened by longer exposure
to 5°C. In their study, flowering was considered to be when the
first flower bud was visible to the unaided eye and stem diameter
was used as an indication of the termination of the juvenile phase
(Cheng and Moore, 1968).

As mentioned, the members of B, campestris differ from those

 

of B, oleracea in their vernalization response. Lorenz (1946)
studied the effect of low temperatures and daylength on flowering in
3 cultivars of Chinese cabbage: Wong Bok, Pe-tsai, and Chili.
Plantings were made during the cool part of the year when as few as
2 true leaves were present which resulted in varying degrees of
bolting, dependingcuithe cultivar. He observed that bolting
increased in all these Chinese cabbage cultivars when grown at tem-
peratures between 10° - 15°C as compared to 15° - 20°C. Lorenz
(1946) suggested that optimum bolting in Chinese cabbage could be
obtained by exposing 2 week old plants to 5°C for a period of 2
weeks. Nagagawa and Henmi (1955) and Permadi (1974) induced flower-
ing in Chinese cabbage seedlings by exposing them to 0°C for 32 days
immediately following germination. Kagawa (1966) observed that the
longer the duration of low temperature, the greater the acceleration
of bolting and flowering in Chinese cabbage. Yamazaki (1956) inves-
tigated the effect of low temperatures on flower bud formation in
Chinese cabbage and developed the following formulae: (13°C - X) Y =
87°C, where X is the temperature below 13°C and Y is the number of

days with minimum temperatures below 13°C. When the sum of this

11

equation reached or exceeded 87°C flower induction occurred.
Nakamura (1976) suggested that this equation is a general rule and
that cultivars differ in the duration of low temperatures required
for flower induction. It was suggested that many cultivars could
be induced to flower with 30 days with minimum temperatures of 13°C,
15 days with minimum temperatures of 10°C, or 10 days with minimum
temperatures of 5°C. Honma (1981) reported that 14 days at 5°C
resulted in bolting of standard cultivars of Chinese cabbage and
that no known slow bolting heading Chinese cabbage cultivars exist
at present. However, in screening heat tolerant Chinese cabbage
lines at the Asian Vegetable Research and Development Center (AVRDC,
1979) resistance to bolting was observed when vernalized for 10 to‘
30 days at 10°C.

In B. campestris ssp. chinensis cultivar To-pe-tsai, Wang

 

(1969) observed that exposing 12 day old (1 true leaf) plants for 3
days at 4°C induced flower bud development. Insufficient low tem-
perature accumulation or exposure to high temperatures after the cold
period produced abnormal flowering in this cultivar (Wang, 1969).
Hester and Magruder (1938) investigated cultivar differences
in bolting of turnips. They observed that a period of low tempera-
tures followed by a period of warmer temperatures was required for
seed stalk development in turnips, with longer durations of low tem—
peratures reducing time to express bolting. A plant was considered
a bolter when the central axis had elongated sufficiently to distin-

guish internodes. Cultivars differed for number of bolters with

12

some strains bolting prior to reaching marketable size while the
majority bolted only after reaching a marketable size (Hester and
Magruder, 1938). Sakr (1944) observed that turnips were sensitive
to low temperature induced bolting from the early stages of seed-
ling development to the mature plant stage.

The species B, ngg§_also requires cold for flower induction.
Peto (1934) reported that exposing rutabaga plants to 12°C for a
period of 30 to 50 days following sowing date resulted in bolting
approximately 70 days later. It has been reported that B, BBBBB
can be seed vernalized (Shinohara, 1959), and Kagawa (1971) reported
that the F1 between cabbage and Chinese cabbage had the Chinese
cabbage early flowering habit and could be vernalized during all
stages of development. Honma and Heeckt (1960) reported that the F1
between Siberian kale and Chinese cabbage did not flower when seed-
lings were vernalized 5 weeks, however, all the hybrids flowered when
given an additional 4 weeks of low temperature.

The vernalization process can be considered to include two
distinct phases. The first phase in which low temperatures are the
critical environmental parameter, involves the transition from vege-
tative to reproductive development, while the second phase involves
the expression of the reproductive phase in the form of flower stalk
development and flowering (Curtis and Clark, 1956). Although this
study is concerned with the first phase of vernalization, the
relationship between the vernalized state and the environment during

the second phase of the vernalization process is also important.

13

Purvis (1966) suggested that daylength and temperature during the
post-vernalization period can effect the development of flowers.

The majority of plants with a cold requirement flower more rapidly
if long days and warm temperatures are present during the time
period immediately following vernalization (Lang, 1965). Reports

on vernalization of the Brassica species considered in this study
are in agreement that long days and warm temperatures tend to hasten
flowering of vernalized plants. Heide (1970) observed optimum
flowering in cabbage if conditions following vernalization were

long days at temperatures between 12° and 18°C. Thomas found a
similar response in Brussels Sprouts (1980). In Chinese cabbage,
long days following the cold period also hasten flowering (Lorenz,
1946, and Kagawa, 1966). Mori et al. (1979) treated 15-day-old
Chinese cabbage plants with 2° or 8°C for 5, 10, or 15 days and then
placed the plants at 20°C under 8, 12, 16, 20, or 24 hour daylengths.
They concluded from this study that longer duration of cold period
and longer daylength following vernalization shortened the delay in
flowering. A similar effect of long days was observed in B, chinensis
(Hang, 1969). Purvis (1966) suggests that the promotive effect of
long days on flowering in vernalized plants is quantitative in that
with increased photosynthesis as a result of longer daylength allows
for a more rapid transition to reproductive develOpment. Presumably

the increased temperatures are acting in a similar manner.

14

Interspecific Hybridization

 

Karpechenko (1922), Pearson (1928), Morinaga (1929), and
U (1935) were responsible for developing the relationships between
members of the genus Brassica. Cytological observations on chromo-
some associations during pollen development and success of attempted
hybridizations between members of this genus were the basis for the
relationships proposed by U (1935) which has become known as the

"triangle of U." There are 3 primary species: B, campestris (n=10),

 

é genome; B, fliflEE (n=8), B_ genome; and B, oleracea (n=9), 9_ genome.
Through natural hybridization between these primary species, 3 sec-
ondary species were formed: B, carinata (n=17), Bg_genome; B.

jggggg (n=18), gB_genome; and B, DEEEE (n=19), gg_genome. The basic
chromosome number of the genus Brassica as determined from chromo-
some morphology and secondary associations is x = 6 and the differ-
ences in chromosome number between primary species resulted from
aneuploidy of different chromosomes (Richharia, 1937; Robbelen, 1960;
Nwankiti, 1970; Kamala, 1978). Many studies have been reported in
which various cross combinations within and between species were
attempted. In general, crossing between individuals with a common
primary genome resulted in at least partial fertility (Morinaga,
1929). Success in crossing between individuals with different genomic
compliments was dependent on the cultivars or forms used, the
environment, plant age, and number of pollinations (Yarnell, 1956).
Due to the large amount of literature on inter-crossing in the genus
Brassica, this review will be limited to crosses between B, oleracea

and B. campestris, and between B, campestris and B, napus.

 

 

15

In 1920, Ragonieri reported on attempts to cross the Chinese

cabbage cultivar Pe-tsai (B, campestris ssp. pekinensis) with B,

 

oleracea, I'Cows Fodder," B, oleracea var. acephala, green scotch
kale, and B. oleracea var. botrytis, cauliflower. Using Chinese
cabbage as the female a few seeds were obtained from crosses with
Cows Fodder and cauliflower, however, the crosses with kale were
unsuccessful. Morinaga (1929) demonstrated that B, gng§_was a

naturally occurring amphidiploid between the B_genome (B, campestris)

 

and the g genome (B, oleracea). U (1935) reported success in cross-
ing these two genomes using the B, oleracea vars. gemmifera and
acephala. He produced 4F3_hybrids which differed from each other

in chormosome number, having 19, 28, 29, and 38 chromosomes. The

genomic constitutions of these F 's were gg_(sterile), acc and aac

1
(partially fertile) and aacc (fertile) respectively. Frandsen (1947)
succeeded in crossing tetraploid forms of B, oleracea var. capitata

and B, campestris ssp. rapifera with the resulting F1 having 2n = 38

 

chromosomes. Hosoda (1950) excised portions of the style to improve

the crossability of B. oleracea var. capitata and B, campestris ssp.

 

pekinensis. The F1 hybrids were sterile; however, applications of
colchicine restored fertility. By crossing B, campestris ssp.
rapifera and ssp. oleifera with vars. acephala, capitata, gongylodes,

 

 

and gemmifera of B, oleracea, Olsson (1960) obtained 16 hybrids. No
morphological description was given, but karyological studies
revealed that the majority of the Fl's had 29 chromosomes which

formed 10 bivalents and 9 univalents during meiosis resulting in

16

partial fertility. With the aid of n-meta-tolyphalamic acid applied
to the base of flowers, Honma and Heeckt (1960) obtained F1 hybrids

between B. campestis ssp. pekinensis cv. Mandarin and B, oleracea

 

var. acephala cv. Siberain kale. This interspecific hybrid was
partially fertile and was intermediate in morphological characteris-
tics, however, no cytological analysis was made. The F2 population
segregated for both parental types with the majority of the plant
characters observed showing continuous variation (Honma and Heeckt,
1960). Nishi et al. (1962) crossed reciprocally, B, oleracea var.

capitata and B. campestris ssp. pekinensis and the hybrid embryos

 

were successfully cultured in vitro. In a further study, Nishi

et al. (1970) attempted to transfer soft rot resistance from heading
cabbage to Chinese cabbage. To develop head forming B, EEEEE culti-
vars, they crossed B, campestris ssp. pekinensis, chinensis, oleifera,
rapifera and narinosa with different varieties of the cabbage group.
In most all cases the hybrids were sterile and failed to backcross
with common cabbage and Chinese cabbage (Nishi, 1980). Doubling the
chromosomes with colchicine restored fertility. The amphidiploid
was selfed to develop "Hakuran" a heading B, gng§_and was back-
crossed to Chinese cabbage to transfer soft rot resistance (Nishi,
1980). Sarashima (1964), with the objective of producing forage
types, crossed B, campestris ssp. including pekinensis with B,
oleracea vars. acephala, capitata, botrytis, italica, and gongylodes.
The hybrids were treated with colchicine to restore fertility and

selection was made for improved forage types. Namai (1971) grafted

17

.B. campestris ssp. onto B. oleracea_var. acephala and used this plant

 

as the female in a cross with B, oleracea var. acephala. The graft
combination improved the success of crossing these two species; how-
ever, applications of a sugar and agar mixture to the flower bases
had no noticeable effect. Inomata (1980) conducted a cytological
investigation of interspecific hybrids between B, campestris ssp.

rapifera, oleifera, chinensis, and pekinensis with B, oleracea vars.

