I

I
II

 

THS_

ENVIRONMENTAL, GENETIC. AND
DEVELOPMENTAL $TUDIES OF THE HYPOCOTYL
ELO‘NGA'I‘IQN TRAIT IN PHASEOLUS VULGARIS

(VAR.e 211-V AND SANILAC)

Thesis for H19 Degree OI M. S.
MICHIGAN STATE UNIVERSITY

Antonio M. Pinchinat
1960

LIBRARY

Michigan Sm:
University

 

ENVIRONMENTAL, GENETIC, AND DEVELOPMENTAL
STUDIES OF THE HYPOCOTYL ELONGATION
TRAIT IN PHASEOLUS VULGARIS

(Var. le-V and Sanilac)

BY
ANTONIO M. PINCHINAT

AN ABSTRACT

Submitted to the College of Agriculture of Michigan
State University of Agriculture and Applied
Science in partial fulfillment of
the requirements for the
degree of

MASTER OF SCIENCE

Department of Farm Crops

Year 1960

Approved _% %/%W—/

1
Antonio M. Pinchinat

ABSTRACT

To investigate on the differential hypocotyl elongation
of two commercially grown varieties of Navy beans, environ-
mental, genetic, and developmental studies were carried out.

Following application of alternative combinations of
day length and light intensity treatments upon seedlings of
Sanilac and le—V from germination to complete development,
it has been noticed that, although differences in average
hypocotyl length between the bush type, Sanilac, and the viny
type, le-V, were more pronounced under long day, high light
conditions than under any other treatment, difference in
hypocotyl elongation was primarily attributable to intrinsic
factors rather than to environmental influences.

To study the mode of inheritance of the short hypocotyl
trait in Sanilac and its opposite, the long hypocotyl character
in le-V, appropriate crosses and backcrosses between the
parent materials and their F1 progeny were performed. Common
beans (Phaseolus vulqaris) being a naturally self-pollinated
crop, F2 seedlings were obtained by growing seeds harvested
from F1 plants. Analyses of data gathered for this study
suggest that transmission of the hypocotyl elongation trait
in Navy bean seedlings could be interpreted on the basis of
a duplicate recessive epistasis mode of inheritance in which
the long hypocotyl trait would be caused by recessivity in

two different genes, both in dominant condition being necessary to

2

Abstract Antonio M. Pinchinat
prevent the length growth of the hypocotylary axis.

Observations made on cortical cell layers of hypocotyl
sections of the seedlings of Sanilac and 211-V disclose no
appreciable difference in average cell length of both varieties.
implying, therefore, the occurrence of a larger number of
cells in a given hypocotylary cortical strand of 211-V as
compared to a corresponding strand in Sanilac. Referring to
recent studies on phytohormones and their relation to growth
and development, it has been assumed that gibberellic or
gibberellin-like sybstances might be taken into account for the
marked increase in cell division rate of le-V, the long
hypocotyl variety. The results from the genetic and develop-
mental studies, considered together, may suggest the following
alternative hypotheses:

1. All bean varieties possess gibberellin-like substances
which tend to promote hypocotyl elongation, but most varieties,
like Sanilac, also possess a genetic inhibitory system which,
when two non-allelic genes are present in dominant condition
effectively suppress the phytohormonal growth action.

2. Or, only those varieties of beans with recessive
alleles in the homozygous state at both the postulated loci
involved are able to produce the gibberellin-like material

and no direct inhibiting system is present.

ENVIRONMENTAL, GENETIC, AND DEVELOPMENTAL
STUDIES OF THE HYPOCOTYL ELONGATION
TRAIT IN PHASEOLUS VULGARIS

(Var. le-V and Sanilac)

By
ANTONIO M. PINCHINAT

A THESIS

Submitted to the College of Agriculture of Michigan
State University of Agriculture and Applied
Science in partial fulfillment of
the requirements for the
Degree of

MASTER OF SCIENCE

Department of Farm Crops

Year 1960

ii

ACKNOWLEDGEMENTS

The author wishes to express his sincere gratitude to
Dr. M. Wayne Adams for his guidance in this research project
and for his helpful advice in the preparation of the
manuscript.

Indebtedness and appreciation are also extended to
Mr. Hubert M. Brown for his precious aid in the statistical
analysis of the experiments, to Dr. Carter M. Harrison for
his assistance and counsel in the microtechnic assays required
for this study, and to Agronome Renan Fontus for his
unfailing inspiration and encouragement.

The writer deeply appreciates the financial support
of the Department of Agriculture of Haiti and the scholar-
ship granted by the International Cooperation Administration,

which enabled him to undertake this investigation.

TABLE OF CONTENTS

INTRODUCTI ON . O C O O O O O O O O O O C O O C O O O 0
REVIEW OF LITERATURE . . . . . . . . . . . . . . . . .

A. Environmental Studies
B. Genetic Studies
C. Developmental Studies

SECTION I. ENVIRONMENTAL STUDIES, EXPERIMENT I . . .

Materials and Methods
Experimental Results and Discussion
Summary and Conclusions

SECTION II. 'GENETIC STUDIES . . . . . . . . . . . . .

A. Experiment II--Part I
Materials and Methods
Experimental Results
Discussion of Results
Summary

B. Experiment II--Part II
Materials and Methods
Experimental Results
Discussion of Results
Summary and Conclusions

SECTION III. DEVELOPMENTAL STUDIES, EXPERIMENT III .

Materials and Methods
Results

Discussion of Results
Summary and Conclusions

GENERAL SUMMARY AND CONCLUSIONS . . . . . . . . . .

LITEMTUM CITED 0 O O O O O O O O O O O O O O O O O 0

iii

55
55
55
56
60
61

63

10.

ll.

12.

13.

LIST OF TABLES

Hypocotyl lengths of Sanilac and 211-V under
four day lengths and light intensity treatments

Analysis of variance of hypocotyl lengths of
Sanilac under four day and light treatments . .

Analysis of variance of hypocotyl lengths of
le-V under four day and light treatments . . .

A t—test for effect of day length upon Sanilac
A t-test for effect of day length upon 211—V .

Testing variability of Sanilac and le-V to day
and light treatments . . . . . . . . . . . . .

Hypocotyl lengths and means lengths of Sanilac,

le-V, and Fl 0 o o o o o o o o o o o o o o o 0

Comparing mean lengths of Sanilac, le-V, and

F1 . . . . . . . . . . . . . . . . . . . . . .

Mean and standard deviation of Sanilac,
Experiment II, Part II . . . . . . . . . . . .

Means and standard deviation of le—V,
Experiment II, Part II . . . . . . . . . . . .

Mean and standard deviation of F1, Experiment
II, Part II 0 O O O O O O O O O O O O O O O 0

Mean and standard deviation of BCS*, Experiment
II, Part II 0 O O O O O O O O O O O O O O O 0

Mean and standard deviation of BCV,**
Experiment II, Part II . . . . . . . . . . . .

*BCS: Back cross of F to Sanilac.

1

**BCV: Back cross of F to 211-V.

1

iv

Page

18

23

23
26

26

27

34

36

41

42

43

44

45

Table Page
14. Results obtained for F2 . . . . . . . . . . . . . 46
15. X? for an expected 3:1 ratio in BCv . . . . . . . 51
16. X? for an expected 9:7 ratio in F2 . . . . . . . 51
17. Hypocotyl cortical cell lengths of Sanilac

and 211—V . . . . . . . . . . . . . . . . . . . . 57
18. Analysis of variance for hypocotyl cortical

cell lengths of Sanilac and 211-V . . . . . . . . 57

LIST OF FIGURES

Plate showing seedling growth of Sanilac and
le-v o o o o o o o a o o o o o o o o o o o 0

Graphs showing response of Sanilac and 211-V
to light intensity . . . . . . . . . . . . . .

