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Cytogenetic analysis of translocation heterozygotes and trisomics isolated from their progenies in common bean (Phaseolus vulgaris L.)

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Title:
Cytogenetic analysis of translocation heterozygotes and trisomics isolated from their progenies in common bean (Phaseolus vulgaris L.)
Added title page title:
Common bean
Added title page title:
Phaseolus vulgaris
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Ashraf, Mohammad, 1945-
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English
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xi, 121 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Chromosome translocation ( jstor )
Chromosomes ( jstor )
Disomics ( jstor )
Genetics ( jstor )
Heterozygotes ( jstor )
Planting ( jstor )
Pollen ( jstor )
Seed pods ( jstor )
Tetrasomics ( jstor )
Trisomics ( jstor )
Beans -- Genetics ( lcsh )
Dissertations, Academic -- Horticultural Science -- UF
Horticultural Science thesis Ph. D
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1985.
Bibliography:
Bibliography: leaves 115-119.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Mohammad Ashraf.

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CYTOGENETIC ANALYSIS OF TRANSLOCATION HETEROZYGOTES
AND TRISOMICS ISOLATED FROM THEIR PROGENIES IN COMMON BEAN
(PHASEOLUS VULGARIS L.)






BY






MOHAMMAD ASHRAF


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


UNIVERSITY OF FLORIDA


1985
























To those who devoted their lives

for the welfare of mankind.















ACKNOWLEDGMENTS


There are many people whom I would like to thank for providing

help, infusion of ideas, understanding, friendship and love. These

are the people who supported me through events that will be

remembered as cherished memories. Special thanks go to Mark J.

Bassett, Professor, Vegetable Crops Department, who not only served

as committee chairman and shared his expertise, but also provided

untold hours in consultation and providing guidance to shape a

growing mind which had no previous exposure to cytogenetics.

I also thank Drs. Ken H. Quesenberry, Associate Professor of

Agronomy; Paul M. Lyrene, Professor, Fruit Crops Department; L.C.

Hannah, Professor, Vegetable Crops Department; Daniel A. Roberts,

Professor, Plant Pathology Department; and Gloria A. Moore, Assistant

Professor, Fruit Crops Department, for furthering my knowledge of

cytogenetics, genetics, plant breeding and plant pathology.

I am especially appreciative to my wife and kids, who, for the

past 3 years, have provided much in the way of spiritual and moral

support as well as exhibited exemplary patience and understanding

necessary for the family of a graduate student. This degree is as

much theirs as it is mine, and I hope our future adventures may be as

pleasant and successful as this one has been.









Finally, the Quaid-e-Azam/Merit scholarship awarded by the

Ministry of Education, Government of Pakistan, for completion of this

study is highly acknowledged. I also acknowledge the research grant

provided for this project through Dr. M.J. Bassett, without which it

would not have been possible to work on the project reported in this

dissertation.















TABLE OF CONTENTS


Page

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

LIST OF TABLES..... ....... ..................................vii

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

ABSTRACT................................... ................... x

SECTIONS


I INTRODUCTION ........................... ....... ....... 1

II CYTOGENETIC ANALYSIS OF TRANSLOCATION HETEROZYGOSITY
IN COMMON BEAN (PHASEOLUS VULGARIS L.)..................3

Introduction.................................................3
Materials and Methods...............................15
Results and Discussion...............................16

III TERTIARY TRISOMICS AND TETRASOMICS FROM TRANSLOCATION
HETEROZYGOTES IN COMMON BEAN (PHASEOLUS
VULGARIS L.) ...........................................23

Introduction..........................................23
Materials and Methods...................................35
Results and Discussion................................37

IV ORIGIN, CYTOLOGY, MORPHOLOGY AND TRANSMISSION OF
THE EXTRA CHROMOSOME IN FIVE PRIMARY TRISOMICS OF
COMMON BEAN (PHASEOLUS VULGARIS L.)....................67

Introduction.........................................67
Materials and Methods.................................73
Results and Discussion...............................74

V SUMMARY AND CONCLUSIONS..............................102


APPENDICES

1


MODIFICATION OF THE SLIDE PREPARATION TECHNIQUE
OF CHENG AND BASSETT (1981)..........................104










2 OFF-TYPE PLANTS OBSERVED AMONG THE PROGENIES OF
TRANSLOCATION HETEROZYGOTES DURING THE FIRST FIELD
PLANTING (SUMMER, 1984)..............................107

3 ANEUPLOIDS RECOVERED FROM THE PROGENIES OF
TRANSLOCATION HETEROZYGOTES DURING THE SECOND
FIELD PLANTING (SUMMER, 1985)........................110

4 PROCEDURES TO IDENTIFY PRIMARY AND TERTIARY
TRISOMICS ORIGINATING AMONG THE SELF PROGENY OF
A TRANSLOCATION HETEROZYGOTE.........................113

5 FIELD PLOT NUMBERS OF PLANT PROGENIES GROUPED
ACCORDING TO THEIR SIMILARITY OF SEGREGATION
PATTERNS; TABLE 111-2 (SECOND FIELD PLANTING,
SUMMER, 1985)........................................114

LITERATURE CITED...............................................115

BIOGRAPHICAL SKETCH............................................120














LIST OF TABLES


Table Page

II-1 Chromosome configurations at M-I at meiosis observed
in PMCs of FI progeny from crosses of translocation
homozygote stocks.......................................... 17

II-2 Comparison of the mean number of seeds per pod and the
distribution of seeds per pod in F1s of each cross
combination showing abnormal'M-I configurations
compared to a standard normal bean breeding line
0-181................. ..... ..... ... .......... ...... ........20

III-1 Transmission of the extra chromosome in four trisomics
of common bean (First greenhouse planting, Fall, 1984).....40

111-2 Progeny tests of two tertiary trisomics originally
derived from translocation heterozygote 1-52 (Second
field planting, Summer, 1985).............................41

111-3 Segregation for trisomic progeny from selfed (2n+l)
or cross pollinated [(2n+l) x 2n] trisomics originally
derived from four different translocation heterozygote
progenies (Second field planting, Summer, 1985)............46

III-4 Putative trisomics derived from different translocation
heterozygote progenies; their phenotype and rate of
transmission of extra chromosome in (2n+l) self
(Second field planting, Summer, 1985).....................47

IV-1 Transmission of the extra chromosome in two trisomics
of common bean after crossing [(2n+l) x 2n] (Second
greenhouse planting, Spring, 1985)........................82

IV-2 The frequency of PMCs with different chromosome
configurations at Diplotene to M-I of meiosis in
different plant progenies of Weak Stem Trisomic............93















LIST OF FIGURES


Figure Page

II-1 Illustration of various types of quadruple pairing
derived from a translocation heterozygote with
photomicrographs of these configurations in common
bean ......................................................7

11-2 Coorientation types of quadruples at M-I ..................10

11-3 Illustration of events leading to formation of a ring
of 6 chromosomes.........................................10

11-4 Metaphase-I of a normal disomic and diplotene to M-I
of a translocation heterozygote with one or two
translocations involving common or different
chromosomes in common bean...............................14

III-1 A table giving all the types of trisomics that can
result from selfing a translocation heterozygote...........28

III-2 Pentavalent configurations expected in a tertiary
trisomic according to various positions of chiasmata.......32

III-3 Meiotic diplotene to anaphase-I as observed in
tertiary trisomics of common bean.........................52

III-4 Meiotic diakinesis to anaphase-I as observed in a
tetrasomic of common bean.................................57

III-5 Photographs of tertiary trisomic and tetrasomic
plants............. ............................ ..... .60

III-6 Plant photographs of suspected trisomics (genetic
evidence only)......................................... ..64

IV-1 Meiotic diplotene to anaphase-I as observed in the
primary trisomics of common bean..........................78

IV-2 Meiotic diplotene to anaphase-I as observed in the
primary trisomics of common bean..........................80

IV-3 Plant photographs of primary trisomics at the
seedling stage............................................84


viii




LIST OF FIGURES
Figure Page
11 -1 Illustration of various types of quadruple pairing
derived from a translocation heterozygote with
photomicrographs of these configurations in common
bean 7
11 -2 Coorientation types of quadruples at M-I 10
11-3 Illustration of events leading to formation of a ring
of 6 chromosomes 10
II4 Metaphase-I of a normal disomic and diplotene to M-I
of a translocation heterozygote with one or two
translocations involving common or different
chromosomes in common bean 14
III-1 A table giving all the types of trisomics that can
result from selfing a translocation heterozygote 28
III-2 Pentavalent configurations expected in a tertiary
trisomic according to various positions of chiasmata 32
III-3 Meiotic diplotene to anaphase-I as observed in
tertiary trisomics of common bean 52
III-4 Meiotic diakinesis to anaphase-I as observed in a
tetrasomic of common bean 57
III-5 Photographs of tertiary trisomic and tetrasomic
plants 60
III-6 Plant photographs of suspected trisomics (genetic
evidence only) 64
IV-1 Meiotic diplotene to anaphase-I as observed in the
primary trisomics of common bean 78
IV2 Meiotic diplotene to anaphase-I as observed in the
primary trisomics of common bean 80
IV-3 Plant photographs of primary trisomics at the
seedling stage 84
viii












Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

CYTOGENETIC ANALYSIS OF TRANSLOCATION HETEROZYGOTES
AND TRISOMICS ISOLATED FROM THEIR PROGENIES IN COMMON BEAN
(PHASEOLUS VULGARIS L.)

BY

MOHAMMAD ASHRAF

December, 1985


Chairman: Mark J. Bassett
Major Department: Horticultural Science (Vegetable Crops)


Common bean, Phaseolus vulgaris L., does not have either a

translocation tester set or aneuploid series available for use in

linkage studies. In 1982 Bassett and Hung attempted to develop a set

of translocations by means of pollen irradiation. Genetic tests gave

an indication that the resulting semisterility mutations might be due

to translocation heterozygosity. In this study, an attempt was made

to 1) investigate cytologically these semisterility mutations; 2) if

they are translocations, determine whether they have common or

different chromosomes involved in the interchanges; 3) quantify

reduction of seed set due to translocation heterozygosity; 4) search

for trisomics among the SI progenies of translocation heterozygotes,

assuming that they are phenotypically distinguishable; and 5) develop

primary trisomics, 2n + 1 = 23, on normal chromosome background.









This work has proved that the five semisterility mutations

studied are chromosome translocations. The five stocks used in this

study have at least 8 different chromosomes involved in their

reciprocal translocations. The cytological behavior and

morphological abnormalities with respect to pollen and ovule abortion

in translocation heterozygotes of common bean as observed in this

study are quite similar to the earlier reports in other crops.

The 11 translocation stocks, used in the search for trisomics,

produced 12 distinct phenotypic groups of plants. Five of these 12

groups have been verified to be trisomics by genetic tests and by

cytological verification. Genetic evidence from the remaining seven

indicates trisomy, but this has not yet been confirmed

cytologically. Five primary trisomics have been identified on the

basis of their cytological behavior. Of these five primaries, three

related tertiaries have been identified on the basis of their

phenotypic relationship. For another primary group, the related

tertiary has been isolated on the basis of cytology. Two trisomic

groups produced tetrasomics at a low frequency. For two of the five

primary trisomic groups, primaries have been developed on a

homozygous normal chromosome background. Physical identification of

the extra chromosome has not yet been done for any of the

translocation and trisomic groups.














SECTION I
INTRODUCTION



Extensive linkage maps have been produced for several crops, and

for many other crops they are being worked out. Their knowledgeable

use can be of considerable advantage in the design of breeding

projects and genetic experiments. Despite their potential value, the

development of linkage maps has been very slow, especially for self-

pollinated crops. Common bean is a self-pollinated crop, for which

26 genes were initially assigned to eight linkage groups (Lamprecht

1961). Since then, very few linkages have been reported although

over 200 genes have been described for common bean (Roberts 1i2).

There are several reasons for the lack of linkage study in

common bean, one of which is that neither a chromosome translocation

tester set nor an aneuploid series, most commonly used for gene

mapping, has yet been developed in common bean.

Chromosome translocations were first reported by Belling (1914)

while working on a breeding program for improvement of Florida velvet

bean. The phenomenon was not well understood until 1925. While

Belling was working with the Blakeslee group at Cold Spring Harbor,

he showed how semisterility could be due to segmental interchange

between nonhomologous chromosomes. He also reported the linkage

between semisterility and several characters in Florida velvet








bean, but it was not known whether these factors belonged to the same

or different linkage groups. Later on, interchanges were reported in

Drosophila (Stern 1926), and they were first induced by x-rays in

Drosophila during 1930 (Muller). In maize, interchanges were first

reported by Brink (1927) and confirmed cytologically by Burnham

(1930). Since then they have been induced in many crops and

extensively used by plant breeders for understanding the gene-

chromosome relationship (Burnham 1956, 1962).

The discovery of the Globe trisomic of Datura stramonium, the

first primary trisomic, was the beginning of the pioneer

investigations of trisomics by Blakeslee and Avery (1919). Trisomics

have appeared spontaneously among progeny of normal diploids of many

other species but are obtained more frequently from asynaptic

mutants, desynaptic mutants, or among the progeny of polyploids, most

notably triploids (Khush 1973). Chromosome translocations, when in a

heterozygous form, also produce aneuploids at a low frequency (Khush

1973). Trisomics are another excellent tool for assigning linkage

groups to specific chromosomes. As a matter of fact, the most

extensive genetic studies done with the aid of aneuploids have been

conducted with trisomics (Schaeffer 1980).














SECTION II
CYTOGENETIC ANALYSIS OF TRANSLOCATION HETEROZYGOSITY IN
COMMON BEAN (PHASEOLUS VULGARIS L.)



Introduction

A mutation in which terminal segments of nonhomologous

chromosomes have exchanged positions is known as chromosomal

interchange, an interchange, a reciprocal translocation or simply a

translocation. All of these terms have been and are still used

interchangeably in the literature to describe the same phenomenon.

Translocations have been and still are among the most productive

tools of cytogenetics. They offer an enormous potential for plant

breeders to engage in chromosome engineering.

Naturally occurring translocations were first reported by

Belling (1914) in Florida velvet bean, followed by Stern (1926) in

Drosophila. Muller (1930) was the first to induce chromosome

translocations by x-rays, in Drosophila. In maize, translocations

were first reported by Brink (1927) and were confirmed cytologically

by Burnham (1930). The classical experiments of Burnham (1930) and

of Brink and Cooper (1931) established that semisterility caused by

translocation has all the characteristics of a gene located at the

translocation breakpoint. The location of the breakpoint in relation

to other genes can therefore be determined and mapped.








As soon as it was established that translocations could be used

successfully for gene mapping, researchers started developing tester

sets of translocations in which the positions of breakpoints with

respect to centromeres and telomeres were known. Maize (Burnham

1954) and barley (Burnham et al. 1954) were the first field crops

followed by English pea (Lamm and Miravalle 1959), rye (Sybenga and

Wolters 1972), and tomato (Gill et al. 1980) for which complete

tester sets of translocations were developed.

We are aware of only one report of chromosome translocations in

common bean before 1982 (Mutschler and Bliss 1980). In this case one

of the bean breeding lines segregated for semisterile and fertile

progeny plants in a 1:1 ratio. The term semisterility has been used

to refer to those plants in which pollen abortion rates fall between

normal (fertile) and sterile plants. The semisterility was

attributed to translocation heterozygosity, but the authors could not

provide any cytological proof, stating that tetravalent formations

were not observed at meiosis, probably due to the very small size of

common bean chromosomes. Furthermore, they reported no reduction in

seed set in the semisteriles. In 1982, Bassett and Hung induced

semisterility mutations (probably translocations) in common bean.

They used irradiated pollen to pollinate normal, untreated plants.

Twelve semisterile mutant lines were produced and various genetic

tests supported the hypothesis that the semisterility was due to

translocation heterozygosity. Five out of these 12 translocation

lines were used in this study.








The photomicrographs provided alongside each drawing in Figure

II-1 show the cytological behavior of a translocation heterozygote in

common bean at different stages of meiosis. Figure II-1A shows the

mutual exchange of chromosome segments between 2 nonhomologous

chromosome pairs. If the ends of chromosomes are numbered (1-2,

3-4), the 4 chromosomes that all have different end combinations

(1-2, 2-3, 3-4, 4-1) will result. No 2 chromosomes of this group can

pair along their entire length but all 4 can come together in a

pairing configuration (quadruple) that allows partial pairing of

homologous chromosome segments. Quadruples (Sybenga 1972) resemble

quadrivalents, which are formed when all 4 chromosomes are homologous

or equivalent to each other. An organism in which quadruple pairing

occurs is called a translocation heterozygote. In pachytene such a

configuration can appear as a cross (Fig. II-1B), which sometimes may

be prevalent even in diplotene.

Translocation breaks can occur at any point along the

chromosome. The position of the breakpoints will determine the

future fate of the translocation quadruple. Different quadruple

configurations can arise depending on the formation of crossovers in

interstitial or distal chromosome segments. Possible interstitial

crossovers can lead to duplication and deficiency gametes and

consequently are not recovered (Burnham 1962). Possible diakinesis

configurations originating from different pachytene situations are

shown in Figure II-1C,D,E. If for instance, crossovers occur in

locations 1, 2, 3, 4, 5 and 6, diakinesis configurations resemble a

number 8. If chiasmata occur only in locations 1, 3, 5 and 6, a ring

















Fig. II-1 Illustration of various types of quadruple pairing
derived from a translocation heterozygote with
photomicrographs of these configurations in common bean
(x 1000) (modified from Schaeffer 1980).

A. Two nonhomologous chromosome pairs involved in
reciprocal translocation.

B. Pachytene configuration appearing as a cross.
Numbers 1 to 6 designate chiasmata.

C. If chiasmata form only at 1, 3, 5 and 6, the
quadruple appears as a ring at diplotene to M-I.

D. If chiasmata form at all six locations, the quadruple
appears as a figure 8.

E. If chiasmata form only at 1, 3 and 5, the quadruple
appears as a chain. The thinner arrow in (e) that
points downward indicates a chain of 4 chromosomes.
The thicker arrow indicates a noncooriented
quadruple.

F. Noncoorientation of a quadruple, which gives rise to
nondisjunction and consequently an aneuploid gamate.










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of four chromosomes is formed in diakinesis. If chiasmata occur in

locations 1, 3, and 5, a chain of four chromosomes can form.

Chromosome orientation of quadruples in M-I is critical.

Bivalents in normal meiosis have only 2 centromeres that are arranged

in coorientation and that distribute the 2 chromosomes involved to

opposite poles. However, several types of orientation on the

metaphase plate are possible when a quadruple is formed, since not 2

but 4 centromeres are involved.

Figure II-2 illustrates the coorientation types of quadruples in

metaphase-I. Alternate-1 and alternate-2 give rise to viable gametes

because 2 translocated chromosomes pass to one pole and the 2

nontranslocated chromosomes pass to the other, giving balanced

chromosome complements in the gametes. On the other hand, adjacent-1

and adjacent-2 give rise to duplication and deficiency gametes, which

cause semisterility in translocation heterozygotes (Endrizzi 1974).

The proportion of fertile and aborted gametes is close to 1:1 in

several species. It is expressed by half of the seeds being missing

in an inflorescence of a plant, e.g., maize, petunia, peas and

sorghum, whereas it is less in others like Hordeum, Secale, Datura,

Triticum and Oenothera (Schaeffer 1980).

In noncoorientation (Fig. II-1F) the 2 centromeres on opposite

sides of the quadruple are cooriented and are positioned equidistant

from the equitorial plate. The 2 intermediate centromeres are

noncooriented and are stretched out between the other 2, seemingly

not attached to the poles. In anaphase-I, there may be a 2:2 or 3:1

segregation, the first one giving rise to a duplication-deficiency




8
of four chromosomes is formed in diakinesis. If chiasmata occur in
locations 1, 3, and 5, a chain of four chromosomes can form.
Chromosome orientation of quadruples in M-I is critical.
Bivalents in normal meiosis have only 2 centromeres that are arranged
in coorientation and that distribute the 2 chromosomes involved to
opposite poles. However, several types of orientation on the
metaphase plate are possible when a quadruple is formed, since not 2
but 4 centromeres are involved.
Figure 11-2 illustrates the coorientation types of quadruples in
metaphase-I. Alternate-1 and alternate-2 give rise to viable gametes
because 2 translocated chromosomes pass to one pole and the 2
nontranslocated chromosomes pass to the other, giving balanced
chromosome complements in the gametes. On the other hand, adjacent-1
and adjacent-2 give rise to duplication and deficiency gametes, which
cause semisteri1ity in translocation heterozygotes (Endrizzi 1974).
The proportion of fertile and aborted gametes is close to 1:1 in
several species. It is expressed by half of the seeds being missing
in an inflorescence of a plant, e.g., maize, petunia, peas and
sorghum, whereas it is less in others like Hordeum, Secale, Oatura,
Triticum and Oenothera (Schaeffer 1980).
In noncoorientation (Fig. 11-IF) the 2 centromeres on opposite
sides of the quadruple are cooriented and are positioned equidistant
from the equitorial plate. The 2 intermediate centromeres are
noncooriented and are stretched out between the other 2, seemingly
not attached to the poles. In anaphase-I, there may be a 2:2 or 3:1
segregation, the first one giving rise to a duplication-deficiency


Fig. 11-2 Coorientation types of quadruples at M-I.
Noncoorientation is shown in Fig. 11 1. Letters
designate centromeres and numbers designate chromosome
ends (modified from Schaeffer 1980).
Fig. 11-3 Illustration of events leading to formation of a ring of
6 chromosomes.
A. The two nonhomologous pairs involved in two
independent reciprocal translocations having one
chromosome in common.
B. Pachytene configuration appearing as a star which
forms a ring or chain of 6 depending upon chiasmata.






(and hence nonviable) gametes and the second giving rise to aneuploid

gametes (Hagberg 1954).

The objectives of this study were to (1) determine cytologically

whether the semisterile stocks developed by Bassett and Hung (1982)

are translocation heterozygotes; (2) if the semisterility in these

stocks is due to chromosome interchanges, determine whether the

translocation lines used in this study have common or different

chromosomes interchanged; and (3) develop quantitative data on the

reduction of seed set in translocation heterozygotes. The

information from objective 2 will help establish a tester set of

translocations for linkage studies if the chromosomes involved in

translocations can be physically identified. This information could

be tested by correlating it with the different types of trisomics

originating in the progeny of each translocation heterozygote.

Since only 1/4 of the progeny from the intercross of two

heterozygous translocation lines should carry both translocations, it

is preferable to isolate homozygous lines for each translocation as a

first step. In intercrosses between such lines or in crosses with

the homozygous normal plants, all the F1 plants can be used (Burnham

et al. 1954). Theoretically, half the normal (fertile pollen) plants

in the progeny derived from self-pollination of a plant heterozygous

for an interchange should be homozygous for that interchange. To

identify translocation homozygotes, the plants having normal pollen

are selfed and also crossed onto a standard normal line. If the

plant being tested is homozygous for the interchange, all the








test-cross F1 plants should be semisterile (Brink and Burnham

1929). When homozygous translocation lines are crossed with a

standard normal line and meiosis of the F1 plants is examined

cytologically, all the cells show either a pachytene cross

configuration or one of the other metaphase-I configurations already

discussed (Fig. II-1C,D,E). When homozygous translocation lines are

intercrossed and the meiosis of F1 plants is examined cytologically,

a ring or a chain of 6 chromosomes (Fig. II-3 and II-4F,G) indicates

that only 1 chromosome is common to the two interchanges, whereas if

separate rings or chains of 4 chromosomes are observed (Fig.

II-4C,E), this indicates that the two interchanges have no

chromosomes in common (Burnham et al. 1954).

In translocation heterozygotes of barley showing a ring of 4

chromosomes at M-I of meiosis, the pollen abortion rate ranges in

different lines from 14 to 58 percent the average being 28.8

percent. It is 51 percent in one line with two separate rings of

4. Ovule abortion on the same plants reported is 39, 92 and 67

percent respectively. The data on ovule abortion are based on seed

set counts of two heads on the original semisterile plants, and may

not necessarily be the true degree of sterility (Burnham et al.

1954). In another study, seed set in translocation heterozygote

barley lines has been found to be between 60 to 70 percent compared

with 97 percent in the original control varieties (Kunzel and

Bretschneider 1981). A negative correlation between the number of

translocated chromosomes and the fertility of the plants has been

observed in Pisum lines heterozygous for these translocations










Fig. 11-4 Metaphase-I of a normal disomic and diplotene to M-I of a
translocation heterozygote with one or two translocations
involving common or different chromosomes in common bean
(x 1000).

A. Diplotene of a translocation heterozygote involving
one translocation. Arrow indicates a ring of 4
chromosomes. Eight bivalents are spread around and
one bivalent is still associated with the nucleolus.

B. Metaphase-I of (A) above. Arrow indicates a ring of
4 chromosomes. The lighter black dot with two
bivalents on either side of it is the disappearing
nucleolus.

C. Metaphase-I of a PMC heterozygous for two independent
translocations. The two arrows each point to a ring
of 4 chromosomes.

D. Metaphase-I of a normal disomic bean PMC showing 11
bivalents.

E. Metaphase-I of a translocation heterozygote involving
two independent translocations. The two arrows
indicate a ring of 4 and a chain of 4 chromosomes.
One bivalent is overlapping the right end of the
chain.

F. Metaphase-I of a translocation heterozygote involving
two translocations having 1 chromosome in common.
The arrow indicates a ring of 6 chromosomes and the
interpretive drawing is to the right (f).

G. The arrow indicates a chain of 6 chromosmes and the
interpretive drawing is to the right (g).

f & g. Please note that in both interpretive drawings (f)
and (g) one pair of normal chromosomes, i.e., abcd,
ijkl and two pairs of translocated chromosomes, i.e.,
ao efcd and ijgh, efkl are involved in multivalent
formation. One pair of normal chromosomes, i.e.,
efgh common to both translocations, is not involved
in multivalent formation.






















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glacial acetic acid and treated for 24 h. Buds were then transferred

to 70% ethanol for preservation. Microscopic slides were prepared

using the technique of Cheng and Bassett (1981) with slight

modification (see Appendix I). A total of 50 pollen mother cells

from pachytene to M-I of meiosis were analyzed for each cross

combination.



Results and Discussion

In crosses 1 to 5 (Table II-1) all the five stocks were

individually crossed to standard normal line, 0-181. PMCs of Fls

showed 1 4 and 9 II during late prophase and metaphase-I (Fig.

II-4A,B). This cytological evidence along with the previous genetic

evidence (Bassett and Hung 1982) demonstrates conclusively that the

semisterile stocks used in this study are translocation

heterozygotes. Meiotic metaphase-I of the standard normal line

(0-181) included in this study was normal. It did not show any type

of multivalent association (Fig. II-4D).