 

acephala, capitata, gemmifera, gongylodes, and italica obtained by
embryo culture. The F1 hybrids with 19 chromosomes generally had
9 bivalents and 1 univalent with low fertility while the Fl's with
38 chromosomes had 19 bivalents and were fertile. Harberd and
McArthur (1980) observed from 3 to 9 bivalents in an F1 hybrid

between B. campestris and B. oleracea. In a cross between B,

 

campestris ssp. pekinensis cv. Granaat and B, oleracea var. acephala

 

 

cv. Normal, Balicka et al. (1980) produced 607 F1 hybrid of which
one was an allohaploid, 2n = 19 chromosomes, but it was sterile. In
a further study with this cross (Zwierzykowska, 1981) embryo culture
was used to rescue the Fl's obtained from several cultivars of
pekinensis and acephala. The number of attempts at crossing these
two genomes is large and the success appears to be dependent to a
large extent on the cultivars used. Tsunoda and Nishi (1968) sug-
gested that the self-incompatibility of the parental strains may
influence the production of F1 embryos and their further growth.
Moue (1979) in a series of studies on interspecific crosses between

Chinese cabbage and cabbage reported that the endosperms disappeared

18

and the embryo had ceased to develop 20 days after pollination when
cabbage was used as the female and 25 days after pollination when
Chinese cabbage was the female. These results may explain the dif-
ference in success between reciprocal crosses and the effectiveness
of embryo culture in rescuing hybrid embryos (Moue, 1979).

Many attempted crosses between B, campestris and B, napus

 

have been reported with variable success (Morinaga, 1929; Frandsen
and Hinge, 1932; U, 1935; Calder, 1937; Mizushima, 1950; Jahr, 1962;
Nwankiti, 1970; Jelinkova, 1971; Lammerink, 1970; McNaughton, 1973;
Mackay, 1977; Snell, 1977; Fantes and Mackay, 1979). Morinaga
(1929) was the first to report successful crosses between B,

campestris ssp. and B. napus. The hybrids obtained from reciprocal

 

crosses had 29 chromosomes (Egg) and formed 10 bivalents and 9 uni—
valents during metaphase I of meiosis. The hybrids were partially
self- and cross-compatible with both parental species. The distribu-
tion of the 9 univalents appeared random and the number of chromo-
somes in the daughter cells ranged from 10 to 19 with a mode of 14-
15. The hybrids (B, ggpgg x.B. campestris) obtained by U (1935),
Calder (1937), Nwankiti (1970), Mackay, (1977), and Fantes and
Mackay (1979) also had 29 chromosomes and were partially fertile.
The morphology of the F1 hybrids was intermediate to the parent spe-
cies (McNaughton, 1973; and Mizushima, 1950). Frandsen and Hinge
(1932) crossed B, ggpgg, swede, and B, campestris ssp. rapifera.

The selfed progeny from this interspecific hybrid had 2n = 58

chromosomes which they attributed to spontaneous doubling of the

19

gametic chromosome compliment (2n = 29). It was proposed that this
auto-allo-hexaploid was a new species which they called B,

napocampestris. In later studies, Jahr (1962) and Nwankiti (1970)

 

produced B. napocampestris by doubling the chromosome number of the

 

hybrid (B, napus x B, campestris) with colchicine. Nwankiti (1970)

 

also backcrossed the hybrid to the B. campestris parent. The

 

chromosome numbers of the hybrid gametes generally were 10, 13, 16,
or 19, suggesting nonrandom distribution. They attributed this to

bivalent formation between chromosomes of the g_genome. The

 

resulting plants from backcrossing the F1 with B, campestris were
aneuploids. Plants which were nearly euploid (2n = 20 or 2n = 38)
were fertile and occurred in the highest frequency. The progeny
from this backcross with 2n = 20 chromosomes formed 10 bivalents at
metaphase I and had high pollen fertility, but poor seed set. Sikka
(1940) observed that the g genome of B, 222 3 had chromosomes lacking

satellite structures characteristic of the B, campestris §_genome.

 

Nwankiti (1970) suggested that this may account for the observed
sterility. On further observation of these backcross progeny,
Nwankiti (1971) observed that after one generation of selfing,
the fertility was restored and the chromosome numbers were stabilized.
Fantes and Mackay (1979) also observed a higher than expected
number of plants with extreme chromosome numbers (2n = 20 and 2n =

38) when they backcrossed the hybrid (B. napus x B, campestris) to

 

the parent species.

MATERIALS AND METHODS

Parent Material

 

Chinese Cabbage

 

Chinese cabbage (B. campestris L. spp. pekinensis) is a

 

 

head forming annual. The leaves are large with a prominent mid-
rib, light green to yellowish green, soft and veiny with undulating
margins (Figure 1). The inflorescence is short with tightly clus-
tered flowers. The following open-pollinated Chinese cabbage nappa
cultivars were used in this study: Chee Hoo, a mid-season large
headed cultivar; Hong Bok, a late-season standard cultivar; Nozaki
Early, an extra-early spring or early summer cultivar; and Mandarin,
an early summer, small headed cultivar. The first 3 cultivars were
obtained from Takii Seed Co., Kyoto, Japan, while the later was
obtained from the National Seed Storage, Fort Collins, Colo. These
cultivars were induced to bolt and flower by exposing 2 week old
plants (2 true leaves, 2 cm long) at 5°C for 2 weeks. Plants were
selfed 4 generations to obtain homozygosity for bolting response.
Cytological observations of pollen mother cells of Chinese cabbage

showed 10 bivalents at metaphase I (Figure 2).

Kale
The kale cultivar, Siberian, is cold tolerant and long

standing. It is a leafy biennial plant having blue green foliage

20

21

Figure 1: Top: Leaves of Chinese cabbage, hybrid, and
Siberian kale (left to right)

Center: Seedling of Siberian kale, hybrid, and
Chinese cabbage (left to right)

Bottom: Inflorescence of Chinese cabbage, hybrid,
and Siberian kale (left to right)

 

23

Figure 2: Metaphase I in Pollen Mother Cells of (Clockwise from
upper left): Siberian Kale (n=19), Chinese Cabbage
(n=10), the F Hybrid, Siberian Kale x Chee Hoo (n=29),
and Chikale (i=10).

24

 

25

which is coarse and crinkled with dentate margins (Figure 1). The
inflorescence is elongated and the flowers become well spread along
the stalk at anthesis. Seeds of Siberian kale were obtained from
Harris Seed Co., Rochester, New York. Plants with 6 mm stem dia—
meters were vernalized for 8 weeks at 5°C to promote flowering.
After 3 generations of selfing, plants appeared homozygous for bolt-
ing response after 8 weeks of vernalization. Individual plants were
hybridized with Chinese cabbage.

In 1960 Honma and Heeckt reported that Siberian kale used
in their study to be B, oleracea var. acephala, a diploid (2n=18).
These authors did not make a cytological study of Siberian kale and
assumed it to have 9 chromosome pairs. Thomas and Crane (1942)
reported that most kale strains belong to the B. oleracea var.
acephala species, however, Late Rape kale, Hungary Gap kale, and
Asparagus kale have 19 chromosome pairs and belong to the B, ngg§
species. These amphidiploid kales are believed to have resulted from

hybridization between B. campestris ssp. rapifera, turnip, (2n=20)

 

and B. oleracea var. acephala, kale, (2n=18). In 1978, Borchers
and Taylor reported that Siberian kale has 19 chromosome pairs which
would suggest it to be B, nggs, Cytological Observations of pollen
mother cells of Siberian kale showed 19 bivalents at metaphase I

which suggest that Siberian kale belongs to B, napus (Figure 2)

Chikale
Quick-bolt and late-bolt lines were developed from an F4

p0pulation derived from an interspecific cross between Siberian kale

26

and Chinese cabbage (cv. Mandarin) made by Honma and Heeckt (1960).
Although they reported that Siberian kale used in their study was
B, oleracea var. acephala, it is possible that the Siberian kale
strain used in their study was also B, nggg, Cytological observa-
tions of the late-bolt segregant used in this study showed 10
bivalents (Figure 2) at metaphase I of pollen mother cells suggest-
ing this Chikale line had 20 chromosome pairs.

' The lines developed from this F4 population will be referred
to as Chikale (CK) lines. This F4 population consisted of 9 sister
lines which had not been screened for bolting response, but were
selected for phenotypic similarity to Chinese cabbage. These lines
were transplanted into a greenhouse ground bed in January, 1979.
The temperature for the first 3 weeks following transplanting was
12°C at night and 17°C during the day. After 3 weeks, the tempera-
ture was raised to 20°C and observations were made on the time of
bolting. Bolting was determined by the visible flower buds at the
apex of the plant. The lines segregated for time of bolting with the
majority of the lines bolting within 50 days after the temperature
was raised to 20°C. Line CK-2 was the first to bolt and line CK-7
failed to bolt. Selections were made from these 2 lines for quick-
and late-bolting types. The nonbolted plants were vernalized for
6 weeks at 5°C to induce flowering and were then self-pollinated.
Progeny from these selections were vernalized for 2 weeks at 5°C
when the second true leaves were 2 cm long. Lighting was provided
by banks of 40 watt cool white fluorescent lamps (approximately

20 H m'z) for 16 hours each day. The vernalized plants were

27

transplanted into a greenhouse ground bed with a minimum night tem-
perature of 20°C. The quick-bolt selections bolted by the 30th
day after transplanting, however, none of the late-bolt selections
bolted. Selections were again made for quick- and late-bolting
types and the selfed progeny from these selections were screened
for bolting response at the second true leaf stage at 5°C for 0, 1,
2, 3, 4, and 5 weeks. Staggered plantings were made so that all
treatments were removed from the vernalization room on the same
date. The treated plants were placed into a lath house for 1 week
to stabilize vernalization prior to transplanting into the field.
The quick-bolt selections appeared homozygous for bolting response
and bolted in every treatment except the control (0 weeks). The
late-bolt selections were also homozygous for bolting response and
bolted after 5 weeks of vernalization. This experiment was repeated
in the greenhouse during the fall (1980) and similar observations
were noted. Individual plants selected from the field experiment
were used in this study and will be referred to as QB-2 (quick-bolt)

and LB-7 (late-bolt).

Turnip
The turnip (B, campestris L. ssp. rapifera) cultivar, Milan

 

Hhite, was obtained from Mikado Seed Co., Chiba-City, Japan. The
root of Milan Hhite is semi-globe shaped, medium sized, and white.
The leaves are green, narrow, and coarse with entire margins. Vernali-
zation was accomplished by exposing 2 week old plants to 5°C for

6 weeks. After 3 generations of selfing, individuals which were

28

phenotypically homozygous for bolting response were hybridized with

Chinese cabbage.