Graph showing responses of Sanilac and 211-V
to day length . . . . . . . . . . . . . . . .

Graph showing magnitudes of difference
between Sanilac and 211-V for four day ,
and light treatments . . . . . . . . . . . . .

Plate illustrating growth of Sanilac and
211-V under high light intensity . . . . . . .

Plate illustrating growth of Sanilac and
le-V under reduced light intensity . . . . .

Photomicrograph of hypocotyl cortical cells
Of sanilac O O O O O O O O O O O O O O O O O C

Photomicrograph of hupocotyl cortical cells of
le-V o o o o o o o o o o o o o o o o o o o 0

vi

Page

24

24

24

29

3O

58

58

INTRODUCTION

Extensive and intensive studies have been carried out
to find ways to develop varieties that are high in yield and
quality, resistant to the most economically important plant
diseases, and more fitted to modern crop management purposes
and practices.

The present paper will not endeavor to set forth a list
of the tremendous and innumerable advances which have been
realized in field bean breeding and improvement in North
America or abroad during the past half century. It is merely
a modest contribution to the study of an important agronomic
character of two commercially grown varieties of Navy pea
beans (Phaseolus vulgaris) le-V and Sanilac, which have
directly or indirectly arisen from this invaluable crop
breeding and improvement program.

Released by a private plant breeder, 211-V is apparently
of mutational origin from the parental variety 211, which
is declared to result from a cross of Robust x Black African.
This new strain of Navy pea bean is a late maturing vine type,
susceptible to the common bean mosaic, and characterized by
its unusually elongated hypocOtyl and basal internodes. (1.)

The development of Sanilac has been briefly sketched
by Down and Andersen (3d. _ In 1940, an early bush mutant
segregated from a lot of Michelite bean seeds treated with

x-rays in 1938. This mutant was increased in size by back

crossing to Michelite followed by selection. The recovered
mutant strain was used in a cooperative bean breeding project
begun in 1948 between the Michigan Agricultural Experiment
Station (MAES) and the United States Department of Agriculture
(USDA). This research program has resulted in the introduction
of the bush type variety Sanilac to Michigan agriculture in
1956. Besides simiIarity with Michelite in seed type, good
canning quality, and resistance to common bean mosaic (V-I),
Sanilac is superior to its parent in a six day earliness and
resistance to thetl and 6» strains of the fungus causing
anthracnose. Being an erect bush type, it holds its upper
pods off the ground at harvest and offers mechanical resistance
to white mold disease of beans. It conspicuously differs
from the le-V by its short hypocotyl section. (Plate I)
Considering this last point, in spite of outstanding
agronomic features and its high position in the canning
trade (18 ) further improvement is being viewed in Sanilac to
bring its basal pods higher above ground level through
appropriate breeding techniques. Success in this respect
would result in the possibility of harvesting this crop by
combine without pulling and with the least damage to the
lower pods, and in a significant increase in its yield by
decreasing losses due to the cull material or "pick."

In the present investigation an attempt has been

3
made to study hypocotyl elongation of Sanilac and leAV under
different artificial environmental conditions, and to find
an explanation to the inheritance of this agronomic trait by
means of hybridization. Consideration also has been given to
cell division and cell expansion in the above—mentioned region

of seedlings of both strains of Navy pea beans.

.>IHHN 6cm umHHcmm mo mmcflapmmm masow .H m9<qm

 

 

REVIEW OF LITERATURE

As beans form an extremely important agricultural and
horticultural crop plant all over the world, the existence of
an extensive literature on the subject is quite natural. To
insure optimum conditions for growth, increase yield, and over-
come disease hazards, particular consideration has been devoted
to the study of environmental or genetic factors affecting
plant growth and development. As stated by Norton (1?) in
his experiments on beans, the greatest effect of environment
seems to be upon such characters as length of the plant axis
and probably the twining habit to some extent. These characters
and many other such as increased yield and canning quality are
related to the genetic make-up of the plant as genes on the
chromosome exert their action upon physiological and develop-
mental processes through a controlling effect on the synthesis

of enzymes.

A. Environmental Studies

Environment, by definition, is the "aggregate of all
the external conditions and influences affecting the life and
development of an organism." Among environmental factors
widely investigated, temperature and radiant energy have
received considerable attention. Although it is not intended
to present here a comprehensive review of literature on

responses of crops to temperature and to light intensity,

quality and duration, a brief discussion of some recent
investigations on the subject might be of some interest.

The effects of temperature, photoperiod and age upon
growth are reported by Viglierchio and Went ( 25 ) who found
that, in general, bean plants showed greater stem growth at the
higher temperatures. During the early stages the rate of stem
elongation is a function of night temperature, increasing with
the higher nycto temperatures. They also noticed in their
experiment that plants under long day condition grew faster
than those under short days. Their conclusions suggest that
production of photosynthates may have been limiting growth in
the short day regime.

Highkin ( £3 ) grew Pea plants (giggm_satiuum) under
conditions of constant temperature and artificial light or
in controlled-temperature greenhouses in which there was a
diurnally fluctuating temperature. The results of these
experiments demonstrated that the phenotype of the plant is
the summation of its genetic heritage together with the summation
of the parent and present environment, and that any
environmental effect is inversely proportional to the number
of generations by which that environment is removed from the
generations under consideration.

As reported by de Zeeuw and Leopold ( 27 ), exposure
of etiolated pea stem sections to ultraviolet light priOr to
auxin treatment inhibits the normal growth response preventing

auxin action inside the plants.

7
With the use of a series of wave bands of radiant energy

in the blue, red and far red, and wet weight of leaves, inhibition
of hypocotyl length and stimulation of epicotyl length were-
found by Klein §§_§l, ( 10 ) to be approximately proportional
to the log of the incident energy, with maximum effectiveness
in the red at 630.700 millimicrons (nap), which was markedly
more effective than the far red. They also noticed that the
increase in relative anthocyanin concentration in the bean
hypocotyl similarly was about prOportional to the 109 of
irradiance, with the red more effective than the blue and markedly
more so than the far red. As suggested in their conclusions,
the increased synthesis is coupled in some manner with growth
photoreactions. However, Tisdale and Nelson ( 23 L discussing
the effect of light quality on plant growth, remark that
the results of studies that have been made in this respect
On the whole indicate that the full spectrum of sunlight is

generally most satisfactory for plant growth.

B. Genetingtudies

Inheritance in beans has been investigated for a long
time and in a great extent but still there are many divergent
results that call for further studies to provide more reliable
bases for greater accuracy in the interpretation of observations.
Summary of the literature on Phaseolus vulgaris and breeding
has been presented by Ten Doorkaat-Koolman ( ll ), Fruwirth
( 11 ), and Matsuura ( ll ), cited by Kooiman ( ll ) and

Wade ( 26 ).

8

When classified as to type of vine, beans are referred
in the literature as "bushy" or "viny." A bush bean, as defined
by Wade ( 26 ), is a type in which the inflorescence is at
the tip of the plant; when it appears the plant stOps growing.
In a viny or pole bean, on the other hand, the flowers are
along the stem, which continues to grow indefinitely; its
ultimate length depends on environmental conditions.

To Emerson ( 44 ), pole and bush beans also differ in
height in consequence of the difference in habit of growth.

Bush beans, he comments, have a short axis in part because of
a small number of internodes and in part also because of a
small mean internode length, the latter being due to
termination of the plant axis in the period of growth rate
acceleration. Pole and bush types, then, are characterized
by difference in height, only in so far as height is dependent
upon determinate and indeterminate habits of growth.

Mendel ( 15 ) crossing tall and dwarf forms of beans
found that tallness is dominant to dwarfness and that F2
segregated in a 3:1 ratio of tall to dwarf. With similar crosses
the same results were obtained by McRostie, Tjebbes and Kooiman,
and Doornkaat-Koolman, and quoted by Wade ( 26 ).