The half diallel set of crosses among the five homozygous

translocation lines was examined cytologically to determine whether

the five interchanges might involve any particular nonhomologous

chromosome in more than one interchange. Fls of crosses 6, 7, 10,

11, 13 and 15 (Table II-1) showed 204 + 7 II, indicating that 4

different nonhomologous chromosomes are involved in the two

interchanges (Fig. II-4C,E). In cross 8, we observed 104 and 1

chain of 4 with 7 II chromosomes (Fig. II-4E) instead of 204

chromosomes. When the same translocation stocks involved in cross 8,




16
glacial acetic acid and treated for 24 h. Buds were then transferred
to 70% ethanol for preservation. Microscopic slides were prepared
using the technique of Cheng and Bassett (1981) with slight
modification (see Appendix I). A total of 50 pollen mother cells
from pachytene to M-I of meiosis were analyzed for each cross
combination.
Results and Discussion
In crosses 1 to 5 (Table 11-1) all the five stocks were
individually crossed to standard normal line, 0-181. PMCs of F^s
showed 1 4 and 9 II during late prophase and metaphase-I (Fig.
II-4A.B). This cytological evidence along with the previous genetic
evidence (Bassett and Hung 1982) demonstrates conclusively that the
semisterile stocks used in this study are translocation
heterozygotes. Meiotic metaphase-I of the standard normal line
(0-181) included in this study was normal. It did not show any type
of multivalent association (Fig. II-4D).
The half dial lei set of crosses among the five homozygous
translocation lines was examined cytologically to determine whether
the five interchanges might involve any particular nonhomologous
chromosome in more than one interchange. F^s of crosses 6, 7, 10,
11, 13 and 15 (Table 11 -1) showed 24 + 7 II, indicating that 4
different nonhomologous chromosomes are involved in the two
interchanges (Fig. II-4C.E). In cross 8, we observed 10 4 and 1
chain of 4 with 7 II chromosomes (Fig. II-4E) instead of 204
chromosomes. When the same translocation stocks involved in cross 8,






namely 1-52 and 11-70, were individually crossed with the standard

normal line (0-181), both of the stocks individually showed 104 +

9 II at M-I. The differential behavior of one of the two

translocation stocks at M-I may be attributed to the involvement of

an acrocentric chromosome in reciprocal translocations in that

stock. When the breakpoint is in the short arm, this hinders

chiasmata formation.

Cross 12 shows a chain of 6 and 8 II chromosomes (Fig. II-4G),

indicating that translocations 1-97 and II-121 have 1 chromosome in

common in the two interchanges. Formation of a chain of 6

chromosomes instead of a ring of 6 may possibly be due to the same

reason discussed above for cross 8 (Table II-1). Cross 14 (Table

II-1) showed 106 (Fig. II-4F), indicating the involvement of 1

common chromosome in both of the interchange stocks. All the PMCs

scored in cross 9 (Table II-1) failed to show any clear ring or chain

configuration. In most of the cells there were either 2 or 3

bivalents lying together, from which it was not possible to make any

interpretation.

Keeping in view the cytological results stated in Table II-1,

one can conclude that the five homozygous translocation lines used in

this study involve 8 (out of 11 total) chromosomes in their

interchanges. It would be quite desirable to have all 11 chromosomes

involved in the 12 interchanges present in these lines because the

ultimate aim is to develop a complete set of primary trisomics from

these stocks. Results of cross 9 involving 1-52 x 11-121 were

inconclusive, but this did not affect our ability to establish that a









minimum of 8 chromosomes are involved. When we cross 11-121 with

I-97 or 11-3, we see a chain or ring of 6 chromosomes proving that

II-121 has one common chromosome involved in interchange with respect

to both 1-97 and 11-3. When 1-97 is crossed with 11-3, 204 result,

indicating that both interchanges involve unique pairs of

chromosomes. Assigning arbitrary chromosome numbers to the stocks

for illustration, if 11-3 has 1 and 2 interchanged and 1-97 has 3 and

4 interchanged chromosomes involved in translocations, then 11-121,

on the basis of our results, has 1 of the 2 interchanged chromosomes

in 11-3 (either chromosome 1 or 2) and 1 of the 2 interchanged

chromosomes in 1-97 (either chromosome 3 or 4) involved in its

interchange. Crosses 6 and 7, in which 1-52 has been crossed with

1-97 and II-3 respectively, tell us that 1-52 neither has its

chromosomes involved in the translocations 1-97 and 11-3. Therefore,

interchange 1-52 can be assigned 5 and 6 for its chromosomes.

Because interchange 11-70 has no chromosome in common with 11-3,

1-97, 11-121 and 1-52, therefore a thorough analysis of all the data

in Table II-1 indicates that the two interchanges in cross 9 probably

involve unique chromosome pairs and that 8 chromosomes (in total) are

involved in reciprocal translocations in the five stocks used in this

study. The percent reduction in seed set for cross 9 (Table II-2)

also indicates that 204 should have formed, but for some reason

typical rings or chains were not apparent.

Average pollen abortion rates for crosses 1 to 5, which showed

104 chromosomes at M-I of meiosis, ranged from 35 to 63 percent;

whereas in intercrosses 6 to 15, involving two interchanges, it was













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Table 11-2
Comparison of the mean number of seeds per pod and the distribution of seeds per pod in F^s of each cross
combination showing abnormal M-I configurations compared to a standard normal bean breeding line 0-181.
Cross
combination
Chromosome
configuration
at M-I
Mean seeds
per pod
Percent pods in each
number of
I 5 3 4
category with respect
seeds per pod
~5 6 7 Q~
to
9
Percent
reduction
in
seed set
1.
1-52 x
0-181
104 + 9
II
2.28
31.26
30.76
23.68
13.15
0.72
0.43
_
_
_
60.75
2.
1-97 x
0-181
104 + 9
II
3.63
9.4
15.2
19.2
26.6
21.1
7.5
1.0
-
-
37.52
3.
11 -3 x
0-181
104 + 9
II
2.91
15.3
27.3
23.49
22.54
7.53
3.28
0.46
-
-
49.91
4.
11-70 x
: 0-181
104 + 9
II
2.86
18.38
27.35
21.99
19.28
8.96
2.69
1.34
-
-
50.77
5.
11-121
x 0-181
104 + 9
II
2.20
36.21
31.6
18.34
8.85
5.0
-
-
-
-
62.13
6.
1-52 x
1-97
2@4 + 7
II
1.72
48.27
34.48
13.8
2.9
0.53
-
-
-
-
70.39
7.
1-52 x
11-3
24 + 7
II
1.52
59.60
29.50
9.73
0.88
0.29
-
-
-
-
73.83
8.
1-52 x
11-70
14 + 1
of 4 + 7
chai n
II
1.52
60.23
29.38
8.88
0.78
0.49
0.24


73.83
9.
1-52 x
11-121
Results
inconclusive
1.47
64.12
25.88
8.52
1.48


74.69
10.
1-97 x
11-3
204 + 7
II
2.0
36.66
36.04
19.65
5.75
1.3
0.3
0.3
-
-
65.57
11.
1-97 x
11-70
204 + 7
II
2.05
31.6
42.25
16.90
7.39
1.76
-
-
-
-
64.71
12.
1-97 x
11-121
1 chain of 6
+ 8 II
2.16
36.56
33.94
17.76
5.75
1.56
3.39
1.04

62.82
13.
11-3 x
11-70
204 + 7
II
1.96
43.6
29.8
16.70
6.37
3.53
-
-
-
-
66.26
14.
11-3 x
11-121
106 + 8
II
1.52
62.0
28.0
8.0
1.5
0.5
-
-
-
-
73.83
15.
16.
11-70 >
0-181
; 11-121
204 + 7
11 II
II
1.54
5.31
56.92
2.03
32.2
3.39
9.97
6.33
0.91
10.85
12.66
21.94
27.8
14.1
0.9
73.49
ro
o



22



these interchanges, pachytene analysis is difficult (Krishnan and De

1970) but not impossible (Cheng and Bassett 1981). New techniques

like Giemsa staining (Mok and Mok 1976) or use of biotin labelled

probes to map specific DNA sequences on chromosomes (Rayburn and Gill

1985) may hopefully help with identifying not only the chromosomes

but also the breakpoints.














SECTION III
TERTIARY TRISOMICS AND TETRASOMICS FROM TRANSLOCATION
HETEROZYGOTES IN COMMON BEAN (PHASEOLUS VULGARIS L.)



Introduction

Trisomic individuals have 1 more chromosome than the diploid or

somatic number. Since the trisome modifies the genetic ratios for

genes located on that chromosome, trisomics offer one of the most

efficient methods of associating genes with their linkage groups.

Tertiary trisomics are those trisomics that have a translocated

chromosome in addition to the normal somatic chromosome complement.

They rank among the most useful genetic tools because they can be

used to determine the arm location of marker genes, the position of

centromeres, and the orientation of linkage maps. They are also a

good source of the related primary trisomics, which regularly appear

in their selfed progenies (Khush and Rick 1967). Despite their

utility for such cytogenetic analysis, they have been investigated in

only a few species.

Tertiary trisomics were first discovered in the genus Datura and

described at length over a number of years (Belling and Blakeslee

1926, Avery et al. 1959). The other plant species for which a few

tertiary trisomics have been reported are Zea (Burnham 1930),

Nicotiana (Goodspeed and Avery 1939), Pisum (Sutton 1939), Godetia








(Hakansson 1940), Hordeum (Ramage 1960), Secale (Sybenga 1966) and

Lycopersicon (Khush and Rick 1967). Most of the reports are

fragmentary and in no way compare to the systematic studies carried

out in Datura and tomato. No reports of tertiary trisomics in common

bean (2n + 1 = 23) have so far appeared in the literature. In

general, common bean lacks every type of aneuploid series to be used

for gene mapping, which is the main reason why few of the more than

200 genes described in the literature so far (Roberts 1982) have been

assigned to their respective linkage groups (Lamprecht 1961, Nagata

and Bassett 1984). The only previous report of aneuploids in common

bean was by Mok and Mok (1977) in which they reported two monosomics.

Tertiary trisomics appear spontaneously in the progenies of

normal disomics at an extremely low frequency. Asynaptic mutants,

triploids, and monosomics can also produce tertiary trisomics at a

very low frequency; however, translocation heterozygote progenies

produce not only the tertiaries at a comparatively higher frequency,

but also the primary trisomics (Khush 1973). With these

considerations in view, a project was started by Bassett and Hung

(1982) in which reciprocal chromosome translocations were induced by

gamma irradiation. Twelve translocation lines were established and

genetically verified. Five of the 12 translocation stocks were also

verified cytologically (Section II). Eleven of these 12

translocation stocks were used to produce trisomics in this study.

Trisomics are morphologically distinguishable in certain species

like Datura (Blakeslee 1934), L. esculentum (Khush and Rick 1967),

Capsicum annuum (Pochard 1970), Avena sativa (Azael 1973), Avena




24
(Hakansson 1940), Hordeum (Ramaga 1960), Secale (Sybenga 1966) and
Lycopersicon (Khush and Rick 1967). Most of the reports are
fragmentary and in no way compare to the systematic studies carried
out in Datura and tomato. No reports of tertiary trisomics in common
bean (_2n_ + 1 = 23) have so far appeared in the literature. In
general, common bean lacks every type of aneuploid series to be used
for gene mapping, which is the main reason why few of the more than
200 genes described in the literature so far (Roberts 1982) have been
assigned to their respective linkage groups (Lamprecht 1961, Nagata
and Bassett 1984). The only previous report of aneuploids in common
bean was by Mok and Mok (1977) in which they reported two monosomies.
Tertiary trisomics appear spontaneously in the progenies of
normal disomies at an extremely low frequency. Asynaptic mutants,
triploids, and monosomies can also produce tertiary trisomics at a
very low frequency; however, translocation heterozygote progenies
produce not only the tertiaries at a comparatively higher frequency,
but also the primary trisomics (Khush 1973). With these
considerations in view, a project was started by Bassett and Hung
(1982) in which reciprocal chromosome translocations were induced by
gamma irradiation. Twelve translocation lines were established and
genetically verified. Five of the 12 translocation stocks were also
verified cytologically (Section II). Eleven of these 12
translocation stocks were used to produce trisomics in this study.
Trisomics are morphologically distinguishable in certain species
like Datura (Blakeslee 1934), L. esculentum (Khush and Rick 1967),
Capsicum annuum (Pochard 1970), Avena sativa (Azael 1973), Avena






Chromosome orientation of a quadruple in M-I of a translocation

heterozygote having 4 centromeres is critical. In addition to the

four principal types of coorientation (Fig. II-2) the quadruple can

also appear in noncoorientation (Fig. II-1F and III-1), where 2

centromeres on opposite sides of the quadruple are cooriented and are

positioned equidistant from the equitorial plate. The 2 intermediate

centromeres are noncooriented and are stretched out between the other

2 (Fig. III-1), which gives these centromeres the appearance of not

being attached to the poles. In anaphase-I, the 2 cooriented

chromosomes pass to opposite poles while the noncooriented ones

either pass to the same pole (3:1 segregation) or pass to opposite

poles (2:2 segregation). In 3:1 segregation of the quadruple, the

gametes become aneuploid. After fertilization, this leads to trisomy

or monosomy (Schaeffer 1980). The rates of chromosomal

nondisjunction (3:1 segregation) differ rather widely for the

translocation stocks. The observed rates of nondisjunction are

probably affected by the orientation of the translocation complex and

the viability of the aneuploid gametes or zygotes (Khush and Rick

1967).

Any of the 4 chromosomes of a quadruple that consists of 2

primary and 2 tertiary chromosomes may participate in the

nondisjunction. Thus four different n+1 spores and gametes may be

produced by an individual heterozygous for one reciprocal

translocation. In addition to the n+1 gametes of four different

types, two types of viable n gametes are produced by the

translocation heterozygotes, i.e., one with normal chromosomes and




26
Chromosome orientation of a quadruple in M-I of a translocation
heterozygote having 4 centromeres is critical. In addition to the
four principal types of coorientation (Fig. 11-2) the quadruple can
also appear in noncoorientation (Fig. 11-IF and 111-1), where 2
centromeres on opposite sides of the quadruple are cooriented and are
positioned equidistant from the equitorial plate. The 2 intermediate
centromeres are noncooriented and are stretched out between the other
2 (Fig. 111-1), which gives these centromeres the appearance of not
being attached to the poles. In anaphase-I, the 2 cooriented
chromosomes pass to opposite poles while the noncooriented ones
either pass to the same pole (3:1 segregation) or pass to opposite
poles (2:2 segregation). In 3:1 segregation of the quadruple, the
gametes become aneuploid. After fertilization, this leads to trisomy
or monosomy (Schaeffer 1980). The rates of chromosomal
nondisjunction (3:1 segregation) differ rather widely for the
translocation stocks. The observed rates of nondisjunction are
probably affected by the orientation of the translocation complex and
the viability of the aneuploid gametes or zygotes (Khush and Rick
1967).
Any of the 4 chromosomes of a quadruple that consists of 2
primary and 2 tertiary chromosomes may participate in the
nondisjunction. Thus four different n+1 spores and gametes may be
produced by an individual heterozygous for one reciprocal
translocation. In addition to the n+1 gametes of four different
types, two types of viable n gametes are produced by the
translocation heterozygotes, i.e., one with normal chromosomes and







other with translocated chromosomes. All the six types of gametes

may be functional on the female side but n+1 gametes are not

generally transmitted through pollen (Khush 1973). If n+1 gametes

are transmitted through pollen, the frequency is very low.

Therefore, when an interchange heterozygote is selfed, three types of

disomics are produced--standard normal homozygotes, interchange

heterozygotes, and interchange homozygotes. In addition, eight

different types of trisomics may also be expected in the progeny. In

Figure III-1 (modified from Khush 1973), trisomic types 1 to 4 result

from male gametes with a standard normal chromosome arrangement while

trisomics 5 to 8 are fertilized by male gametes with translocated

chromosomes. The extra chromosome in trisomics 1, 2, 5 and 6 is the

tertiary chromosome; while in trisomics 3, 4, 7 and 8, it is the

primary chromosome. Trisomics 1 and 2 are homozygous for the

standard chromosome arrangement while trisomics 3, 4, 5 and 6 are

heterozygous for the interchange. Trisomics 7 and 8 are homozygous

for the translocation (Fig. III-1).

To distinguish the tertiary trisomics 1 and 2 from 5 and 6 (Fig.

III-1) it is necessary to grow and examine their selfed progenies and

their progenies from crosses with normal disomics. Tertiary

trisomics 1 and 2 when selfed or crossed with a normal disomic,

produce disomics in the progeny with normal meiosis, whereas

trisomics in the progeny show abnormal meiosis, i.e., predominantly a

pentavalent at M-I. Tertiary trisomics 5 and 6 whether selfed or

crossed with a normal disomic, produce some disomics in the progeny

showing a multivalent of four chromosomes at M-I and others with








normal meiosis. As regards trisomic progeny, some of them show

predominantly a pentavalent association at M-I, whereas others show a

multivalent of 4 plus 1 univalent as well as a pentavalent.

Figure III-2 (modified from Khush and Rick 1967) shows

pentavalent configurations expected in a tertiary trisomic according

to various positions of chiasmata. Six different configurations can

appear in different situations; however, chiasma terminalization or

failure of chiasma formation may reduce the association to such

lesser configurations as 1 II + 1 III, 2 II + 1 I, 1 IV + 1 I, 1 III

+ 2 I, 1 II + 3 I or 5 I (Khush and Rick 1967). As translocation

heterozygosity does not alter the plant phenotype, trisomic 1 should

be identical to trisomic 5 and trisomic 2 to trisomic 6 (Fig.

III-1). If the morphology of the primary trisomics is known, they

can be easily identified in segregating progeny, because the

morphology of a tertiary is suggested by a combination of the various

morphological characters of the two related primaries (Khush and Rick

1967).

Since the procedure for identifying tertiaries cytologically is

quite laborious, especially for crops like common bean, pollen

fertility analysis may be employed at a preliminary stage. Because

translocation heterozygosity produces nearly 50 percent pollen

abortion in common bean (Section II), tertiary trisomics of types 1

and 2 (Fig. III-1) having standard normal chromosome background, when

selfed or crossed with normal disomics, yield disomics in the progeny

with normal pollen and tertiary trisomics in the progeny with the

pollen abortion rates higher than the normal plants and lower than




30
normal meiosis. As regards trisomic progeny, some of them show
predominantly a pentavalent association at M-I, whereas others show a
multivalent of 4 plus 1 univalent as well as a pentavalent.
Figure III2 (modified from Khush and Rick 1967) shows
pentavalent configurations expected in a tertiary trisomic according
to various positions of chiasmata. Six different configurations can
appear in different situations; however, chiasma terminalization or
failure of chiasma formation may reduce the association to such
lesser configurations as 1 II + 1 III, 2 II + 1 1, 1 IV + 1 1, 1 III
+21, 1 II + 3 I or 5 I (Khush and Rick 1967). As translocation
heterozygosity does not alter the plant phenotype, trisomic 1 should
be identical to trisomic 5 and trisomic 2 to trisomic 6 (Fig.
111 -1). If the morphology of the primary trisomics is known, they
can be easily identified in segregating progeny, because the
morphology of a tertiary is suggested by a combination of the various
morphological characters of the two related primaries (Khush and Rick
1967).
Since the procedure for identifying tertiaries cytologically is
quite laborious, especially for crops like common bean, pollen
fertility analysis may be employed at a preliminary stage. Because
translocation heterozygosity produces nearly 50 percent pollen
abortion in common bean (Section II), tertiary trisomics of types 1
and 2 (Fig. 111-1) having standard normal chromosome background, when
selfed or crossed with normal disomies, yield disomies in the progeny
with normal pollen and tertiary trisomics in the progeny with the
pollen abortion rates higher than the normal plants and lower than



























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50 percent, assuming that trisomy causes less pollen abortion

compared with translocation heterozygosity. The progenies of

tertiary trisomics 5 and 6 (Fig. III-1) having translocation

heterozygote chromosome background, when selfed or crossed with

normal disomics, should segregate approximately one normal

(fertile): one translocation heterozygote in both the progeny

trisomics as well as normal disomics. Pollen abortion rates of

normal progeny disomics and trisomics with standard normal chromosome

background should be less than 50 percent in either case; whereas in

progeny disomics and trisomics with translocation heterozygote

chromosome background, it should be 50 percent and higher than 50

percent, respectively. Pollen fertility analysis is easy compared to

the cytology of PMCs but classification based on pollen fertility is

often difficult and one must be very careful in drawing conclusions

based on it because (1) most of the translocation heterozygotes

differ in pollen abortion rates (Bassett and Hung 1982); (2) in the

beginning it is not known what effect on pollen abortion the extra

chromosome has (Burnham 1962); (3) there will be an effect of genetic

background (Khush and Rick 1967); and (4) environmental fluctuations

will alter the pollen abortion rates. However, if pollen abortion

rates are supplemented with cytological examination one can get

fairly dependable results.

Selfed progenies of a trisomic normally segregate into normal

diploids and trisomics if there is no male transmission of the extra

chromosome; however, if the extra chromosome is transmitted through

the pollen, a third class of plants, i.e., tetrasomics, will also









arise among the progeny. The frequency of tetrasomics depends upon

the frequency of transmission of an extra chromosome in a male gamete

that fertilizes a female gamete already carrying the same extra

chromosome. The physical presence of tetrasomics in a trisomic

progeny may under represent the actual transmission rate of an extra

chromosome in the pollen because (1) the extra chromosome may be

eliminated during megasporogenesis; (2) the viability of n+1 gametes

may be reduced; (3) the development of 2n+2 zygotes, endosperms, and

embryos may be subnormal or abnormal; and (4) the germination of

tetrasomic seedlings may be poor or delayed.

Tetrasomics among the progeny of a trisomic may also arise by

another process, i.e., begin with a trisomic plant which still has a

translocation heterozygote chromosome background. After a quadruple

forms at M-I and given noncoorientation and nondisjunction of the

quadruple, various possibilities arise. Please note that we are

dealing here with a trisomic plant that already has an extra

chromosome in every cell. At anaphase-I, this extra chromosome will

go to one of the two poles. At the same time, if due to

nondisjunction of a quadruple originating due to translocation

heterozygosity of that trisomic plant, 3 of the group of 4

chromosomes also go to the same pole that has already received the

extra chromosome, two gametes arising from this embryo sac mother

cell should be tetrasomic, with the condition that the extra

chromosome due to nondisjunction must be the same as that already in

triplicate in the trisomic plant (otherwise the gametes will be

double trisomic).








Tetrasomics can be easily distinguished from their corresponding

trisomics if the trisomic morphology is known. In general, the

characters of trisomics are accentuated in tetrasomics (Burnham

1962).



Materials and Methods

Preparation of Seed Stocks

Eleven out of 12 translocation stocks established by Bassett and

Hung (1982) were first developed into homozygous translocations

(Section II). These homozygous translocation stocks were then

crossed with a standard normal bean breeding line and F1 seed

collected. The F1 generation was grown to F2 seed. For each

translocation, the F2 seed from each F1 plant was separated into

light (lower 30 percent of seed weight distribution) and heavy

(remaining 70 percent of seed weight distribution).

First Field Generation (Summer, 1984)

These weight classes were planted separately in adjacent plots

from each F1 plant. The F2 progeny plots were searched for any

plants that were phenotypically distinguishable from each other and

from normals. We did not cross-pollinate putative trisomics with the

normal disomics in the field because it is too difficult under

Florida conditions. The off-type plants were classified into

phenotypic groups in each translocation stock and seed from every

tagged plant was harvested separately.








First Greenhouse Generation (Fall, 1984)

The S1 seed from four different phenotypic classes, namely, Weak

Stem, Puckered Leaf, Dark Green Leaf, and Convex Leaf was planted in

the greenhouse. Immature flower buds were collected from the

abnormal S1 plants that were identical to the seed parent and handled

as in Section II. Descriptive notes and photographs were taken from

the putative trisomics. These plants were also crossed with pollen

from normal disomics.

Second Greenhouse Generation (Spring, 1985)

Only the FI seed from two of the four groups, namely, Weak Stem

and Puckered Leaf from the S1 greenhouse planting, was selected for

further study in the next greenhouse generation. F1 trisomic

segregates were backcrossed with normal disomic plants in the second

greenhouse generation. These crosses to disomics were actually a

part of our scheme to isolate primaries on homozygous normal

chromosome background. It will be dealt with in Section IV. Seed

from only the abnormal segregates in each group was replanted and the

necessary data recorded.

Second Field Generation (Summer, 1985)

All the remnant seed from abnormal plants in the first field

generation and the first and second greenhouse generations was

planted in the second field generation where immature flower buds

were collected from a few of the phenotypic groups for cytological

analysis (see cytology section, page 51). Detailed descriptive notes

and photographs were taken. Abnormal plants arising in each

translocation stock were re-examined and put into specific phenotypic




36
First Greenhouse Generation (Fall, 1934)
The seed from four different phenotypic classes, namely, Weak
Stem, Puckered Leaf, Dark Green Leaf, and Convex Leaf was planted in
the greenhouse. Immature flower buds were collected from the
abnormal plants that were identical to the seed parent and handled
as in Section II. Descriptive notes and photographs were taken from
the putative trisomics. These plants were also crossed with pollen
from normal disomies.
Second Greenhouse Generation (Spring, 1985)
Only the F^ seed from two of the four groups, namely, Weak Stem
and Puckered Leaf from the S^ greenhouse planting, was selected for
further study in the next greenhouse generation. F^ trisomic
segregates were backcrossed with normal disomic plants in the second
greenhouse generation. These crosses to disomies were actually a
part of our scheme to isolate primaries on homozygous normal
chromosome background. It will be dealt with in Section IV. Seed
from only the abnormal segregates in each group was replanted and the
necessary data recorded.
Second Field Generation (Summer, 1985)
All the remnant seed from abnormal plants in the first field
generation and the first and second greenhouse generations was
planted in the second field generation where immature flower buds
were collected from a few of the phenotypic groups for cytological
analysis (see cytology section, page 51). Detailed descriptive notes
and photographs were taken. Abnormal plants arising in each
translocation stock were re-examined and put into specific phenotypic






different translocation heterozygotes is somewhat arbitrary. Out of

6600 seeds, 2100 seeds were separated into the lower (30 percent)

seed weight class, i.e., nearly one third of the total seed

harvested.

First Field Generation

All the light and heavy seeds were planted separately in

adjacent plots. Ninety-six phenotypically distinct plants (off-type)

originated among the progeny and all 96 appeared in the light weight

seed class. On the basis of these results, it can be suggested that

in common bean or closely related crops if one separates nearly 1/4

of the total seeds produced from translocation heterozygotes that are

lighter in weight, he can enrich the progenies with aneuploids with

minimal risks of loosing any distinctive trisomics (off-type), while

saving much searching labor and field space. These off-type plants

were allowed to self-pollinate. Sixty out of 96 plants set pods and

seeds. These plants were divided into 17 different phenotypic

classes which were distinguishable from each other and from normal

plants.

First Greenhouse Generation

The seed from four phenotypic classes was planted in the first

greenhouse generation (Table III-1). In the first two groups, i.e.,

Puckered Leaf and Weak Stem, the trisomic segregates were easily

differentiated from normals at the seedling stage. In the third and

fourth groups, i.e., Dark Green Leaf and Convex Leaf, it was very

difficult to distinguish (in the greenhouse) between trisomic and

normal segregates in the seedling stage. Cytological proof, that the









four phenotypic groups mentioned above are trisomics, will be

presented later on page 51. Thirty days after planting, the plants

that looked slightly different from normals were kept. Their number

was 54 in the Dark Green Leaf group and 26 in Convex Leaf group. The

rest were discarded to save the greenhouse space. These plants were

progeny tested in the summer 1985 field planting where some of them

bred true. Thirty-nine in the Dark Green Leaf group and 17 in the

Convex Leaf group segregated into normals and aneuploids. Moreover,

under field conditions it was quite easy to differentiate between

normals and aneuploids. It is possible that the transmission rate

for these two classes given in Table III-1 may be underestimated

because during classification in the greenhouse we probably discarded

some trisomic segregates along with the normal disomics, a hypothesis

supported by our segregation data for Dark Green Leaf trisomic in

Table III-2.

A few plants in the progeny of each trisomic were phenotypically

different from normal segregates as well as trisomics (Table

III-1). They flowered profusely and were sterile, although some of

them set pods that produced no seed. Some of them were weaker while

others had almost the same vigor as trisomic plants. They were not

analyzed cytologically. It is assumed that the weaker plants may

probably be the tetrasomics because the monosomics reported so far in

common bean set seed and cannot be distinguished from normal disomics

on the basis of morphological characteristics (Mok and Mok 1977).

Cytologically validated tetrasomic segregates were later observed in

the trisomic progeny tests in the field (Table III-2). As regards















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the sterile segregates having the same vegetative vigor as trisomic

plants, these were two in number and emerged in the progeny

classified as Dark Green Leaf. The plant phenotype was Chlorotic

Leaf, which will be described in Section IV, under primary trisomics.