Hybridization

 

Inheritance studies on bolting in the interspecific cross,
Siberian kale x Chinese cabbage were made from 2 separate hybridiza-
tions. One study involved Siberian kale and Chinese cabbage while
the other study involved Chinese cabbage and Chikale. The Siberian
kale x Chinese cabbage study included the following unilateral
crosses:

Siberian x Chee Hoo
Siberian x Nozaki Early
Mandarin x Siberian

Approimately 500 recirocal pollinations between Siberian and
3 Chinese cabbage cultivars failed to produce viable seeds, suggest-
ing interspecific incompatibility. Therefore, the flower setting
hormone, n-meta tolyphalamic acid at 201nm1was used to circumvent the
incompatibility (Honma and Heeckt, 1960). A small piece of cotton
was attached to the base of individual flowers and was soaked peri-
odically with this hormone solution until the flowers abScissed or
pods began to develop. Results of unilateral crosses produced the
following number of viable seeds: 110 seeds of Siberain x Chee H00,
4 seeks of Siberian x Nozaki Early, and 2 seeds of Mandarin x
Siberian. The F1 hybrids were intermediate to the parents for leaf
shape and leaf color (Figure 1). All of the F1 plants appeared

phenotypically similar regardless of the Chinese cabbage cultivar

29

used as the parent. The F1 hybrids with 6 mm stem diameters were
vernalized for 8 weeks at 5°C. The inflorescence of the hybrid was
also intermediate to the parents (Figure 1) for flower color, flower
size, and flower spacing. The length of time for the appearance of
flower buds after 8 weeks of vernalization was less for the hybrids
(30 days) than for Siberian (50 days). The F1 hybrids showed vary-
ing degrees of fertility and cross compatibility with Siberian and
Chinese cabbage, although the anthers appeared plump and shed
abundant pollen. Cytological observations of the hybrids were made
by staining pollen mother cells with lacto-propionic orcein. Pollen
viability was estimated by examining 500 pollen grains from each
hybrid by staining with acetic carmine.

Three methods were used to obtain F2 and backcross seeds:
(A) the use of n-meta-tolyphalamic acid to obtain F2 and backcross
seeds resulted in limited success, (B) F1 hybrids with similar pheno-
types from a specific cross were placed in groups of 3 plants and
sib-mated with the aid of bees; and (C) increasing the number of
plants asexually of one Siberian x Chee Hoo hybrid which were then‘
pollinated by bees. Due to the high degree of sterility only
limited backcrosses were obtained. Two F3 families from the cross
Siberian x Chee H00 and 6 F3 families from the croSs Siberian x
Nozaki Early were obtained by selfing individual F2 plants.

The second study involved Chikale and Chinese cabbage and

included the following reciprocal crosses:

3O

LB-7 x 08-2

LB-7 X Wang Bok

QB-2 x Hong Bok
Hybridization was accomplished by bud emasculation followed by
brushing pollen from the male parent onto the stigmatic surface of
the female parent, then covering with a glassine bag until pods
began to develop. All hybridizations were successful and the result-
ing F1 hybrids were fertile and backcrossed successfully to both
parents. The parents were maintained by leaf cuttings and were
used as the female parent in backcrosses with the F1 hybrids.

An intra-specific cross between Chinese cabbage and turnip
was also made to study the inheritance of bolting. The cultivars
used were Mandarin (Chinese cabbage) and Milan Hhite (turnip).
Hybridization was accomplished bytnuipollination and the F1 hybrid
was selfed and backcrossed to Chinese cabbage only since flowering
plants of Milan Hhite were not available at the time the F1 was
in bloom. The F1 was intermediate to the parents for both leaf and

root morphology (Figure 3).

Vernalization

 

All artificial vernalization in this study was conducted
in a cold room at a temperature of 5°C i 1°. The plants were pro-
vided with a 16 hour day using banks of fluorescent lamps (approxi-
mately 20 H m'z). Except for experiments conducted in the green-

house, all plants were placed in a lath house immediately following

31

Figure 3: Leaf and taproots of Chinese cabbage (left), hybrid
(center), and turnip (right).

32

 

33

vernalization for 1 week prior to transplanting into the field to
prevent devernalization.

A natural field vernalization experiment was conducted in
order to compare bolting response under artificial vernalization
at a constant temperature with vernalization with fluctuating
temperatures. Yamasaki (1956) reported that the critical temperature
for vernalization of Chinese cabbage was 13°C and proposed the
following formula for predicting flower primorida induction:
(13°C - X) Y = 87°C, where X is the temperature below 13°C and Y
is the number of days with minimum temperatures below 13°C. Hhen
the sum of this equation equals or exceeds 87°C, then flower pri-
mordia formation is induced. This formula has proved to be adequate
for predicting bolting in Chinese cabbage under artificial vernali-
zation at a constant temperature of 5°C, followed by temperatures
above 20°C. It has been observed that the Chinese cabbage cultivars
Chee Hoo, Nozaki Early, Mandarin, and Matsushima did not bolt after
1 week, but bolted after 2 weeks at 5°C in the vernalization chamber.
According to Yamasaki's formula, the sum after 1 week of vernalization
at 5°C is 56°C, which is insufficient to induce bolting, while the
sum after 2 weeks is 112°C which exceeded the level necessary to
induce bolting.

Determination of Plant Age Effect on
Vernalization Response
Chinese cabbage (Nakamura, 1976) and turnip (Sakr, 1944)

have been reported to be sensitive to vernalization from seed

34

germination to mature plants. Although no specific information on
the effect of plant age on vernalization response was found for
Siberian kale (B. ggpgg),Cheng and Moore (1968) reported that the
collard QB. oleracea var. acephala) cultivar Yates requires stem
diameters of at least 4 mm before it can be vernalized by low tem—
peratures. Since B, EEEEE is an amphidiploid involving B. oleracea

and B. campestris (Morinaga, 1929), a preliminary investigation was

 

made to determine the effect of plant age (leaf number) on vernali-
zation response for Chikale, Chinese cabbage, turnip, and Siberian
kale in order to establish the proper plant age for screening popu-
lations for bolting. Plants of each of the parents were vernalized
when they were 2 weeks (2 true leaves), 3 weeks (3-4 true leaves),
and 4 weeks (5-7 true leaves) old. Chikale, Chinese cabbage, and
turnip plants bolted regardless of plant age. None of the 2 week

old Siberian plants bolted while only some of the 3 week old Siberian
plants bolted, and all of the 4 week old plants bolted.

The effect of plant age or leaf number on vernalization of
Siberian kale was further investigated. Plants with 2-3, 4-5, 6-7,
and 7&8 true leaves were vernliazed for 5, 6, 7, or 8 weeks. The
experimental design was a ramdomized complete block with 4 replicates.
Each treatment combination had 16 plants and were transplanted into
the field June 30, 1981. The number of bolted plants as determined
by the visibility of flower buds at the apex of the plants, was
recorded 55 days after removal from the cold room. Analysis of

variance showed highly significant effect (p = .01) for plant size

35

and number of weeks of vernalization. The interaction between plant
size and length of vernalization and blocks effects were not signifi-
cant (p = .05). The results are presented in Table 1. After 5 weeks
of vernalization none of the plants bolted regardless of leaf

number. The treatment combination, 2-3 true leaves and 6 weeks
vernalization, only produced 66% bolters, while all the other treat-
ment combinations produced 100% bolters. These results suggest that
Siberian kale with a minimum of 4 true leaves can be induced to

bolt after 6 weeks of vernalization. Since the parents Chinese
cabbage, Chikale, turnip, and Siberian kale produce 5 to 7 true
leaves in 4 weeks when grown in the greenhouse, all vernalization

treatments were initiated when the plants were 4 weeks old.

Establishing‘Bolting,Criteria

 

Nakamura (1976) reported that Chinese cabbage seedlings with
20-25 outer leaves developed prior to vernalization form heads before
the flower stalk protrudes frOm the head and, therefore, are still
marketable. Therefore, the length of time for the flower stalk to
develop in relation to the heading becomes important. In the process
of developing homozygous parent material, a plant was considered to
have bolted when flower buds were visible at the apex. It was
observed that the length of time between removal from the cold room
to the visible appearance of flower buds was shorter for Chinese
cabbage than for Siberian kale. This made it difficult to determine
the proper time to recdrd the data on bolting for the segregating

populations from this interspecific cross.

36

Table 1. Effect of plant size (leaf number) and duration of exposure
to 5°C on the percent bolting in Siberian kale.

 

 

Number of Heeks at 5°C Number of True Leaves Percent Bolting
5 2-3 0
6 2-3 66
7 2-3 100
8 2-3 100
5 4-5 0
6 4-5 100
7 4-5 100
8 4-5 100
5 6-7 0
6 6-7 100
7 6-7 100

6-7 100
5 7-8 0
6 7-8 100
7 7-8 100
8 7-8 100

 

37

A preliminary vernalization experiment was conducted in 1981
to observe bolting in response to 0, 1, 2, 3, 4, 5, 6, or 7 weeks
of vernalization of Siberian, Chee Hoo,_ and an F2 population
(Siberian x Chee Hoo). The visibility of flower buds was the
criterion used to classify plants as bolters. Data was recorded
weekly for 8 weeks following vernalization. Chinese cabbage bolted
in all treatments with 2 or more weeks of vernalization. The mean
number of days after removal from the cold room to bolting for the
cultivar Chee Hoo was significantly (p = .05) lower after 6 weeks
of vernalization (10 i .45 days) than after 2 weeks of vernalization
(32 t 1.37 days). Siberian kale bolted after 6 or more weeks of
vernalization. The mean number of days after removal from the cold
room to bolting of Siberian after 6 weeks of vernalization (26 i 1.82
days) was significantly greater (p = .05) than for Chee Hoo vernalized
for 6 weeks (10 1 .45). The lesser number of days to visible flower
buds in Chee Hoo after 6 weeks of vernalization as compared to 2
weeks of vernalization suggest that when the threshold or minimum
number of hours of thermo-induction is reached, vegetative growth
ceases and reproductive growth begins and continues while the plants
remain at the vernalizing temperatures. If this assumption is cor-
rect, the mean number of days to bolting in Chinese cabbage after 2
weeks of vernalization and the mean number of days to bolting in
Siberian after 6 weeks of vernalization should be similar. The mean
number of days to bolting in Chee Hoo vernalized for 2 weeks (32 i
1.37) was not significantly different (p = .05) from the mean number

of days to bolting in Siberian vernalized for 6 weeks (26 i 1.82).

38

None of the F2 plants bolted after 0, 1, 2, or 3 weeks of
vernalization. The percentage of F2 plants bolting after 4, 5, 6,
and 7 weeks of vernalization was 15%, 41%, 48%, and 70% respectively.
The absence of bolted F2 plants in the 2 and 3 week treatments and
the presence of some nonbolted plants after 6 and 7 weeks of
vernalization suggested transgressive segregation or for a need of
a more precise method for determining bolting. In an inheritance
study on bolting in celery, Bouwkamp and Honma (1970) made longi-
tudinal cuts through the apex of nonbolted plants to observe the
growing point for elongation when plants reached a marketable size.
Plants that showed pointed apices were classified as bolters. These
authors suggested that this criterion f0r bolting eliminated the
variability for rate of seed stalk development which is dependent on
post-vernalization environmental factors.