Dealing with the determinate versus the indeterminate
habit of growth as a separate character instead of limiting his
investigation to the general character of tall versus short
habit of growth, Emerson ( ‘4 ) shed more light on the subject.

He showed that there are three factors involved in plant height,

namely:

1. determinate versus indeterminate habit of growth;

2. number of internodes, which in pole beans nevertheless
depends largely on environmental conditions;

3. internode length, which is greater in the hypocotyl
than the epicotyl which in turn is longer than the second
internode.

Indeterminate habit of growth is found to be fully dominant to
determinate habit. Sharp segregation occurs in F2 resulting
in a 3:1 ratio of indeterminate to determinate habit. This
recessive trait is constant in F while some indeterminate F2

3

plants breed true in F and others segregate again into pole

3
and bush beans. Difference in habit of growth, therefore,

appears to be a unit character. This opinion is also held

by Sax ( 21 ) and Makarem ( 14 ) reporting Hilpert's

conclusion on the subject. Emerson also observed that while
number of internodes is directly, and internode length indirectly
related to habit of growth, these characters are in a way distinct
from it. There exist distinct types of bush beans and distinct
types of pole beans with respect to both number of internodes

and internode length. Crossing varieties of bush beans with
different internode lengths seems to result in an intermediate

condition in F1, and F and a wide range of variation in the

2!
latter. This was interpreted by Emerson in accordance with
the multiple factor hypothesis which postulates that hereditary

quantitative differences are due to two or more non-dominant,

10
independently inherited factors.

Norton ( 17 ) asserts that the habit of all the varieties
of beans can be accounted for easily with only three characters:
A, L and T. "A" designates the presence of axial inflorescence
permitting an indefinite growth Of the main axis and main
branches; its recessive counterpart "a" stands for a terminal
inflorescence causing definite growth; "L" the length of axis,
an important factor controlling plant habit and probably
governed by a series of two or more factors which behave after
the fashion of Emerson's hypothesis for the inheritance of
quantitative characters; "T" the climbing habit, is due to a
factor for circumnutation. The cause of the various degrees
of the climbing habit has not been determined with any degree
of certainty, while the contorted stems of erect bush forms
are thought to be probably caused by T. Furthermore it is
assumed that A, L and T may be present in any possible
combination giving rise to the various habit types of beans.

Later Lamprecht ( 12 ) proposed ”Fin" to replace the
symbol A used by Norton. He also replaced T by Tor to describe
the twining habit. Analyzing results obtained from a cross
between a tall type of Phaseolus vulgaris and a slender type,
which differs from the latter by its excessively long internodes,
Lamprecht reached the conclusion that infinite "Fin," long
"L," climbing axis "Tor" are respectively dominant to finite
growth of the axis ”fin," short internodes "1'' and not

climbing axis "tor."

11

He also reported results of crosses made by de Haan
(1927) and Rasmusson (1927) between certain families of dwarf
peas. Simultaneously two ratios of 15:1 and 3:1 dwarfs to
cryptodwarfs were computed in this case. Two symbols
"Cryl" and "Cryz" (Rasmusson) or "La" and "Lb" (de Haan)
were assigned to be responsible for the dwarf-cryptodwarf
character. Besides, a cross of dwarf x dwarf types of Phaseolus
vulgaris made by Lamprecht (.12 ) yielded a ratio of 15:1
dwarfs to slender. Such a result caused him to infer that the
slender type is caused by recesSivity in two genes for the length
of the internodes "Cry" and "La," each in dominant condition
preventing the length growth of the internodes. Moreover,
he pointed out that for Other characters, such as pods, there
also seems to exist in Phaseolus vulgaris,as it is in giggm_
sativum, a number of polymeric genes which as "Cry" and "La"
may be taken as an evidence that Phaseolus vulgaris has arisen
from a tetraploid ancestor by loss of one or more chromosomes.

The foregoing discussion substantiates the statement
that genetics of beans is quite complicated and that penetrating
investigations are required to find an answer to many intriguing

and important questions.

C. Developmental Studies

Whether due to environmental or to genetic influences,
phenotypic dissemblances between or within species may evolve from

striking differences in the anatomical features of the living

12
organisms under comparison. These differences may be related
to cell division, to cell expansion, or to both. Ecological
factors such as light and temperature, and certain natural or
synthetic substances such as gibberellin and 2-4,D are known
to affect plant growth and development

A very abundant literature is available on the study of
cell responses to various treatments with light and growth
hormones. Without any attempt to cover this broad material
in its entirety, a few reports of recent investigations on the
subject might be worthy of being cited here.

Humphries and Wheeler ( 9 ) working with Kinetin,
Gibberellic acid, and light on expansion and cell division in
leaf disks of dwarf beans (Phaseolus vulgaris), found that Kinetin
increased leaf expansion in the dark wholly by increasing cell
size. This effect was eliminated by light. Gibberellic acid
also increased cell size and cell division in the dark but not
in the light. Light similarly increased cell division and
cell size, and although it eliminated the effect of gibberellin
on cell division, it enhanced the action of that hormone on cell
expansion. The authors brought no clear evidence that light
acts by influencing concentration of Kinetin or gibberellin
in the leaf but assumed that both substances depress cell
division in presence of light.

To Feutcht and Watson ( £3 ), application of 20 milligrams
of gibberellin on seedlings of Phaseolus vulgaris not only

appeared to increase the length of the internodes of the seedlings

13
but seemed also to augment the number and length of cells.
In spite of the substantial increase in cell numbers, cell
elongation was assumed to be primarily responsible for the
increase in internode length.

Conclusions presented later by Greulach and Haesloop (7)
on a similar experiment disagree in some extent with Feutcht's
assumptions. Growth promotion by Gibberellic acid, as viewed
by Greulach and Haesloop, involved only cell division and not
cell elongation, and in the pith the growth hormone may have
also influenced the plane of cell division. These authors even
suggested that earlier conclusions that Gibberellic acid growth
promotion is principally due to cell enlargement must be

revised.

SECTION I

14

15
ENVIRONMENTAL STUDIES

Experiment I

Mategials and Methods

Among the commercially grown variety of Navy pea beans,
le-V is conspicuous under certain conditions by its very
long hypocotyl section, which is very short in Sanilac on a
comparative basis. To study the extent in which certain
environmental conditions may affect this agronomic trait the
following experiment was set up.

In the fall of 1959, foundation seeds of Sanilac and
211-V were sown in the Plant Science greenhouses of Michigan
State University, at East Lansing, Michigan. Eight flats filled
with steamed sand were planted with 40 seeds of each variety
in five rows of eight seeds each. Rows were four inches
apart and with a distance of two inches between seeds in
the row. The sand in the flats was leveled with a specially
built wooden device and great care was provided to place seeds
practically at a uniform depth of one inch to insure identical
bedding conditions for uniform germination. After each watering
and application of liquid fertilizer, the sand in the flats
was maintained at the original level by adding small quantities
of wet sand to the flat and working the surface by hand.

Four reciprocal treatments were applied to the materials,
namely:

1. Long day-—high light intensity.

l6
2. Long day——reduced light intensity.
3. Short day--high light intensity.
4. Short day-—reduced light intensity,
so that there were two groups of day length split into two
light intensities. Each treatment comprised a set of two flats
with the variety arranged in.semi-a1ternate fashion between
consecutive flats within a day—group. Varietal arrangement was
reversed between groups.
Long day refers to a lighting period of 15 hOurs
and short day to a 9 hour period. Two parallelepipedic
wooden frames covered with dark brown and perforated cloth on
all but one face provided shading to material under reduced
light treatment. Similar boxes wrapped in black and solid
cloth were used on the materials placed under short day
conditions, after the 9 hour period of exposure to light was
attained. Materials under long day, high light treatment were
kept unshaded throughout the experiment. Day lighting period
in the fall for Michigan more or less lasts 9 hours, the sun
rising at about eight o'clock in the morning and setting at
about five o'clock in the evening. To extend light exposure
to 15 consecutive hours, eight 1250 foot candle bulbs were
mounted on a circuit switched to an automatic clock timer that
turned on the lights at 5 p.m. and cut them off at 11 p.m.
Lights were kept on during dark days to supplement the normal
day light period of 9 hours.