The inability to distinguish between Dark Green Leaf, Convex

Leaf and normal disomic segregates in the first greenhouse planting

may be attributed to the quality and quantity of light which plants

receive in offseason planting in the greenhouse. It is not unusual

to observe that chlorophyll production is often decreased by short

days (Salisbury and Ross 1978). As regards the quality of light, the

red and blue regions of the visible spectrum are mostly absorbed by

the chlorophylls. These two regions are the most effective

wavelengths in inducing chloroplast division and most efficient in

driving photosynthetic reactions (Salisbury and Ross 1978). If at

least one of these two regions has its transmission reduced while

passing through the glass, it may affect the normal development of

chlorophylls in these trisomic classes, because they develop dark

green leaves under field light conditions. It is probably the blue

region of visible spectrum of light which is affected while passing

through glass (personal communication with Prof. R.H. Biggs, Fruit

Crops Department, University of Florida, Gainesville). In future, it

has been planned to plant these two trisomics a bit early in the

fall, keeping all the plant pots outdoors for one month. After

differentiation is possible between normal disomic and trisomic

seedlings, the trisomic plants can be transported into the greenhouse

for the winter.




43
the sterile segregates having the same vegetative vigor as trisomic
plants, these were two in number and emerged in the progeny
classified as Dark Green Leaf. The plant phenotype was Chlorotic
Leaf, which will be described in Section IY, under primary trisomics.
The inability to distinguish between Dark Green Leaf, Convex
Leaf and normal disomic segregates in the first greenhouse planting
may be attributed to the quality and quantity of light which plants
receive in offseason planting in the greenhouse. It is not unusual
to observe that chlorophyll production is often decreased by short
days (Salisbury and Ross 1978). As regards the quality of light, the
red and blue regions of the visible spectrum are mostly absorbed by
the chlorophylls. These two regions are the most effective
wavelengths in inducing chloroplast division and most efficient in
driving photosynthetic reactions (Salisbury and Ross 1978). If at
least one of these two regions has its transmission reduced while
passing through the glass, it may affect the normal development of
chlorophylls in these trisomic classes, because they develop dark
green leaves under field light conditions. It is probably the blue
region of visible spectrum of light which is affected while passing
through glass (personal communication with Prof. R.H. Biggs, Fruit
Crops Department, University of Florida, Gainesville). In future, it
has been planned to plant these two trisomics a bit early in the
fall, keeping all the plant pots outdoors for one month. After
differentiation is possible between normal disomic and trisomic
seedlings, the trisomic plants can be transported into the greenhouse
for the winter.







among each trisomic progeny (Table III-2). They were all

phenotypically distinguishable from each other and from normal

disomics as well as trisomics. In fact, trisomic phenotype ws more

accentuated in tetrasomics. Translocation heterozygote stock 1-97

yielded two phenotypic groups, i.e., Weak Stem (Table III-3) and

Pinched Leaflet Tip (Table III-4). Weak Stem has been verified

cytologically as a trisomic whereas Pinched Leaflet Tip lacks

cytological confirmation. However, genetic tests give an indication

of trisomy. The primary trisomic for Weak Stem has been isolated

from the tertiary Weak Stem by cytological examination (Section IV)

because the primary is not morphologically distinct from the

tertiary.

Discussing the overall segregation data in Table III-2, 16 off-

type plants falling into two phenotypic groups, Dark Green Leaf and

Convex Leaf, were tagged in F2 progeny of the translocation

heterozygote 1-52 in the first field generation. Selfed progenies

from 8 out of these 16 plants, 6 in Dark Green Leaf and 2 in Convex

Leaf, were tested in the first greenhouse generation (Table III-1).

On the basis of information we recorded from the second field

generation (Table III-2), we hypothesize that all eight plants we

started with were tertiary trisomics of two types, i.e., Dark Green

Leaf tertiary and Convex Leaf tertiary. They segregated into

tertiaries and related primaries in the first greenhouse

generation. If the Dark Green Leaf trisomic progenies are examined

(Table III-2), it seems that some of the plants segregated into both




















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Putative trisomics derived from different translocation heterozygote progenies; their phenotype and rate of
transmission of extra chromosome in (2n+l) self (Second field planting, Summer, 1985).
Translocation
stock
Trisomica
phenotype
Plants
grown
Trisomics
scored in
the progeny
Progeny
trisomic
phenotype
Percent
transmission
Other
remarks
I -85b
Weak Plant
13
5
Weak Plant
38
No pod or seed
set
1-97
Pinched Leaflet Tip
21
4
3
True to seed parent
Phenotype more
accentuated
19
14
May be tetra -
somic. No
pod or seed
set
11-6
Small Leaf
32
7
True to seed parent
22
11-14
Epinastic Leaf
167
26
8
True to seed parent
Phenotype more
accentuated
16
5
No pod or seed
set. May be
tetrasomic
11-109
Leaf Margins Curl Down
26
7
True to seed parent
27
11-121
Leaf Tip Curl
6
3
True to seed parent
50
a These phenotypic classes have not yet been verified cytologically as trisomics and that is why they are
called putative trisomics.
b One plant of Extreme Lance type originated in the light weight seed class among translocation
heterozygote 1-85 progeny. Another plant of the same type appeared among Weak Plant putative trisomic
progeny. Both of them were sterile.


48
related primaries in and produced uniform primary trisomics in S2
(Table 111-2, a and b). They also produced identical tertiaries in
their segregating progenies in S^. These tertiaries either produced
identical tertiaries and one related primary in S2 (Table III-2, c),
or they produced identical tertiaries along with both the related
primaries in S2 (Table 111-2, d). A similar explanation seems true
for Convex Leaf tertiary trisomic. The only difference is that the
Convex Leaf did not segregate for either primary in S^; but in S2, it
segretaed for one related primary trisomic, i.e., Dark Green Leaf
(Table III-2, f). Because the phenotype was more difficult to
differentiate in the greenhouse, these could not be identified in S^.
One of the unique features of the breeding behavior by which
tertiary trisomics differ from other types of trisomics is the
regular appearance of the two related primary trisomics in their
progenies (Khush and Rick 1967) which is illustrated by the
segregating progenies of Dark Green Leaf and Convex Leaf tertiary
trisomics (Table 111-2). Unrelated primary trisomics also appear at
low frequencies in some of the trisomic progenies (Khush and Rick
1967), which is illustrated by Chlorotic Leaf segregates appearing at
a low frequency throughout the trisomic progenies in Table III-2.
Extreme Lance, another morphologically distinct type, also appeared
in two plant progenies of Dark Green Leaf trisomic (footnote, Table
III-2). It also appeared in F2 progenies of translocation
heterozygote 1-85, and among S2 progeny of Weak Plant, a putative
trisomic (footnote, Table III-4). By the same analogy, Extreme Lance
type may be another unrelated trisomic, it could not be analyzed






Cytology

During the preliminary cytological analysis in the first

greenhouse generation to see whether the abnormal segregates were

trisomics or not, 25 PMCs were analyzed which clearly show 11

bivalents and one univalent chromosome at diakinesis or M-I (Fig.

III-3A), 23 chromosomes at early anaphase-I (Fig. III-3B), or 11 V/S

12 chromosomes at late anaphase-I (Fig. III-3D) in all the four

phenotypic classes (Table III-1). During this search, certain

anaphase-I cells with 11 chromosomes at each pole and with one

laggard still in the middle were also observed at a very low

frequency (Fig. III-3C). Buds from all the off-type plants falling

in one group were collectively picked and preserved. As the

phenotype of Dark Green Leaf and Convex Leaf was not very distinctive

in the first greenhouse planting, they were re-examined in the second

field generation. Some of the plants bred true and there was not

even a single off-type plant in their progeny, giving an indication

that they were normal disomic plant progenies and that it was not

possible to discard them in the greenhouse screening. The numbers of

true breeding parent plants were 15 in the Dark Green Leaf group and

9 in the Convex Leaf group. Dark Green Leaf produced the following

plant progeny classes: an extremely uniform class (a) of Dark Green

Leaf plants along with normal disomics (Table III-2, a); a uniform

class (b) of Convex Leaf trisomics along with Convex Leaf tetrasomics

and normal disomics (Table III-2, b); class (c) of Dark Green Leaf

trisomics whose phenotypes were a mixture of characteristics of

classes (a) and (b) with characters of class (a) predominant

















Fig. III-3


Meiotic diplotene to anaphase-I as observed in tertiary
trisomics of common bean (x 1000).

A. A PMC at diakinesis showing 11 bivalents and a
univalent (arrow).

B. Early anaphase-I showing 23 univalents.

C. Anaphase-I showing 11 univalents on each pole with
one laggard (arrow) in the middle.

D. Late anaphase-I showing 11 univalents on 1 pole and
12 on the other (right).

E. Diplotene showing a pentavalent association (arrow)
along with 9 bivalents.

F. Metaphase-I showing a pentavalent (arrow) and 9
bivalents.

G. Diakinesis showing a trivalent (arrow) and 10
bivalents.























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(Table 111-2, c) along with disomics, rare tetrasomics and segregates

that were phenotypically identical to class (a); and a fourth class

(d) that produced all the plant types in (a) through (c) detailed

above (Table III-2, d) along with their tetrasomics and normal

disomics. When one observes a mixture of the phenotypic

characteristics from the two related primaries in a tertiary trisomic

in which the characteristics of one primary predominate, it is

interesting to speculate that the longer of the two segments

composing the extra chromosome in the tertiary is homologous with the

triplicated chromosome in the related primary trisomic having closer

phenotypic resemblance (Khush and Rick 1967).

The cytology of Dark Green Leaf in class (a) and Convex Leaf in

class (b) gave an indication that they were primary trisomics

(Section IV). It becomes fairly easy to identify tertiary trisomics

if the morphology of the two related primaries is known because the

nature of tertiaries is suggested by a combination of various

morphological characteristics of the two related primaries (Khush and

Rick 1967). Keeping this fact in view, the Dark Green Leaf

segregates in class (c) having the combination of characteristics of

both related primaries with dominant characteristics of Dark Green

Leaf were classified as Dark Green Leaf tertiary. Similarly, Convex

Leaf trisomic plant progenies produced a class (a) of Convex Leaf

trisomic with a quite uniform phenotype within the group but

different from the Convex Leaf primary trisomic which originated in

Dark Green Leaf progenies. Having the combined characteristics of

both related primaries and having the dominant morphological








similarities of the Convex Leaf primary, it was named Convex Leaf

tertiary trisomic (Table 111-2, e). Another class (b) originating

among Convex Leaf trisomic progenies was the Dark Green Leaf primary

trisomic along with the normal disomics (Table 111-2, f).

Puckered Leaf trisomic was fairly uniform in the beginning. In

the second field planting when primary trisomic plants were

identified cytologically (Section IV), it was easier to identify a

morphologically different class within Puckered Leaf trisomic plant

progenies which were thought to be tertiaries. It is appropriate to

emphasize that all the three tertiary trisomic groups, i.e., Dark

Green leaf, Convex leaf and Puckered Leaf have been classified as

tertiaries only on the basis of their morphological relationship with

their related primaries and by the fact that they continue producing

related primaries in their progenies, whereas primaries segregate

almost exclusively for the same primary. We have no cytological

proof that they are tertiaries. This is because common bean cytology

is fairly difficult, confusing and time consuming due to the small

size of chromosomes. The Weak Stem trisomic class was the only one

in which tertiaries were isolated from primaries cytologically. This

was because no morphological distinction existed between primary and

tertiary in this trisomic class. Four plant progenies were examined

cytologically (Section IV). Three of these, i.e., GK #22, 23, 25,

were classified as tertiary. Our results are based on 50 analyzable

PMCs from each progeny at diplotene, diakinesis, or M-I of meiosis

(Table IV-2). On an average 21 PMCs showed a pentavalent association

(Fig. 111-2) with the rest of the chromosomes as bivalents, 20 showed








a trivalent configuration (Fig. III-3G), and 9 PMCs appeared having

11 bivalents and 1 univalent (Fig. III-3A). Certain pentavalent

configurations as illustrated in Figure III-2, helped to reach the

conclusion that they are tertiary trisomics. Otherwise, a primary

trisomic still having a translocation heterozygote background can

form pentavalent associations.

Immature flower buds from Convex Leaf tetrasomic plants were

collected and analyzed cytologically. PMCs showed 11 bivalents and 2

univalents at diakinesis (Fig. III-4A), 24 chromosomes at early

anaphase-1 (Fig. III-4B) and 12 V/S 12 at two poles at late

anaphase-1 (Fig. III-4C) of meiosis. All other tetrasomics were

identified from their phenotypic relationship with the seed parent

phenotype and were not analyzed cytologically. None of the

tetrasomic plants from any class set seed.

Morphology of Known Tertiary Trisomics

The following description of the Puckered Leaf trisomic is based

on observation from one generation, while Dark Green leaf and Convex

Leaf trisomic descriptions are based on two generations, and trisomic

suspects and tetrasomics are based on one generation only. Weak Stem

trisomic morphology is described in Section IV because of the

nonexistence of morphological distinction between tertiary and

primary in this class. For each of the tertiary trisomics listed

below, the name given to it is derived from its "related" primary

trisomic because of the strong resemblance, e.g., the tertiary and

primary Puckered Leaf trisomics are quite similar in appearance;

hence the same phenotype-derived name is used for both.

























Fig. III-4


Meiotic diakinesis to anaphase-I as observed in a
tetrasomic of common bean (x 1000).

A. A PMC at diakinesis showing 11 bivalents and 2
univalents that overlap (arrow).

B. Early anaphase showing 24 univalents.

C. Late anaphase showing 12 univalents at each pole.
The arrow points to 2 chromosomes lying close to each
other.











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Puckered Leaf. This trisomic was closely examined in three

selfed and two crossed (with 2n) generations for isolation of

primaries. It was only in the last generation in which the plants

having these morphological characteristics (Fig. III-5C) were marked

as tertiaries because the phenotype of the related primary was

established cytologically (Section IV). Puckered Leaf tertiary

trisomic is different from normal as well as Puckered Leaf primary.

The terminal leaflet of the trifoliate leaf of the tertiary is

semilanceolate. Leaf petioles form a wider angle with the stem

resulting in a wider plant canopy. Leaves are less puckered than

primary, and the stem and pods have more anthocyanin pigment in

them. Leaves do not have a glossy appearance on attaining full size

like the primary and they are slightly darker green in color compared

to normal or primary. Pod and seed set is better under field

conditions compared to the primary but less than disomic

segregates. Many missing seeds can be seen in pods. It is slightly

later maturing than the normal disomic and related primary trisomic.

Dark Green Leaf. Plants are smaller than the related primary

and have more recessed veins and smaller leaves. The leaves look

darker green than the related primary. They set equally well under

greenhouse and field conditions. Pod, seed shape and all other

characteristics are similar to those in the related primary. It is

easy to classify in the field about 20 days after planting whereas

classification in the greenhouse is difficult and subject to serious

levels of error.




53
Puckered Leaf. This trisomic was closely examined in three
sel fed and two crossed (with 2n) generations for isolation of
primaries. It was only in the last generation in which the plants
having these morphological characteristics (Fig. III-5C) were marked
as tertiaries because the phenotype of the related primary was
established cytologically (Section IV). Puckered Leaf tertiary
trisomic is different from normal as well as Puckered Leaf primary.
The terminal leaflet of the trifoliate leaf of the tertiary is
semilanceolate. Leaf petioles form a wider angle with the stem
resulting in a wider plant canopy. Leaves are less puckered than
primary, and the stem and pods have more anthocyanin pigment in
them. Leaves do not have a glossy appearance on attaining full size
like the primary and they are slightly darker green in color compared
to normal or primary. Pod and seed set is better under field
conditions compared to the primary but less than disomic
segregates. Many missing seeds can be seen in pods. It is slightly
later maturing than the normal disomic and related primary trisomic.
Dark Green Leaf. Plants are smaller than the related primary
and have more recessed veins and smaller leaves. The leaves look
darker green than the related primary. They set equally well under
greenhouse and field conditions. Pod, seed shape and all other
characteristics are similar to those in the related primary. It is
easy to classify in the field about 20 days after planting whereas
classification in the greenhouse is difficult and subject to serious
levels of error.

























0


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r,= \ S S ^


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Convex Leaf. Plants of this trisomic can be distinguished from

disomics at the seedling stage, but there are chances of error.

However, at 25 days after planting, the leaves of the tertiary

trisomic start forming a convex shape with recessed veins, and the

plants can be identified for classification 30 days after planting

(Fig. III-581). In this respect it differs from the related primary,

which can be identified easily at 20 days of age. Leaves of the

tertiary are darker green with comparatively less recessed veins and

are less convex than the primary. Plants of this trisomic have

shorter internodes than disomics or related primary trisomic and

remain bushy, but they can grow as tall as diploid sibs and still

remain compact in appearance. Pod and seed set is equally good in

either greenhouse or field conditions. This class is also difficult

to classify in the greenhouse but not so in the field. Pods and

seeds are smaller than in disomics but similar to the related

primary.

Convex Leaf tetrasomic. Tertiary tetrasomic plants in this

class are extremely dwarfed with very short internodes, highly convex

leaves, highly recessed veins and dark green leaf color (Fig.

III-SB2). They have very small buds, flower profusely, but rarely

set any pods or seed. They differ from the primary tetrasomic, which

has a lighter green leaf color and comparatively taller plants. All

other morphological characteristics are similar.

Dark Green Leaf tetrasomics. Tetrasomic plants in this class

look like trisomic parents at the seedling stage. Only after one

month of vegetative growth when trisomics attain considerable size,








the tetrasomics appear comparatively stunted. Also, the leaves show

more recessed veins, darker green color and smaller size compared to

the trisomics (Fig. III-5D). The flower color of tetrasomics is more

intense than trisomics, and they flower profusely but do not set any

pods. Tetrasomic plants of this class grow taller compared to Convex

Leaf tetrasomics.

Morphology of Trisomic Suspects

Weak Plant. The plants are short in stature, and the leaves are

pale green and slightly smaller than normal (Fig. III-6F). This

trisomic flowers profusely but sometimes without pod and seed set.

The apparent sterility in the 1985 season may probably be due to very

hot weather conditions (max. temps. at 1000F) in the field. The

original plants of Weak Plant were able to set seed in the field

during the milder summer of 1984.

Pinched Leaflet Tip. Progenies of the putative trisomic

segregated for two types of plants besides the normal disomics. The

first type was extremely abnormal and had round leaves. In these

plants there was single broad leaf instead of a trifoliate leaf. The

plants were vegetatively vigorous and tall and they flowered

profusely and set pods with aborted ovules. The second type of

plants was intermediate in phenotype between disomics and the first

type. They produced medium-sized leaves with a round tip (Fig.

III-6C). The terminal leaflet sometimes had a point in early stages,

but the tip was blunt or rounded at full maturity of the leaf. The

plants had small pods with very small seeds.
























Fig. III-6


Plant photographs of suspected trisomics (genetic
evidence only).

Al. Leaf Margins Curl Down, 30 days after planting.

A2. A normal common bean plant, 30 days after planting.

B. Leaf Tip Curl Down, 70 days after planting.

C. Pinched Leaflet Tip, 30 days after planting.

D. Extreme Lanceolate Leaf, 30 days after planting.

E. Small Leaf, 30 days after planting.

F. Weak plant, 30 days after planting.

G. Epinastic Leaf, 30 days after planting.




64



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."O




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Small Leaf. Plants of this putative trisomic have smaller

leaves and weaker stems compared to disomics. They also have weaker

leaf petioles and a wider plant canopy (Fig. III-6E). The leaves are

normal in color. Trisomic segregates can be distinguished 25 days

after planting. Pods are very long and look like normal. Seeds are

big and flat. Pod and seed set is late compared to disomics.

Epinastic Leaf. This class also segregated into three classes

of plants: normal disomics (a); very weak, small plants (b) with

epinastic leaves and without pods and seed set; and a third plant

phenotype falling between (a) and (b). The leaf epinasty of this

third plant class looks like mosaic virus effects and the plants are

of medium height (Fig. III-6G) and set medium-sized pods with some

missing seeds at random positions.

Leaf Margins Curl Down. The leaves are smaller than in disomics

and their margins curl down. Plants are slow growing, smaller than

normal segregates, and can be distinguished 40 days after planting

(Fig. III-6A1). Plants grow spindly branches, have anthocyanin

pigment in their branches, are late maturing, and set medium-sized

pods with missing seeds at random positions.

Leaf Tip Curl. Plants are slow growing and weaker than

disomics. Leaf tips curl down. The branches and pods have more

anthocyanin pigment in them than disomics. The pods are

morphologically similar to Puckered Leaf tertiary trisomic pods.

There are many missing seeds in the pods. It is a late blooming and

late maturing class. Plants can be distinguished from normals 30 to

40 days after planting (Fig. III-6B).




65
Small Leaf. Plants of this putative trisomic have smaller
leaves and weaker stems compared to disomies. They also have weaker
leaf petioles and a wider plant canopy (Fig. III-6E). The leaves are
normal in color. Trisomic segregates can be distinguished 25 days
after planting. Pods are very long and look like normal. Seeds are
big and flat. Pod and seed set is late compared to disomies.
Epinastic Leaf. This class also segregated into three classes
of plants: normal disomies (a); very weak, small plants (b) with
epinastic leaves and without pods and seed set; and a third plant
phenotype falling between (a) and (b). The leaf epinasty of this
third plant class looks like mosaic virus effects and the plants are
of medium height (Fig. Ill-6G) and set medium-sized pods with some
missing seeds at random positions.
Leaf Margins Curl Down. The leaves are smaller than in disomies
and their margins curl down. Plants are slow growing, smaller than
normal segregates, and can be distinguished 40 days after planting
(Fig. Ill-6A^). Plants grow spindly branches, have anthocyanin
pigment in their branches, are late maturing, and set medium-sized
pods with missing seeds at random positions.
Leaf Tip Curl. Plants are slow growing and weaker than
disomies. Leaf tips curl down. The branches and pods have more
anthocyanin pigment in them than disomies. The pods are
morphologically similar to Puckered Leaf tertiary trisomic pods.
There are many missing seeds in the pods. It is a late blooming and
late maturing class. Plants can be distinguished from normals 30 to
40 days after planting (Fig. III-6B).






tertiary, telo, and compensating (Khush 1973). Out of these five

types, primary and tertiary trisomics have been used most extensively

for linkage studies. Tertiary trisomics are among the most useful

cytogenetic variants because they can be used to determine the arm

location of marker genes, position of centromeres, and orientation of

linkage maps. These special applications of tertiaries are only

possible if the gene in question is first assigned to a particular

linkage group by use of the primary trisomic series and the pachytene

chromosome analysis of the particular species is possible. Pachytene

chromosome analysis helps in physically identifying the two

nonhomologous chromosomes involved in an interchange and the exact

position of breakpoints. Pachytene analysis has been successfully

used in maize (Rhoades and McClintock 1935) and tomato (Rick and

Barton 1954), but has not yet been successful in locating the exact

position of a translocation breakpoint in common bean. There has

been a paucity of techniques reported (Cheng and Bassett 1981; Mok

and Mok 1976) that will permit the identification of the extra

chromosome in a primary trisomic.

Primary trisomics have appeared spontaneously among the progeny

of normal diploids of many species, but are obtained more frequently

from asynaptic and desynaptic mutants, from the progeny of polyploids

(most notably triploids), and from the progeny of translocation

heterozygotes (Khush 1973). Among the different sources of primary

trisomics mentioned above, triploids are the best and most dependable

source (Khush 1973). The most common route to get triploids is the

use of colchicine to produce tetraploids, which are then crossed with








diploids to get triploid seed. Triploid plants, when selfed or

backcrossed, usually produce many types of aneuploids, but primary

trisomics are produced at a high frequency.

Producing trisomics from the progenies of 3n x 2n crosses is

difficult in common bean as the study of Braak and Kooistra (1975)

shows. Amphidiploids were first developed by crossing Phaseolus

vulgaris with Phaseolus ritensis. Obtaining F1 plants was only

possible by artificial embryo culture. Amphidiploids were obtained

by doubling the chromosomes of the interspecific hybrid. These

amphidiploids were then backcrossed with (2n) Phaseolus vulgaris.

Out of 25,000 pollinations, 8 shrivelled seeds were obtained. With

the help of embryo culture, the seedlings were raised. Seven of the

eight seedlings died and one was grown to maturity. It proved to be

a triploid. Vegetative propagation was the only way to propagate

this plant. There was no success when the triploid was backcrossed

with Phaseolus vulgaris. Out of 1600 pollinations with Phaseolus

ritensis, the other parent, only one vigorous plant was obtained, a

trisomic 2n + 1 = 23 chromosomes. S1 progeny of this trisomic

produced a total of 159 plants, including a small number of

semisterile plants. These semisterile plants proved to be

trisomics. All of the progeny were very similar to the Phaseolus

vulgaris parent. S2 and S3 progenies of trisomic segregates were

grown. The article did not give any further details, but indicates

that additional information about the trisomic progeny would be

published in a separate article in the near future. Such a report

has not appeared. No spontaneous occurrence of trisomics in disomic








progenies or information regarding the presence of asynaptic or

desynaptic mutants has been reported in common bean. Keeping these

facts in view, it was decided to follow another route, i.e.,

aneuploid segregates due to nondisjunction appearing in the progenies

of translocation heterozygotes.

A translocation heterozygote upon selfing produces eight types

of trisomics. These eight types will fall into four different

phenotypic classes, provided that each chromosome (including its

translocated segment) has some effect on the morphology of the

trisomic plant that carries it as the extra chromosome (Fig. III-1

and Appendix 4). Four out of these eight trisomic types are primary

and four tertiary. The four primary trisomic types do not have a

normal chromosome background. Two out of these four are on

translocation heterozygote background and the other two are on

homozygous translocation background (Appendix 4). To get primaries

on a homozygous normal chromosome background, trisomic plants are

backcrossed with normal (2n) pollen. The primaries with homozygous

translocation background should yield FI progeny (disomics as well as

trisomics) all having semisterile pollen, whereas tertiaries with

homozygous normal chromosome background yield F1 progeny all having

fertile pollen (Appendix 4). Primaries and tertiaries with

heterozygous translocation background, after being crossed with

normals, should yield a progeny segregating for either fertile or

semisterile plants. So these two classes are not distinguishable

solely on the basis of pollen fertility analysis (Appendix 4). As

already stated in Section III, common bean cytology is quite








confusing and time consuming probably due to the small size of the

chromosomes. It was employed only whenever it was necessary to make

proofs or distinctions.

The semisterile primary trisomic plants originating in the F1

progeny of a primary trisomic (with homozygous translocation

background) crossed with a normal disomic, if backcrossed with the

normal disomics, can be converted to a homozygous normal primary

trisomic. In BC1F1 trisomic progeny, only fertile trisomic plants

are selected (personal communication with G.S. Khush, IRRI,

Philippines). Fertile trisomic plants in BC1F1 progeny can then be

verified cytologically. If a high percentage of PMCs show a

trivalent association with all other chromosomes appearing as

bivalents, the plants in question are primary trisomic with a

homozygous normal chromosome background (Khush 1973). This procedure

avoids a lot of cytological analysis during preliminary stages.

In case one is unable to recover any primary trisomics from the

progeny of a translocation heterozygote, the best alternative is to

grow a few selfed progenies of a tertiary trisome and in each progeny

look for any deviates from the tertiary trisomic phenotype. One of

the unique features of the breeding behavior by which tertiary

trisomics differ from other types of trisomics is the regular

appearance of the two related primaries in their progenies (Khush and

Rick 1967).