An experiment was conducted in the greenhouse in 1981 to
determine if a similar procedure for determination of bolting would
be applicable for this study. Eighty plants of Siberian, Nozaki
Early, and 160 F2 plants (Siberian x Nozaki Early) were grown at
20°C for 4 weeks prior to vernalization. The F2 and Siberian plants
were vernalized for 6 weeks while Nozaki Early was vernalized for 2
weeks. The vernalized plants were transplanted into a greenhouse
ground bed and the greenhouse temperature was maintained at 20°C.
Thirty days after removal from the cold room, plants with visible
flower buds were observed in all the populations. 0n the 30th, 37th,

44th, and Slst days after removal from the cold room, twenty parental

39

plants and 40 F2 plants were observed either for visible bolting or
were cut longitudinally to observe apex elongation. Data were
recorded on the percentage of plants visibly bolted and total per-
cent bolted (visible and plants with pointed apices) (Figure 4).
The results (Table 2) suggested that by cutting the plants, it was
possible to classify bolted plants that would have been classified
as nonbolters if only the visible bud technique was used. Since
the number of bolted plants as determined by cutting through the
apex did not change beyond 37 days in this study, it provided an
accurate estimation of bolting.

Based on these findings, the following procedure was imple-
mented for classifying bolting and nonbolting. Data was recorded
at weekly intervals for number of visibly bolted plants until the
bolt resistant parent visibly bolted or reached marketable size.
All nonbolted plants were cut longitudinally through the apex to
observe. for elongation. Plants with pointed apices were classified
as bolters while those with rounded apices were classified as non-
bolters. The data on percent bolters as determined from the sum of
visible bolters and those with pointed apices, for the various popu-
lations were used to develop genetic models for inheritance of bolt-
ing in the various crosses. Chi-square tests for goodness of fit
and statistical tests were conducted as outlined in Little and Hills

(1972).

40

Figure 4: Stages in seedstalk development. Left, not bolting;
center and right, bolting; growing points outlined
for ease of identification.

41

 

42

Table 2. Percentage of bolted plants at weekly intervals after

 

 

vernalization
Percent Bolted Plants
Generation 30 days 37 days 44 days 51 days
A B A B A B A B

 

Siberian kale 30 80 70 100 75 100 80 100
Nozaki Early 95 100 95 100 100 100 100 100
F2 75 95 84 95 88 9O 95 95

 

A = percent plants with visible flower buds at the apex.

B = total percent bolted plants, visible and from macroscopic
observation of longitudinal sections of apices.

Vernalization Experiments

 

Experiment 1: Natural Vernalization
of Siberian Kale, Chinese Cabbagei
an F2 Population (Siberian x Chee
Hop);and Chikale

 

 

In the spring of 1982 an experiment was conducted to
observe bolting under natural field conditions. This experiment
included an F2 p0pulation from the cross Siberian x Chee Hoo
(derived from sib-mating 3 F1 plants), Siberian kale, commercial
Chinese cabbage cultivars Chee Hoo, Michihili, and Spring A-1, and
4 late-bolt Chikale lines (LB-129A, LB-129B, LB-217, LB-219). Seeds
were sown in vermiculite on April 4, 1982. Seedlings with 1 true
leaf were transplanted into No. 24 PCV growing trays filled with a
mixture of] peat: 1 perlite: 1 vermiculite. The trays were placed

under G.E. High Intensity Discharge lamps with 1000 watt multivapor

43

bulbs (approximately 24llm'2) in a greenhouse maintained at 20°C.
Hhen the plants were 4 weeks old (5 to 7 true leaves), they were
transplanted into the field in a randomized complete block design,
with 4 replicates. Each replicate included 48 F2 plants and 6
plants of each of the other cultivars. The spacing within rows was
44 cm and between rows PBS 120 cm. After transplanting all plants
were watered with 120 ml of a starter solution and insecticide
(Diazinon) mixture. llarplot was irrigated, sprayed, and cultivated
as necessary. A pre-plant application of 1135 kg per hectare of
12-12-12 was made and one sidedressing of ammonium nitrate at 113.5
kg per hectare was applied 2 weeks after transplanting. The air
temperature was monitored with a Weather Hawk thermograph.
Experiment II: Artificial Vernaliza-

tion Studies Siberian Kale x
Chinese Cabbage

 

 

 

In 1982, an artificial vernalization experiment was con-
ducted to determine the bolting response of parents, F1, F2, F3 and
backcross p0pulations from the interspecific cross Siberian kale x
Chinese cabbage after various amounts of vernalization. Asexual
propagation of one Siberian x Chee Hoo F1 hybrid plant was used to
produce F2 and backcross populations. This experiment also included
F2 and backcross populations from the cross Siberian x Nozaki Early
and Mandarin x Siberian. Due to a limited amount of seeds, part of
the populations were only grown in selected treatments. The vernali-
zation treatments were 0, 2, 3, 4, 5, and 6 weeks. All treatments

were removed from the cold room August 24, and transplanted into the

44

field 1 week later. Planting in the field was a randomized complete
block design with 3 replicates and cultural practices were similar
to Experiment I. The experiment was terminated on October 18, 1982,
when Siberian plants that were vernalized for 6 weeks visible bolted.
Experiment III: Artificial Vernali-

zation Studies of Siberian x
Nozaki Early 53 Families

 

 

Observation of bolting response of Siberian, Nozaki Early,
and F3 families derived from selfing individual F2 plants form the
cross Siberian x Nozaki Early were made in the greenhouse during
the Fall (1982). These F2 plants all bolted after 6 weeks vernaliza-
tion. Due to partial sterility, the number of plants used in each
of the vernalization treatments (0, 2, 4, and 6 weeks) varied. After
vernalization treatments, the plants were tranSplanted into a green-
house ground bed at a temperature of 20°C. The experiment was
terminated in December when Siberian plants after 6 weeks of vernali-
zation visibly bolted.

Experiment IV: Artificial Vernaliza-
tion Studies of Chinese Cabbage x

Turnip

 

Inheritance studies of Chikale crosses with Hong Bok were
made “11982. Parents F1, F2 and backcross populations from recipro-
cal crosses: LB-7 x 08-2, LB-7 x Hong Bok, and QB-2 x Hong Bok were
vernalized for 0, 1, 2, 3, 4, and 5 weeks. Each vernalization treat-
ment included 12 plants of each parent and F1, 24 plants of each
backcross, and 66 plants of each F2. The plants were randomized

in 3 replicates, and transplanted into the field July 16, 1982.

45

The cultural practices were similar to Experiment I and the experi-
ment was terminated September 10, 1982, when the late-bolt Chikale
parent (LB-7) visibly bolted in the 5 week treatment.

Experiment V: Artificial Vernaliza-
tion Studies of Chinese Cabbage x

Turnip
This phase of the study on the inheritance of bolting

involved the intra-specific cross between Chinese cabbage (Mandarin)
and the turnip cultivar, Milan White. The various populations

were vernalized for 0, 1, 2, 3, 4, and 5 weeks. Each treatment
included 18 plants of each parent and F1, 72 F2 plants and 36 back-
cross plants which were divided into 3 replicates. This experiment
was terminated on October 20, 1982, when Milan Hhite after 5 weeks
of vernalization visibly bolted. Cultural practices were similar

to Experiment I.

RESULTS AND DISCUSSION

Experiment 1: Natural Vernalization Studies of Siberian
Kale,,Chinese Cabbage, an F2 Population (Siberian X Chee
H00) and Chikale

 

 

The data on the percent bolters for the various populations
grown under natural field conditions (May 4 to June 21, 1982) are
presented in Table 3. The Chinese cabbage cultivars and the Chikale
late-bolt (LB) lines had formed marketable heads by June 21, there-
fore, the experiment was terminated. Heekly observations were made
for the visual appearance of flower buds at the apex of the plants,
however, due to the formation of heads, it was not possible without
damaging the plant. By June 21 none of the plants showed visible
flower buds. All of the plants were cut longitudinally to observe
for apex elongation. All of the Michihili and Chee Hoo cultivars had
elongated apices with flower stalks of various lengths. Of the
cultivar Spring A-1, 44 percent showed elongation of their apices,
while the remaining 56% showed no signs of elongation. None of the
plants from the late-bolt Chikale lines, Siberian kale, and the F2
population (Siberian x Chee Hoo) showed signs of apical elongation.

The minimum-maximum air temperatures 30 cm from ground level
for each day from May 4 to June 20, 1982, are shown in Figure 5.
Using Yamasaki's formula and the temperature data recorded for this

natural vernalization experiment, the degree units obtained was

46

47

Table 3. Percentage of bolters under natural field conditions.

 

 

Population Percent Bolters*
Chee H00 100
Michihili 100
Spring A-I ‘ 44
Siberian kale 0
LB-129A 0
LB-129B 0
LB-217 0
LB-219 0
(Siberain x Chee Hoo) F2 0

 

*Percent bolters on June 21, 1982, 48 days from transplanting.

48

Figure 5. Minimum-Maximum air temperature chart for the period
May 4 to June 21, 1982.

49

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50

99.3°C which exceeded the amount (87°C) necessary to induce flower
primordia in Chinese cabbage. Yamasaki's formula was adequate in
predicting bolting in the cultivars Chee Hoo, Michihili, and

Spring A-l under natural vernalization even with fluctuating tem-
peratures. Nakamura (1976) suggested that minimum night temperature
was the important factor for induction of flower stalks in Chinese
cabbage. He observed that 10 days with minimum temperatures of 5°C,
15 days with minimum temperatures of 8-9°C and 30 days with minimum
temperatures of 13-14°C induced flower stalks in most Chinese cabbage
cultivars. In this experiment the plants were exposed to 1, 11, and
26 days with minimum temperatures of 5°, 8-9°, and 13-14°C, respec-
tively. The total cumulative cold units received by the Chinese
cabbage cultivars used in this exerpiment were sufficient to induce
flower stalks.

The fact that no visible bolting occurred in this experi-
ment is explained as follows. The temperature after transplanting
into the field was not conducive to initiate the development of the
floral primorida, but continued to favor the vegetative phase which
meant the development of 35 or more leaves which was sufficient to
form a head and mask the visible flower stalks. It is also possible,
as Nakamura (1976) suggested, that larger plants are more suscepti-
ble to low temperatures than smaller (younger) plants. Lorenz
(1946) observed that the cultivar Pe-tsai bolted before forming a
head if grown at temperatures between 10 and 15°C, while they bolted

after forming a head if grown at temperatures between 15° and 20°C.

51

The absence of bolters in the late-bolt Chikale lines and
the F2 population suggest that bolt resistance from Siberian kale was
transferred to Chinese cabbage. Although the F2 population did not
segregate for bolting response, the morphological variability observed
suggested that hybridization of Siberian x Chee Hoo was successful.

Experiment 11: Artificial Vernalization Studies of
Siberian Kale x Chinese Cabbage

 

' The percent of bolters in the parental, F1, F2, and backcross
populations for the cross Siberian kale x Chee Hoo are presented in
Table 4. Siberian kale required 6 weeks vernalization while Chee
Hoo bolted after 2 weeks vernalization. Due to the limited number
of F1 seeds, samples of the F1 population were vernalized for only
2, 4, and 6 weeks. Only F1 plants which were vernalized for 6 weeks
bolted, suggesting dominance for bolt resistance. Since no F1 plants
were included in the 5-week vernalization treatment, it was not
possible to distinguish between partial and complete dominance. It
was observed that after 6 weeks vernalization, the F1 plants bolted
30 days after removal from the cold room, while the Siberian plants
did not bolt until the 50th day. As discussed in the material and
methods, the lower number of days to visible bolting in the F1
population as compared to Siberian with the same degree of vernaliza-
tion suggest that the F1 plants required less cold than the Siberian
plants.