For the sake of convenience, 22 days after emergence

17
of the seedlings, the hypocotyl section was considered to have
reached full development. The plants were then pulled up and
measurements of their hypocotyl taken in half centimeters
with a graduated ruler. Abnormal seedlings were discarded,
and to deal with a same number of individuals in each treatment,
a random sample of 25 seedlings of each variety in each flat
was selected and mean hypocotyl lengths of each variety in

each treatment calculated on a basis of 50 measurements.

Experimental Results and Discussion

Plate II and Plate III illustrate typical behaviors of
both Sanilac and 211-V under full light and reduced light
conditions for a lehour period of illumination corresponding
to the long day treatments. Actual measurements and computed
average hypocotyl lengths in this first experiment are recorded
in Table 1.

From a mean length of 5.00 cms under long day and
5.14 cms under short day treatments with high light intensity
applied, the hypocotyl section of Sanilac reached an average
height of 7.76 cms under long day and of 8.17 cms under short
day treatments when light intensity is reduced by shading.

Exposed to full light cOndition, 211-V decreases from
an average hypocotyl length of 11.22 cms in the long day to a
mean length value of 9.52 cms in the short day regimes. Under
reduced light treatment this variety still decreases with day

length, shifting from an average hypocotyl length of 12.87 cms

18

 

 

 

 

 

 

 

NN.HH 00.0 mammz
0.NH 0.0a 0.HH 0.0a 0.0a 0.0 0.¢ 0.v 0.¢ 0.0
0.0a 0.HH 0.HH 0.HH 0.NH 0.0 0.0 0.¢ 0.¢ 0.0
0.0a 0.0a 0.0a 0.0a 0.0a 0.0 0.0 0.0 0.0 0.0
0.HH 0.HH 0.NH 0.0a 0.HH 0.0 0.v 0.0 0.0 0.0
0.HH 0.NH 0.HH 0.0a 0.HH 0.0 0.¢ 0.0 0.0 0.v N
0.HH 0.0a 0.HH 0.0a 0.HH 0.0 0.0 0.? 0.0 0.0
0.0a 0.HH 0.0a 0.NH 0.NH 0.0 0.0 0.0 0.0 0.0
0.HH 0.0a 0.NH 0.NH 0.HH 0.0 0.0 0.0 0.0 0.0
0.0a 0.HH 0.0a 0.0a 0.NH 0.0 0.v 0.0 0.v 0.0
0.0a 0.NH 0.0a 0.HH 0.HH 0.0 0.0 0.0 0.0 0.0 H umHm
>IHHN omaflcmm
.mucwfiummnu How >IHHN 0cm OMHHsmm mo aumcma hauooomhm .H usmfiflnwmxm .H mamfie

l9

 

 

 

 

5m.NH 5H.m Com:
0.NH 0.NH 0.MH 0.¢H 0.¢H 0.0 0.0 0.5 0.5 0.0a
0.¢H 0.NH 0.0a 0.0a 0.mH 0.0 0.0 0.5 0.0 0.0
0.0a 0.HH 0.NH 0.HH 0.0a 0.5 0.5 0.0 0.0 0.0
0.NH 0.0a 0.HH 0.0a 0.NH 0.0 0.5 0.0 0.0 0.0
0.NH 0.0a 0.NH 0.HH 0.NH 0.5 0.5 0.0 0.0 0.0a
0.NH 0.NH 0.HH 0.0a 0.?H 0.0a 0.5 0.5 0.0 0.0
0.HH 0.NH 0.0a 0.0a 0.¢H 0.0 0.0 0.0 0.0 0.5
0.0a moaa 0.¢H 0.NH 0.0a 0.m 0.5 0.0 0.0 0.0a
0.HH 0.HH 0.NH 0.0a 0.va 0.5 0.5 0.0 0.0 0.0a
0.NH 0.MH 0.vH 0.NH 0.NH 0.5 0.0 0.0 0.0 0.0a H umam
>IHHN UMHHGmm

 

Mam: omosswmuémc anon

 

 

 

©05GHDGOU H aflmdfi

20

 

 

 

 

N0.0 fia.0 mcwmz
0.0 0.HH 0.0 0.0 0.0 0.¢ 0.¢ 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.¢ 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.¢ 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.v 0.0 N
0.0 0.HH 0.0 0.0 0.0a 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.HH 0.0 0.¢ 0.0 0.¢ 0.0 0.0
0.0 0.0a 0.0 0.HH 0.HH 0.0 0.0 0.0 0.0 0.0
0.0 0.0a 0.0a 0.0 0.0a 0.0 0.0 0.0 0.0 0.0
0.0 0.0a 0.0 0.0a 0.HH 0.0 0.0 0.0 0.0 0.0 H umHm
>IHHN UMHHcmm

 

 

 

 

smssflusoo H.mqm¢e

21

 

 

 

 

v0.ma 05.5 mommz
0.HH 0.HH 0.ma 0.HH 0.HH 0.5 0.5 0.5 0.5 0.0
0.NH 0.NH 0.mH 0.NH 0.ma 0.5 0.5 0.0 0.0 0.5
0.ma 0.HH 0.HH 0.HH 0.0a 0.5 0.5 0.5 0.0 0.0
0.HH 0.¢H 0.HH 0.HH 0.NH 0.5 0.0 0.5 0.5 0.0
0.0a 0.0a 0.HH 0.NH 0.va 0.0 0.5 0.5 0.0 0.0 m
0.ma 0.~H 0.ma 0.NH 0.NH 0.5 0.5 0.5 0.0 0.0
0.~H 0.0a 0.NH 0.~H 0.6a 0.0 0.5 0.0 0.0 0.5
0.NH 0.NH 0.NH 0.NH 0.mH 0.5 0.0 0.0 0.0 0.0
0.NH 0.NH 0.NH 0.¢H 0.0a 0.0 0.5 0.5 0.0 0.0
0.NH 0.ma 0.NH 0.ma 0.0a 0.5 0.5 0.0 0.5 0.5 H umHm
>IHHN omaaamm

 

 

 

 

vmficflucoo H aflmdB

22

in long day condition to a corresponding value of 12.64 cms
under short day treatment.

Analyzing these observations, reduced light intensity
seems to have a greater effect than full light in increasing
the hypocotyl section of either variety. Actually as
indicated by analyses of variance set up in Table 2 and Table
3, differences due to light intensity are highly significant
in both cases. Variations of Sanilac and 211-V in relation
to light treatment are plotted in Figure 2.

These results though striking are not surprising
and were rather normally expected as it is generally observed
and reported that leaf size and stem elongation of many
species are at a maximum at low light intensities.i
Most plants when shaded are taller and thinner than when
exposed to full light. Etiolation of seedlings in crowded
stand is a very common phenomenon.

Considering effect of photoperiodism on Sanilac
and 211-V with respect totheir hypocotyl elongation, it can
be easily seen in Figure 3 that while 211—V constantly
decreases when passing from long day to short day conditions,
Sanilac on the other hand, displays greater hypocotyl lengths
in short day than in long day when submitted to high light
intensities but decreases from long day to short day when

under reduced light intensity. As it is revealed

23

TABLE 2. Analysis of variance of hypocotyl elongation of
Sanilac under four day and light treatments.