Tertiary trisomics usually show pentavalent configurations at

M-I of meiosis, depending upon the chiasmata. If there is a

trivalent having a pair of one chromosome and an extra translocated








chromosome, two types of n+1 gametes can result from the segregation

of such a trivalent. As an example, alternate disjunction from the

1-2, 1-2, 1-3 trivalent can produce n+1 gametes in which the tertiary

1-3 is the extra chromosome. On the other hand, adjacent disjunction

could yield the primary with 1-2 as the extra chromosome. In the

same fashion, the tertiary trisome may form a trivalent with 3-4,

3-4, 1-3 chromosomes, adjacent disjunction from which could yield 3-4

as the extra, which means the other related primary. Adjacent

segregation from a pentavalent could give similar results. Thus, a

tertiary trisomic might regularly produce three kinds of n+1 gametes

and 2n+1 zygotes, one of the latter being the parental tertiary

trisomic and the other two being the related primary trisomics. The

frequency of related primaries yielded must depend, however, upon the

frequency of different types of associations formed at M-I, the types

of disjunction at anaphase-I, and the viability of the trisomic with

the extra primary chromosome. The frequency of two related primary

trisomics might also differ, depending upon the relative length of a

tertiary chromosome (Khush and Rick 1967). As an example, if arm 1

of the tertiary chromosome 1-3 is longer than arm 3, it will

preferentially pair with the homologue pair 1-2, 1-2 rather than 3-4,

3-4, because the greater length of the arm 1 will provide greater

opportunity to pair and form chiasmata. The adjacent disjunction

from such a trivalent will accordingly yield a primary trisome for

chromosome 1-2 at a higher frequency than a primary trisome for

chromosome 3-4 (Khush and Rick 1967).









Transmission rates of trisomics on crossing with 2n plants or on

crossing with marker stocks during linkage studies have been reported

to be higher than rates observed after selfing. This has been

attributed to the genetic heterozygosity that is created by crossing

(Khush and Rick 1967).



Materials and Methods

S1 seed from four phenotypic classes of off-type plants

identified from translocation heterozygote progenies grown in the

first field generation was planted in the first greenhouse generation

(Table III-1). Convex Leaf and Dark Green Leaf classes were allowed

to self-pollinate, and some of the plants picked at random from both

of these classes were also crossed with 2n. All the off-type plants

in Puckered Leaf and Weak Stem were searched for male-fertile

trisomic plants and, wherever found, were crossed with pollen from 2n

plants. If male-fertile plants with the trisomic phenotype were not

present in the progeny, semisterile trisomic plants were selected for

crossing with 2n pollen. FI seed from these two phenotypic classes

was grown in the second greenhouse generation and all the F1

progenies of male-fertile trisomic parents (including trisomic as

well as disomic segregates) were tested for pollen fertility. The

progenies having all semisterile plants were selected and trisomic

segregates were backcrossed with 2n pollen from a normal disomic. On

the other hand, the F1 progenies derived by crossing the semisterile

trisomic plants with 2n pollen in the first greenhouse generation

were searched for male-fertile trisomic plants, which were tagged and








allowed to self-pollinate. F2 seed from these plants, BC1F1 seed

from the other semisterile trisomic groups, and S2 and F1 seed from

Dark Green Leaf and Convex Leaf from the first greenhouse generation

were all planted in the second field generation. Male-fertile

trisomic plants were identified in BC1F1 progenies and immature

flower buds were collected from them. Similarly, flower buds were

also collected from F2 trisomic segregates of Weak Stem (see Appendix

4). From S2 progenies of Dark Green Leaf and Convex Leaf, trisomic

plant progenies showing morphological dissimilarities between them

(segregates for disomics and trisomics) but uniformity within the

class (segregates for only one type of trisomic) with the least

number of off-type segregates, were selected for meiotic analysis.

Another phenotypic class originating among most of the trisomic

progenies at a very low frequency, was analyzed cytologically.

Plant, pod and seed photographs were made wherever possible, and

other descriptive notes for all five groups were recorded in the

field along with the transmission and ovule abortion data.



Results and Discussion

Because the method of isolation of these primary trisomics

differs slightly from one to the other (with one exception that is

explained under one heading), they are treated under separate

headings.

Puckered Leaf Trisomic

Out of 100 S1 seeds of Puckered Leaf, planted in the first

greenhouse generation, 26 plants were identified as phenotypically








identical to the seed parent. Five of these 26 plants were

identified as male-fertile on the basis of pollen abortion rates. In

these five male-fertile trisomics the pollen abortion rates ranged

from 20 to 36 percent. This gave an indication that these

male-fertile trisomic plants can either be tertiary or primary, both

either on homozygous normal or homozygous translocation background.

When the off-type plants originated in the field among the

selfed progeny of a translocation heterozygote, the off-type plants

in each phenotypic group were principally of four types (Table

III-1). Male-fertiles, either tertiaries with homozygous normal

background or primaries with homozygous translocation background.

Similarly, semisteriles, either primaries or tertiaries both on

translocation heterozygote background (Fig. III-1). When these were

allowed to self-pollinate in the first field generation, the male-

fertiles bred true with respect to their background, but the

semisteriles (having translocation heterozygote background)

segregated into 1 homozygous normal (fertiles), 1 homozygous

translocation (fertiles) and 2 heterozygous translocation

(semisteriles) both in primaries and tertiaries. So the male-

fertiles from tertiaries having homozygous translocation background

also appeared in the S1 progeny, although perhaps at a low

frequency. Now here it is worthwhile to point out that if one

identifies male-fertile trisomic plants by pollen abortion analysis

in the first field generation (selfed progeny of a translocation

heterozygote), he can eliminate two classes of trisomics among the

Puckered Leaf phenotypic group and end up with only two classes that









are very easily identifiable by progeny test of their Fls. These can

be primary with homozygous translocation background and tertiary with

homozygous normal background. Keeping all these theoretical aspects

in view, an attempt was made to identify the trisomic plants with

homozygous translocation background. Principally they should be

primary, though contaminated with tertiary at a low frequency. These

five plants were crossed (male-fertile trisomic segregates) with 2n

pollen and their progenies grown. In the second greenhouse

generation, one out of these five male-fertile plant progenies

produced an all semisterile F1. It produced 23 plants that

segregated into 12 trisomics and 11 normal disomics. Pollen abortion

in these trisomes ranged from 50 to 65 percent, whereas in all the

semisterile disomics it was 40 to 45 percent. This gave an

indication that the plant progeny may be primary or tertiary with

translocation heterozygote background. These 12 trisomic plants from

semisterile progeny were selected for backcrossing with 2n pollen.

BC1F1 seed produced on these plants was grown in the second field

generation. The progeny produced 283 plants in total with 97

trisomic segregates. Due to unfavorable climatic conditions in the

field during the summer of 1985, the mortality rate of trisomic

plants was high. Nevertheless, 19 male-fertile trisomic plants from

5 plant progenies were tagged on the basis of pollen abortion

analysis. Three of the male-fertile plant progenies having a maximum

of surviving trisomic plants were analyzed cytologically. Thirty-six

out of 50 analyzable PMCs at diplotene to M-I showed a trivalent

association with 10 bivalents (Fig. IV-2C,E). Twelve PMCs showed one




76
are very easily identifiable by progeny test of their F]_s. These can
be primary with homozygous translocation background and tertiary with
homozygous normal background. Keeping all these theoretical aspects
in view, an attempt was made to identify the trisomic plants with
homozygous translocation background. Principally they should be
primary, though contaminated with tertiary at a low frequency. These
five plants were crossed (male-fertile trisomic segregates) with 2n
pollen and their progenies grown. In the second greenhouse
generation, one out of these five male-fertile plant progenies
produced an all semisterile Fi. It produced 23 plants that
segregated into 12 trisomics and 11 normal disomies. Pollen abortion
in these trisomes ranged from 50 to 65 percent, whereas in all the
semisterile disomies it was 40 to 45 percent. This gave an
indication that the plant progeny may be primary or tertiary with
translocation heterozygote background. These 12 trisomic plants from
semisterile progeny were selected for backcrossing with 2n pollen.
BCj^ seed produced on these plants was grown in the second field
generation. The progeny produced 283 plants in total with 97
trisomic segregates. Due to unfavorable climatic conditions in the
field during the summer of 1985, the mortality rate of trisomic
plants was high. Nevertheless, 19 male-fertile trisomic plants from
5 plant progenies were tagged on the basis of pollen abortion
analysis. Three of the male-fertile plant progenies having a maximum
of surviving trisomic plants were analyzed cytologically. Thirty-six
out of 50 analyzable PMCs at diplotene to M-I showed a trivalent
association with 10 bivalents (Fig. IV-2C.E). Twelve PMCs showed one



















































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univalent with 11 bivalents (Fig. IV-1A,B). Two pentavalent

associations were also observed that were probably due to overlapping

of chromosomes. Each plant progeny was analyzed separately.

Transmission rates of the extra chromosme after selfing (2n+1)

or crossing [(2n+l) x 2n] have been given in detail in Table III-3

for progenies classified in the second field generation. A total of

5567 progeny plants from selfed (2n+1) parents yielded 2025

trisomics, giving a 36 percent transmission rate. A total of 528 F1

progeny plants from crosses yielded 201 trisomics, giving a 38

percent transmission rate. Similarly in first greenhouse generation

(Table III-1), a total of 100 progeny plants from selfed trisomic

parents yielded 26 trisomics, giving a 26 percent transmission

rate. A total of 91 F1 progeny plants from crosses (Table IV-1) in

second greenhouse generation, yielded 46 trisomics, giving a 50

percent transmission rate. Here again we observe the same trend,

i.e., transmission is higher in cross progenies than self

progenies. Higher rates from crossing have been attributed to the

heterozygosity created by crossing (Khush and Rick 1967).

The phenotype of Puckered Leaf primary trisomic can be

distinguished 15-20 days after planting or after the 2nd true leaf

emergence (Fig. IV-3A). Leaves are puckered in shape and give a

glazed appearance after attaining full size. Plants are weaker than

diploid sibs during the early stage of growth but attain the same

size later (Fig. IV-4B). Also, puckered plants show a tendency for

accentuated semi-vining growth habit. They flower nearly at the same

time as diploids do. There is no difference in flower color. The














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Photographs of five types of primary trisomic plants.

Al. A normal disomic plant, 30 days after planting.

A2. Dark Green Leaf trisomic of the same age.

B. Puckered Leaf trisomic, 30 days after planting. The
glossy leaf surface gives a light color (pseudo-
chlorosis) in black and white photographs.

C. Weak Stem trisomic, 30 days after planting.

D. Convex Leaf trisomic, 45 days after planting.

E. Chlorotic Leaf trisomic, 30 days after planting.


Fig. IV-4




Fig. IV-4 Photographs of five types of primary trisomic plants.
A}. A normal disomic plant, 30 days after planting.
A£. Dark Green Leaf trisomic of the same age.
B. Puckered Leaf trisomic, 30 days after planting,
glossy leaf surface gives a light color (pseudo
chlorosis) in black and white photographs.
C. Weak Stem trisomic, 30 days after planting.
D. Convex Leaf trisomic, 45 days after planting.
E. Chlorotic Leaf trisomic, 30 days after planting.
The






pods are broader and shorter with an unfilled basal portion, and they

have slightly longer stylar tips with a pronounced curvature (Fig.

IV-5B). Seeds are larger and have slight wrinkles on the test (Fig.

IV-6E). Because this trisomic can be identified easily at the

seedling stage (same for Weak Stem trisomic), all or most of the

undesired diploids can be rogued from stocks or Fls in early stages,

thus saving greenhouse space. It is also sensitive to heat and

drought (but less than the Weak Stem trisomic) which affects the pod

and seed set adversely. It sets very well in the greenhouse where

mean seeds per pod in trisomic plants is 3.35 1.63 compared to 5.61

* 1.46 in normal diploids (means from 10 plants). A 43 percent

reduction in seed set or ovule abortion under greenhouse conditions

has been observed. Trisomic plants may have improved seed set when

developed on a homozygous normal chromosome background.

Weak Stem Trisomic

Nineteen S1 seeds recovered from two plants of the Weak Stem

trisomic originating in the selfed progeny of a translocation

heterozygote in the first field generation were planted in the first

greenhouse generation. Six plants were identified as trisomic from

their phenotype (Table III-1). Pollen fertility analysis showed that

they were all semisteriles. Pollen abortion rates ranged from 60 to

75 percent in these trisomic plants. It gave an indication that

these plants are either primary or tertiary trisomics still having a

translocation heterozygote background. To convert them to homozygous

normal chromosome background, they were crossed with pollen from

normal (2n) plants. One thing which should be emphasized here is



























Photographs of pods from primary trisomic plants.

A. Normal disomic plant pods.

B. Puckered Leaf pods.

C. Weak Stem pods.

0. Convex Leaf pods.

E. Dark Green Leaf pods.

(For details, please see text.)


Fig. IV-5













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A. Dark Green Leaf seeds.

B. Normal disomic seeds.

C. Convex Leaf seeds.

D. Weak Stem seeds.

E. Puckered Leaf seeds.

(For details, please see text.)


Fig. IV-6




Full Text
CYTOGENETIC ANALYSIS OF TRANSLOCATION HETEROZYGOTES
ANO TRISOMICS ISOLATED FROM THEIR PROGENIES IN COMMON BEAN
(PHASEOLUS VULGARIS L.)
BY
MOHAMMAD ASHRAF
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1985

To those who devoted their lives
for the welfare of mankind.

ACKNOWLEDGMENTS
There are many people whom I would like to thank for providing
help, infusion of ideas, understanding, friendship and love. These
are the people who supported me through events that will be
remembered as cherished memories. Special thanks go to Mark J.
Bassett, Professor, Vegetable Crops Department, who not only served
as committee chairman and shared his expertise, but also provided
untold hours in consultation and providing guidance to shape a
growing mind which had no previous exposure to cytogenetics.
I also thank Drs. Ken H. Quesenberry, Associate Professor of
Agronomy; Paul M. Lyrene, Professor, Fruit Crops Department; L.C.
Hannah, Professor, Vegetable Crops Department; Daniel A. Roberts,
Professor, Plant Pathology Department; and Gloria A. Moore, Assistant
Professor, Fruit Crops Department, for furthering my knowledge of
cytogenetics, genetics, plant breeding and plant pathology.
I am especially appreciative to my wife and kids, who, for the
past 3 years, have provided much in the way of spiritual and moral
support as well as exhibited exemplary patience and understanding
necessary for the family of a graduate student. This degree is as
much theirs as it is mine, and I hope our future adventures may be as
pleasant and successful as this one has been.

Finally, the Quaid-e-Azam/Merit scholarship awarded by the
Ministry of Education, Government of Pakistan, for completion of this
study is highly acknowledged. I also acknowledge the research grant
provided for this project through Dr. M.J. Bassett, without which it
would not have been possible to work on the project reported in this
dissertation.
TV

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES vii
LIST OF FIGURES viii
ABSTRACT x
SECTIONS
I INTRODUCTION 1
II CYTOGENETIC ANALYSIS OF TRANSLOCATION HETEROZYGOSITY
IN COMMON BEAN (PHASEOLUS VULGARIS L.) 3
Introduction 3
Materials and Methods 15
Results and Discussion 16
III TERTIARY TRISOMICS AND TETRASOMICS FROM TRANSLOCATION
HETEROZYGOTES IN COMMON BEAN (PHASEOLUS
VULGARIS L.) 23
Introduction 23
Materials and Methods 35
Results and Discussion 37
IV ORIGIN, CYTOLOGY, MORPHOLOGY AND TRANSMISSION OF
THE EXTRA CHROMOSOME IN FIVE PRIMARY TRISOMICS OF
COMMON BEAN (PHASEOLUS VULGARIS L.) 67
Introduction 67
Materials and Methods 73
Results and Discussion 74
V SUMMARY AND CONCLUSIONS 102
APPENDICES
1 MODIFICATION OF THE SLIDE PREPARATION TECHNIQUE
OF CHENG AND BASSETT (1981) 104
V

2OFF-TYPE PLANTS OBSERVED AMONG THE PROGENIES OF
TRANSLOCATION HETEROZYGOTES DURING THE FIRST FIELD
PLANTING (SUMMER, 1984) 107
3 ANEUPLOIDS RECOVERED FROM THE PROGENIES OF
TRANSLOCATION HETEROZYGOTES DURING THE SECOND
FIELD PLANTING (SUMMER, 1985) 110
4 PROCEDURES TO IDENTIFY PRIMARY AND TERTIARY
TRISOMICS ORIGINATING AMONG THE SELF PROGENY OF
A TRANSLOCATION HETEROZYGOTE 113
5 FIELD PLOT NUMBERS OF PLANT PROGENIES GROUPED
ACCORDING TO THEIR SIMILARITY OF SEGREGATION
PATTERNS; TABLE 111-2 (SECOND FIELD PLANTING,
SUMMER, 1985) 114
LITERATURE CITED 115
BIOGRAPHICAL SKETCH 120
VI

LIST OF TABLES
Table Page
11-1 Chromosome configurations at M-I at meiosis observed
in PMCs of Fj progeny from crosses of translocation
homozygote stocks 17
II-2 Comparison of the mean number of seeds per pod and the
distribution of seeds per pod in F^s of each cross
combination showing abnormal'M-I configurations
compared to a standard normal bean breeding line
0-181 20
III-1 Transmission of the extra chromosome in four trisomics
of common bean (First greenhouse planting, Fall, 1984) 40
III-2 Progeny tests of two tertiary trisomics originally
derived from translocation heterozygote 1-52 (Second
field planting, Summer, 1985) 41
III-3 Segregation for trisomic progeny from selfed (2n+l)
or cross pollinated [(2n+l) x 2n] trisomics originally
derived from four different translocation heterozygote
progenies (Second field planting, Summer, 1985) 46
III-4 Putative trisomics derived from different translocation
heterozygote progenies; their phenotype and rate of
transmission of extra chromosome in (2n+l) self
(Second field planting, Summer, 1985) 47
IV-1 Transmission of the extra chromosome in two trisomics
of common bean after crossing [(2n+l) x 2n] (Second
greenhouse planting, Spring, 1985) 82
IV-2 The frequency of PMCs with different chromosome
configurations at Diplotene to M-I of meiosis in
different plant progenies of Weak Stem Trisomic 93
vi i

LIST OF FIGURES
Figure Page
11 -1 Illustration of various types of quadruple pairing
derived from a translocation heterozygote with
photomicrographs of these configurations in common
bean 7
11 -2 Coorientation types of quadruples at M-I 10
11-3 Illustration of events leading to formation of a ring
of 6 chromosomes 10
II—4 Metaphase-I of a normal disomic and diplotene to M-I
of a translocation heterozygote with one or two
translocations involving common or different
chromosomes in common bean 14
III-1 A table giving all the types of trisomics that can
result from selfing a translocation heterozygote 28
III-2 Pentavalent configurations expected in a tertiary
trisomic according to various positions of chiasmata 32
III-3 Meiotic diplotene to anaphase-I as observed in
tertiary trisomics of common bean 52
III-4 Meiotic diakinesis to anaphase-I as observed in a
tetrasomic of common bean 57
III-5 Photographs of tertiary trisomic and tetrasomic
plants 60
III-6 Plant photographs of suspected trisomics (genetic
evidence only) 64
IV-1 Meiotic diplotene to anaphase-I as observed in the
primary trisomics of common bean 78
IV—2 Meiotic diplotene to anaphase-I as observed in the
primary trisomics of common bean 80
IV-3 Plant photographs of primary trisomics at the
seedling stage 84
viii

IV-4 Photographs of five types of primary trisomic plants 86
IV-5 Photographs of pods from primary trisomic plants 89
IV-6 Photographs of seeds from primary trisomic plants 91
IX

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
CYTOGENETIC ANALYSIS OF TRANSLOCATION HETEROZYGOTES
AND TRISOMICS ISOLATED FROM THEIR PROGENIES IN COMMON BEAN
(PHASEOLUS VULGARIS L.)
BY
MOHAMMAD ASHRAF
%
December, 1985
Chairman: Mark J. Bassett
Major Department: Horticultural Science (Vegetable Crops)
Common bean, Phaseolus vulgaris L., does not have either a
translocation tester set or aneuploid series available for use in
linkage studies. In 1982 Bassett and Hung attempted to develop a set
of translocations by means of pollen irradiation. Genetic tests gave
an indication that the resulting semisteri 1 ity mutations might be due
to translocation heterozygosity. In this study, an attempt was made
to 1) investigate cytologically these semisteri 1 ity mutations; 2) if
they are translocations, determine whether they have common or
different chromosomes involved in the interchanges; 3) quantify
reduction of seed set due to translocation heterozygosity; 4) search
for trisomics among the progenies of translocation heterozygotes,
assuming that they are phenotypically distinguishable; and 5) develop
primary trisomics, 2n + 1 = 23, on normal chromosome background.
x

This work has proved that the five semisteri1ity mutations
studied are chromosome translocations. The five stocks used in this
study have at least 8 different chromosomes involved in their
reciprocal translocations. The cytological behavior and
morphological abnormalities with respect to pollen and ovule abortion
in translocation heterozygotes of common bean as observed in this
study are quite similar to the earlier reports in other crops.
The 11 translocation stocks, used in the search for trisomics,
produced 12 distinct phenotypic groups of plants. Five of these 12
groups have been verified to be trisomics by genetic tests and by
cytological verification. Genetic evidence from the remaining seven
indicates trisomy, but this has not yet been confirmed
cytologically. Five primary trisomics have been identified on the
basis of their cytological behavior. Of these five primaries, three
related tertiaries have been identified on the basis of their
phenotypic relationship. For another primary group, the related
tertiary has been isolated on the basis of cytology. Two trisomic
groups produced tetrasomics at a low frequency. For two of the five
primary trisomic groups, primaries have been developed on a
homozygous normal chromosome background. Physical identification of
the extra chromosome has not yet been done for any of the
translocation and trisomic groups.
xi

SECTION I
INTRODUCTION
Extensive linkage maps have been produced for several crops, and
for many other crops they are being worked out. Their knowledgeable
use can be of considerable advantage in the design of breeding
projects and genetic experiments. Despite their potential value, the
development of linkage maps has been very slow, especially for self-
pollinated crops. Common bean is a self-pollinated crop, for which
26 genes were initially assigned to eight linkage groups (Lamprecht
1961). Since then, very few linkages have been reported although
over 200 genes have been described for common bean (Roberts 1982).
There are several reasons for the lack of linkage study in
common bean, one of which is that neither a chromosome translocation
tester set nor an aneuploid series, most commonly used for gene
mapping, has yet been developed in common bean.
Chromosome translocations were first reported by Belling (1914)
while working on a breeding program for improvement of Florida velvet
bean. The phenomenon was not well understood until 1925. While
Belling was working with the Blakeslee group at Cold Spring Harbor,
he showed how semisteri1ity could be due to segmental interchange
between nonhomologous chromosomes. He also reported the linkage
between semisterility and several characters in Florida velvet
1

2
bean, but it was not known whether these factors belonged to the same
or different linkage groups. Later on, interchanges were reported in
Drosophila (Stern 1926), and they were first induced by x-rays in
Drosophila during 1930 (Muller). In maize, interchanges were first
reported by Brink (1927) and confirmed cytologically by Burnham
(1930). Since then they have been induced in many crops and
extensively used by plant breeders for understanding the gene-
chromosome relationship (Burnham 1956, 1962).
The discovery of the Globe trisomic of Datura stramonium, the
first primary trisomic, was the beginning of the pioneer
investigations of trisomics by Blakeslee and Avery (1919). Trisomics
have appeared spontaneously among progeny of normal diploids of many
other species but are obtained more frequently from asynaptic
mutants, desynaptic mutants, or among the progeny of polyploids, most
notably triploids (Khush 1973). Chromosome translocations, when in a
heterozygous form, also produce aneuploids at a low frequency (Khush
1973). Trisomics are another excellent tool for assigning linkage
groups to specific chromosomes. As a matter of fact, the most
extensive genetic studies done with the aid of aneuploids have been
conducted with trisomics (Schaeffer 1980).

SECTION II
CYTOGENETIC ANALYSIS OF TRANSLOCATION HETEROZYGOSITY IN
COMMON BEAN (PHASEOLUS VULGARIS L.)
Introduction
A mutation in which terminal segments of nonhomologous
chromosomes have exchanged positions is known as chromosomal
%
interchange, an interchange, a reciprocal translocation or simply a
translocation. All of these terms have been and are still used
interchangeably in the literature to describe the same phenomenon.
Translocations have been and still are among the most productive
tools of cytogenetics. They offer an enormous potential for plant
breeders to engage in chromosome engineering.
Naturally occurring translocations were first reported by
Belling (1914) in Florida velvet bean, followed by Stern (1926) in
Drosophila. Muller (1930) was the first to induce chromosome
translocations by x-rays, in Drosophila. In maize, translocations
were first reported by Brink (1927) and were confirmed cytologically
by Burnham (1930). The classical experiments of Burnham (1930) and
of Brink and Cooper (1931) established that semisterility caused by
translocation has all the characteristics of a gene located at the
translocation breakpoint. The location of the breakpoint in relation
to other genes can therefore be determined and mapped.
3

4
As soon as it was established that translocations could be used
successfully for gene mapping, researchers started developing tester
sets of translocations in which the positions of breakpoints with
respect to centromeres and telomeres were known. Maize (Burnham
1954) and barley (Burnham e_t aj_. 1954) were the first field crops
followed by English pea (Lamm and Miravalle 1959), rye (Sybenga and
Wolters 1972), and tomato (Gill et al. 1980) for which complete
tester sets of translocations were developed.
We are aware of only one report of chromosome translocations in
common bean before 1982 (Mutschler and Bliss 1980). In this case one
of the bean breeding lines segregated for semisterile and fertile
progeny plants in a 1:1 ratio. The term semisteri1ity has been used
to refer to those plants in which pollen abortion rates fall between
normal (fertile) and sterile plants. The semisteri1ity was
attributed to translocation heterozygosity, but the authors could not
provide any cytological proof, stating that tetravalent formations
were not observed at meiosis, probably due to the very small size of
common bean chromosomes. Furthermore, they reported no reduction in
seed set in the semisteriles. In 1982, Bassett and Hung induced
semisteri1ity mutations (probably translocations) in common bean.
They used irradiated pollen to pollinate normal, untreated plants.
Twelve semisterile mutant lines were produced and various genetic
tests supported the hypothesis that the semisteri1ity was due to
translocation heterozygosity. Five out of these 12 translocation
lines were used in this study.

5
The photomicrographs provided alongside each drawing in Figure
11—1 show the cytological behavior of a translocation heterozygote in
common bean at different stages of meiosis. Figure II-1A shows the
mutual exchange of chromosome segments between 2 nonhomologous
chromosome pairs. If the ends of chromosomes are numbered (1-2,
3-4), the 4 chromosomes that all have different end combinations
(1-2, 2-3, 3-4, 4-1) will result. No 2 chromosomes of this group can
pair along their entire length but all 4 can come together in a
pairing configuration (quadruple) that allows partial pairing of
homologous chromosome segments. Quadruples (Sybenga 1972) resemble
quadrivalents, which are formed when all 4 chromosomes are homologous
or equivalent to each other. An organism in which quadruple pairing
occurs is called a translocation heterozygote. In pachytene such a
configuration can appear as a cross (Fig. II-1B), which sometimes may
be prevalent even in diplotene.
Translocation breaks can occur at any point along the
chromosome. The position of the breakpoints will determine the
future fate of the translocation quadruple. Different quadruple
configurations can arise depending on the formation of crossovers in
interstitial or distal chromosome segments. Possible interstitial
crossovers can lead to duplication and deficiency gametes and
consequently are not recovered (Burnham 1962). Possible diakinesis
configurations originating from different pachytene situations are
shown in Figure II-1C,D,E. If for instance, crossovers occur in
locations 1, 2, 3, 4, 5 and 6, diakinesis configurations resemble a
number 8. If chiasmata occur only in locations 1, 3, 5 and 6, a ring

Fig. 11-1 Illustration of various types of quadruple pairing
derived from a translocation heterozygote with
photomicrographs of these configurations in common bean
(x 1000) (modified from Schaeffer 1980).
A. Two nonhomologous chromosome pairs involved in
reciprocal translocation.
B. Pachytene configuration appearing as a cross.
Numbers 1 to 6 designate chiasmata.
C. If chiasmata form only at 1, 3, 5 and 6, the
quadruple appears as a ring at diplotene to M-I.
D. If chiasmata form at all six locations, the quadruple
appears as a figure 8.
E. If chiasmata form only at 1, 3 and 5, the quadruple
appears as a chain. The thinner arrow in (e) that
points downward indicates a chain of 4 chromosomes.
The thicker arrow indicates a noncooriented
quadruple.
F. Noncoorientation of a quadruple, which gives rise to
nondisjunction and consequently an aneuploid gamate.