The percent of bolters in the F2 population increased with

increased duration of vernalization. The percent bolters observed

52

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53

after 2 weeks (10.9%) and 3 weeks (11%) were similar. The percent
bolters after 4 weeks (57%) and 5 weeks (62.3%) of vernalization were
also similar and were both significantly greater (p = .05) than
after 3 weeks. The percent bolters after 6 weeks vernalization
(82.8%) was significantly greater (p = .05) than after 5 weeks, how-
ever, 27.2% of the F2 plants were more bolt resistant than Siberian,
the bolt resistant parent.

. In the backcross to Siberian, no bolters were observed
until after a minimum of 4 weeks vernalization (Table 4). The per-
cent bolters observed after 4 weeks (58%), 5 weeks (61.5%), and
6 weeks (53.9%) of vernalization were not significantly different
(p = .05). Similar to that observed in the F2 population, plants
requiring a longer period of vernalization than Siberian were
observed. Of the plants in the backcross to Siberian population,
46% did not bolt after 6 weeks of vernalization.

In the backcross to Chee Hoo the percent bolters observed
after 2 weeks vernalization was 13.6% (Table 4). There was a sig-
nificant increase (p = .05) in percent of bolters after 3 and 4
weeks of vernalization (53.6% and 55.4%, respectively). The percent
of bolters also increased between 4 weeks (55.4%) and 5 weeks (76.9%)
vernalization and between 5 weeks and 6 weeks (89.9%) of vernaliza-
tion. In this population. 10.1% of the plants were more bolt
resistant than Siberian after 6 weeks vernalization.

This experiment also included F2 and backcross progeny from

the cross Siberian x Nozaki Early (Table 5) and Mandarin x Siberian

54

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55

(Table 6). The observed segregation for percent bolters for the
various durations of vernalization were similar for all 3 crosses in
that (1) the percentage of bolters increased with increased duration,
(2) the backcross to Siberian populations had fewer bolters in all
treatments than the F2 populations, (3) the backcross to Chinese
cabbage populations had larger percentage of bolters in all treat-
ments than the F2 populations, and (4) plants were observed in some
of the populations vernalized for 6 weeks which required longer dura-
tions of vernalization than Siberian. However, there were signifi-
cant differences between these 3 crosses for percent bolters in
certain treatments. For example, the percent bolters after 6 weeks
vernalization in the Mandarin x Siberian F2 p0pulation (100%) was
significantly greater (p = .05) than in the Siberian x Chee Hoo F2
population (82.8%) and the Siberian x Nozaki Early F2 population
(83.3%). The percent of bolters after 2 weeks vernalization in the
Mandarin x Siberian F2 p0pulation was also significantly greater

(p = .05) than the percent of bolters observed in the Siberian x Chee
Hoo F2 population and the Siberian x Nozaki Early F2 population.
Significant difference (p = .05) for percent of bolters were observed
in the backcross to Siberian populations for all 3 crosses after

3 weeks vernalization; the percent of bolters in the backcross popu-
lation Siberian x (Siberian x Chee Hoo) after 4 weeks of vernaliza-
tion was greater (p = .05) than the percent of bolters in the
corresponding p0pulations from the other 2 crosses, while after 5

and 6 weeks of vernalization, the percentage of bolters in the

56

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57

backcross to Siberian populations from the Siberian x Chee Hoo cross
were less (p = .05) than in the other 2 crosses. The percentage of
bolters were also significantly different (p = .05) in the backcross
to Chinese cabbage populations between at least 2 of the 3 crosses

in all of the treatments. It appears that the Chinese cabbage culti-
var used as a parent in crosses with Siberian affected the segrega—
tion for vernalization response. Although no differences were
observed for percent bolters between these 3 cultivars after I and 2
weeks of vernalization, they may'have shown differences if vernalized
for 8, 9, 10, ll, 12, and 13 days, due to minor genes. Minor genes
that effect bolting response may be responsible for the observed
differences for percent bolting in the populations produced from
crossing these 3 cultivars with Siberian.

The differences in the basic chromosome number between
Siberian kale (n = 19) and Chinese cabbage (n = 10) may also explain
the segregation ratios observed. Nwankiti (1970) and Mackay (1977)
previously reported that the resulting hybrids from crossing B, papp;

and B. campestris had 29 chromosome pairs, 10 from B. campestris

 

 

(p_genome) and 19 from B. napus (afp_genome). The pairing at meta-
phase I in these hybrids included 10 bivalents (presumably the

campestris a_genome and the napus afgenome) and 9 univalents (p

 

genome). Nwankiti (1970) observed some multivalent associations and
therefore the number of univalents was variable. Pollen mother cells
from the hybrids used in this study were observed to determine

chromosome number and pairing at metaphase I. All the hybrids had

58

29 chromosomes and the most common association at metaphase I was 10
bivalents and 9 univalents (Figure 2). In general, the bivalents
were centered along the equatorial plate and the 9 univalents were
scattered randomly in the cell. Occasionally, only a portion of the
9 univalents were separated from the bivalents which suggest some
multivalent associations. These observations agree with the reports
by Nwankiti (1970)'and Mackay (1977). Therefore, one would expect
chromosome numbers in the gametes to range from 10 to 19 and they
should occur in equal frequencies. The unequal chromosome numbers
in the gametes probably explains the reduced fertility observed in
the hybrids. Pollen viability as estimated by staining with acetic
carmine ranged from 44 to 58%, which agrees with that reported by
Mackay (1977). No attempt was made in this study to determine the
chromosome numbers in the F2 and backcross progenies. However,

both Nwankiti (1970) and Mackay (1977) observed a greater than
expected number of plants with chromosome numbers near to the euploid
level (20 and 38). They attributed this to selection for gametes
with extreme numbers of chromosomes, which Nwankiti (1970) suggested
was the result of their competitive ability in achieving fertiliza—
tion. They also observed sterility in the F2 and backcross plants
with aneuploid chromosome numbers which may help explain the steril-
ity of many F2 and backcross selections noted in this study. Since
the 2 species used in this study, and observations on chromosome
numbers, chromosome pairing, and fertility of the hybrids were

similar to that reported by Nwankiti (1970) and Mackay (1977), it is

59

possible that selection for euploid gametes may also have occurred
in the hybrids produced in this study.

A hypothesis for genetic control of bolting in this inter-
specific cross, Siberian kale x Chinese cabbage, is based on the
following: (1) Chinese cabbage, g_genome, bolts after 2 weeks
vernalization; (2) common cabbage, p_genome, bolts after 8 weeks
vernalization; (3) Siberian kale, a;p_genome, bolts after 6 weeks
vernalization, and (4) the sesqui-diploid hybird, EELS genome,
bolts after 5 or 6 weeks of vernalization. According to the hypothe-
sis of Purvis (1966) on vernalization and gene action, plants
requiring thermo-induction are lacking genes or their products that
are able to bring about flowering in related species without a cold
requirement. That is, the cold period compensates physiologically
for the absence of these genes' products. Such a hypothesis suggest
that plants with a greater cold requirement have more negative or
inactive alleles while those with a lesser cold requirement have
more positive or active alleles. One can only speculate as to the
nature of these genes or gene products. Melchers and Lang (Lang,
1965) and Purvis (1966) favor the hypothesis of a specific substance,
"vernalin," which is produced through a series of biochemical inter-
mediates and that the enzymes that catalyse these reactions leading
to the formation of "vernalin" have lower temperature optimums or
are activated at lower temperatures. Therefore, at low temperatures
"vernalin" formation is favored. Assuming that the substrates for

these hypothetical enzymes are not limiting, the production of more

60'

enzyme would hasten the reactions thereby shortening the length of
cold period required to produce sufficient levels of "vernalin" to
allow for bolting and flowering. Increased levels of enzyme could
result from (1) changes in regulator genes which allows for increased
production of these enzymes; (2) loss of suppressors that limit pro-
duction of these enzymes; or (3) increased number of structural genes
which code for these enzymes. It is difficult to determine the
actual mechanism underlying the segregation of degree of vernaliza-
tion requirement, however, it is possible that varying levels of
catalytic enzymes effect the rate of "vernalin" formation. It is
suggested that the rate of vernalin formation as effected by genotype
is the basis for variable degrees of vernalization requirement in the
progeny produced from crossing Siberian kale and Chinese cabbage.

Selfing the F1 hybrid could produce progeny with 20, 29, or
38 chromosomes as well as some aneuploids, assuming selection for
euploid gametes. The homology between chromomes from the a_and a}
genomes would make it possible for chiasmata formation and crossing
over to occur, as well as allow for random assortment of the chromo-
somes from the a and a; genomes. Crossing over and random assort-
ment of the chromosomes would allow for recombination of genes which
condition the degree of vernalization requirement and could produce
individuals with various levels of bolt resistance in each of the
ploidy levels. This recombination of both major and minor genes may
have resulted in the segregants that require greater periods of

vernalization than Siberian kale. The majority of individuals

61

selected for vernalization requirements greater than Siberian kale
were sterile which could have resulted from aneuploidy as suggested
by Nwankiti (1970) and Mackay (1977). Presumably, the individuals
with greater vernlization requirements than Siberian kale are lacking
genes present in Siberian kale and Chinese Cabbage which may have
conditioned reduced vernalization requirement and that the absence
of these genes may have resulted from either recombination, crossing
over, or aneuploidy.

A similar relationship appears to exist in the backcross
populations. In the backcross to Chinese cabbage (§_genome), the
majority of the individuals should have either 20 or 29 chromosomes,
therefore the percent of bolters expected after 6 weeks of vernaliza-
tion is 100%. In the backcross to Nozaki Early (Table 5) and
Mandarin (Table 6) populations, 100% of the plants bolted after 6
weeks of vernalization. However, in the backcross to Chee Hoo popu-
lation only 89.9% of the plants bolted after 6 weeks of vernalization.
As mentioned, these nonbolters could be attributed to recombination
or aneuploidy.

In the backcross to Siberian (2.19. genome), the majority of
the plants would have either 29 or 38 chromosomes, therefore would
yield only a few bolters after vernalization of less than 4 weeks.
In the Siberian x (Siberian x Chee Hoo) population no bolters were
observed after 2 or 3 weeks of vernalization (Table 4). In the
Siberian x (Siberian x Nozaki Early) population, 18.2 percent of the

plants bolted after 3 weeks of vernalization (Table 5) and is

62

probably due to individuals with 29 chromosomes which may have
resulted from gametes which carried Chinese cabbage genes which
conditioned shorter vernalization requirements. In the backcross
p0pulation, Siberian x (Manarin x Siberian), 13% and 40% of the
plants bolted after 2 and 3 weeks of vernalization, respectively
(Table 6). These bolters are also probably due to aneuploidy.
The frequency of these genotypes was higher than expected if chromo-
some distribution was at random. One possible explanation for this
apparent nonrandom distribution of chromosomes is that the cytOplasm
effected the ability of certain gametes to produce balanced pollen
grains (Nwankiti, 1970). Another possible explanation for the
higher than expected percentage of bolters in some of these popula-
tions may be due to cytoplasmic factors which influence the vernaliza-
tion processes (Bouwkamp and Honma, 1970). The increased number of
bolters observed when Manarin is used as the female parent (Table 6)
as compared to when Siberian is used as the female parent (Table 4
and Table 5) may be attributed to one or both of these factors.