 

 

Source of Variation D.F.
Total 199
Day 1
Treatments Light 1
D x L 1
Error 196

S. Sq.
-522.69
.91
419.05
3.78

98.94

M. Sq. F:
.91 1.82
419.05 838.10**
3.78 7.56**
.50

 

**Significant at the 1%»level.

 

TABLE 3.

Analysis of variance of hypocotyl elongation of

le-V under four day and light treatments.

 

 

Source of Variation D.F.
Total 199
Day 1
Treatments Light 1
D x L 1
Error. 196

S.'Sq.
531.47
46.56
284.41
27.01

173.49

M. Sq. F.
46.56 52.90**
284.41 323.19**
27.01 30.69**
.88

 

**Significant at the 1%»1evel.

 

EXPERIMENT

I 24

Hypocotyl Elongation of Sanilac and 211-V

 

 

 

 

 

 

 

 

Treatments

Figure 2 Figure 3
Response to light intensity Response to day length
14 -- 14 ._
. ,’\ / /~'—‘ "‘
12 of I I \\ // 12 j“ /
'l/ \ / //
\ \
m /' \ /
"G 10 -- \\ / 10 ~r- \\ /
3‘ v’ \\/
C‘.
.33
8 “- 8 4r-
0
01
E
g 6-~ 6i-
0
4~P 4._
Sanilac Sanilac
2 ._ 2 __
——-— 211-V —- —- 211-V
O J l L i O s 5 9 ALL
(L)H (L) R (S)H (S)R "L(H) S (H) L(R) S (R)
Treatments Treatments
Figure 4
7'F Mean Differences for Treatments
6 -_ \‘
\\
5 ~— \.
\- ,/"
.Ll-“u p,,
m V
5 4 ._
c
-H
.C 3*-
D
01
5
A 24-
1 .—
0 I l l l
LH LR sH s‘R

25

by t-tests in Table 4 and Table 5, there is no
indication that day length affects hypocotyl growth rate of
Sanilac while 211-V fails to manifest consistent responses
in this sense. The latter strain of Navy bean appears to
be indifferent to duration of exposure to light under shading
but shows a significantly higher hypocotyl average length in
long day than in short day when full light conditions are
prevailing.

A curve plotting differences between varieties for
each type of day-light treatment is made available in Figure 4.
An analysis of variance for the whole experiment clearly indicates
in Table 6 that there are highly significant differences
between mean hypocotyl lengths of Sanilac and 211-V for treat-
ments and using the Student Range Method to test differences
among means, it has been found that any mean value of le-V is
always significantly superior to the corresponding mean value

of Sanilac for any given treatment.

Summary and Conclusions

By testing variations in hypocotyl lengths of Sanilac
and 211-V for significant differences between their relative
mean values under the various treatments applied, it has been
deducted that both varieties develop a higher hypocotyl section
with reduced light intensity than under full light condition.

The bush variety shows little change with respect to

length of day whereas the vine type behaves rather inconsistently,

26

TABLE 4. A t-test of effect of day length treatment upon

 

 

 

 

 

 

 

 

Sanilac. j
_ 4647.75 - 4336.22 _
SL ‘ w ’ V 99 ‘ 3'14
_ 370.50 - 4160.25
SS _ '\/ 99 T — V2.12
S_ ___ V2.12. = ‘Vi-ii
L 100 10
S- = V2.12 = 2.12
S 100 10
3.14 + 2.12 _
SE ,- 's') ’ V 100 ’ '23
t — 6'58 “16'45 = .56 N.S. (3)
.23
SL = Standard deviation for long day.
SS = Standard deviation for short day.
N.S. =- Not significant.

 

j

 

TABLE 5. A t-test of effect day length treatment on 211-V.

-
4

 

 

 

 

 

 

 

 

 

S V14672.25 - 14508.20 ___ V1733
L 99
_ 12597.50 - 12276.64 _

SS - '\/ 99 — 'V3.24
S— = V1.66 = 1.66
L 100 10
S- = 'V3.24 = V3.24
S 100 10
_ 1.66 + 32;4 _

S(L - §) ‘ V 100 ’ '22
t = 12:04 - 11.08 = 4.36**
.22
SL = Standard deviation for long day.
SS = Standard deviation for short day

** Significant at the 1% level.

 

27

TABLE 6. General analysis of variance of Sanilac and 211-V
under different treatments of day length and light

 

 

 

intensity.

Source of Variation D.F. S.Sq. .M.Sq F.
Total 399 3599.36

Day length (D) 1 30.25 30.25 21.92**(1)
Treatments Light intensity (L) 1 696.96 696.96 505.04**

D x L l 5.29 5.29 3.83
Varieties (V) 1 2545.20 2545.20 l844.35**
D x V l 17.22 17.22 12.48**
L x V 1 6.50 6.50 4.71
D x L x V 1 25.51 25.51 18.48**
Error within 8 11.03 1.38
Other errors 384 261.40

 

ANNEX TO TABLE 6. Student range test for hypocotyl means
of Sanilac and 211-V.

 

 

 

S.D. of a treatment average: ST = léég- = .83
Significant studentized ranges for d. = 8 (P/NZ)
[2] 4.74 x .83 = 3.93

Treatments Variety Means Difference
Long day high light 211-V - Sanilac 11.22-5.00 6.22**
Long day reduced light 211-V - Sanilac 12.87-8.17 4.70**
Short day high light 211-V - Sanilac 9.52-5.14 4.38**
Short day reduced 1ight21l-V - Sanilac 12.64-7.76 4.88**(l)

 

(l)**Significant at the 1% level.

 

28
revealing a Significantly greater hypocotyl mean height during
long day than during short day when maintained under full light
exposure but standing practically unaffected by photoperiodicity
when shaded.

Comparing hypocotyl elongation of these two varieties
for response to combined effect of day length and light
intensity, greater difference between their mean hypocotyl
length has been noticed to occur under long day high light
intensity than under any one of the three remaining combinations.
Nevertheless, throughout the experiment hypocotyl mean sections
of 211-V have never failed to be significantly longer than
that of Sanilac irrespectively of the treatment considered.

This observation may suggest that besides environmental influences
and more than them intrinsic factors are responsible for this
tangible and consistent difference in hypocotyl growth rate

of these two varieties.

29

.usmfiummnu

sunmcmucfl usmfla Hash >66 maoH
Hwocs >IHHN mo mmcfiapwmm 0:505

m

 

 

.ucmsummuu muflmcwusfl unmfla
Hadm >00 mCOH Hows: undecmm
mo mmcflasmmm annoy .HH mumHm .m mmoon

¢

    
  

- 5.2:... 13 _E
- I

3O

.ucosummnp
wpflmcmusfl unmfla omosowu hop
mood Hwocs >Iaam mo mmcflapmmm mono»

 

.ucoEummHu
huflmcmucfl pnmwa 000500“
woo chH Amoco OMHflcmm mo
mmcHHUomm mono» .HHH mpmam

.0 MMDOHm

 

SECTION II

31

32

GENETIC STUDIES, EXPERIMENT II, PART I

Materials and Methods

In 1959 crosses between Sanilac and 211-V were
made and the seeds obtained from such crosses preserved for
genetic studies. In March, 1960, an experiment to compare

growth habit of F seedlings to that of the parents was

1
undertaken. Eight pots filled with steamed sand and organic
matter received each: four Fl seeds and two seeds of either
parental varieties under no special environmental condition

in the greenhouse room. Liquid fertilizer was supplied twice

to the pots. When seedlings had achieved a fairly advanced
stage of development their hypocotyl section was measured

in a single operation with a ruler graduated in half-centimeters.
Then each plant was supplied with an iron wire to allow

twining and keep them from entanglement. Care was provided

in handling materials to avoid damage as further crosses

and harvest were planned. Beans are somewhat difficult to
hand-pollinate since the curled and brittle style of the

flowers is easily broken during the process of opening the

keel. Wade (.26 ) states that the time required to make

crosses has prevented genetic studies in Phaseolus vulgaris
involving back-crosses. Nevertheless, remarked he, keeping
atmosphere near saturation point a few days after artificial
pollination has been effected may yield fairly successful
crosses.