L

8
of four chromosomes is formed in diakinesis. If chiasmata occur in
locations 1, 3, and 5, a chain of four chromosomes can form.
Chromosome orientation of quadruples in M-I is critical.
Bivalents in normal meiosis have only 2 centromeres that are arranged
in coorientation and that distribute the 2 chromosomes involved to
opposite poles. However, several types of orientation on the
metaphase plate are possible when a quadruple is formed, since not 2
but 4 centromeres are involved.
Figure 11-2 illustrates the coorientation types of quadruples in
metaphase-I. Alternate-1 and alternate-2 give rise to viable gametes
because 2 translocated chromosomes pass to one pole and the 2
nontranslocated chromosomes pass to the other, giving balanced
chromosome complements in the gametes. On the other hand, adjacent-1
and adjacent-2 give rise to duplication and deficiency gametes, which
cause semisteri1ity in translocation heterozygotes (Endrizzi 1974).
The proportion of fertile and aborted gametes is close to 1:1 in
several species. It is expressed by half of the seeds being missing
in an inflorescence of a plant, e.g., maize, petunia, peas and
sorghum, whereas it is less in others like Hordeum, Secale, Da tura,
Triticum and Oenothera (Schaeffer 1980).
In noncoorientation (Fig. 11-IF) the 2 centromeres on opposite
sides of the quadruple are cooriented and are positioned equidistant
from the equitorial plate. The 2 intermediate centromeres are
noncooriented and are stretched out between the other 2, seemingly
not attached to the poles. In anaphase-I, there may be a 2:2 or 3:1
segregation, the first one giving rise to a duplication-deficiency

Fig. 11-2
Coorientation types of quadruples at M-I.
Noncoorientation is shown in Fig. 11 —1. Letters
designate centromeres and numbers designate chromosome
ends (modified from Schaeffer 1980).
Fig. 11-3 Illustration of events leading to formation of a ring of
6 chromosomes.
A. The two nonhomologous pairs involved in two
independent reciprocal translocations having one
chromosome in common.
B. Pachytene configuration appearing as a star which
forms a ring or chain of 6 depending upon chiasmata.

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11
(and hence nonviable) gametes and the second giving rise to aneuploid
gametes (Hagberg 1954).
The objectives of this study were to (1) determine cytologically
whether the semisterile stocks developed by Bassett and Hung (1982)
are translocation heterozygotes; (2) if the semisteri1ity in these
stocks is due to chromosome interchanges, determine whether the
translocation lines used in this study have common or different
chromosomes interchanged; and (3) develop quantitative data on the
reduction of seed set in translocation heterozygotes. The
information from objective 2 will help establish a tester set of
translocations for linkage studies if the chromosomes involved in
translocations can be physically identified. This information could
be tested by correlating it with the different types of trisomics
originating in the progeny of each translocation heterozygote.
Since only 1/4 of the progeny from the intercross of two
heterozygous translocation lines should carry both translocations, it
is preferable to isolate homozygous lines for each translocation as a
first step. In intercrosses between such lines or in crosses with
the homozygous normal plants, all the plants can be used (3urnham
et al. 1954). Theoretically, half the normal (fertile pollen) plants
in the progeny derived from self-pollination of a plant heterozygous
for an interchange should be homozygous for that interchange. To
identify translocation homozygotes, the plants having normal pollen
are sel fed and also crossed onto a standard normal line. If the
plant being tested is homozygous for the interchange, all the

12
test-cross Fj plants should be semisterile (Brink and Burnham
1929). When homozygous translocation lines are crossed with a
standard normal line and meiosis of the plants is examined
cytologically, all the cells show either a pachytene cross
configuration or one of the other metaphase-I configurations already
discussed (Fig. II-1C,D,E). When homozygous translocation lines are
intercrossed and the meiosis of F^ plants is examined cytologically,
a ring or a chain of 6 chromosomes (Fig. 11-3 and II-4F.G) indicates
that only 1 chromosome is common to the two interchanges, whereas if
separate rings or chains of 4 chromosomes are observed (Fig.
II-4C,E), this indicates that the two interchanges have no
chromosomes in common (Burnham e_t al_. 1954).
In translocation heterozygotes of barley showing a ring of 4
chromosomes at M-I of meiosis, the pollen abortion rate ranges in
different lines from 14 to 58 percent the average being 28.3
percent. It is 51 percent in one line with two separate rings of
4. Ovule abortion on the same plants reported is 39, 92 and 67
percent respectively. The data on ovule abortion are based on seed
set counts of two heads on the original semisterile plants, and may
not necessarily be the true degree of sterility (Burnham et al.
1954). In another study, seed set in translocation heterozygote
barley lines has been found to be between 60 to 70 percent compared
with 97 percent in the original control varieties (Kunzel and
Bretschneider 1981). A negative correlation between the number of
translocated chromosomes and the fertility of the plants has been
observed in Pi sum lines heterozygous for these translocations

Fig. 11-4 Metaphase-I of a normal disomic and diplotene to M-I of a
translocation heterozygote with one or two translocations
involving common or different chromosomes in common bean
(x 1000).
A. Diplotene of a translocation heterozygote involving
one translocation. Arrow indicates a ring of 4
chromosomes. Eight bivalents are spread around and
one bivalent is still associated with the nucleolus.
B. Metaphase-I of (A) above. Arrow indicates a ring of
4 chromosomes. The lighter black dot with two
bivalents on either side of it is the disappearing
nucleolus.
C. Metaphase-I of a PMC heterozygous for ttfo independent
translocations. The two arrows each point to a ring
of 4 chromosomes.
D. Metaphase-I of a normal disomic bean PMC showing 11
bivalents.
E. Metaphase-I of a translocation heterozygote involving
two independent translocations. The two arrows
indicate a ring of 4 and a chain of 4 chromosomes.
One bivalent is overlapping the right end of the
chain.
F. Metaphase-I of a translocation heterozygote involving
two translocations having 1 chromosome in common.
The arrow indicates a ring of 6 chromosomes and the
interpretive drawing is to the right (f).
G. The arrow indicates a chain of 6 chromosmes and the
interpretive drawing is to the right (g).
f & g. Please note that in both interpretive drawings (f)
and (g) one pair of normal chromosomes, i.e., abed,
ijkl and two pairs of translocated chromosomes, i.e.,
abgh, efed and ijgh, efkl are involved in multivalent
format!on. One pair of normal chromosomes, i.e.,
efgh common to both translocations, is not involved
in multivalent formation.

14
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15
(Gottschalk 1978). Burnham (1962) has reported nearly 50 percent
pollen abortion with translocation heterozygosity in maize.
Materials and Methods
Five semisteri1ity mutants (suspected chromosome translocation
lines) developed by Bassett and Hung (1982) were used in this
study. Their seed was planted and the plants were classified into
semisteriles and fértiles in each line on the basis of aborted pollen
counts using iodine stain. The plants classified as fértiles were
allowed to self and were also testcrossed (as pollinators) to another
standard normal bean breeding line (0-181) carrying the recessive
marker round leaf (rnd). The seed from the pollen parents and
testcrosses was harvested. testcross progeny seed was planted and
the plants classified into semisteriles or fértiles. Homozygous
translocation lines were identified by observing F^ testcross progeny
that were all semi sterile.
The five different homozygous translocation lines recovered,
i.e., 1-52, 1-97, 11 —3, 11-70 and 11-121, were crossed with a
standard normal bean breeding line, 0-181, and were also intercrossed
in a half diallel set. Fj seed was planted and immature flower buds
were collected individually from five plants each of all the ten half
diallel cross combinations of homozygous translocation lines, five
cross combinations of homozygous translocation lines with 0-131 and
one 0-181 selfed. Immature flower buds were collected every other
day between 10 to 12 A.M. (EST), put immediately in Carnoy's fixative
solution containing 6:3:1 by volume of 95? ethanol, chloroform, and

16
glacial acetic acid and treated for 24 h. Buds were then transferred
to 70% ethanol for preservation. Microscopic slides were prepared
using the technique of Cheng and Bassett (1981) with slight
modification (see Appendix I). A total of 50 pollen mother cells
from pachytene to M-I of meiosis were analyzed for each cross
combination.
Results and Discussion
In crosses 1 to 5 (Table 11-1) all the five stocks were
individually crossed to standard normal line, 0-181. PMCs of F^s
showed 1 4 and 9 II during late prophase and metaphase-I (Fig.
II-4A.B). This cytological evidence along with the previous genetic
evidence (Bassett and Hung 1982) demonstrates conclusively that the
semisterile stocks used in this study are translocation
heterozygotes. Meiotic metaphase-I of the standard normal line
(0-181) included in this study was normal. It did not show any type
of multivalent association (Fig. II-4D).
The half dial lei set of crosses among the five homozygous
translocation lines was examined cytologically to determine whether
the five interchanges might involve any particular nonhomologous
chromosome in more than one interchange. F^s of crosses 6, 7, 10,
11, 13 and 15 (Table 11 -1) showed 204 + 7 II, indicating that 4
different nonhomologous chromosomes are involved in the two
interchanges (Fig. II-4C.E). In cross 8, we observed 10 4 and 1
chain of 4 with 7 II chromosomes (Fig. II-4E) instead of 204
chromosomes. When the same translocation stocks involved in cross 8,

17
Table 11-1
Chromosome configurations at M-I of meiosis observed in PMCs of
progeny from crosses of translocation homozygote stocks.
Cross combination
Chromosome configuration
1.
1-52 x 0-131
1 © 4 + 9 II
2.
1-97 x 0-181
1 ©4 + 9 II
3.
11-3 x 0-181
1 © 4 + 9 II
4.
11-70 x 0-181
1 ©4 + 9 II
5.
11-121 x 0-181
1© 4 + 9 II
6.
1-52 x 1-97
2®4 + 7 II
7.
1-52 x 11-3
2©4 + 7 II
8.
1-52 x 11-70
1©4 + 1 chain of 4 + 7 II
9.
1-52 x 11-121
Results inconclusive
10.
1-97 x 11-3
2© 4 + 7 II
11.
1-97 x 11-70
2©4 + 7 II
12.
1-97 x 11-121
1 chain of 6+3 II
13.
II—3 x 11-70
2 ©4 + 7 II
14.
11-3 x 11-121
1 ©6 + 3 II
15.
11-70 x 11-121
2©4 + 7 II
16.
0-181 (Standard normal)
11 II
Note: 0-181 is a standard normal line; all other code numbers in the
above intercrosses refer to homozygous translocation lines.

13
namely 1-52 and 11-70, were individually crossed with the standard
normal line (0-181), both of the stocks individually showed 1®4 +
9 II at M-I. The differential behavior of one of the two
translocation stocks at M-I may be attributed to the involvement of
an acrocentric chromosome in reciprocal translocations in that
stock. When the breakpoint is in the short arm, this hinders
chiasmata formation.
Cross 12 shows a chain of 6 and 8 II chromosomes (Fig. II-4G),
indicating that translocations 1-97 and 11-121 have 1 chromosome in
common in the two interchanges. Formation of a chain of 6
chromosomes instead of a ring of 6 may possibly be due to the same
reason discussed above for cross 8 (Table 11-1). Cross 14 (Table
11 -1) showed 106 (Fig. II-4F), indicating the involvement of 1
common chromosome in both of the interchange stocks. All the PMCs
scored in cross 9 (Table 11-1) failed to show any clear ring or chain
configuration. In most of the cells there were either 2 or 3
bivalents lying together, from which it was not possible to make any
interpretation.
Keeping in view the cytological results stated in Table II—1,
one can conclude that the five homozygous translocation lines used in
this study involve 8 (out of 11 total) chromosomes in their
interchanges. It would be quite desirable to have all 11 chromosomes
involved in the 12 interchanges present in these lines because the
ultimate aim is to develop a complete set of primary trisomics from
these stocks. Results of cross 9 involving 1-52 x 11-121 were
inconclusive, but this did not affect our ability to establish that a

19
minimum of 8 chromosomes are involved. When we cross 11-121 with
I-97 or 11-3, we see a chain or ring of 6 chromosomes proving that
II-121 has one common chromosome involved in interchange with respect
to both 1-97 and 11-3. When 1-97 is crossed with II-3, 2 04 result,
indicating that both interchanges involve unique pairs of
chromosomes. Assigning arbitrary chromosome numbers to the stocks
for illustration, if 11-3 has 1 and 2 interchanged and 1-97 has 3 and
4 interchanged chromosomes involved in translocations, then 11-121,
on the basis of our results, has 1 of the 2 interchanged chromosomes
in 11-3 (either chromosome 1 or 2) and 1 of the 2 interchanged
chromosomes in 1-97 (either chromosome 3 or 4) involved in its
interchange. Crosses 6 and 7, in which 1-52 has been crossed with
1-97 and 11-3 respectively, tell us that 1-52 neither has its
chromosomes involved in the translocations 1-97 and 11-3. Therefore,
interchange 1-52 can be assigned 5 and 6 for its chromosomes.
Because interchange 11-70 has no chromosome in common with 11—3,
1-97, 11-121 and 1-52, therefore a thorough analysis of all the data
in Table 11-1 indicates that the two interchanges in cross 9 probably
involve unique chromosome pairs and that 8 chromosomes (in total) are
involved in reciprocal translocations in the five stocks used in this
study. The percent reduction in seed set for cross 9 (Table 11-2)
also indicates that 2©4 should have formed, but for some reason
typical rings or chains were not apparent.
Average pollen abortion rates for crosses 1 to 5, which showed
1©4 chromosomes at M-I of meiosis, ranged from 35 to 63 percent;
whereas in intercrosses 6 to 15, involving two interchanges, it was

Table 11-2
Comparison of the mean number of seeds per pod and the distribution of seeds per pod in F^s of each cross
combination showing abnormal M-I configurations compared to a standard normal bean breeding line 0-181.
Cross
combination
Chromosome
configuration
at M-I
Mean seeds
per pod
Percent pods in each
number of
I 5 3 4
category with respect
seeds per pod
~5 6 7 Q~
to
9
Percent
reduction
in
seed set
1.
1-52 x
0-181
104 + 9
II
2.28
31.26
30.76
23.68
13.15
0.72
0.43
_
_
_
60.75
2.
1-97 x
0-181
104 + 9
II
3.63
9.4
15.2
19.2
26.6
21.1
7.5
1.0
-
-
37.52
3.
11 -3 x
0-181
104 + 9
II
2.91
15.3
27.3
23.49
22.54
7.53
3.28
0.46
-
-
49.91
4.
11-70 x
: 0-181
104 + 9
II
2.86
18.38
27.35
21.99
19.28
8.96
2.69
1.34
-
-
50.77
5.
11-121
x 0-181
104 + 9
II
2.20
36.21
31.6
18.34
8.85
5.0
-
-
-
-
62.13
6.
1-52 x
1-97
2@4 + 7
II
1.72
48.27
34.48
13.8
2.9
0.53
-
-
-
-
70.39
7.
1-52 x
11-3
2©4 + 7
II
1.52
59.60
29.50
9.73
0.88
0.29
-
-
-
-
73.83
8.
1-52 x
11-70
104 + 1
of 4 + 7
chai n
II
1.52
60.23
29.38
8.88
0.78
0.49
0.24
—
73.83
9.
1-52 x
11-121
Results
inconclusive
1.47
64.12
25.88
8.52
1.48
“
—
74.69
10.
1-97 x
11-3
204 + 7
II
2.0
36.66
36.04
19.65
5.75
1.3
0.3
0.3
-
-
65.57
11.
1-97 x
11-70
204 + 7
II
2.05
31.6
42.25
16.90
7.39
1.76
-
-
-
-
64.71
12.
1-97 x
11-121
1 chain of 6
+ 8 II
2.16
36.56
33.94
17.76
5.75
1.56
3.39
1.04
“
62.82
13.
11-3 x
11-70
204 + 7
II
1.96
43.6
29.8
16.70
6.37
3.53
-
-
-
-
66.26
14.
11-3 x
11-121
106 + 8
II
1.52
62.0
28.0
8.0
1.5
0.5
-
-
-
-
73.83
15.
16.
11-70 >
0-181
; 11-121
204 + 7
11 II
II
1.54
5.31
56.92
2.03
32.2
3.39
9.97
6.33
0.91
10.85
12.66
21.94
27.8
14.1
0.9
73.49
ro
o

21
60 to 85 percent. Under the same conditions, the standard normal
bean breeding line with no meiotic disturbance had 10 to 15 percent
pollen abortion. Table 11-2 gives a detailed picture of percent pods
with respect to number of seeds in each cross combination. In the
F^s of intercrosses between homozygous translocation lines showing
2 ©4 or 1©6 at M—I of meiosis, 70 to 90 percent of the pods had 1 to
2 seeds, whereas only 25 to 68 percent of the pods had 1 to 2 seeds
when the same homozygous translocation stocks were crossed with a
standard normal and the F^s (showing 104 at M-I of meiosis) were
analyzed for seed set. In comparison, only 5 percent of the pods had
1 to 2 seeds in the standard normal line 0-181 (Table 11-2). Percent
reduction in seed set ranged from 63 to 75 percent for crosses of two
translocations showing 2©4 or 10 6 at M-I and ranged from 38 to 62
percent for crosses involving only one translocation showing 10 4 at
M-I of meiosis. The wide ranges in pollen and ovule abortion are due
to the differences between translocation heterozygote stocks with
respect to the varying frequencies of four principal orientations of
a quadruple (Fig. 11-2) at M-I (Burnham 1962).
Five stocks used in this study have been verified cytologically
as chromosome translocations. Cytological analysis of a half dial lei
set of crosses indicates that these five stocks involve 8 different
chromosomes in reciprocal translocations. Cytological behavior and
morphological abnormalities, with respect to pollen and ovule
abortion in translocation heterozygotes of common bean as observed in
this study, are nearly the same as reported in other crops (Burnham
1962). As regards physical identification of chromosomes involved in

22
these interchanges, pachytene analysis is difficult (Krishnan and De
1970) but not impossible (Cheng and Bassett 1981). New techniques
like Giemsa staining (Mok and Mok 1976) or use of biotin labelled
probes to map specific DNA sequences on chromosomes (Rayburn and Gill
1985) may hopefully help with identifying not only the chromosomes
but also the breakpoints.

SECTION III
TERTIARY TRISOMICS AND TETRASOMICS FROM TRANSLOCATION
HETEROZYGOTES IN COMMON BEAN (PHASEOLUS VULGARIS L. )
Introduction
Trisomic individuals have 1 more chromosome than the diploid or
somatic number. Since the trisóme modifies the genetic ratios for
genes located on that chromosome, trisomics offer one of the most
efficient methods of associating genes with their linkage groups.
Tertiary trisomics are those trisomics that have a translocated
chromosome in addition to the normal somatic chromosome complement.
They rank among the most useful genetic tools because they can be
used to determine the arm location of marker genes, the position of
centromeres, and the orientation of linkage maps. They are also a
good source of the related primary trisomics, which regularly appear
in their selfed progenies (Khush and Rick 1967). Despite their
utility for such cytogenetic analysis, they have been investigated in
only a few species.
Tertiary trisomics were first discovered in the genus Datura and
described at length over a number of years (Belling and Blakeslee
1926, Avery e_t al_. 1959). The other plant species for which a few
tertiary trisomics have been reported are Zea (Burnham 1930),
Nicotiana (Goodspeed and Avery 1939), Pi sum (Sutton 1939), Godetia
23

24
(Hakansson 1940), Hordeum (Ramaga 1960), Secale (Sybenga 1966) and
Lycopersicon (Khush and Rick 1967). Most of the reports are
fragmentary and in no way compare to the systematic studies carried
out in Datura and tomato. No reports of tertiary trisomics in common
bean (_2n_ + 1 = 23) have so far appeared in the literature. In
general, common bean lacks every type of aneuploid series to be used
for gene mapping, which is the main reason why few of the more than
200 genes described in the literature so far (Roberts 1982) have been
assigned to their respective linkage groups (Lamprecht 1961, Nagata
and Bassett 1984). The only previous report of aneuploids in common
bean was by Mok and Mok (1977) in which they reported two monosomies.
Tertiary trisomics appear spontaneously in the progenies of
normal disomies at an extremely low frequency. Asynaptic mutants,
triploids, and monosomies can also produce tertiary trisomics at a
very low frequency; however, translocation heterozygote progenies
produce not only the tertiaries at a comparatively higher frequency,
but also the primary trisomics (Khush 1973). With these
considerations in view, a project was started by Bassett and Hung
(1982) in which reciprocal chromosome translocations were induced by
gamma irradiation. Twelve translocation lines were established and
genetically verified. Five of the 12 translocation stocks were also
verified cytologically (Section II). Eleven of these 12
translocation stocks were used to produce trisomics in this study.
Trisomics are morphologically distinguishable in certain species
like Datura (Blakeslee 1934), L. esculentum (Khush and Rick 1967),
Capsicum annuum (Pochard 1970), Avena sativa (Azael 1973), Avena

25
strigosa (Rajhathy 1975), Potentilla argéntea (Asker 1976),
Pennisetum (Manga 1976), Oryza sativa (Khush et_ aj_. 1984) and many
other species. Morphological differences between trisomics are not
large enough to be distinguished in Clarkia (Vasek 1956, 1963),
Coll insia (Dhillon and Garber 1960, Garber 1964), Triticum (Sears
1954), Nicotiana (Clausen and Goodspeed 1924) and soybeans (Palmer
1976). In maize only two trisomics, triplo-3 and triplo-5, could be
identified morphologically. The rest could not be distinguished from
each other or from the disomies (McClintock 1929, McClintock and Hill
%
1931, Rhoades and McClintock 1935). In this experiment, it was
decided to make an intensive search for trisomics only if they were
morphologically distinguishable.
Translocation heterozygotes should be developed into homozygous
translocations so that when crossed with a standard normal line,
every seed in carries translocation heterozygosity (Burnham et al.
1954). Most of the trisomics produced by barley interchange
heterozygotes were found in the light weight seeds (Ramage and Day
1960). In desynaptic mutant lines of English pea, when only the
lighter seeds were selected and grown to search for trisomics, it
enriched the proportion of trisomics recovered (Personal
communication with Prof. Davies, John Innes Inst., Norwich).
According to these studies if the seeds produced on translocation
heterozygote plants are separated into heavier and lighter seed-
weight classes and only the lighter seeds planted, it would save a
lot of space and might yield a higher percentage of trisomic plants
in the progeny.

26
Chromosome orientation of a quadruple in M-I of a translocation
heterozygote having 4 centromeres is critical. In addition to the
four principal types of coorientation (Fig. 11-2) the quadruple can
also appear in noncoorientation (Fig. 11-IF and 111-1), where 2
centromeres on opposite sides of the quadruple are cooriented and are
positioned equidistant from the equitorial plate. The 2 intermediate
centromeres are noncooriented and are stretched out between the other
2 (Fig. 111-1), which gives these centromeres the appearance of not
being attached to the poles. In anaphase-I, the 2 cooriented
chromosomes pass to opposite poles while the noncooriented ones
either pass to the same pole (3:1 segregation) or pass to opposite
poles (2:2 segregation). In 3:1 segregation of the quadruple, the
gametes become aneuploid. After fertilization, this leads to trisomy
or monosomy (Schaeffer 1980). The rates of chromosomal
nondisjunction (3:1 segregation) differ rather widely for the
translocation stocks. The observed rates of nondisjunction are
probably affected by the orientation of the translocation complex and
the viability of the aneuploid gametes or zygotes (Khush and Rick
1967).
Any of the 4 chromosomes of a quadruple that consists of 2
primary and 2 tertiary chromosomes may participate in the
nondisjunction. Thus four different n+1 spores and gametes may be
produced by an individual heterozygous for one reciprocal
translocation. In addition to the n+1 gametes of four different
types, two types of viable n gametes are produced by the
translocation heterozygotes, i.e., one with normal chromosomes and

Fig. III-l A table giving all the types of trisomics that can result
from selfing a translocation heterozygote (modified from
Khush 1973).

29
other with translocated chromosomes. All the six types of gametes
may be functional on the female side but n+1 gametes are not
generally transmitted through pollen (Khush 1973). If n+1 gametes
are transmitted through pollen, the frequency is very low.
Therefore, when an interchange heterozygote is sel fed, three types of
disomies are produced--standard normal homozygotes, interchange
heterozygotes, and interchange homozygotes. In addition, eight
different types of trisomics may also be expected in the progeny. In
Figure 111-1 (modified from Khush 1973), trisomic types 1 to 4 result
from male gametes with a standard normal chromosome arrangement while
t+isomics 5 to 3 are fertilized by male gametes with translocated
chromosomes. The extra chromosome in trisomics 1, 2, 5 and 6 is the
tertiary chromosome; while in trisomics 3, 4, 7 and 8, it is the
primary chromosome. Trisomics 1 and 2 are homozygous for the
standard chromosome arrangement while trisomics 3, 4, 5 and 6 are
heterozygous for the interchange. Trisomics 7 and 8 are homozygous
for the translocation (Fig. 111-1).
To distinguish the tertiary trisomics 1 and 2 from 5 and 6 (Fig.
111-1) it is necessary to grow and examine their selfed progenies and
their progenies from crosses with normal disomies. Tertiary
trisomics 1 and 2 when selfed or crossed with a normal disomic,
produce disomies in the progeny with normal meiosis, whereas
trisomics in the progeny show abnormal meiosis, i.e., predominantly a
pentavalent at M-I. Tertiary trisomics 5 and 6 whether selfed or
crossed with a normal disomic, produce some disomies in the progeny
showing a multivalent of four chromosomes at M-I and others with

30
normal meiosis. As regards trisomic progeny, some of them show
predominantly a pentavalent association at M-I, whereas others show a
multivalent of 4 plus 1 univalent as well as a pentavalent.
Figure III—2 (modified from Khush and Rick 1967) shows
pentavalent configurations expected in a tertiary trisomic according
to various positions of chiasmata. Six different configurations can
appear in different situations; however, chiasma terminalization or
failure of chiasma formation may reduce the association to such
lesser configurations as 1 II + 1 III, 2 II + 1 1, 1 IV + 1 1, 1 III
+21, 1 II + 3 I or 5 I (Khush and Rick 1967). As translocation
heterozygosity does not alter the plant phenotype, trisomic 1 should
be identical to trisomic 5 and trisomic 2 to trisomic 6 (Fig.
111 -1). If the morphology of the primary trisomics is known, they
can be easily identified in segregating progeny, because the
morphology of a tertiary is suggested by a combination of the various
morphological characters of the two related primaries (Khush and Rick
1967).
Since the procedure for identifying tertiaries cytologically is
quite laborious, especially for crops like common bean, pollen
fertility analysis may be employed at a preliminary stage. Because
translocation heterozygosity produces nearly 50 percent pollen
abortion in common bean (Section II), tertiary trisomics of types 1
and 2 (Fig. 111-1) having standard normal chromosome background, when
selfed or crossed with normal disomies, yield disomies in the progeny
with normal pollen and tertiary trisomics in the progeny with the
pollen abortion rates higher than the normal plants and lower than

Fig. Ill-2 Pentavalent configurations expected in a tertiary
trisomic according to various positions of chiasmata
(modified from Khush and Rich 1967). Numbers in the
photomicrographs (x 1000) represent the type designation
in the illustrative drawing.