Observations made of the F3 families tend to support the
theory that the genomic constitution of the parents may have influ-
enced the segregation for vernalization response. All of the F2
plants selected that failed to bolt after 6 weeks of vernalization
were nearly sterile which suggest that these plants had aneuploid
chromosomes.

Only 2 F3 families from the Siberian x Chee Hoo cross were

observed for vernalization response. The percent bolters after

63

varius durations of vernalization for these F3 families are pre-

sented in Table 7. One of these F3 families (SC-3) was produced

Table 7. Percent bolters after five durations of vernalization in
two F3 families from the cross Siberian kale x Chee Hoo

 

Heeks at 50°C

 

 

F3 Family

2 1 3 ‘ 4 5 6
SC-3 ‘ 20 83 100
SC-6 O O O 16 36

 

from an F2 plant that did not bolt after 2 weeks of vernalization
while SC-6 was produced from an F2 plant that did not bolt after 5
weeks of vernalization. The observed segregation for SC-3 suggest
one major gene is segregating for vernalization requirement. The
expected percent of bolters assuming one major additive gene is

25% after 2 weeks of vernalization. Using the gene symbols [_for
bolt susceptibility and y for bolt resistance, these 25% bolters
after 2 weeks of vernalization have the genotype xx, The 75%
bolters expected after 4 weeks vernalization are the sum of the geno-
types 1! and 1!: while the 100% bolters expected after 6 weeks
vernalization are the sum of the genotypes BB, 1!: and pp, Although
the chromosome number of this F2 plant was not determined cytologi-
cally, its fertility in crosses with Chinese cabbage suggest it has

20 chromosomes. The observed segregation for bolting response can

64

be explained if it is assumed that this F2 plant has one y_allele
from Chinese cabbage and one y_allele from Siberian kale.

The F3 family SC-6 has 16% bolters after 5 weeks vernaliza-
tion and 36% bolters after 6 weeks vernalization (Table 7). The F2
parent of this p0pulation was not fertile in crosses with Chinese
cabbage suggesting that it was polyploid. The absence of bolters
after 2, 3, or 4 weeks of vernalization suggest that the F2 parent
did not have any major genes from Chinese cabbage that conditioned
reduced vernalization requirement. The observed segregations sug-
gest that the F2 parent was heterozygous for major and minor genes
from Siberian and that through recombination, individuals that
require 5, 6, or more weeks of vernalization were produced. Possibly
variation of ploidy level may also have effected the observed segre-
gations.

Experiment III: Artificial Vernalization Studies
of Siberian x Nozaki Early F3 Families

The data for percent bolting after 2, 4, or 6 weeks of
vernalization for 6 (Siberian x Nozaki Early) F3 families are pre-
sented in Table 8. Although the population sizes were small, the
observed segregations suggest 2 or 3 different F2 genotypes, which
produced these families. The F3 families SN-2, SN-18, SN-20, and
SN-25 segregated for 1 major additive gene, 1 (less vernalization),
and ! (greater vernalization). Individuals that bolted after 2 weeks
vernalization have the genotype 1!, those that bolt after 4 weeks

vernalization have the genotypes B! or ii: and all three genotypes

65

Table 8. Percent bolters after three durations of vernalization of
F3 families from the cross Siberian x Nozaki Early

 

Heeks at 5°C

 

 

F3 Family
2 4 6
SN-2 33 (12) 67 (12) 100 (12)
SN-7 O (12) 37 (12) 100 (12) E“
SN-17 O (12) 67 (12) 100 (12) E
SN-IB - 28 (22) 100 (12)
SN-20 I7 (12) 100 (12)
SN-25 23 (48) 96 (48) h

 

 

*Number in parenthesis represents the total number of plants.

L!!9.!!1.!!) bolt after 6 weeks vernalization. The few nonbolters
observed after 6 weeks vernalization in SN-25 may be due to minor or
modifier genes. Chi-square tests for goodness of fit to a single
additive gene model for inheritance of bolting response in the F3
families SN-2, SN-18, SN-20, and SN-25 all suggest a good fit

(Table 9). The high degree of self-fertility of these F2 plants

and their observed fertility in crosses with Chinese cabbage suggest
they had 10 chromosome pairs.

The 2 remaining F3 families (SN-7 and SN-17) failed to yield
bolters after 2 weeks of vernalization. The percent bolting of these
2 families differed after 4 weeks of vernalization (Table 8) sug—
gesting that the individual F2 plants were of different genotypes.
SN-17 had 67% and 100% bolters after 4 and 6 weeks of vernalization,

66

 

1 ..
d1..1.\i - In . I.

 

 

 

 

 

-1 11 o N we we mN1zm o
- - o o NH NH 0N1zm o
- 1- o o NH NH wH1zm m
- 1- o 0 NH NH N-zm o
m. 1 N ¢¢.o m a m m N1zm e
N. - m HH.o om Nm NH HH mN-zm N
m. 1 N e¢.o m 0H m N QN-zm N
m. 1 m no.0 m.oH oH m.m o wH1zm N
m. - N ¢¢.o m m m e N-zm N
a x umaumaxm um>emmno umuomnxu nm>tmmno coHHmHsaoa coHHNHmeumw»
memHHoncoz weep—om
NHLmN me~oz
x cavemawm mmoeu we» sage mmHHHEaN mu mo :oHHmNHHmcem> No mcompmezu woes» Loewe
mcwupon so» Hmvos memo m>HuHuua mHacwm a on awe No mmwcuoom Low Ham» menacm-ng .m mHamN

67

respectively. The similarity in percent bolters after 4 and 6 weeks
of vernalization between SN-17 and SN-2 and the observed fertility
when SN-17 was crossed with Chinese cabbage suggest that this F3
parent was an euploid with 10 chromosome pairs and that the segre-
gation of modifiers genes produced all nonbolters in the 2-week
treatment. Perhaps the absence of bolters in this population may s:
also have been due to the small population size (12 plants).

' The F3 family, SN-7 had only 36% bolters after 4 weeks
vernalization and was not grown in the 6-week treatment due to

limited number of seeds. The F2 parent was highly sterile and

 

failed to produce any viable seeds in crosses with Chinese cabbage,
suggesting polyploidy. Since there were no plants in the 6-week
treatment, the determination of the parental genotype is not possible.
The observed segregation in this F3 family is attributed to both

gene and chromosome number differences. It appears that the segre-
gation for bolting response noted from Siberian kale x Nozaki Early
F3 families tends to support the theory that recombination of parental
genes conditioning vernalization requirement and the variation in

ploidy level were responsible for the segregation of this character.

Experiment IV: Artificial Vernalization of Chikale
This experiment included crosses between Chikale lines devel-
oped for quick-bolting habit (1 week of vernalization) and late-
bolting habit (4 weeks of vernalization) and the Chinese cabbage

cultivar Hong Bok.

68

Chikale LB-7 x Chikale QB-Z

 

The data for percent bolters after 0, 1, 2, 3, and 4 weeks of
vernalization for the parents, F1, F2, and backcross populations
from reciprocal crosses between Chikale LB-7 and Chikale QB-2 are
presented in Table 10. When Chikale QB-2, Chikale LB-7, and Hong

Bok plants were placed in a greenhouse with a temperature of 20°C

5 - 11.2? J'Afi

following artificial vernalization, bolting was observed after 1, 4,
and 2 weeks of vernalization, respectively. However, since QB-2
plants in the control (0 weeks) treatment bolted and LB-7 bolted l

after 3 weeks of vernalization, it was evident that further thermo-

 

induction occurred after the plants were removed from the cold room.
While the plants were in the lath house awaiting transplanting into
the field, the minimum night temperatures ranged from 9° to 11°C for
6 days, and this additional low temperature may have caused the bolt-
ing. Since all of the plants received similar post-vernalization
temperatures, the data were interpreted as noted.

The quick-bolt parent, QB-2, bolted in all of the treatments
while the late-bolt parent, LB-7, required a minimum of 3 weeks
vernalization to bolt (Table 10). The F1 population from reciprocal
crosses differed in the duration of vernalization required to induce
bolting, suggesting cytoplasmic influence in the vernalization
responses; however, no significant reciprocal differences (p = .05)
between the F2 populations were observed. The intermediate vernaliza-
tion requirement of the F1 plants and the increase in percentage of

bolters with increased duration of vernalization suggest additive

69

Table 10. Bolting percentages after five durations of vernalization
for the various populations from the cross LB-7 x 08-2.

 

Heeks at 5°C

 

 

 

 

Generations
O 1 2 3 4

LB-7 O 0 O 100 100

08-2 100 100 100 100 100

(LB-7 x OBp2) F1 0 0 100 100 100 3
(QB-2 x LB-7) F1 0 100 100 100 100 g
Fl pooled 0 50 50 100 100 E
(LB-7 x 08-2) F2 27 68 92 100 100 Z
(AB-2 x LB-7) F2 21 53 91 100 100 1
F2 pooled 24 60 91.5 100 100

(LB-7 x 08-2) x LB-7 0 0 83 100 100

(QB-2 x LB-7) x LB-7 0 0 46 100 100

F1 x LB-7 pooled 0 O 64.5 100 100

(LB-7 x QB-Z) x QB-2 83 92 100 100 100

(08-2 x LB-7) x 08-2 78 92 100 100 100

F1 x 08-2 pooled 80.5 92 100 100 100

 

70

gene action, which supports the observations made in Experiment II
and 111. Based on the observed segregations for botling response,
the following gene model is proposed. The model involves two coup-
ling phase additive gene pairs. The gene !_is used for reduced
vernalization requirements, and l for greater vernalization require-
ment. Different gene pairs are designated by use of subscripts.

The quick-bolt parent having the least cold requirement has

the genotype-BLAZE!2 and bolts after 0 weeks of vernalization. The

 

late-bolt parent has the genotypellllpzp2 and bolts after 3 weeks of
vernalization. The F1 is heterozygous for both 1°C1’-!1!1—2!2 and is
phenotypically intermediate of the parents bolting after 1 or 2
weeks of vernalization. Based on the hypothesis on vernalization
(Purvis, 1966, and Lang, 1965) that the cold period leads to the
formation of a specific flower-inducing substance, vernalin, which
brings about changes in the cells of the meristematic region leading
to flowering, the cytOplasmic differences noted may be attributed

to differential sensitivity of the cytoplasm to intermediate levels
of the gene products, if it is assumed that the cytOplasm is the
receptor of the vernalizing stimulus and that the chemical and bio-
chemical changes which lead to bolting and flowering take place in
the cytoplasm (Bouwkamp and Honma, 1970). The absence of reciprocal
differences in the F2 populations probably resulted from recombina-
tion of nuclear genes which effected the cytoplasm, thereby reducing
cytoplasmic differences in the reciprocal F‘2 populations. The back-

cross populations were produced by using the recurrent parent as the

71

female in crosses with the F1 hybrids, therefore, the cytoplasm of
the recurrent parent makes up the majority of the cytoplasm in the
backcross progeny, and may also have effected the vernalization
response of this population. The Chinese cabbage parent from which
these Chikale lines resulted was the cultivar Mandarin which was
the parent used in crosses with Siberian kale (Experiment II). In
this cross cytoplasmic differences were also noted.