To produce F seeds, Sanilac was used as

1

33
the maternal parent and 211—V as pollen donor. BaCk crosses
to Sanilac in the present studies are termed BCS and to
211-V, BCV.

Crosses were made by selecting flowers of the female
parent before eclosion to minimize possibility of selfing.
Emasculation was effected by pinching the base of the flower
and forcing the reproductory organs to protrude and, using
a pair of fine tweezers, carefully driving out the anthers.
Stamens, together with the pistil of the male parent, were
picked up with tweezers and rubbed onto the stigma of the
emasculated flower. Only flowers with mature viable pollen
were chosen for fertilization. Pollination was carried
out in the early morning with a high moisture level in the
atmosphere of the greenhouse room. Tags were placed around
the stem of the pollinated flower to indicate the parents
involved in the cross and the date of the operation. The
female parent was always mentioned first on the tag. Hybrids
and selfed seeds harvested from this experiment were

saved for further studies.

Experimental Results

Measurements taken on the seedlings and average hypocotyl
lengths of each type of plant in the experiment are reported

in Table 7. Three seedlings in the F group upon development

1

34

TABLE 7. Experiment 11 (a). Genetic'Study.
Hypocotyl lengths of Sanilac, 211-V, and F .

 

 

 

 

 

 

 

 

 

 

 

 

1
Entries Sanilac Fl 211-V

1* 10.0 10.0 14.0
10.0 8.0 14.0

8.5

8.5
2 5.5 8.5 14.5
6.0 9.0 14.0

10.0
3 6.0 8.0 14.0
7.0 9.5 13.5

7.5

8.5
4* 10.5 10.5 13.5
9.5 8.0 13.5

10.0

10.5'

5* 8.5 10.5 14.0
11.5 10.5 14.5

10.5

11.5
6 7.5 9.0 14.0
7.5 8.0 14.5

8.5

7.5
7 6.0 14.0
5.0 9.0 14.0

9.5
8 7.5 8.0 14.5
6.5 9.0 14.0

8.5

7.5
Total i124.5 262.5 224.5
Means 7.78 9.05 14.03

 

*Plots placed very close to hot pipes in the greenhouse.

35
turned out to be accidental selfed plants and were disregarded
in computing average values of either group. Averages obtained

were 9.05 cms for F 7.78 cms for Sanilac, and 14.03 cms for

1!
le-V.

All the true F plants showed the viny character of

l
le-V but indicated a mean hypocotyl height closer to that
of Sanilac than to that of 211-V. Crosses and back crosses of
F1 to either parent were satisfactorily successful and yield
obtained from each group of plants was abundant and supplied
enough material for the next experiments.

An analysis of variance carried out in Table 8, reveals
that there is highly significant difference among hypocotyl
mean lengths of the three classes in this experiment. Using the
Student Range Method for testing significance among average
measurements it has been obtained, as shown in the Annex Table
to Table 8, that while hypocotyl mean length of 211-V is
significantly greater than that of either Sanilac or F1, there
is no significant difference between the bush parents and F1
with regard to their average hypocotyl growth rate.

Plants growing in pots placed near the heating pipes
on the benches.h1the greenhouse room have been found to elongate

more than those grown in pots located farther from the heat

source .

36

TABLE 8. Analysis of Variance for hypocotyl elongation of

 

 

Sanilac, F1, 211-V.
Source of Variation D.F. SS M.sq F
Total 60 460.25
Between classes 2 95.38 47.69 7.58**
Error 58 364.87 6.29

 

**Significant at the 1% level.

 

 

m

ANNEX TO TABLE 8. Student Range Test for hypocotyl means of
Sanilac, F1, 211-V.

 

 

l 1363

N0 = 5' (61 ~ ‘_6I—) = 19.41

S.D. of a class average = {%f%% = .57

Means of classes 1 : 2 : 3
211—V Fl Sanilac
14.03 9.05 7.78

Significant Studentized Ranges at the 1% level with 58. d.f.

[2] 3.76 x .57 = 2.14

[3] 3.92 x .57 = 2.23

Comparisons Respective means Differences
F1 - Sanilac 9.05 - 7.78 1.27 N.S.
211-V - Fl 14.03 - 9.05 4.98**
211-V - Sanilac 14.03 - 7.78 6.25**

 

N.S. Not significant.
**Significant at the 1% level.

 

 

37

Discussion of Results

Inheritance of the character "internode length" and
specially hypocotyl length in common beans is less strongly
established than that of the agronomic character "determinate
versus indeterminate" type of growth, another component of
plant height. Experimental papers on the subject, such as
those published by Emerson (4), Norton (1?), and others have

reported that F plants of crosses between parents of different

1
internode lengths are of an intermediate condition. Emerson
has adopted the multiple factor hypothesis to explain his
findings. However, there is no complete agreement as to the
number of genes responsible for the transmission of the trait.
Lamprecht (12), backing Rasmusson and de Haangs conclusions,
stated that long internode in beans (Phaseolus vulgaris) is due
to recessivity in two genes for the length of the internode,
each in dominant condition preventing the length growth of the
internode. Rasmusson and de Haan (1927) have respectively
suggested the symbols Cryl, Cryz, and La, Lb to designate these
factors, which later were represented as Cry La by Lamprecht
(12). None of the reports consulted in the review of literature
for the preparation of the present paper offers special mention
to the hypocotyl section of the plant, which is not considered

as a true internode by many plant anatomists, as pointed out by

Esau (5).

38

As it happens that there is no significant difference
between Sanilac and F1 in their respective hypocotyl mean
heights while both are significantly shorter than 211-V, and
taking into account the slight but appreciable advantage of
F1 to Sanilac in average hypocotyl length growth, it could
be assumed that short hypocotyl is partially dominant to
the long hypocotyl trait. This slight difference between

mean lengths of Sanilac and F could also be due to hybrid

l
vigor in the latter. So far the hypothesis of non-dominance,
which would result in an intermediate condition in the first
generation of plants may be disregarded. At this stage of
the study, however, it is too early to assign any specific
number of genes as responsible for the transmission of this
character. Subsequent generations of selfing of the hybrids

and appropriate back crossings may lead to more information

and more accuracy in this respect.

Summary

Crosses between Sanilac, a bush bean with short hypocotyl
and 211-V, a viny type with long hypocotyl, have resulted in
viny Fl plants which affected a mean hypocotyl length very close
to that of the short internode parent. This and other pertinent
considerations call for culturing subsequent generations

of selfed hybrid and back crossed plants to test the possibility

39
of a partial or complete dominance of short hypocotyl to long
hypocotyl section in Phaseolus vulgaris. No evidence is found
to sustain a non-dominance hypothesis. At this stage of the
study no specific number of factors can be assigned as
responsible for the inheritance of hypocotyl elongation in

common beans.

EXPERIMENT II, PART II

Materials and Methods

 

In late spring, 1960, seeds of Sanilac, 211-V, Fl'

BCS, and BCV were sown in ten flats similar to those used in
the experiment on environmental studies the preceding fall.
Each‘flat was furrowed into eight rows, containing eight seeds

each. There were 15 rows of F 10 of each parent, and

2'

five of F BCS, and BCV, respectively. The parent variety

1’
stood for check materials. Even level of sand in the flats
was realized and same depth of planting observed to insure
uniform germination. When satisfactorily developed, all
seedlings were measured the same day for hypocotyl elongation.
Afterwards F2 and back cross seedlings were retransplanted in
large pots filled with sand and organic matter so that

distinction could be made among segregates as to the bushy

or viny types of growth.