32

33
50 percent, assuming that trisomy causes less pollen abortion
compared with translocation heterozygosity. The progenies of
tertiary trisomics 5 and 6 (Fig. 111-1) having translocation
heterozygote chromosome background, when sel fed or crossed with
normal disomies, should segregate approximately one normal
(fertile): one translocation heterozygote in both the progeny
trisomics as well as normal disomies. Pollen abortion rates of
normal progeny disomies and trisomics with standard normal chromosome
background should be less than 50 percent in either case; whereas in
progeny disomies and trisomics with translocation heterozygote
chromosome background, it should be 50 percent and higher than 50
percent, respectively. Pollen fertility analysis is easy compared to
the cytology of PMCs but classification based on pollen fertility is
often difficult and one must be very careful in drawing conclusions
based on it because (1) most of the translocation heterozygotes
differ in pollen abortion rates (Bassett and Hung 1982); (2) in the
beginning it is not known what effect on pollen abortion the extra
chromosome has (Burnham 1962); (3) there will be an effect of genetic
background (Khush and Rick 1967); and (4) environmental fluctuations
will alter the pollen abortion rates. However, if pollen abortion
rates are supplemented with cytological examination one can get
fairly dependable results.
Selfed progenies of a trisomic normally segregate into normal
diploids and trisomics if there is no male transmission of the extra
chromosome; however, if the extra chromosome is transmitted through
the pollen, a third class of plants, i.e.,
tetrasomics, will also

34
arise among the progeny. The frequency of tetrasomics depends upon
the frequency of transmission of an extra chromosome in a male gamete
that fertilizes a female gamete already carrying the same extra
chromosome. The physical presence of tetrasomics in a trisomic
progeny may under represent the actual transmission rate of an extra
chromosome in the pollen because (1) the extra chromosome may be
eliminated during megasporogenesis; (2) the viability of n+1 gametes
may be reduced; (3) the development of 2n+2 zygotes, endosperms, and
embryos may be subnormal or abnormal; and (4) the germination of
%
tetrasomic seedlings may be poor or delayed.
Tetrasomics among the progeny of a trisomic may also arise by
another process, i.e., begin with a trisomic plant which still has a
translocation heterozygote chromosome background. After a quadruple
forms at M-I and given noncoorientation and nondisjunction of the
quadruple, various possibilities arise. Please note that we are
dealing here with a trisomic plant that already has an extra
chromosome in every cell. At anaphase-I, this extra chromosome will
go to one of the two poles. At the same time, if due to
nondisjunction of a quadruple originating due to translocation
heterozygosity of that trisomic plant, 3 of the group of 4
chromosomes also go to the same pole that has already received the
extra chromosome, two gametes arising from this embryo sac mother
cell should be tetrasomic, with the condition that the extra
chromosome due to nondisjunction must be the same as that already in
triplicate in the trisomic plant (otherwise the gametes will be
double trisomic).

35
Tetrasomics can be easily distinguished from their corresponding
trisomics if the trisomic morphology is known. In general, the
characters of trisomics are accentuated in tetrasomics (Burnham
1962).
Materials and Methods
Preparation of Seed Stocks
Eleven out of 12 translocation stocks established by Bassett and
Hung (1982) were first developed into homozygous translocations
(Section II). These homozygous translocation stocks were then
crossed with a standard normal bean breeding line and seed
collected. The Fj generation was grown to F2 seed. For each
translocation, the ?2 seed ^rom each Fl Plant was separated into
light (lower 30 percent of seed weight distribution) and heavy
(remaining 70 percent of seed weight distribution).
First Field Generation (Summer, 1984)
These weight classes were planted separately in adjacent plots
from each F^ plant. The F2 progeny plots were searched for any
plants that were phenotypically distinguishable from each other and
from normals. We did not cross-pollinate putative trisomics with the
normal disomies in the field because it is too difficult under
Florida conditions. The off-type plants were classified into
phenotypic groups in each translocation stock and seed from every
tagged plant was harvested separately.

36
First Greenhouse Generation (Fall, 1934)
The seed from four different phenotypic classes, namely, Weak
Stem, Puckered Leaf, Dark Green Leaf, and Convex Leaf was planted in
the greenhouse. Immature flower buds were collected from the
abnormal plants that were identical to the seed parent and handled
as in Section II. Descriptive notes and photographs were taken from
the putative trisomics. These plants were also crossed with pollen
from normal disomies.
Second Greenhouse Generation (Spring, 1985)
Only the F^ seed from two of the four groups, namely, Weak Stem
and Puckered Leaf from the S^ greenhouse planting, was selected for
further study in the next greenhouse generation. F^ trisomic
segregates were backcrossed with normal disomic plants in the second
greenhouse generation. These crosses to disomies were actually a
part of our scheme to isolate primaries on homozygous normal
chromosome background. It will be dealt with in Section IV. Seed
from only the abnormal segregates in each group was replanted and the
necessary data recorded.
Second Field Generation (Summer, 1985)
All the remnant seed from abnormal plants in the first field
generation and the first and second greenhouse generations was
planted in the second field generation where immature flower buds
were collected from a few of the phenotypic groups for cytological
analysis (see cytology section, page 51). Detailed descriptive notes
and photographs were taken. Abnormal plants arising in each
translocation stock were re-examined and put into specific phenotypic

37
classes. They were further classified into tertiary or primary
classes where possible, based on their morphology, genetic tests,
pollen fertility analysis, cytological evidence (see Appendix 4) and
previous literature about trisomics in other species.
Results and Discussion
Preparation of Seed Stock
F£ seed of homozygous translocation stocks crossed with a normal
bean line was divided into light and heavy classes on an individual
plant basis. On the average the individual seed weight ranged from
46 to 287 mg in the 30 percent seed weight class and from 107 to 670
mg in the 70 percent seed weight class. The wide range in each class
(note the overlap) is probably due to two main reasons: a)
individual plant differences due to environment; b) genetic
differences between different translocation heterozygote stocks with
respect to the rate of ovule abortion. In the translocation stocks
used in this study, ovule abortion rates ranged from 19 to 60 percent
(Bassett and Hung 1982 and Table 11-2). In lines with higher ovule
abortion, the number of seeds per plant is reduced whereas individual
seed weight increases. As a result, the lower and upper limits of
the seed weight distribution both change; i.e., the whole
distribution for each plant is shifted. The same type of
compensation has been observed in translocation heterozygotes of
soybean (Palmer 1976). For this reason any seed weight that is
chosen as the dividing line between heavy and light seeds to increase
the efficiency of searching for trisomics within the progenies of

38
different translocation heterozygotes is somewhat arbitrary. Out of
6600 seeds, 2100 seeds were separated into the lower (30 percent)
seed weight class, i.e., nearly one third of the total seed
harvested.
First Field Generation
All the light and heavy seeds were planted separately in
adjacent plots. Ninety-six phenotypically distinct plants (off-type)
originated among the progeny and all 96 appeared in the light weight
seed class. On the basis of these results, it can be suggested that
in common bean or closely related crops if one separates nearly 1/4
of the total seeds produced from translocation heterozygotes that are
lighter in weight, he can enrich the progenies with aneuploids with
minimal risks of loosing any distinctive trisomics (off-type), while
saving much searching labor and field space. These off-type plants
were allowed to self-pollinate. Sixty out of 96 plants set pods and
seeds. These plants were divided into 17 different phenotypic
classes which were distinguishable from each other and from normal
plants.
First Greenhouse Generation
The seed from four phenotypic classes was planted in the first
greenhouse generation (Table 111-1). In the first two groups, i.e.,
Puckered Leaf and Weak Stem, the trisomic segregates we^e easily
differentiated from normals at the seedling stage. In the third and
fourth groups, i.e., Dark Green Leaf and Convex Leaf, it was very
difficult to distinguish (in the greenhouse) between trisomic and
normal segregates in the seedling stage. Cytological proof, that the

39
four phenotypic groups mentioned above are trisomics, will be
presented later on page 51. Thirty days after planting, the plants
that looked slightly different from normals were kept. Their number
was 54 in the Dark Green Leaf group and 26 in Convex Leaf group. The
rest were discarded to save the greenhouse space. These plants were
progeny tested in the summer 1985 field planting where some of them
bred true. Thirty-nine in the Dark Green Leaf group and 17 in the
Convex Leaf group segregated into normals and aneuploids. Moreover,
under field conditions it was quite easy to differentiate between
normals and aneuploids. It is possible that the transmission rate
for these two classes given in Table III-l may be underestimated
because during classification in the greenhouse we probably discarded
some trisomic segregates along with the normal disomies, a hypothesis
supported by our segregation data for Dark Green Leaf trisomic in
Table III-2.
A few plants in the progeny of each trisomic were phenotypically
different from normal segregates as well as trisomics (Table
111-1). They flowered profusely and were sterile, although some of
them set pods that produced no seed. Some of them were weaker while
others had almost the same vigor as trisomic plants. They were not
analyzed cytologically. It is assumed that the weaker plants may
probably be the tetrasomics because the monosomies reported so far in
common bean set seed and cannot be distinguished from normal disomies
on the basis of morphological characteristics (Mok and Mok 1977).
Cytologically validated tetrasomic segregates were later observed in
the trisomic progeny tests in the field (Table 111-2). As regards

Table III-l
Transmission of
1984).
the extra chromosome
in four trisomics of common
bean (First greenhouse planting, Fall,
Translocation
stock
Seed parent
phenotype
Number
of seeds
planted
Number of
trisomics
scored
Percent
transmission
on selfing
Other remarks
11-70
Puckered Leaf
100
26
26
One sterile plant in
the progeny
1-97
Weak Stem
19
6
32
One sterile plant
1-52
Dark Green Leaf
125
39
31a
Five sterile plants
Convex Leaf
50
17
34a
Two sterile plants
a
The transmission rate of Dark Green Leaf and Convex Leaf is based on the progeny test of these classes
in the second field generation. For details, please see text.
4^
O

Table 111-2
Progeny tests of two tertiary trisomics originally derived from translocation heterozygote 1-52 (Second
field planting, Summer, 1985).
Progenyy
Number of
plants grown
Tertiary trisomic
Number of
aneuploids
in the
Percent
Code
(2n+l) self
parent phenotype
Classes of progeny
progeny
transmission
a
1758
Dark Green Leaf2
Dark Green Leaf (Primary)
718
41
Chlorotic Leaf (Primary, unrelated)
23
1
b
211
Dark Green Leaf
Convex Leaf (Primary)
64
30
Chlorotic Leaf (Primary, unrelated)
3
1
Convex Leaf, tetrasomics
3
1
c
523
Dark Green Leaf
Dark Green Leaf (Tertiary with rare
primary)
209
40
Dark Green Leaf, tetrasomics
7
1
d
1032
Dark Green Leaf
Dark Green Leaf (Tertiary with rare
primary)
365
35
Convex Leaf (Primary)
53
5
Chlorotic Leaf (Primary, unrelated)
22
2
Convex Leaf, tetrasomics
16
2
Dark Green Leaf, tetrasomics
10
1
e
1530
Convex Leaf
Convex Leaf (Tertiary)
428
28
Chlorotic Leaf (Primary, unrelated)
9
1
Convex Leaf, tetrasomics
94
6
f
434
Convex Leaf
Convex Leaf (Tertiary)
87
20
Dark Green Leaf (Primary)
27
6
Chlorotic Leaf (Primary, unrelated)
6
1
Convex Leaf, tetrasomics
26
6

Table III-2--continued.
Progenyy
Code
Number of
plants grown
[(2n+l) x 2n]
cross
Tertiary trisomic
parent phenotype
Classes of progeny
Number of
aneuploids
i n the
progeny
Percent
transmission
9
266
Dark Green Leaf2
Dark Green Leaf (Primary)
73
32
h
67
Dark Green Leaf
Dark Green Leaf (Tertiary and Primary) 15
22
Convex Leaf (Primary)
11
16
Chlorotic Leaf (Primary, unrelated)
2
3
i
101
Convex Leaf
Convex Leaf (Tertiary)
24
24
Chlorotic Leaf (Primary, unrelated)
3
3
Convex Leaf, tetrasomics
2
2
j
25
Convex Leaf
Convex Leaf (Tertiary)
4
16
Dark Green Leaf (Primary)
7
28
Chlorotic Leaf (Primary, unrelated)
3
12
y Each code refers to a group of S2 trisomic plant progenies (Appendix 5) derived from trisomic plant
progenies in the first greenhouse planting, Fall, 1984. The members of each group of progenies had
similar segregation patterns.
z Two Extreme Lanceolate Leaf (ELL) type plants originated in Dark Green Leaf trisomic progenies, one ELL
plant in progeny a_ and one in progeny c¡_. Both plants were sterile.
4^
no

43
the sterile segregates having the same vegetative vigor as trisomic
plants, these were two in number and emerged in the progeny
classified as Dark Green Leaf. The plant phenotype was Chlorotic
Leaf, which will be described in Section IY, under primary trisomics.
The inability to distinguish between Dark Green Leaf, Convex
Leaf and normal disomic segregates in the first greenhouse planting
may be attributed to the quality and quantity of light which plants
receive in offseason planting in the greenhouse. It is not unusual
to observe that chlorophyll production is often decreased by short
days (Salisbury and Ross 1978). As regards the quality of light, the
red and blue regions of the visible spectrum are mostly absorbed by
the chlorophylls. These two regions are the most effective
wavelengths in inducing chloroplast division and most efficient in
driving photosynthetic reactions (Salisbury and Ross 1978). If at
least one of these two regions has its transmission reduced while
passing through the glass, it may affect the normal development of
chlorophylls in these trisomic classes, because they develop dark
green leaves under field light conditions. It is probably the blue
region of visible spectrum of light which is affected while passing
through glass (personal communication with Prof. R.H. Biggs, Fruit
Crops Department, University of Florida, Gainesville). In future, it
has been planned to plant these two trisomics a bit early in the
fall, keeping all the plant pots outdoors for one month. After
differentiation is possible between normal disomic and trisomic
seedlings, the trisomic plants can be transported into the greenhouse
for the winter.

44
Immature flower buds of the plants identical to the seed parent
(putative trisomic) from each class were collected separately. Their
meiotic analysis has been discussed under cytology. The trisomic
plants emerging in all the four classes of plants (Table 111-1) were
allowed to self-pollinate and also crossed with the pollen from
normal disomies.
Second Greenhouse Generation
Only seed from two trisomic groups, Puckered Leaf and Weak
Stem, was planted in the second greenhouse generation where trisomic
segregates were backcrossed to the normal disomies. It was actually
a part of our plan to isolate primary trisomics from tertiaries,
which is dealt with in detail in the next section.
Second Field Generation
The remnant seed from all the trisomics of two greenhouse
plantings and from all the off-type plants in the first field
planting were replanted in the second field generation during the
summer of 1985. Translocation heterozygote 1-52 was the only stock
yielding all the four expected types of trisomics, i.e., two
primaries and two tertiaries.
In tomato, Khush and Rick (1967) obtained tertiary trisomics
from four translocation stocks. None of the four stocks yielded all
four expected trisomic categories. T5-7 and T7-11 each produced one
tertiary and two related primaries whereas T9-12 yielded both
tertiaries but one related primary. Tl-11 yielded just one tertiary
trisomic. Along with all types of trisomics, translocation
heterozygote stock 1-52 also produced tetrasomics of different types

45
among each trisomic progeny (Table III-2). They were all
phenotypically distinguishable from each other and from normal
disomies as well as trisomics. In fact, trisomic phenotype ws more
accentuated in tetrasomics. Translocation heterozygote stock 1-97
yielded two phenotypic groups, i.e., Weak Stem (Table III-3) and
Pinched Leaflet Tip (Table III-4). Weak Stem has been verified
cytologically as a trisomic whereas Pinched Leaflet Tip lacks
cytological confirmation. However, genetic tests give an indication
of trisomy. The primary trisomic for Weak Stem has been isolated
from the tertiary Weak Stem by cytological examination (Section IV)
because the primary is not morphologically distinct from the
tertiary.
Discussing the overall segregation data in Table III-2, 16 off-
type plants falling into two phenotypic groups, Dark Green Leaf and
Convex Leaf, were tagged in F2 progeny of the translocation
heterozygote 1-52 in the first field generation. Sel fed progenies
from 8 out of these 16 plants, 6 in Dark Green Leaf and 2 in Convex
Leaf, were tested in the first greenhouse generation (Table 111-1).
On the basis of information we recorded from the second field
generation (Table 111-2), we hypothesize that all eight plants we
started with were tertiary trisomics of two types, i.e., Dark Green
Leaf tertiary and Convex Leaf tertiary. They segregated into
tertiaries and related primaries in the first greenhouse
generation. If the Dark Green Leaf trisomic progenies are examined
(Table III-2), it seems that some of the plants segregated into both

Table 111-3
Segregation for trisomic progeny from selfed (2n+l) or cross-pollinated [(2n+l) x 2n] trisomics originally
derived from four different translocation heterozygote progenies (Second field planting, Summer, 1985).
Translocation
stock
Trisomic
phenotype
Number of plants grown
2n+l self (2n+l) x 2n
Number of
trisomic
progeny
Progeny
phenotype
Percent
transmiss
11-70
Puckered Leaf3
5567
2025
Puckered Leaf
36
528
201
Puckered Leaf
38
1-99
Puckered Leaf
147
57
Puckered Leaf
39
37
Chlorotic Leaf*3
0.6
1-97
Weak Stem0
2373
851
Weak Stem
36
85
35
Weak Stem
41
11-121
Weak Stem
4
Seeds did
not germinate
a Morphological
distinction exists
between the primary and
tertiary forms
of Puckered Leaf.
h Chlorotic Leaf is a primary trisomic (relationship undetermined) that originated in all the above
progenies.
c
No morphological distinction exists between the primary and tertiary forms of Weak Stem.
-P*

Putative trisomics derived from different translocation heterozygote progenies; their phenotype and rate of
transmission of extra chromosome in (2n+l) self (Second field planting, Summer, 1985).
Translocation
stock
Trisomica
phenotype
Plants
grown
Trisomics
scored in
the progeny
Progeny
trisomic
phenotype
Percent
transmission
Other
remarks
I -85b
Weak Plant
13
5
Weak Plant
38
No pod or seed
set
1-97
Pinched Leaflet Tip
21
4
3
True to seed parent
Phenotype more
accentuated
19
14
May be tetra -
somic. No
pod or seed
set
11-6
Small Leaf
32
7
True to seed parent
22
11-14
Epinastic Leaf
167
26
8
True to seed parent
Phenotype more
accentuated
16
5
No pod or seed
set. May be
tetrasomic
11-109
Leaf Margins Curl Down
26
7
True to seed parent
27
11-121
Leaf Tip Curl
6
3
True to seed parent
50
a These phenotypic classes have not yet been verified cytologically as trisomics and that is why they are
called putative trisomics.
b One plant of Extreme Lance type originated in the light weight seed class among translocation
heterozygote 1-85 progeny. Another plant of the same type appeared among Weak Plant putative trisomic
progeny. Both of them were sterile.

48
related primaries in and produced uniform primary trisomics in S2
(Table 111-2, a and b). They also produced identical tertiaries in
their segregating progenies in S^. These tertiaries either produced
identical tertiaries and one related primary in S2 (Table III—2, c),
or they produced identical tertiaries along with both the related
primaries in S2 (Table 111-2, d). A similar explanation seems true
for Convex Leaf tertiary trisomic. The only difference is that the
Convex Leaf did not segregate for either primary in S^; but in S2, it
segretaed for one related primary trisomic, i.e., Dark Green Leaf
(Table III-2, f). Because the phenotype was more difficult to
differentiate in the greenhouse, these could not be identified in S^.
One of the unique features of the breeding behavior by which
tertiary trisomics differ from other types of trisomics is the
regular appearance of the two related primary trisomics in their
progenies (Khush and Rick 1967) which is illustrated by the
segregating progenies of Dark Green Leaf and Convex Leaf tertiary
trisomics (Table 111-2). Unrelated primary trisomics also appear at
low frequencies in some of the trisomic progenies (Khush and Rick
1967), which is illustrated by Chlorotic Leaf segregates appearing at
a low frequency throughout the trisomic progenies in Table III-2.
Extreme Lance, another morphologically distinct type, also appeared
in two plant progenies of Dark Green Leaf trisomic (footnote, Table
III-2). It also appeared in F2 progenies of translocation
heterozygote 1-85, and among S2 progeny of Weak Plant, a putative
trisomic (footnote, Table III-4). By the same analogy, Extreme Lance
type may be another unrelated trisomic, it could not be analyzed

49
cytologically. All the plants of this phenotype that have appeared
so far have failed to reproduce; i.e., flower buds have had arrested
development at a rudimentary stage.
Translocation stock 11-121 has also produced the Weak Stem
trisomic as did 1-97 (Table III-3), indicating that both these
translocation stocks involve a common chromosome in the
interchanges. A triple dose of this chromosome gives rise to the
Weak Stem phenotype. The presence of a common chromosome has already
been confirmed cytologically. When translocation stock 11-121 was
crossed with 1-97, cytology showed a chain of 6 chromosomes and 8
bivalents (Table 11-1). Two other translocation stocks, 1-99 and
11-70, segregated for the Puckered Leaf trisomic, indicating that
these two stocks also have one common chromosome involved in their
interchanges. In a triple dose, this chromosome could be responsible
for alteration of the normal phenotype to that of Puckered Leaf.
Cytogenetic analysis of translocation stocks (Section II) did not
include 1-99 and no direct cytological test was possible. However,
the appearance of the same trisomic group in two translocation
heterozygote stocks is substantial proof that these two stocks have a
common chromosome involved in their interchanges.
Trisomic suspects (Table III-4) have not been examined
cytologically, however, their genetic segregation data and the fact
that they all appeared among the light weight seed classes of
translocation heterozygotes suggests that they may be trisomics.
They have been discussed briefly under morphology.

50
Cytology
During the preliminary cytological analysis in the first
greenhouse generation to see whether the abnormal segregates were
trisomics or not, 25 PMCs were analyzed which clearly show 11
bivalents and one univalent chromosome at diakinesis or M-I (Fig.
III-3A), 23 chromosomes at early anaphase-I (Fig. III-3B), or 11 V/S
12 chromosomes at late anaphase-I (Fig. Ill-3D) in all the four
phenotypic classes (Table 111-1). During this search, certain
anaphase-I cells with 11 chromosomes at each pole and with one
laggard still in the middle were also observed at a very low
frequency (Fig. 111 -3 C). Buds from all the off-type plants falling
in one group were collectively picked and preserved. As the
phenotype of Dark Green Leaf and Convex Leaf was not very distinctive
in the first greenhouse planting, they were re-examined in the second
field generation. Some of the plants bred true and there was not
even a single off-type plant in their progeny, giving an indication
that they were normal disomic plant progenies and that it was not
possible to discard them in the greenhouse screening. The numbers of
true breeding parent plants were 15 in the Dark Green Leaf group and
9 in the Convex Leaf group. Dark Green Leaf produced the following
plant progeny classes: an extremely uniform class (a) of Dark Green
Leaf plants along with normal disomies (Table III-2, a); a uniform
class (b) of Convex Leaf trisomics along with Convex Leaf tetrasomics
and normal disomies (Table III—2, b); class (c) of Dark Green Leaf
trisomics whose phenotypes were a mixture of characteristies of
classes (a) and (b) with characters of class (a) predominant

Fig. Ill-3 Meiotic diplotene to anaphase-I as observed in tertiary
trisomics of common bean (x 1000).
A. A PMC at diakinesis showing 11 bivalents and a
univalent (arrow).
B. Early anaphase-I showing 23 univalents.
C. Anaphase-I showing 11 univalents on each pole with
one laggard (arrow) in the middle.
D. Late anaphase-I showing 11 univalents on 1 pole and
12 on the other (right).
E. Diplotene showing a pentavalent association (arrow)
along with 9 bivalents.
F. Metaphase-I showing a pentavalent (arrow) and 9
bivalents.
G. Diakinesis showing a trivalent (arrow) and 10
bivalents.

52

53
(Table III-2, c) along with disomies, rare tetrasomics and segregates
that were phenotypically identical to class (a); and a fourth class
(d) that produced all the plant types in (a) through (c) detailed
above (Table III-2, d) along with their tetrasomics and normal
disomies. When one observes a mixture of the phenotypic
characteristics from the two related primaries in a tertiary trisomic
in which the characteristics of one primary predominate, it is
interesting to speculate that the longer of the two segments
composing the extra chromosome in the tertiary is homologous with the
triplicated chromosome in the related primary trisomic having closer
phenotypic resemblance (Khush and Rick 1967).
The cytology of Dark Green Leaf in class (a) and Convex Leaf in
class (b) gave an indication that they were primary trisomics
(Section IV). It becomes fairly easy to identify tertiary trisomics
if the morphology of the two related primaries is known because the
nature of tertiaries is suggested by a combination of various
morphological characteristics of the two related primaries (Khush and
Rick 1967). Keeping this fact in view, the Dark Green Leaf
segregates in class (c) having the combination of characteristics of
both related primaries with dominant characteristies of Dark Green
Leaf were classified as Dark Green Leaf tertiary. Similarly, Convex
Leaf trisomic plant progenies produced a class (a) of Convex Leaf
trisomic with a quite uniform phenotype within the group but
different from the Convex Leaf primary trisomic which originated in
Dark Green Leaf progenies. Having the combined characteristics of
both related primaries and having the dominant morphological

54
similarities of the Convex Leaf primary, it was named Convex Leaf
tertiary trisomic (Table III-2, e). Another class (b) originating
among Convex Leaf trisomic progenies was the Dark Green Leaf primary
trisomic along with the normal disomies (Table III-2, f).
Puckered Leaf trisomic was fairly uniform in the beginning. In
the second field planting when primary trisomic plants were
identified cytologically (Section IV), it was easier to identify a
morphologically different class within Puckered Leaf trisomic plant
progenies which were thought to be tertiaries. It is appropriate to
emphasize that all the three tertiary trisomic groups, i.e., Dark
Green leaf, Convex leaf and Puckered Leaf have been classified as
tertiaries only on the basis of their morphological relationship with
their related primaries and by the fact that they continue producing
related primaries in their progenies, whereas primaries segregate
almost exclusively for the same primary. We have no cytological
proof that they are tertiaries. This is because common bean cytology
is fairly difficult, confusing and time consuming due to the small
size of chromosomes. The Weak Stem trisomic class was the only one
in which tertiaries were isolated from primaries cytologically. This
was because no morphological distinction existed between primary and
tertiary in this trisomic class. Four plant progenies were examined
cytologically (Section IV). Three of these, i.e., GK #22, 23, 25,
were classified as tertiary. Our results are based on 50 analyzable
PMCs from each progeny at diplotene, diakinesis, or M-I of meiosis
(Table IV-2). On an average 21 PMCs showed a pentavalent association
(Fig. 111-2) with the rest of the chromosomes as bivalents, 20 showed

55
a trivalent configuration (Fig. III-3G), and 9 PMCs appeared having
11 bivalents and 1 univalent (Fig. III-3A). Certain pentavalent
configurations as illustrated in Figure 111-2, helped to reach the
conclusion that they are tertiary trisomics. Otherwise, a primary
trisomic still having a translocation heterozygote background can
form pentavalent associations.
Immature flower buds from Convex Leaf tetrasomic plants were
collected and analyzed cytologically. PMCs showed 11 bivalents and 2
univalents at diakinesis (Fig. III-4A), 24 chromosomes at early
anaphase-1 (Fig. III-4B) and 12 Y/S 12 at two poles at late
anaphase-1 (Fig. III-4C) of meiosis. All other tetrasomics were
identified from their phenotypic relationship with the seed parent
phenotype and were not analyzed cytologically. None of the
tetrasomic plants from any class set seed.
Morphology of Known Tertiary Trisomics
The following description of the Puckered Leaf trisomic is based
on observation from one generation, while Dark Green leaf and Convex
Leaf trisomic descriptions are based on two generations, and trisomic
suspects and tetrasomics are based on one generation only. Weak Stem
trisomic morphology is described in Section IV because of the
nonexistence of morphological distinction between tertiary and
primary in this class. For each of the tertiary trisomics listed
below, the name given to it is derived from its "related" primary
trisomic because of the strong resemblance, e.g., the tertiary and
primary Puckered Leaf trisomics are quite similar in appearance;
hence the same phenotype-derived name is used for both.