- Based on the proposed 2 additive gene model, the F2 popula-
tion would have l/16 (4 "BB alleles): 4/16 (3 "V" alleles): 6/16
(2"V“ alleles): 4/16 (l "V" allele) and l/16 (no "V" alleles). The
observed frequency of individuals with the Quick-bolt parental
phenotype is approximately 4/16 which suggest that individuals with
3 and 4 "V" allele bolt after similar durations of vernalization.
Therefore, it is proposed that individuals with 3 or 4 "V" alleles
bolted after 0 weeks, plants with 2 or more "V9 alleles bolt after 1
week, plants with 1 "V" allele bolt after 2 weeks and plants with
no "V" alleles at these 2 loci bolt after 3 weeks of vernalization.
Chi-square tests were conducted to determine if the observed ratios
in the segregating populations for each duration of vernalization
fit the 2 additive gene model. The observed percent bolters sug-
gested a good fit to the proposed model in all populations except
the backcross to LB-7 populations (Table 11). The poor fit to the
eXpected ratios in the backcross to LB-7 population after 1 week of

vernalization may be due to cytoplasmic factors.

72

1. .11 3.... .12....)13HH

 

 

 

 

1- -- 00.0 0 00 00 N100 x H: 0
1- 1- 00.0 0 00 00 N-0H x H: 0
11 -- 00.0 0 NNH NNH N: 0
-1 1- 00.0 0 00 00 N-00 x H: N
00. 1 H. 0N.N 00.NH NH 00 H0 N100 x H: N
0. - 0. 00.0 0N.0 HH 0N.NNH HNH N: N
-- -1 00.0 0 00.00 00 N-00 x H: H
H0. v 0 00.0H 00.00 00 00.NH 0 N-0H x H0 H
00. 1 H. 00.0 0N.H0 N0 0N.00 00 N: H
H. 1 N. 00.H 00.00 0N 00.00 00H N100 x H0 0
1- -- 00 00 00.0 0 N104 x H: 0
m0. 1 H. N0.N 0N.00 00H 0N.H0 N0 N0 0
cmuumaxm 00>:mmno cmyumgxm uw>emmno
0 Nx 00H0000000 0.0 00 0:003
emaHoaeoz 000,00

 

.N-mo x N-mH mmoeu m:u so» :oH00NHH0::m> No 0:0Humtau Lacy Lope:
0:HuHo: Low Hence 0:00 m>Hchum exp 0 ca ppm No 000:0000 sow mummy meoaam1H:u

.HH mpnmh

73

Chikale LB-7 x HongBok
The proposed 2 additive gene model is further supported by
the segregation for bolting response in the cross Chikale LB-7 x

Hong Bok (Table 12). The Chinese cabbage parent, Hong Bok, bolted

Table 12. Percentage of bolters* after five durations of vernaliza-
tion in the various populations from the cross LB-7 x

 

 

 

Hong Bok.
' Heeks at 5°C
Generations
0 1 2 3 4
LB-7 0 0 0 100 100
Hong Bok 0 100 100 100 100
(LB-7 x Hong Bok) F1 0 O 100 100 100
(LB-7 X Wang Bok) F2 0 26.5 80.5 100 100
F1 x LB-7 0 O 33 96 100
0 73 100 100 100

F1 X Wang Bok

 

*Data from reciprocal populations pooled.

in all treatments with 1 or more weeks of vernalization while the
late-bolt parent, LB-7, bolted in all treatments with 3 or more

weeks of vernalization. The data were pooled since no significant
differences (p = .05) were observed between reciprocal populations.
The F1 was intermediate for bolting response and bolted after 2 weeks
of vernalization (Table 12). The F2 and backcross populations segre-
. gated for bolting response. The percent bolters in the F2 (26.5%)
and the backcross to Hong Bok (73%) p0pulations after 1 week of

74

vernalization suggested that only one gene was segregating in this
cross. The following gene model is proposed based on the model
developed for the previous cross (LB-7 x QB-2). Hong Bok has the
genotype.y_111.y'_2.!2 and bolts after 1 week of vernalization. LB-7

has the genotype 11111212 and bolts after 3 weeks of vernalization.
The F1 has the genotypehglgex2 and bolts after 2 weeks vernaliza-
tion. The expected F2 segregation is 25% bolters after 1 week,

75% bolters after 2 weeks, and 100% bolters after 3 weeks of vernaliz-
tion. Chi-square tests (Table 13) for goodness of fit to these
expected ratios suggest a good fit. The deficiency of bolters in

the backcross to Chikale LB-7 population after 2 weeks of vernaliza-
tion and the presence of 2 nonbolted plants after 3 weeks of vernali-
zation may have resulted from recombination of modifier genes or
crossing over.

Chi-square analysis of goodness of fit to the expected ratio
of 75% bolters to 50% nonbolters in the backcross to Hong Bok popu-
lation after 1 week of vernalization showed a good fit (p > .50) and
all of the plants bolting after 2 or 3 weeks of vernalization supports

the single additive gene model proposed (Table 13).

Hong Bok x Chikale QB-2

The segregation for bolting for the parents, F1, F2, and
backcross populations from the cross Hong Bok x QB-2 also supports
the proposed additive gene model (Table 14). Chikale QB-2 has the
genotype BLBLBZBZ and bolts after 0 weeks of vernalization. Hong Bok

has the 99"°t7P9.1}!}!z!2 and bolts after 1 week of vernalization.

75

 

 

 

 

 

- - 0 0 00 00 000 0003 x H0 0
- - 0 N 00 00 N10H x H: 0
1- - 0 0 NNH NNH N0 0
- - 0 0 00 00 000 0003 x H: N
N0. - 00. 00.0 0N N0 0N 0H N-0H x H: N
H. - N. 00.H 00 0N 00 00H N: N
N. 1 0. HH.0 NH 0H 00 00 000 0003 x H0 H
- - 00 00 0 0 N-0H x H0 H
0. - N. 0H.0 00 N0 00 00 N: H
umuu0axm 00>:0mno 000000xm u0>e0mno
0 NH 00HH000000 0.0 00 00003

.000H00002

000H00

 

.300 0:03 x N1>H 000:0 0:» :00 :oH00NHH0::0> mo 0:0H00::0 m
:0000 0:N0Hon :0» H0005 0:00 0>H0H000 0:0 0 op up» mo 000:0000 :00 0000» 0:0:am1p:u .mH 0H00N

76

Table 14. Percentage of bolters* after five durations of vernaliza-
tion in the various populations from the cross Hong Bok

 

 

 

x QB-2
Heeks at 5°C

Generations

O 1 2 3 4
Hong Bok O 100 100 100 100
QB-2 100 100 100 100 100
(Hong Bok x QB-2) F1 100 100 100 100 100
(Hong Bok x QB-2) F2 63 93 97 100 100
F1 x Hong Bok 71 100 100 100 100
F1 x 08-2 100 100 100 100 100

 

*Data from reciprocal populations were pooled.

The data were pooled since no significant reciprocal differences were
observed (p = .05). The F1 has the genotype-Blah!2 and bolts after
0 weeks of vernalization. The vernalization treatments plus the
additional post vernalization in this series of experiments did not
make it possible to differentiate plants with the genotypesllyllz!2
and XIXIXZXZ' The percent bolters expected in the F2 p0pulation
after 0 weeks of vernalization is 75%. Chi-square tests (Table 15)
suggest a poor fit (p < .01). This poor fit and the presence of 9
nonbolting plants after 1 week and 4 nonbolting plants after 2 weeks
of vernalization in the F2 population are attributed to recombina-
tion or crossing over.

The segregation for bolting in the backcross populations

supports the proposed additive gene model. After 0 weeks of

77

 

 

 

 

- - 0 0 00 00 N-00 x H: N
- - 0 0 00 00 000 0003 x H: N
-1 1- 0 0 N0H 0NH N: N
11 - 0 0 00 00 N-00 x H0 H
1- - 0 0 00 00 000 000: x H: H
- - 0 0 NNH 0NH N: H
1- - 0 0 00 00 N-00 x H: 0
0. - N. 00.0 NH 0H 00 00 000 000: x H: 0
H0. v 0 H0.N 00 00 00 00 N: 0
00pu00xm 00>:00ao 000000xm 00>:00no
0 Nx :0000:0:0w com 00 0x00:

.000H00002

000H00

 

.N1mo x :00 0:03 000:0 0:0 :00 :0H00NHH0::0> 00 0:0000:00 00::u
:0000 0:HHH0: :00 H0002 0:00 0>000000 0:0 0 00 0H0 00 000:0000 :00 0000p 0:00001_:u .mH 0H00N

78

vernalization the expected percent bolters in the backcross to Hong
Bok population is 75% and Chi-square tests suggested a good fit
(Table 15). The absence of nonbolters after 1 and 2 weeks of
vernalization in the backcross to Hong Bok populations and in all
of the backcross to Chikale QB-2 populations was expected based on
the proposed single additive gene model.

Based on the observed bolting segregations from these 3
crosses following various durations of vernalization and the origin
of the Chiklae lines, it appears that genes for lesser vernalization
requirement were contributed by both Siberian kale and Chinese
cabbage. The segregations observed in the Siberian kale x Chinese
cabbage crosses (Experiment II and III) and in the Chikale x Chinese
cabbage crosses are explainable if 4 major genes control vernaliza-
tion requirement. As mentioned previously, the gene symbol "Bf con-
ditions a reduced vernalization requirement while {1" conditions a
greater vernalization requirement. Alleles of these 4 genes have
an equal and additive effect on vernalization requirement. Chinese
cabbage has the ge"°tYPe.Xll}!2!2!3!3!4!4 and bolts after 2 weeks of
vernalization. Siberian kale has the genotype X1113222!3!3!4!4 and
bolts after 6 weeks of vernalization. It is suggested that these
genes for reduced vernalization requirement are on chromosomes of the
a_and.a' genomes since cabbage (p_genome) requires 8 weeks of vernali-
zation. The F1 hybrid from the cross Siberian kale (female) x Chee

Hoo has the genotype V1!1!2!H!3!3quq and bolts after 5 weeks of

vernalization. Based on additive gene action, the hybrid with 4 "V'I

79

alleles should bolt after 4 weeks vernalization. Since reciprocal
differences have been observed in this study, the increased vernaliza-
tion requirement of this Fl may be attributed to cytoplasmic factors.
Due to pairing of the a_and a] genomes in the hybrid and assuming
random assortment, individuals with 10 chromosome pairs in the
offspring of this hybird have between 8 9V“ alleles and no "V" alleles.
As suggested previously, polyploidy could also produce individuals
with more or less "V" alleles than the parents. That is, individuals
which resulted from the pairing of gametes which both were lacking
chromosomes from the parents carrying the "V“ alleles would have a
vernalization requirement greater than Siberian kale. If these
gametes had chromosomes from the p_genome substituted for chromo-
somes from the a or_a' genomes they would theoretically be func-
tional and able to complete fertilization, however, the resultant
zygote would be aneuploid for specific chromosomes. This aneuploidy
could explain the observed association between greater vernalization
requirement and sterility.