40

Experimental Results

Measurements were taken for 80 seedlings of Sanilac,

80 of 211-V, 26 of F 39 of back cross of F to Sanilac,

1’ 1
40 of back cross of F1 to 211-V, and 117 of F2 seedlings.
Some bushy plants were found in the F1 lot. Possibly they

might have arisen from selfed seeds during the process of
hybridization and consequently were disregarded as pertaining
to the F1 group. Experimental results are shown in Table 9
through Table 14. For each group of plants hypocotyl lengths
are distributed in classes of one cm of interval and their mean
values computed on the basis of actual measurements. The
following averages were calculated in accordance with this
procedure: 5.66 cms for Sanilac, 12.91 for 211-V, 7.03 for

F1' 6.14 for BCS, 11.61 for BCV, 8.58 for F2. Standard
deviations were very small in the case of Sanilac and F1'
being smaller than 1 cm. It was relatively high for BCV,

reaching nearly 3 cms.

Discussion of Results

 

A consideration of the mean hypocotyl lengths together
with the appropriate standard deviations for Sanilac and
211-V in relation to the mean elongation growth of the hypocotyl
of the F1 progeny, may bring out some indication regarding
the mode of inheritance involved in the transmission of this

trait.

By its mean value, the F1 generation is closer to the

41

TABLE 9. Experiment II (b). Hypocotyl Elongation of Sanilac.

 

 

1. 4 0 6.0 6.5 6.0 6.0 6.0 4.5 4.0
2 4 5 5 5 6 5 7 5 5.0 5 0 4 0 4 0
3 5.0 5.0 6.5 7 5 5.0 6 0 5.0 5 O
4. 4 O 5 5 7 5 7.0 6.0 6 5 6.0 7 O
5 4 5 5 5 6 5 6.5 5 5 5 5 6 5 5.0
6. 4 O 5.5 6 O 6.5 5 5 4.0 5.5 5.0
7 5.0 7.0 6.0 6 5 5 5 6.0 6.0 5.5
8 5.0 6.0 6.0 5.5 7.0 4.5 7.0 5.5
9. 7.0 5.0 6.0 6 5 6.0- 6.0 6.0 4 0
10. 6.5 4 5 6.5 7 5 4.0 5.0 6.5 5 0
Mean E. = 5 66
S.D. = .96385
Distribution
Class Frequency
3.5 - 4.4 9
4.5 - 5.4 19
5.5 - 6.4 29
6.5 - 7.4 19
7.5 - 8.4 4

 

Total 80

 

. 42

TABLE 10. Experiment 11 (b). Hypocotyl Elongation of 211-V.

 

 

1. 11.5 13.0 11.0 14.0 15.5 14.0- 14.0 13.51
2. 11.0 12.0 14.0 12.0‘ 12.0 10.5 14.0 14.0
3. 10.5 12.5 12.0 13.5' 10.5- 13.0 14.0 13.5
4. 11.5 13.0 15.0 15.0 10.5 14.5 14.5 11.0
5. 11.0 12.0 13.0 13.5 12.0 14.0 13.0 11.0'
6. 10.5 13.0 14.0 15.0 11.0 14.0 11.0- 15.5
7. 11.0 15.5 14.0 15.0 15.5 13.5. 13.0“ 11.5
8. 12.5 13.0 14.0 15.0 11.0‘ 14.0 12.5“ 12.0
9. 14.0 14.5 11.0 12.5‘ 14.5- 11.5 11.5' 10.5

10. 13.0 14.5 12.0 15.0 14.0 13.5 11.0“ 13.0-

Mean—X = 12.91
S.D. = 1.4967
Distribution
Class Frequency

10.5 - 11.4 17
11.5 - 12.4 13
12.5 - 13.4 14
13.5 - 14.4 21
14.5 - 15.4 11
15.5 - 16.4 4

Total 80

 

TABLE

43

11. Experiment II (b). Hypocotyl Elongation of F

 

 

1'
F1
1 8.5 8 0 6 5 7.5 6 5 7 0
2 6.0 8.0 2.0 5 5 6.5
3 6.0 6 5 8.5 8 5 6 5
4. 7 5 7 0 7.0 6 5 7 5
5. 7 5 7 5 5.5 7.0 7 0
Mean X = 7.03
S.D = .847
Distribution
Class Frequency
5.5 - 6.4 4
6.5 - 7.4 12
7.5 - 8.4 7
8.5 - 9.4 3

Total 26

 

TABLE 12. Hypocotyl Elongation of BCs'

 

 

BCS
l 5 5 7 0 8.0 6.0 6 0 6 O 7 0
2 7 0 8 0 6 5 6.0 6 5 4.5 5 5
3 5 O 7 0 4 5 4.0 6 0 7.0 6 O
4 4.0 6.5 7.0 6.0 4.0 7 0 7.0
5. 8 5 7 0 6 O 5.0 6.0 8.0 7.5
Mean X = 6.14
S D = 1.17
Distribution
Class Frequency
3.5 - 4.0 3
4.5 - 5.4 6
5 5 - 6.4 13
6.5 - 7.4 12
7.5 - 8.4 4
8.5 — 9.4 1

Total 39

 

45

 

 

TABLE 13. Experiment II (b). Hypocotyl Elongation of BCV.
1. 10.0 10.0 14.5 7.0 12.0 13.0 13. 12.
2. 11.0 6.5 13.5 12.5 14.5 13.0 14. 13.
3. 12.5 11.5 14.0 14.5 13.5 6.0 7. ll.
4. 14.0 12.0 7.0 15.0 15.5 11.0 5. l4.
5. 13.0 7.0 11.0 14.5 7.0 14.5 14. 8.
Mean X. = 11.61
= 2.9343
Distribution
Class Frequency
5.5 - 6.4 2
6.5 - 7.4 5
7.5 - 8.4 2
8.5 - 9.4 0
9.5 -10.4 2
10.5 -1l.4 4
11.5 ~12.4 4
12.5 ~13.4 5
13.5 -l4.4 8
14.5 -15.4 7
15.5 -16.4 1
Total 40

Ratio

9:

31

 

 

46

 

 

TABLE 14. Hypocotyl elongation of F2.

1. 7.0 7.0 11.0 8.5 5.5 8.0 14.0
2. 6.0 12.5 7.0 11.5 7.5 6.5 7.0
3. 11.0 7.0 7.0 13.0 8.0 12.5 14.0
4. 5.0 7.0 7.0 8.5 10.5 7.0 8.0
5. 7.0 8.5 7.0 7.5 5.5 7.0 13.0
6. 7.5 6.0 7.0 7.0 6.0 7.0 13.0
7. 10.0 13.5 7.0 4.0 12.5 6.0 6.0
8. 6.0 4.0 7.0 12.0 6.5 6.0 8.5
9. 6.0 8.5 10.0 9.0 9.0 12.0 6.5
10. 13.0 4.0 10.0 6.5 6.0 10.0 6.5
11. 5.0 4.5 12.5 12.0 9.0 12.5 5.0
12. 5.0 11.5 13.0 9.5 7.0 6.0 9.0
13. 5.0 5.5 13.0 5.5 7.0 12.5 7.5
14. 6.0 4.0 12.5 6.0 7.0 7.5 7.5
15. 4.0 10.0 4.0 7.0 5.0 5.5 14.0
16. 5.5 10.5 12.0 7.0 6.5 4.0 10.0
17. 10.0 5.0 10.0 5.5 12.0 8.0 12.0
18. 6.0 5.5 4.0 8.0 7.0 5.5 9.0
19. 10.0 10.5 13.0 12.0 10.0 12.5 12.5
20. 8.0 5.0 11.0 5.5 11.5 7.0 13.0
21. 11.0 5.5 8.0 8.0 8.5 6.0 7.0
22. 7.0 7.0 8.0 5.5 7.5 13.5 7.0
23. 7.0 8.0 6.0 6.0 8.0 14.0 10.5
25. 12.0 6.0 12.0 12.5 5.5 6.0 6.0