Fig. Ill-4 Meiotic diakinesis to anaphase-I as observed in a
tetrasomic of common bean (x 1000).
A. A PMC at diakinesis showing 11 bivalents and 2
univalents that overlap (arrow).
B. Early anaphase showing 24 univalents.
C. Late anaphase showing 12 univalents at each pole.
The arrow points to 2 chromosomes lying close to each
other.

57
«

53
Puckered Leaf. This trisomic was closely examined in three
sel fed and two crossed (with 2n) generations for isolation of
primaries. It was only in the last generation in which the plants
having these morphological characteristics (Fig. III-5C) were marked
as tertiaries because the phenotype of the related primary was
established cytologically (Section IV). Puckered Leaf tertiary
trisomic is different from normal as well as Puckered Leaf primary.
The terminal leaflet of the trifoliate leaf of the tertiary is
semilanceolate. Leaf petioles form a wider angle with the stem
resulting in a wider plant canopy. Leaves are less puckered than
primary, and the stem and pods have more anthocyanin pigment in
them. Leaves do not have a glossy appearance on attaining full size
like the primary and they are slightly darker green in color compared
to normal or primary. Pod and seed set is better under field
conditions compared to the primary but less than disomic
segregates. Many missing seeds can be seen in pods. It is slightly
later maturing than the normal disomic and related primary trisomic.
Dark Green Leaf. Plants are smaller than the related primary
and have more recessed veins and smaller leaves. The leaves look
darker green than the related primary. They set equally well under
greenhouse and field conditions. Pod, seed shape and all other
characteristics are similar to those in the related primary. It is
easy to classify in the field about 20 days after planting whereas
classification in the greenhouse is difficult and subject to serious
levels of error.

Fig. 111-5 Photographs of tertiary trisomic and tetrasomic plants.
A. A normal common bean plant, 30 days after planting
B^. Convex Leaf tertiary trisomic, 30 days after
planting.
$2' Convex Leaf tetrasomic, 30 days after planting.
C. Puckered Leaf tertiary trisomic, 70 days after
planting.
0. Dark Green Leaf tetrasomic, 45 days after planting

60

61
Convex Leaf. Plants of this trisomic can be distinguished from
disomies at the seedling stage, but there are chances of error.
However, at 25 days after planting, the leaves of the tertiary
trisomic start forming a convex shape with recessed veins, and the
plants can be identified for classification 30 days after planting
(Fig. Ill —5B ]_). In this respect it differs from the related primary,
which can be identified easily at 20 days of age. Leaves of the
tertiary are darker green with comparatively less recessed veins and
are less convex than the primary. Plants of this trisomic have
shorter internodes than disomies or related primary trisomic and
remain bushy, but they can grow as tall as diploid sibs and still
remain compact in appearance. Pod and seed set is equally good in
either greenhouse or field conditions. This class is also difficult
to classify in the greenhouse but not so in the field. Pods and
seeds are smaller than in disomies but similar to the related
primary.
Convex Leaf tetrasomic. Tertiary tetrasomic plants in this
class are extremely dwarfed with very short internodes, highly convex
leaves, highly recessed veins and dark green leaf color (Fig.
Ill-5B2)• They have very small buds, flower profusely, but rarely
set any pods or seed. They differ from the primary tetrasomic, which
has a lighter green leaf color and comparatively taller plants. All
other morphological characteristies are similar.
Dark Green Leaf tetrasomics. Tetrasomic plants in this class
look like trisomic parents at the seedling stage. Only after one
month of vegetative growth when trisomics attain considerable size,

62
the tetrasomics appear comparatively stunted. Also, the leaves show
more recessed veins, darker green color and smaller size compared to
the trisomics (Fig. Ill-5D). The flower color of tetrasomics is more
intense than trisomics, and they flower profusely but do not set any
pods. Tetrasomic plants of this class grow taller compared to Convex
Leaf tetrasomics.
Morphology of Trisomic Suspects
Weak Plant. The plants are short in stature, and the leaves are
pale green and slightly smaller than normal (Fig. III-6F). This
trisomic flowers profusely but sometimes without pod and seed set.
The apparent sterility in the 1985 season may probably be due to very
hot weather conditions (max. temps, at 100°F) in the field. The
original plants of Weak Plant were able to set seed in the field
during the milder summer of 1984.
Pinched Leaflet Tip. Progenies of the putative trisomic
segregated for two types of plants besides the normal disomies. The
first type was extremely abnormal and had round leaves. In these
plants there was single broad leaf instead of a trifoliate leaf. The
plants were vegetatively vigorous and tall and they flowered
profusely and set pods with aborted ovules. The second type of
plants was intermediate in phenotype between disomies and the first
type. They produced medium-sized leaves with a round tip (Fig.
III-6C). The terminal leaflet sometimes had a point in early stages,
but the tip was blunt or rounded at full maturity of the leaf. The
plants had small pods with very small seeds.

Fig. Ill-6 Plant photographs of suspected trisomics (genetic
evidence only).
Aj. Leaf Margins Curl Down, 30 days after planting.
A£. A normal common bean plant, 30 days after planting.
B. Leaf Tip Curl Down, 70 days after planting.
C. Pinched Leaflet Tip, 30 days after planting.
D. Extreme Lanceolate Leaf, 30 days after planting.
E. Small Leaf, 30 days after planting.
F. Weak plant, 30 days after planting.
G. Epinastic Leaf, 30 days after planting.

V®
64

65
Small Leaf. Plants of this putative trisomic have smaller
leaves and weaker stems compared to disomies. They also have weaker
leaf petioles and a wider plant canopy (Fig. III-6E). The leaves are
normal in color. Trisomic segregates can be distinguished 25 days
after planting. Pods are very long and look like normal. Seeds are
big and flat. Pod and seed set is late compared to disomies.
Epinastic Leaf. This class also segregated into three classes
of plants: normal disomies (a); very weak, small plants (b) with
epinastic leaves and without pods and seed set; and a third plant
phenotype falling between (a) and (b). The leaf epinasty of this
third plant class looks like mosaic virus effects and the plants are
of medium height (Fig. Ill-6G) and set medium-sized pods with some
missing seeds at random positions.
Leaf Margins Curl Down. The leaves are smaller than in disomies
and their margins curl down. Plants are slow growing, smaller than
normal segregates, and can be distinguished 40 days after planting
(Fig. Ill-6A^). Plants grow spindly branches, have anthocyanin
pigment in their branches, are late maturing, and set medium-sized
pods with missing seeds at random positions.
Leaf Tip Curl. Plants are slow growing and weaker than
disomies. Leaf tips curl down. The branches and pods have more
anthocyanin pigment in them than disomies. The pods are
morphologically similar to Puckered Leaf tertiary trisomic pods.
There are many missing seeds in the pods. It is a late blooming and
late maturing class. Plants can be distinguished from normals 30 to
40 days after planting (Fig. III-6B).

66
Extreme Lanceolate Leaf. Plants are small and
tall. The internodes are very short and the leaves
lanceolate in shape (Fig. III-6D). The plant forms
and sets no pods.
only grow 6 in.
are extremely
rudimentary buds

SECTION IV
ORIGIN, CYTOLOGY, MORPHOLOGY AND TRANSMISSION OF THE
EXTRA CHROMOSOME IN FIVE PRIMARY TRISOMICS OF COMMON BEAN
(PHASEOLUS VULGARIS L.)
Introduction
A primary trisomic is one having its extra chromosome as a
normal chromosome (not translocated). Since the trisóme modifies
genetic ratios for the genes located on that chromosome, they offer
one of the most efficient methods of associating genes with their
linkage groups. Efforts have been made during the past three decades
to produce the trisomic series in an increasing number of crops.
This is because the most extensive genetic studies done so far with
the aid of aneuploids have been conducted with trisomics (Schaeffer
1980).
The discovery of the Globe trisomic of Datura stramonium, the
first primary trisomic, was the beginning of the pioneer investiga¬
tions of trisomics by Blakeslee and Avery (1919). Maize (McClintock
1929), tobacco (Goodspeed and Avery 1939), wheat (Sears 1939), tomato
(Rick and Barton 1954), Depper (Pochard 1970), Dearl millet (Gill et_
al. 1970), jute (Das and Iyer 1972), sorghum (Schertz 1974), and rice
(Khush e_t al_. 1984) are some of the crops in which primary trisomics
have been developed and are being used for genetic studies.
Trisomics are named after the type or morphology of the extra
chromosome. The types of trisomics are primary, secondary,
67

68
tertiary, telo, and compensating (Khush 1973). Out of these five
types, primary and tertiary trisomics have been used most extensively
for linkage studies. Tertiary trisomics are among the most useful
cytogenetic variants because they can be used to determine the arm
location of marker genes, position of centromeres, and orientation of
linkage maps. These special applications of tertiaries are only
possible if the gene in question is first assigned to a particular
linkage group by use of the primary trisomic series and the pachytene
chromosome analysis of the particular species is possible. Pachytene
chromosome analysis helps in physically identifying the two
nonhomologous chromosomes involved in an interchange and the exact
position of breakpoints. Pachytene analysis has been successfully
used in maize (Rhoades and McClintock 1935) and tomato (Rick and
Barton 1954), but has not yet been successful in locating the exact
position of a translocation breakpoint in common bean. There has
been a paucity of techniques reported (Cheng and Bassett 1981; Mok
and Mok 1976) that will permit the identification of the extra
chromosome in a primary trisomic.
Primary trisomics have appeared spontaneously among the progeny
of normal diploids of many species, but are obtained more frequently
from asynaptic and desynaptic mutants, from the progeny of polyploids
(most notably triploids), and from the progeny of translocation
heterozygotes (Khush 1973). Among the different sources of primary
trisomics mentioned above, triploids are the best and most dependable
source (Khush 1973). The most common route to get triploids is the
use of colchicine to produce tetraploids, which are then crossed with

69
diploids to get triploid seed. Triploid plants, when selfed or
backcrossed, usually produce many types of aneuploids, but primary
trisomics are produced at a high frequency.
Producing trisomics from the progenies of 3n x 2n crosses is
difficult in common bean as the study of Braak and Kooistra (1975)
shows. Amphidiploids were first developed by crossing Phaseolus
vulgaris with Phaseolus ritensis. Obtaining plants was only
possible by artificial embryo culture. Amphidiploids were obtained
by doubling the chromosomes of the interspecific hybrid. These
amphidiploids were then backcrossed with (2n) Phaseolus vulgaris.
Out of 25,000 pollinations, 8 shrivelled seeds were obtained. With
the help of embryo culture, the seedlings were raised. Seven of the
eight seedlings died and one was grown to maturity. It proved to be
a triploid. Vegetative propagation was the only way to propagate
this plant. There was no success when the triploid was backcrossed
with Phaseolus vulgaris. Out of 1600 pollinations with Phaseolus
riten sis, the other parent, only one vigorous plant was obtained, a
trisomic 2n_ + 1 = 23 chromosomes. progeny of this trisomic
produced a total of 159 plants, including a small number of
semisterile plants. These semisterile plants proved to be
trisomics. All of the progeny were very similar to the Phaseolus
vulgaris parent. S2 and S3 progenies of trisomic segregates were
grown. The article did not give any further details, but indicates
that additional information about the trisomic progeny would be
published in a separate article in the near future. Such a report
has not appeared. Wo spontaneous occurrence of trisomics in disomic

70
progenies or information regarding the presence of asynaptic or
desynaptic mutants has been reported in common bean. Keeping these
facts in view, it was decided to follow another route, i.e.,
aneuploid segregates due to nondisjunction appearing in the progenies
of translocation heterozygotes.
A translocation heterozygote upon selfing produces eight types
of trisomics. These eight types will fall into four different
phenotypic classes, provided that each chromosome (including its
translocated segment) has some effect on the morphology of the
trisomic plant that carries it as the extra chromosome (Fig. III-l
and Appendix 4). Four out of these eight trisomic types are primary
and four tertiary. The four primary trisomic types do not have a
normal chromosome background. Two out of these four are on
translocation heterozygote background and the other two are on
homozygous translocation background (Appendix 4). To get primaries
on a homozygous normal chromosome background, trisomic plants are
backcrossed with normal (2n) pollen. The primaries with homozygous
translocation background should yield F^ progeny (disomies as well as
trisomics) all having semisterile pollen, whereas tertiaries with
homozygous normal chromosome background yield F^ progeny all having
fertile pollen (Appendix 4). Primaries and tertiaries with
heterozygous translocation background, after being crossed with
normals, should yield a progeny segregating for either fertile or
semisterile plants. So these two classes are not distinguishable
solely on the basis of pollen fertility analysis (Appendix 4). As
already stated in Section III, common bean cytology is quite

71
confusing and time consuming probably due to the small size of the
chromosomes. It was employed only whenever it was necessary to make
proofs or distinctions.
The semisterile primary trisomic plants originating in the Fj
progeny of a primary trisomic (with homozygous translocation
background) crossed with a normal disomic, if backcrossed with the
normal disomies, can be converted to a homozygous normal primary
trisomic. In BC^ trisomic progeny, only fertile trisomic plants
are selected (personal communication with G.S. Khush, IRRI,
Philippines). Fertile trisomic plants in BCjF^ progeny can then be
verified cytologically. If a high percentage of PMCs show a
trivalent association with all other chromosomes appearing as
bivalents, the plants in question are primary trisomic with a
homozygous normal chromosome background (Khush 1973). This procedure
avoids a lot of cytological analysis during preliminary stages.
In case one is unable to recover any primary trisomics from the
progeny of a translocation heterozygote, the best alternative is to
grow a few selfed progenies of a tertiary trisóme and in each progeny
look for any deviates from the tertiary trisomic phenotype. One of
the unique features of the breeding behavior by which tertiary
trisomics differ from other types of trisomics is the regular
appearance of the two related primaries in their progenies (Khush and
Rick 1967).
Tertiary trisomics usually show pentavalent configurations at
M-I of meiosis, depending upon the chiasmata. If there is a
trivalent having a pair of one chromosome and an extra translocated

72
chromosome, two types of n+1 gametes can result from the segregation
of such a trivalent. As an example, alternate disjunction from the
1-2, 1-2, 1-3 trivalent can produce n+1 gametes in which the tertiary
1-3 is the extra chromosome. On the other hand, adjacent disjunction
could yield the primary with 1-2 as the extra chromosome. In the
same fashion, the tertiary trisóme may form a trivalent with 3-4,
3-4, 1-3 chromosomes, adjacent disjunction from which could yield 3-4
as the extra, which means the other related primary. Adjacent
segregation from a pentavalent could give similar results. Thus, a
tertiary trisomic might regularly produce three kinds of n+1 gametes
and 2n+l zygotes, one of the latter being the parental tertiary
trisomic and the other two being the related primary trisomics. The
frequency of related primaries yielded must depend, however, upon the
frequency of different types of associations formed at M-I, the types
of disjunction at anaphase-I, and the viability of the trisomic with
the extra primary chromosome. The frequency of two related primary
trisomics might also differ, depending upon the relative length of a
tertiary chromosome (Khush and Rick 1967). As an example, if arm 1
of the tertiary chromosome 1-3 is longer than arm 3, it will
preferentially pair with the homologue pair 1-2, 1-2 rather than 3-4,
3-4, because the greater length of the arm 1 will provide greater
opportunity to pair and form chiasmata. The adjacent disjunction
from such a trivalent will accordingly yield a primary trisóme for
chromosome 1-2 at a higher frequency than a primary trisóme for
chromosome 3-4 (Khush and Rick 1967).

73
Transmission rates of trisomics on crossing with 2n plants or on
crossing with marker stocks during linkage studies have been reported
to be higher than rates observed after selfing. This has been
attributed to the genetic heterozygosity that is created by crossing
(Khush and Rick 1967).
Materials and Methods
Sx seed from four phenotypic classes of off-type plants
identified from translocation heterozygote progenies grown in the
first field generation was planted in the first greenhouse generation
(Table 111-1). Convex Leaf and Dark Green Leaf classes were allowed
to self-pollinate, and some of the plants picked at random from both
of these classes were also crossed with 2n. All the off-type plants
in Puckered Leaf and Weak Stem were searched for male-fertile
trisomic plants and, wherever found, were crossed with pollen from 2n
plants. If male-fertile plants with the trisomic phenotype were not
present in the progeny, semisterile trisomic plants were selected for
crossing with 2n pollen. seed from these two phenotypic classes
was grown in the second greenhouse generation and all the
progenies of male-fertile trisomic parents (including trisomic as
well as disomic segregates) were tested for pollen fertility. The
progenies having all semisterile plants were selected and trisomic
segregates were backcrossed with 2n pollen from a normal disomic. On
the other hand, the Fj^ progenies derived by crossing the semisterile
trisomic plants with 2n pollen in the first greenhouse generation
were searched for male-fertile trisomic plants, which were tagged and

74
allowed to self-poll inate. F2 seed from these plants, BC]^ seed
from the other semisterile trisomic groups, and S2 and Fj_ seed from
Dark Green Leaf and Convex Leaf from the first greenhouse generation
were all planted in the second field generation. Male-fertile
trisomic plants were identified in BC^F^ progenies and immature
flower buds were collected from them. Similarly, flower buds were
also collected from F2 trisomic segregates of Weak Stem (see Appendix
4). From S2 progenies of Dark Green Leaf and Convex Leaf, trisomic
plant progenies showing morphological dissimilarities between them
(segregates for disomies and trisomics) but uniformity within the
class (segregates for only one type of trisomic) with the least
number of off-type segregates, were selected for meiotic analysis.
Another phenotypic class originating among most of the trisomic
progenies at a very low frequency, was analyzed cytologically.
Plant, pod and seed photographs were made wherever possible, and
other descriptive notes for all five groups were recorded in the
field along with the transmission and ovule abortion data.
Results and Discussion
Because the method of isolation of these primary trisomics
differs slightly from one to the other (with one exception that is
explained under one heading), they are treated under separate
headings.
Puckered Leaf Trisomic
Out of 100 seeds of Puckered Leaf, planted in the first
greenhouse generation, 26 plants were identified as phenotypically

75
identical to the seed parent. Five of these 26 plants were
identified as male-fertile on the basis of pollen abortion rates. In
these five male-fertile trisomics the pollen abortion rates ranged
from 20 to 36 percent. This gave an indication that these
male-fertile trisomic plants can either be tertiary or primary, both
either on homozygous normal or homozygous translocation background.
When the off-type plants originated in the field among the
selfed progeny of a translocation heterozygote, the off-type plants
in each phenotypic group were principally of four types (Table
111-1). Male-fertiles, either tertiaries with homozygous normal
background or primaries with homozygous translocation background.
Similarly, semisteriles, either primaries or tertiaries both on
translocation heterozygote background (Fig. 111-1). When these were
allowed to self-pol1inate in the first field generation, the male-
fertiles bred true with respect to their background, but the
semisteriles (having translocation heterozygote background)
segregated into 1 homozygous normal (fértiles), 1 homozygous
translocation (fértiles) and 2 heterozygous translocation
(semisteriles) both in primaries and tertiaries. So the male-
fertiles from tertiaries having homozygous translocation background
also appeared in the S^ progeny, although perhaps at a low
frequency. Now here it is worthwhile to point out that if one
identifies male-fertile trisomic plants by pollen abortion analysis
in the first field generation (selfed progeny of a translocation
heterozygote), he can eliminate two classes of trisomics among the
Puckered Leaf phenotypic group and end up with only two classes that

76
are very easily identifiable by progeny test of their F^s. These can
be primary with homozygous translocation background and tertiary with
homozygous normal background. Keeping all these theoretical aspects
in view, an attempt was made to identify the trisomic plants with
homozygous translocation background. Principally they should be
primary, though contaminated with tertiary at a low frequency. These
five plants were crossed (male-fertile trisomic segregates) with 2n
pollen and their progenies grown. In the second greenhouse
generation, one out of these five male-fertile plant progenies
produced an all semisterile Fi. It produced 23 plants that
segregated into 12 trisomics and 11 normal disomies. Pollen abortion
in these trisomes ranged from 50 to 65 percent, whereas in all the
semisterile disomies it was 40 to 45 percent. This gave an
indication that the plant progeny may be primary or tertiary with
translocation heterozygote background. These 12 trisomic plants from
semisterile progeny were selected for backcrossing with 2n pollen.
BCj^ seed produced on these plants was grown in the second field
generation. The progeny produced 283 plants in total with 97
trisomic segregates. Due to unfavorable climatic conditions in the
field during the summer of 1985, the mortality rate of trisomic
plants was high. Nevertheless, 19 male-fertile trisomic plants from
5 plant progenies were tagged on the basis of pollen abortion
analysis. Three of the male-fertile plant progenies having a maximum
of surviving trisomic plants were analyzed cytologically. Thirty-six
out of 50 analyzable PMCs at diplotene to M-I showed a trivalent
association with 10 bivalents (Fig. IV-2C.E). Twelve PMCs showed one

Fig. IV-1 Meiotic diplotene to anaphase-I as observed in the
primary trisomics of common bean (x 1000).
A. A PMC at diplotene showing 11 bivalents and 1
univalent (arrow). One bivalent is still associated
with the nucleolus.
B. Diakinesis showing 11 bivalent and 1 univalent
(arrow).
C. Early anaphase showing 23 chromosomes.
D. Anaphase-I showing 11 chromosomes at one pole and 12
at the other pole (top).
E. Anaphase-I showing 11 chromosomes at each pole. The
thicker arrow points to 2 chromosomes overlapping
each other. The thinner arrow points to a univalent
away from the poles, appearing as a micronucleus.
The large size of the PMC made it necessary to take
two photographs to cover the whole PMC, which were
later cut and joined.

78

Fig. IV-2 Meiotic diplotene to anaphase-I as observed in the
primary trisomics of common bean (x 1000).
A. A PMC at anaphase-I, showing a laggard (arrow).
B. Diakinesis showing a trivalent association
(arrow). One bivalent is still associated with the
nucleolus. Nine bivalents are spread apart.
C. Diakinesis showing a trivalent association (arrow)
along with 10 bivalents.
0. A PMC at diplotene. The thicker arrow points to a
ring of 4 chromosomes. The thinner arrow points to
a trivalent.
E. Diplotene with a trivalent (arrow) and 10 bivalents.

80

81
univalent with 11 bivalents (Fig. IV-1A.B). Two pentavalent
associations were also observed that were probably due to overlapping
of chromosomes. Each plant progeny was analyzed separately.
Transmission rates of the extra chromosme after selfing (2n+l)
or crossing [(2n+l) x 2n] have been given in detail in Table III-3
for progenies classified in the second field generation. A total of
5567 progeny plants from selfed (2n+l) parents yielded 2025
trisomics, giving a 36 percent transmission rate. A total of 528
progeny plants from crosses yielded 201 trisomics, giving a 38
percent transmission rate. Similarly in first greenhouse generation
(Table 111-1), a total of 100 progeny plants from selfed trisomic
parents yielded 26 trisomics, giving a 26 percent transmission
rate. A total of 91 Fj progeny plants from crosses (Table IV-1) in
second greenhouse generation, yielded 46 trisomics, giving a 50
percent transmission rate. Here again we observe the same trend,
i.e., transmission is higher in cross progenies than self
progenies. Higher rates from crossing have been attributed to the
heterozygosity created by crossing (Khush and Rick 1967).
The phenotype of Puckered Leaf primary trisomic can be
distinguished 15-20 days after planting or after the 2nd true leaf
emergence (Fig. IV-3A). Leaves are puckered in shape and give a
glazed appearance after attaining full size. Plants are weaker than
diploid sibs during the early stage of growth but attain the same
size later (Fig. IV-4B). Also, puckered plants show a tendency for
accentuated semi-vining growth habit. They flower nearly at the same
time as diploids do. There is no difference in flower color. The

Table IV-1
Transmission of the
greenhouse planting
extra chromosome in
, Spring, 1985).
two trisomics of
common bean after crossing
[(2n+l) x 2n] (Second
Trisomic
Number of
Number of
Percent transmission
Translocation
plant
progeny
trisomic
on crossing
stock
phenotype
classified
segregates3
(2n+l) x 2n
11-70
Puckered Leaf
91
46
50
1-97
Weak Stem
71
31
44
a No off-type segregates were observed in either progeny.
00
i\3

Fig. IV-3 Plant photographs of primary trisomics at the seedling
stage.
Aj_. Puckered Leaf trisomic, 15 days after planting.
A normal plant of the same age.
B^. A normal plant, 10 days after planting.
Weak Stem trisomic of the same age.
C^. A normal plant, 10 days after planting.
Í2- Convex Leaf trisomic of the same age.

00
-c*

Fig. IV-4 Photographs of five types of primary trisomic plants.
A}. A normal disomic plant, 30 days after planting.
A£. Dark Green Leaf trisomic of the same age.
B. Puckered Leaf trisomic, 30 days after planting,
glossy leaf surface gives a light color (pseudo¬
chlorosis) in black and white photographs.
C. Weak Stem trisomic, 30 days after planting.
D. Convex Leaf trisomic, 45 days after planting.
E. Chlorotic Leaf trisomic, 30 days after planting.
The

86

87
pods are broader and shorter with an unfilled basal portion, and they
have slightly longer stylar tips with a pronounced curvature (Fig.
IV-5B). Seeds are larger and have slight wrinkles on the testa (Fig.
IV-6E). Because this trisomic can be identified easily at the
seedling stage (same for Weak Stem trisomic), all or most of the
undesired diploids can be rogued from stocks or F-^s in early stages,
thus saving greenhouse space. It is also sensitive to heat and
drought (but less than the Weak Stem trisomic) which affects the pod
and seed set adversely. It sets very well in the greenhouse where
mean seeds per pod in trisomic plants is 3.35 ± 1.63 compared to 5.61
± 1.46 in normal diploids (means from 10 plants). A 43 percent
reduction in seed set or ovule abortion under greenhouse conditions
has been observed. Trisomic plants may have improved seed set when
developed on a homozygous normal chromosome background.
Weak Stem Trisomic
Nineteen seeds recovered from two plants of the Weak Stem
trisomic originating in the selfed progeny of a translocation
heterozygote in the first field generation were planted in the first
greenhouse generation. Six plants were identified as trisomic from
their phenotype (Table 111-1). Pollen fertility analysis showed that
they were all semisteriles. Pollen abortion rates ranged from 60 to
75 percent in these trisomic plants. It gave an indication that
these plants are either primary or tertiary trisomics still having a
translocation heterozygote background. To convert them to homozygous
normal chromosome background, they were crossed with pollen from
normal (2n) plants. One thing which should be emphasized here is

Fig. IV—5 Photographs of pods from primary trisomic plants.
A. Normal disomic plant pods.
B. Puckered Leaf pods.
C. Weak Stem pods.
0. Convex Leaf pods.
E. Dark Green Leaf pods.
(For details, please see text.)


Fig. IV-6 Photographs of seeds from primary trisomic plants.
A. Dark Green Leaf seeds.
B. Normal disomic seeds.
C. Convex Leaf seeds.
D. Weak Stem seeds.
E. Puckered Leaf seeds.
(For details, please see text.)