Both Chikale lines used in this study appear to have 10
chromosome pairs based on fertility in crosses with Chinese cabbage
(n = 10). Cytological observation of the pollen mother cells from
Chikale LB-7 revealed 10 chromosome pairs (Figure 2). The follow-
ing genotype is proposed for Chikale LB-7, glylyzgzgayay4g4, however,
other genotypes with 4 "V" alleles are possible. The vernalization
requirement of Chikale LB-7 is 4 weeks. Chikale QB-2 has the geno-
type 11!}!2!2!3!3!4!4 and it bolts after 1 week of vernalization.

80

The proposed genotypes and phenotypes for diploid individuals with

10 chromosome pairs are presented in Table 16.

Table 16. Proposed genotypes and phenotypes for bolting response
produced from the cross Siberian kale x Chinese cabbage.

 

 

Genotype Phenotype
V1V1V2V2V3V3V4V4 Bolts after 1 week of vernalization
V1V2V2V2V3V3v4v4 Bolts after 2 weeks of vernization
V1V1V2V2v3v3v4v4 Bolts after 4 weeks of vernalization
V1V1v2v2v3v3v4v4 Bolts after 6 weeks of vernalization
v1v1v2v2v3v3v4v4 Bolts after 8 weeks of vernalization

 

Experiment V: Artificial Vernalization of Chinese
cabbage x Turnip Populations

The results of the vernalization response from the cross
between Chinese cabbage (Mandarin) and turnip (Milan Hhite) are
shown in Table 17.

Mandarin bolted after 2 weeks of vernalization while Milan
Hhite bolted after 5 weeks vernalization. The F1 hybrids from the
unilateral cross Mandarin x Milan Hhite bolted after 3 weeks of
vernalization. The intermediate vernalization requirement of the
F1 and the observed increase in percent of bolters with increased
vernalization in the F2 and backcross populations suggest additive

gene action with a few number of genes. The segregating populations

81

Table 17. Percentage of bolters after four durations of vernaliza-
tion in the various p0pulations from the cross Mandarin
x Milan Hhite.

 

Heeks at 5°C

 

 

Generations

2 3 4 5
Mandarin 100 100 100 100
Milan Hhite O 0 O 100
F1 0 96 100 100
F2 25 67 95 100
Mandarin x F1 88 100 98 100

 

showed variation for leaf type, heading tendencies, and root shape.
Although most plants were intermediate for these characters, both
parental types were observed. No apparent association was noted
between the morphological characteristics and bolting response; how-
ever, the 4 nonbolting plants in the F2 population after 4 weeks
vernalization had turnip-like roots which were phenotypically simi-
lar to Milan Hhite, while their leaf morphology was intermediate to
the parents. Hester and Magruder (1938) reported that most turnip
cultivars, including Milan Hhite, did not bolt until reaching market-
able size even after prolonged exposure to low field temperatures.
This type of phasic development has also been reported in radish
(Yun and Pyo, 1977). The development of an enlarged root prior to
bolting even under thermo-inductive conditions suggest a complex

phasic development. This complex phasic development may explain the

 

82

absence of recombinants in the segregating populations from this
cross having Chinese cabbage leaf morphology and head formation with
bolt resistance equal to turnip (nonbolting after 4 weeks of vernali-
zation). Since the desired recombinant was not found, it would
appear that hybridization between Chinese cabbage and turnip to
develop bolt resistance in Chinese cabbage may be difficult.

Crane (1943) suggested that kale cultivars with 38 chromo-
somes were produced by natUral hybridization between turnip (2n = 20)
and kale (2n = 18). Thus the similarity between Siberian kale
(glaipp) and turnip (pp) for vernalization requirement suggest that
inheritance for vernalization response in the cross Mandarin x Milan
Hhite is similar to Chinese cabbage x Siberian kale. Based on the
observed segregations for bolting response, the following additive
gene model is proposed. Mandarin has the genotype glylyzy2v3v3qu4
and bolts after 2 weeks of vernalization. Milan Hhite has the geno-
type V1V1v2v2v3v3v4v4 and bolts after 5 weeks of vernalization. The
lesser vernalization requirement of Milan Hhite than Siberian kale
with the same number of "V" alleles could result from the differences
in genomic constitution.

The F1 hybrid is heterozygous at the 12 and B3 loci and has
4 "V“ alleles, which condition bolting after 3 weeks of vernalization.
The F1 hybrid from crossing Mandarin and Milan Hhite was also inter-
mediate for leaf morphology and degree of fleshy root (Figure 3).
Since no reciprocal cross was made, determination of cytoplasmic

effects are not possible in this population.

83

The expected percentage of bolters in the F2 population after
2, 3, 4, and 5 weeks of vernalization are 31%, 69%, 94%, and 100%,
respectively. Chi-square tests (Table 18) show a good fit to the
proposed additive gene model. The backcross to Mandarin is expected
to have 75% bolters after 2 weeks of vernalization and 100% bolters
after 3 or more weeks of vernalization. The Chi-square value for the
backcross to Mandarin after 2 weeks of vernalization (6.35) suggest
a poor fit to this additive gene model. The percent of bolters
in this population may be attributable to recombination of modifier

genes and also to the complex phasic development in turnips.

84

 

 

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- - 00.0 0 00 00 H: x EHLaeeaz 0
0. 1 N. 00.0 00.0 0 0.H0 00 N: 0
- - 00.0 0 00 00 H0 0 EHteecaz 0
N. - 0. HH.0 00.0H 0H 0N 0N NH 0
H0. - N0. 00.0 00.NH 0 00.H0 00 H: x 0::0000: N
N. - 0. . 00.H 0N.H0 00 0N.0H 0H. N: N
000000xm 00>:00ao 000000xm 00>:0000 :0000:0:00 000 00 0x00:

 

 

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000:3 :0HHz x :0:00:02 000:0 0:0 :00 :0000NNH0::0> No 0:0000:00 :000
:0000 0:00H00 :00 H0005 0:00 0>H00000 030 0 00 000 00 000:0000 :00 00000 0:0000-H:u .mH 0H000

SUMMARY AND CONCLUSIONS

The inheritance of bolting in Chinese cabbage (Brassica

campestris L. ssp. pekinensis) was investigated by hybridizing

 

 

Chinese cabbage with Siberian kale (B, napus), Chikale (B, campestris

 

L. ssp. pekinensis x B, napus), and turnip (B, campestris L. ssp.

 

 

rapifera). The inheritance model was developed from segregation
ratios observed in segregating p0pulations from these crosses after
various durations of vernalization at 5°C and 16 hour daylength.
Percent of bolters was determined by the sum of visible bolters and
longitudinally cut plants with pointed apices, observed when the
bolt-resistant parent either visibly bolted or reached a marketable
size.

Segregation for bolting under natural field vernalization
was observed in Chinese cabbage, Chikale late-bolt lines, Siberian
kale, and an F2 population (Siberian kale x Chee Hoo). The observed
bolting in the Chinese cabbage cultivars while Chikale, Siberian
kale and the F2 population failed to bolt suggested (1) differences
in bolting response observed after artificial vernalization at a
constant temperature (5°C) are also expressed after natural vernali-
zation with fluctuating temperatures and (2) bolt resistance from
Siberian kale was transferred to Chikale and the F2 population.

The genetic model was based on observations from a series of

hybridizations and included four major genes, modifier genes, and

85

 

86

cytoplasmic factors. Segreation for bolting response in the progeny
from the crosses Siberian kale x Chee Hoo, Siberian kale x Nozaki
Early, and Mandarin x Siberian kale suggested bolting response was
conditioned by a few major additive genes and that percent of bolters
was dependent on the Chinese cabbage cultivar. An increase in the
percent bolters in crosses where Chinese cabbage was used as the
female suggested cytoplasmic factors. The difference in chromosome
number between the two parents (Chinese cabbage, n = 10 and Siberian
kale, n = 19) produced varying degrees of fertility in the progeny.

Variable ploidy level, random assortment, and probable crossing over

 

of chromosomes may have produced the observed segregations.

Results from selfing F2 plants assumed to have euploid chromo-
some numbers based on their fertility suggested one or more additive
genes were conditioning bolting response.

Results from crossing quick-bolt and late-bolt Chikale lines
with Chinese cabbage, cultivar Hong Bok, suggested 2 major additive
genes were conditioning bolting response. Cytoplasmic influences
were noted in certain crosses.

Results from crossing Chinese cabbage, cultivar Mandarin,
with turnip, cultivar Milan Hhite, also suggested 2 major additive
genes and modifiers were conditioning bolting response.

The gene model was expanded from the 2 major additive gene
theory to 4 major additive genes to hypothesize the probable geno-
types of the parents based on the above crosses. The following geno-

types and phenotypes are proposed for the parents: Chinese cabbage

87‘

has the genotype V1V1V2121313_4_4 and bolts after 2 weeks of vernali-
zation. Siberian kale has the genotype l}_}_2!z!3_3y4y4 and bolts
after 6 weeks of vernalization. Chikale LB-7 has the genotype
11!1!2!2!3!3!4!4 and bolts after 4 weeks of vernalization, while
Chikale 08- 2 has the genotype V1V1V2_2V3_3_4_4 and bolts after 1

week of vernalization. The turnip cultivar Milan White has the
genotype V1V1v2v2v3v3v4v4 and bolts after 5 weeks of vernalization.
Recombination of these 4 major additive genes and modifier genes
accounts for most of the observed segregations.

The complex nature of the vernalization processes and the
interplay between environmental and genetic factors would suggest
quantitative inheritance. Apparently in Brassica, variation in the
vernalization requirement was evolved through selection for specific
genes which had major effects on the vernalization processes. As
mentioned in the literature review, the primary genomes of Brassica
have developed through duplication of specific chromosomes of the
basic set of 6 chromosomes. If these major genes are situated on
a specific chromosome which is found in higher dosage in one species
as compared to others, then variation for vernalization requirement
between these species could result from a dosage effect of particu-
lar chromosomes. For example, Chinese cabbage (a genome) has three
_[ chromosomes while cabbage (p_genome) has only one B chromosome
(Robbelen, 1960). A similar dosage effect was observed by Nwankiti
(1971) for leaf serration in progeny from a cross between B,

campestris ssp. chinensis and B. napus. Therefore, if such a dosage

 

88

effect does exist, breeding for bolt resistant Chinese cabbage
through interspecific crosses with B, pppp§_should be possible as
noted in this study of the late-bolt Chiklae line LB-7. Based on
the additive gene action theory on vernalization requirement, a
system to concentrate the alleles into the inbreds for use in hybrid
production is suggested. Interspecific hybridization drastically
reduces the horticultural quality required in Chinese cabbage and
therefore recombining desirable quantitative traits such as heading,
flavor, and texture with genes for bolt resistance will require time

and patience.

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89

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