92
that out of six trisomic segregates the five that set seed were all
from one of the two original Weak Stem trisomics. The 6th trisomic
plant, derived from the other original parent, set no seed.
F^ seed from five trisomic plants, all from the same trisomic
plant progeny in the first greenhouse generation, was planted in the
second greenhouse generation (Table IV-1). A total of 71 self
progeny segregated for 31 trisomic phenotype plants. These were
tested for pollen fertility; and seven trisomic plants having pollen
abortion rates between 25 to 40 percent were classified as fértiles,
whereas the rest having 50 to 70 percent pollen abortion were
classified as semisteriles. The fertile trisomic plants were tagged
and allowed to self-pollinate.
F2 seed from the fertile trisomic segregates produced 450 plants
in the second field generation, out of which 160 plants were
classified as trisomics. As this trisomic is the most sensitive to
unfavorable climatic conditions, nearly 40 percent of the plants died
at the seedling stage; however, it probably did not affect our
transmission data because of a desirable characteristic of this
trisomic group. It can easily be classified at an early seedling
stage, as early as 10 days after planting. Under greenhouse
conditions no mortality of trisomic seedlings was observed in this
group.
Four out of seven fertile trisomic plant progenies having the
maximum number of trisomic survivors were analyzed cytologically.
Buds were collected from all trisomic segregates within each trisomic

plant progeny. Fifty analyzable PMCs from each plant progeny were
scored (Table IV-2).
93
Table IV-2
The frequency of PMCs with different chromosome configurations at
Diplotene to M-I of meiosis in different plant progenies of Weak Stem
Trisomic.
Progeny
Code
Frequencies of
different
chromosome
configurations
11 II + 1 I
10 II
+ 1 III
9 II + 1 V
GK #19
25
67
8
GK #22
15
75
10
GK #23
22
34
44
GK #25
17
11
72
GK #22, 23 and 25 progenies were classified as tertiaries
because the pentavalent configurations in these three progenies were
clearly indicating them as tertiaries (Fig. Ill-2). GK #19 was
identified as primary because none of its pentavalent configurations
gave an indication of its being a tertiary trisomic. Most of the
pentavalents appeared to be the result of overlapping chromosomes.
Some of the cells showing a univalent or trivalent configuration,
also showed a quadruple association of 4 chromosomes appearing as a
ring, a characteristic of translocation heterozygosity (Fig.
IV-2D). When trisomic plants in this progeny were checked for pollen
fertility, four out of nine plants were fertile, with pollen abortion
rates ranging from 30 to 40 percent. The remaining five semisteriles
had 70 to 85 percent pollen abortion rates. Under the same

94
conditions, a normal plant had 15 to 20 percent pollen abortion.
From these data, it was inferred that the parent plant, which was
originally classified as fertile in Fj progeny of the second
greenhouse planting, was not actually a fertile but a semisterile
that again segregated into nearly 1:1, semisterile:fertile
trisomes. Moreover, the presence of a quadruple at M-I also
indicated semisteri1ity. Hence, in the progeny GK #19, fertile
trisomic segregates were tagged as primary trisomics with a
homozygous normal chromosome background, whereas the semisteriles
with trisomic phenotype were classified as primary trisomics with a
translocation heterozygote chromosome background. Due to unfavorable
climatic conditions, seed was not recovered from any of the plants in
the GK #19 progeny; however, this trisomic can be recovered in the
greenhouse from remnant F2 seed. There was no morphological
distinction between the tertiaries and primaries of this class.
Some of the photomicrographs show a ring of 4 chromosomes + 1
III (Fig. IV-2D). The extra chromosome is either associated or
merely appears associated with another bivalent pair. Usually, the
extra chromosome of a trisomic derived from the nondisjunction
progeny of a translocation heterozygote is one of the 4 chromosomes
forming a quadruple. It is difficult to explain why it appears
associated with another pair with which it has no homology. A
primary trisomic still having a translocation heterozygote background
may also appear in the form of a straight chain of 5 chromosomes,
which if it gets twisted, can appear in strange shapes. This may

95
probably be the reason why we observed pentavalents (Table IV-2) at
such a frequency in this class.
Another thing worth mentioning here is that all Weak Stem
trisomic progeny had been derived from only one trisomic plant. This
plant could either be a primary or a tertiary trisóme. If it is
primary, then all the progeny trisomics should be primary with rare
unrelated primaries. If it is tertiary, the progeny generally
segregates into parental class tertiaries and one or both related
primaries (Khush and Rick 1967). Keeping this fact in view, it was
proceeded further with one plant progeny expecting segregation for
primary trisomics within the progeny of the tertiary trisóme. The
primary trisomic plants isolated on the basis of genetic tests and
cytological analysis discussed above, did not show any morphological
dissimilarities from the parental tertiary phenotype.
This trisomic can be distinguished from diploid sibs in the very
early seedling stage, usually 10 days after planting and sometimes
even before the first true leaf emergence (Fig. IV-3B). The
hypocotyl and the stem from the cotyldons to the primary leaves are
longer than in diploid sibs, and the hypocotyl usually bends aside
because it is weaker than in normal plants (Fig. IV-3B). Plant
internodes and leaf petioles are longer than normal segregates. The
entire plant grows taller than normal and has a spindly appearance.
The weak stem needs support. If the plant apex is broken or support
is not provided, it produces many branches and becomes bushy. Leaf
petioles have a wider angle with the stem than normal plants. The
terminal leaflet of the trifoliate leaf has an angular shape. The

96
plant has a wider or more open plant canopy (Fig. IV-4C). This
trisomic is more sensitive to heat, drought, lodging and sunburn
effects than normal plants, and these effects are very obvious under
Florida field conditions. It is late in maturity, the delay being
about 15 days more than normal diploid sibs. It has poor pod set
under high-temperature field conditions, but pods set adequately in
the greenhouse. Pods are not well filled towards the basal
portion. Constrictions in the pods represent the areas where ovules
aborted and seeds are missing. The pods are broader and their stylar
tips are longer (Fig. IY-5C). Seeds are comparatively longer and
wider than normal diploids (Fig. IV-6D). The data collected from 10
plants chosen at random from the Spring, 1985, greenhouse planting
shows that the average number of seeds per pod is 2.13 ± 1.13
compared to 5.64 ± 1.46 seeds in diploid sibs. This difference can
be attributed to the missing seeds in trisomic plant pods as the
ovule abortion (percent reduction in seed set) data of the same
plants shows 62 percent reduction in seed set. An improvement with
respect to seed set is expected when the trisomics with translocation
heterozygote background are replaced with a homozygous normal
chromosome background. Transmission rates of the extra chromosome on
selfing or crossing have been presented in detail in Table 111-3.
The combined data for both primary and tertiary plants are as
follows. Among 2373 total plants in the progeny of sel fed trisomics,
there were 851 Weak Stem segregates, i.e., 36 percent of the total.
Among a toal of 85 progeny plants from crossing (2n+l) x 2n, there
were 35 trisomic and 50 disomic segregates giving 41 percent

97
transmission through the female. Transmission rates from selfing are
less than from crossing the same trisomic plants with 2n normal
disomies, which can be attributed to the heterozygosity created by
crossing (Khush and Rick 1967).
Dark Green Leaf and Convex Leaf Trisomics
Both of these trisomic phenotypes originated from the
nondisjunction progenies of one translocation heterozygote 1-52.
seed from five off-type plants with Dark Green Leaf phenotype and two
off-type plants with Convex Leaf phenotype was planted in the first
greenhouse generation (Table 111-1). S2 and [(2n+l) x 2n] seed
from these plants was grown in the second field generation. All the
progenies fell into one of the four different types of trisomic plant
progenies observed which are described below. The first type of
plant progeny showed quite uniform (pure type) trisomic segregates
identical to the parent Dark Green Leaf phenotype and also segregated
for normal disomies (Table 111-2, a). The second type of plant
progeny showed uniform trisomic segregates of another type, Convex
Leaf, originating in the progeny of Dark Green Leaf. There were also
a few tetrasomics and the usual abundant disomies (Table 111-2, b).
The third type of progeny had trisomic segregates showing the
dominant morphological characteristics of Dark Green Leaf combined
with Convex Leaf characteristics (Table III-2, c). There were also a
few tetrasomics, numerous disomies and rare segregates of the pure
Dark Green Leaf type among the progeny. The fourth type of plant
progeny had trisomic segregates showing the dominant morphological
characteristics of Convex Leaf combined with characteristics of Dark

98
Green Leaf (Table III-2, f). There were also a few tetrasomics,
abundant normal disomies and rare plants of pure Dark Green Leaf type
among the progeny.
Trisomics in the 3rd and 4th types described above were
classified as tertiaries on the basis of their phenotypic
characteristies and genetic segregations (Section III). Immature
flower buds from trisomic plants of the 1st and 2nd types were
collected separately and analyzed cytologically. Out of 50
analyzable PMCs of Dark Green Leaf, 17 showed a univalent with 11
bivalents (Fig. IY-1A.B) and the remaining 33 showed a trivalent and
10 bivalents at diakinesis to M-I in each PMC (Fig. IY-2C.E).
In 50 PMCs from Convex Leaf Trisomic buds collected from the 2nd
type of trisomic plants, the frequencies of univalents and trivalents
at diakinesis to M-I were equal. On the basis of cytological
evidence, morphological character!sties and genetic segregations, the
two classes described in the 1st and 2nd types were identified as
primaries. These plant progenies might be having translocation
heterozygote or homozygous translocation background, for which no
cytological evidence is available. One to two backcrosses with
normal disomies need to be made to develop them on homozygous normal
chromosome background. Transmission rates of both these classes have
been given in detail in Table II1-2. There is not a clear cut trend
of improvement in transmission by crossing [(2n+l) x 2n] compared to
selfing. This may probably be due to the segregations taking place
among the progenies of tertiary trisomics.

99
Phenotype of Dark Green Leaf
In the field, the trisomic plants are late germinating and slow
growing at the early stage, but they attain the same size as disomies
at maturity. Segregating progenies can easily be classified 15 to 20
days after planting. Trisomic plant leaves are glossy dark green and
smaller in size compared to the normal, but the primary trisomic has
bigger leaves compared to the Dark Green Leaf tertiary. Leaf veins
are slightly recessed (Fig. IV-4A). Flower color is more intense.
The pod and seed set are reduced and a bit late with many missing
seeds under field conditions but less so in the greenhouse. The data
collected from 10 trisomic plants picked at random from the field
showed the mean number of seeds per pod to be 2.09 ± 1.05, compared
to 5.85 ± 1.61 in normal plants under the same conditions. So the
reduction in seed set in trisomic plants was 64 percent. Part of the
reduction may be due to translocation heterozygosity which probably
is still present in the background complement. Pod and seed size are
slightly smaller compared to the diploid segregates (Fig. IV-6A).
Pods possess straight stylar tips that are nearly the same size as in
normal pods (Fig. IV-5E). The phenotype is easily distinguishable in
the field but very difficult to distinguish (indistinct) in the
greenhouse.
Phenotype of Convex Leaf Trisomic
It is difficult to identify at the seedling stage (Fig.
IV-3C). Nearly 20 days after planting, the leaves start turning to a
convex shape with recessed veins. Thirty days after planting, the
phenotype becomes quite obvious in the field but is still difficult

100
(indistinct) in the greenhouse. Leaf color is normal and the leaf
surface is slightly rough. This class sets seed and pods equally
well under field or greenhouse conditions. Primary trisomics have
slightly longer internodes compared to the normal disomies and also
tertiaries, and the branches are weak, needing support. They grow
even taller than normal diploids, and due to late pod set, senesce
late (Fig. IV-4D). Pods are smaller than normal and stylar tips are
normal in length without pronounced curvature (Fig. IV-5D). The
average number of seeds per pod (from 10 plants) in trisomics under
field conditions is 3.84 ± 1.76 compared to 5.85 ± 1.61 in normal
diploids. The reduction in seed set is 34 percent, which is less
than the other trisomics. Seeds are nearly of the same size as
normal seeds, but slightly different in shape (Fig. IV-6C).
Chlorotic Leaf
Trisomic plants with this phenotype originated in three other
types of trisomic plant progenies, namely, Dark Green Leaf, Convex
Leaf and Puckered Leaf, always at a very low frequency in each
progeny (Tables III-2 and 111-3). A total of 108 Chlorotic Leaf
plants were scored in the second field generation, which was less
than one percent of the total population. Immature flower buds were
picked collectively from these plants. Twenty PMCs observed at
diakinesis and M-I of meiosis showed a trivalent and 10 bivalents
(Fig. IV-2C.E). This and the fact that unrelated primaries regularly
appear among the progenies of trisomics at low frequency (Khush 1973)
helped us identify this phenotypic class as a primary trisomic. We
are sure that the Chlorotic Leaf is an unrelated primary segregating

101
in Convex Leaf and Dark Green Leaf progenies because both of the
latter are related primaries recovered only from translocation stock
1-52. For Puckered Leaf, further evidence is needed to determine
whether the Chlorotic Leaf is a related or an unrelated primary
because the transmission rate of Chlorotic Leaf is very low (Tables
III-2 and 111-3). None of the plants in this class set any pods or
seeds. Heterozygosity and genetic background have been reported to
improve the transmission of and tolerance to the extra chromosome
(Khush 1973). If it reappears in the future, it may be pollinated
with a bean line having diverse genetic orign compared to 7-1404,
which has been used as a base for developing translocations and
trisomic stocks.
The leaves of this trisomic are slightly chlorotic. All other
morphological aspects are similar to the normal plants (Fig.
IV-4E). It blooms profusely, fails to set any pods under hot field
conditions, and consequently senesces very late.

SECTION V
SUMMARY AND CONCLUSIONS
Five of the 12 semisterile stocks developed by Bassett and Hung
(1985) have been proved to be translocation heterozygotes by
cytological verification. Cytological analysis of the half dial lei
set has indicated that the five translocation stocks used have in
total at least 8 different chromosomes involved in reciprocal
translocations. Pollen and ovule abortion varies between each
translocation stock.
Eleven of the 12 translocation heterozygote stocks were used to
search for trisomics. For each translocation, the seed from each
heterozygous plant was separated into two weight classes, i.e., heavy
(70 percent) and light (30 percent) seed weight class. These weight
classes were planted separately in adjacent plots. The 11
translocation stocks produced 22 off-type plant groups (Appendix 2)
out of which 12 distinct phenotypic groups were selected on the basis
of genetic tests. Five of these groups have been proved to be
trisomics by genetic tests as well as cytological verification
(Appendix 3). The remaining seven groups need cytological
verification, but their breeding behavior suggests that they may be
trisomics (Appendix 3). Five primary trisomic groups have been
identified on the basis of cytology and morphology. For three
primary trisomic groups, i.e., Puckered Leaf, Dark Green Leaf and
102

103
Convex Leaf, related tertiaries have been identified on the basis of
their phenotypic relationships. For another primary trisomic Weak
Stem, the related tertiary has been identified cytologically because
phenotypic distinction between the primary and related tertiary was
not possible. Two of the five primary trisomic groups, i.e., Dark
Green Leaf and Convex Leaf, produced tetrasomics on selfing at a low
frequency. Another two of the five primary trisomics, i.e., Puckered
Leaf and Weak Stem, have been developed on homozygous normal
chromosome background. We could not recover the seed of Weak Stem
because of its sensitivity to unfavorable climatic conditions in the
summer-1985 field planting.
One of the five primary trisomics, i.e., Chlorotic Leaf, and two
of the seven putative trisomics, i.e., Weak Plant and Extreme
Lanceolate Leaf, did not set seed under field conditions. Physical
identification of the chromosomes involved in the translocations and
trisomics was not achieved.
Our study has proved that nondisjunction progenies of
translocation heterozygotes are an excellent source of aneuploids.
The only difficult aspect of this search is that it needs extensive
cytological examination.

APPENDIX 1
MODIFICATION OF THE SLIDE PREPARATION TECHNIQUE
OF CHENG AND BASSETT (1981)
The time of harvest of immature flower buds and their size have
a direct effect upon the frequency of PMCs at desirable stages of
meiosis one can obtain from slide preparations. During this study we
observed that the best harvest time for buds at diplotene was 10 A.M.
(EST), for maximum cells at M-I it was 12 A.M., and for abundant
anaphase-I cells it was 1 to 2 P.M. As regards the proper size of
the buds to be harvested, pick the smallest buds (whole racemes
really) that one can harvest by hand without damaging the growing
tip. Because every raceme carries a tight cluster of buds (usually
3-4) at various stages of development, one can select the 2 mm size
buds and discard the smaller or larger ones during slide
preparation. We propose another change in the previous technique,
i.e., a change in the duration of the staining period for anthers in
45 percent iron propionic carmine. During this study, we never kept
the anthers in dye solution for more than 30 minutes. The duration
usually ranged from 20 to 30 minutes. Still another modification is
the gentle heating and destaining of the slide.
Stained anthers are taken out of the porcelain well where they
have stayed for 20 to 30 minutes in 2 drops of 45 percent iron
propionic carmine. They are transferred to the slide with a
104

105
restricted quantity of stain solution, i.e., just enough to flow when
the slide is tilted. They are macerated very gently with the help of
a spatula; otherwise the PMCs cell wall breaks. The cover glass is
put on quickly after taking out all the solid material (debris) and
leaving just the stain solution, which is spread out over the entire
area that the cover glass will occupy. Heating and destaining helps
greatly in improving the quality of preparations. Heating helps in
loosening the cell wall and cytoplasm so that when we press the cover
glass gently, all the material underneath spreads out without
breakage. One must be very careful in pressing the cover glass
because if it is pressed slightly harder, it breaks most of the
PMCs. Finally, heating helps to stick most of the cells to the slide
so that when we destain it, they do not come out from underneath the
cover glass along with the destaining agent.
The slide is then gently passed over the flame 3-4 times, being
careful to avoid boiling the stain. The easiest method to know
whether the slide has been heated to its proper temperature is to
look at the lower side of the slide after passing it over the flame
the first time. One can easily observe the water vapors on it, which
may fade away after 5-10 seconds. When we pass the slide over flame
the second time, the water vapors on the underside of the slide will
fade away more quickly than the first time. After passing it over
the flame the third time, they will not be visible, i.e., they will
evaporate as soon as the slide is away from the flame. Now this
process should not be repeated the fourth time, otherwise the stain

106
will start boiling. Sometimes if the flame is too hot, we may need
to pass it over just once or twice.
After heating, the cover glass is pressed gently, keeping
bibulous paper over it to absorb excess stain; and then a drop of 45
percent acetic acid is spread to three sides of the cover slip
leaving the fourth side for the excessive stain to come out. It
takes about 30 seconds to one minute for the destaining agent to flow
under the cover glass. Again, heat the slide as before. Then the
cover glass is again pressed with the thumb, keeping bibulous paper
underneath and starting from the side opposite to the one left for
the stain to come out. If the cells are highly stained, the
destaining process can be repeated twice. The slide is gently heated
again over the flame and finally sealed with wax, keeping it pressed
gently with a cork.

APPENDIX 2
OFF-TYPE PLANTS OBSERVED AMONG THE PROGENIES OF
TRANSLOCATION HETEROZYGOTES DURING THE FIRST FIELD PLANTING
(SUMMER, 1984)
Number of Number
Translocation
stock
seeds
Light
planted3
Heavy Total
Plant phenotypes of plants
observed observed
1-52
43
102
145
Dark Green Leaf
14
Convex Leaf
2
1-85
176
388
564
Weak plantc
2
Rough Leaf Dwarf rnd.°*
1
Extreme Lanceolate Leaf*3
1
Moderate Lanceolate Leaf*3
2
Dark Green Dwarf (pa 1e)e
1
1-97
50
114
164
Weak Stem
7
Pinched Leaflet Tip
1
1-99
118
273
391
Puckered Leaf
3
11-6
213
498
711
Small Leaf
1
Puckered Leaf Tip & Curl0*
1
11-14
360
782
1142
Strong Vining Habit0*
1
Rumple Leafe
2
Leathery Leaf Dwarf-2e
3
107

108
Number of
Translocation seeds planted9 Plant phenotypes
stock Light Heavy Total observed
Number
of plants
observed
11-14 Dwarf-2 type01 3
Epinastic Leaf 2
Leathery Leaf Dwarfb 3
Epinastic Leaf (rough
surface)b 3
Rough Leafb 1
Shiny Leaf Dwarfb 9
11-70
188
430
618
Puckered Leaf
16
Chlorotic Leaf0
1
Plain Dwarfd
2
11-109
368
803
1171
Semi Lanceolate Leafe
1
Savoy Leaflet tip (round)01
1
Weak plantd
1
Leaf Margins Curl Down
1
11-121
84
192
276
Weak Steme
7
Leaf Tip Curl Down
1
Semi Diamond Leafb
1
11-122
199
466
665
Epinastic Leaf Dwarfd
1

109
Number of
Number
Translocation
seeds planted9
Plant phenotypes
of plants
stock
Light Heavy Total
observed
observed
11-3
288 503 791
No off-type segregates
a Before planting, seed was classified into heavy weight (70
percent) and light weight (30 percent) seed classes.
b Plants with these phenotypes did not set seed.
c Plants with these phenotypes did not set seed in Si (summer,
1985).
d Plants with these phenotypes did not segregate in Si (summer,
1985).
e Plants with these phenotypes did not germinate in Si (summer,
1985).

APPENDIX 3
ANEUPLOIDS RECOVERED FROM THE PROGENIES OF
TRANSLOCATION HETEROZYGOTES DURING THE SECOND FIELD PLANTING
(SUMMER, 1985)
Trans-
No. of
Trisomics3
location
plants
Tetra-
stock
screened
Names Classification
somics
1-52
145
Dark Green Leaf
Tertiary
+
Primary (related)
-
Convex Leaf
Tertiary
+
Primary (related)
+
Chlorotic Leaf0
(Extreme
Primary (unrelated)
Lanceolate Leaf0)
Not confirmed
-
1-85
564
(Weak Plant^)
(Extreme
Not confirmed
-
Lanceolate Leaf0)
Not confirmed
-
1-97
164
Weak Steme
Tertiary
-
Primary (related)
-
(Pinched Leaflet
Tip)
Not confirmed
(+)
1-99
391
Puckered Leaf
Tertiary
-
Primary (related)
-
Chlorotic Leaf0
Primary (relationship
-
(undetermined)
11 —6
711
(Small Leaf)
Not confirmed
-
11-14
1142
(Epinastic Leaf)
Not confirmed
( + )
11-70
618
Puckered Leaf
Tertiary
-
Primary (related)
-
Chlorotic Leaf0
Primary (relationship
-
undetermined)
11-109
1171
(Leaf Margins
Curl Down)
Not confirmed
-
110

Ill
Trans¬
location
stock
No. of
plants
screened
Trisomics3
Names
Classification
Tetra-
somicsb
11-121
276
Weak Stern^
(Leaf Tip Curl)
Not confirmed
-
11-122
665
None
No off-type segregates
11-3
791
None
No off-type segregates
a Trisomic suspects are given in parentheses.
b Plus or minus represents the presence or absence of tetrasomics
respectively. Plus in parentheses indicates that tetrasomy is not
confirmed. No tetrasomic plants set seed.
c The plants did not set seed.
^ The plants did not set seed; no remnant seed is available.
e No morphological distinction exists between tertiary and related
primary.
^ Seeds did not germinate in second field planting, summer 1985.

APPENDIX 4
PROCEDURES TO IDENTIFY PRIMARY AND TERTIARY TRISOMICS
ORIGINATING AMONG THE SELFED PROGENY
OF A TRANSLOCATION HETEROZYGOTE

Trisomic classes3
Chromosome Type of
background trisomic
Selfing (2n+l) or
Crossing (2n+l) x 2n
Cytologyb of PMCs from
S^ or Fj of the progeny
trisomics
Pollen fertility analysis0
of the trisomic plant
progeny (including
disomies and trisomics
Homozygous
normal
Tertiary^
Selfing
Crossing
Predominantly a pentavalent
formation at M-I
All Si and Fi fertile
Heterozygous
translocation
Primary
Selfing
Crossing
Predominantly a trivalent or a
ring of 4 + 1 univalent
Si and Fi segregate for
1 fertile:l semisterile
Heterozygous
translocation
Tertiary
Selfing
Crossing
Predominantly a pentavalent
formation or a ring of 4 + 1
Si and Fi segregate for
1 fertile:1 semisterile
Homozygous
translocation
Primary
Selfing
Crossing
Predominantly a trivalent
formation
PMCs show a tetravalent +
1 univalent. A pentavalent
may also appear at a very
low frequency.
All Si fertile
All Fi semisterile
a Trisomic progeny appear with nearly equal frequencies in the four classes listed below.
b Chiasma terminal ization or failure of chiasma formation may reduce the pentavalent or trivalent
associations to smaller configurations (Khush and Rick 1967).
c Pollen fertility of trisomic segregates is generally lower than normal disomies, depending upon extra
chromosome and several other factors (Khush 1973). If pollen fertility analysis is to be employed for
preliminary observations, translocation heterozygosity of the background complement with respect to
pollen abortion, must be taken into account.
^ Selfed progenies of tertiary trisomics regularly produce related primaries (Khush and Rick 1967) which
are another excellent source of primary trisomics in addition to the translocation heterozygote
progenies.

APPENDIX 5
FIELD PLOT NUMBERS OF PLANT PROGENIES GROUPED ACCORDING
TO THEIR SIMILARITY OF SEGREGATION PATTERNS; TABLE II1-2
(SECOND FIELD PLANTING, SUMMER, 1985)
Progeny
code in
Table 111-2
Number of progenies and their field plot numbers
a
19 plant proqenies 158,159,160,161,179,182,183,184,186,
187,207,208,209,220,222,226,227,255,259
b
6 plant progenies 162,163,167,170,219,260
c
9 plant progenies 193,194,196,198,199,201,202,204,205
d
13 plant progenies 108,109,164,165,166,168,169,171,210,
218,221,224,225
e
14 plant progenies 106,107,236,237,238,239,240,244,247,
«2,264,265,268,270
f
4 plant progenies 245,269,272,273
g
18 plant progenies 172,174,176,177,178,188,189,190,191,
152,214,215,216,217,229,230,231,234
h
4 plant progenies 173,175,234,261
i
5 plant progenies 249,250,251,254,278
j
2 plant progenies 252,276
114

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BIOGRAPHICAL SKETCH
Mohammad Ashraf was born in Amritsar (India) on July 23, 1945,
and migrated to Pakistan when it came into being in August, 1947. He
received his B.Sc. (Hons.) Horticulture degree from the University of
Agriculture at Faisalabad during 1968. After receiving his
bachelor's degree, he worked for a private vegetable seed producing
company as farm manager for 3 years. He joined the research wing of
the Agriculture Department in March, 1971, as an Assistant Research
Officer and worked for 9 years. There he was involved mainly in seed
production and breeding of vegetable crops. He began his master's
program in 1976 and, while continuing with his official duties, he
earned his M.Sc. (Hons.) Horticulture degree in 1979 from the same
uni versity.
He joined the Ministry of Higher Education and Scientific
Research in Iraq during 1980 and was involved in teaching
horticultural crops breeding and production courses to undergraduate
students for 2 V2 years.
He won the Quaid-e-Azam/Merit scholarship for Ph.D. studies on
the basis of his master's merit and joined the University of Florida
in the fall of 1982 as a graduate student in the Vegetable Crops
Department under the direction of Dr. Mark J. Bassett. He will be
120

121
awarded the degree of Doctor of Philosophy in December, 1985, in
horticultural science (vegetable crops).
He is married to Sarwar Sultana and has one son and three
daughters.

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
o
Ken H. Quesenberry
Associate Professor of Agronomy
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Paul M. Lyrene
Professor of Horticultural Science
This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School, and was accepted
as partial fulfillment of the requirements for the degree of Doctor
of Philosophy.
December, 1985
'l
Us*
Dean,/T2
ol 1 ege of Ag^i
¡culture
Dean, Graduate School

n
UNIVERSITY OF FLORIDA
3 1262 08556 7542





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