 

47

 

 

TABLE 14 Continued
1. 6.5 8.5 9.0 4.0 6.0 9.0 5.5 12.0
2. 9.0 11.5 9.0 6.0 7.0 13.0 5.5 6.5
3. 13.5 7.5 8.5 10.5 6.0 11.0 7.0 7.0
4. 12.5 13.5 9.0 12.5 8.0 8.0 5.5 7.0
5. 6.5 14.0 10.0 6.5 13.5 6.0 9.0
6. 4.0 10.0 8.0 5.5 10.0 12.0 6.0
7. 7.0 6.0 14.0 8.0 8.0 10.5 6.5
8. 9.0 8.5 6.0 8.0 9.0 6.5 6.5
9. 8.5 13.0 8.5 5.0 5.0 4.5. 13.5
10. 5.5 8.5 8.5 11.0 5.0 6.5 6.0
11. 12.5 7.0 7.5 8.0 8.5 7.0 6.5
12. 6.0 13.5 13.5 7.5 5.5 6.5 14.5
13 12.0 9.0 12.5 13.5 11.0 6.5 11.0
14. 11.5 8.0 10.0 12.0 4.5 12.0 7.5
15 14.0 11.5 7.0 8.0 5.0 7.5 6.5
16. 12.0 7.0 11.5 14.0 6.5 4.0 5.5
17 15.0 9.0 11.0 8.0 9.0 11.5 12.0
18. 6.0 7.5 7.0 7.0 7.0 7.0 6.5
19. 6.0 9.0 4.0 14.5 4.0 4.0 11.0
20. 13.0 9.5 8.0 11.5 5.0 6.5 7.0
21. 6.5 6.5 4.0 7.5 7.0 6.0 5.5
22. 11.0 14.0 7.5 6.0 5.5 7.5 5.5
23. 5.5 7.0 9.0 7.5 12.5 15.0 8.0
24. 9.5 6.5 4.0 7.0 6.5 7.0 5.0
25. 8.5 12.5 5.0 10.5 13.5 14.0 4.5

TABLE 14 Continued

48

 

 

 

Distribution
Class Frequency

3.5 - 4.4 16
4.5 - 5.4 19
5.5 - 6.4 56
6.5 - 7.4 75
6.5 - 8.4 41
8.5 - 9.4 35
9.5 -10.4 17
10.5 -ll.4 18
11.5 —12.4 25
12.5 -l3.4 27
13.5 -l4.4 20
14.5 -15.4
15.5 -16.4 1

354

Mean X} 8.58

Ratio: 207:147

 

49
short than to the elongate parents. In a normal distribution
more than 97 per cent of the area under the curve would be
included between Z values of y.+ 20', where z represents
standard scores, P the average value considered, and<T the
standard deviation of the population. On the basis of the
statistical properties of this distribution the area covered
within the range of variation of Sanilac includes the mean
length of the F1 population while this last value would be
exterior to the limits of 211-V. Extending these considerations
to the F2 phenotypes, it would be noticed that 207 of the
plants in this particular group would fall into the short
category, whereas only 147 of them would belong to the elongate
type.

On the assumption that a partial dominance of short
hypocotyl to long hypocotyl prevails, most of the plants
resulting from a back cross of F1 to the short parent would
show an average hypocotyl length lying within the range of the
latter. This expectation seems to be actually justified by
the distribution of lengths in the BCS as reported in Table 12.

Nevertheless, determining the number of controlling
factors acting upon this trait presents some difficulty as it
is evident in the analysis of data compiled for seedlings
obtained by back crossing F1 to the elongate parent 211-V.

If a "unit character" condition were prevailing, 50 per cent

of these seedlings would have short hypocotyl length and 50

per cent long hypocotyl. The observed results, as presented

50
in Table 13 and analyzed in Table 15, reveal that approximately
only one third of the plants have a relatively short hypocotyl
section While the remaining seem to belong to the long hypocotyl
class. This situation may suggest that more than one pair of
factors are operating in the inheritance of hypocotyl elonga-

tion in Phaseolus vulgaris. Supposing that two pairs of

 

genes (AA, BB), when present simultaneously in dominant
condition are responsible for short hypocotyl but result in
gradually longer hypocotyl as the number of dominant alleles
decreases while that of their recessive counterparts increases,
short hypocotyl and long hypocotyl plants would be in a 9:7
ratio in the F2 population. The proportion of 207 short
hypocotyl to 147 long hypocotyl individuals observed in the
latter generation is approximately a 9:7 ratio, as illustrated
by a chi-square test in Table 16.

Summing up results obtained for the various crosses
that were made, it seems to occur that segregation into short
and long hypocotyl sections in any one of these matings nearly
expresses the type of recombination that possibly could be
expected under a duplicate recessive epistasis hypothesis.

If A— B— results in short hypocotyl, and all other

combinations produce long hypocotyl the following is true:

 

1. In Fl: AABB x aabb > AaBb 100%»short
short long short

 

2. In BCS: AABB x AaBb > AABB, AABb, AaBB, AaBb 100% short
short long short

TABLE 15. Experiment II (b). Computation of x2 for a
3:1 expectation for BCV.

 

 

Class obs exp dev X2
Short 9 10 -l .100
Long 31 30 +1 .033
Tot. 2 40 40 0 .133

Probability 70 to 95%

Remark: Excellent x2 agreement

 

TABLE 16. Computation of x2 for a 9:7 expectation for F2.

 

 

 

Class obs exp dev I X2

Short 207 200 +7 .245
Long 147 154 -7 .318
Tot. 2 354 354 0 .563

Probability 30 to 50%

Remark: Very good x2 agreement

 

52

3. In BCV: aabb x AaBb ——-> AaBb; Aabb, aaBb, aabb 1:3 short to long.
long short short long

4. In F2: AaBb x AaBb ———> 9 APB-, 3 aaB, 3 Abb, 1 aabb 9:7 short to
short short short long long.

Actual results for similar crosses in the present study
are not significantly different from the foregoing, and thus
the data seem to fit the hypothesis above mentioned. Most
geneticists working on related problems agree on assuming a
multiple factor hypothesis to explain internode elongation in

Phaseolus vulgaris.

 

A slight discrepancy may be noticed in the distribution

of hypocotyl lengths among F and BCS seedlings, as it has

1
been found that three plants in the former and one plant in
the latter generation belong to the 8.5 - 9.4 cm class, which
is outside the distribution range of the short parent if two
standard deviations are added to its average length to cover
97 per cent of the area of normal distribution. This excess
length may be due to hybrid vigor or additive effects of the
recessive epistatic factors a and b. Other symbols more
appropriate to depict the genes affecting hypocotyl elongation

in common beans may be chosen to replace the tentative letters

A and B used in the present paper.

Summary and Conclusions

 

The small figures computed as standard deviations of
Sanilac, 2ll-V, and F1 relative to their resepctive hypocotyl
mean length indicate a tendency of individual measurements to

conform closely to a central value. This situation implies

53

that elongation limits of this part of the stem in these
varieties of beans are fairly stable and predictable, environ-
mental conditions being equal. Besides, more likelihood is
achieved in trying to classify offspring as to hypocotyl
length.

Statistical tests carried out in analyzing the data

obtained from parents F and reciprocical back crosses

1' F2.
reveal that there is no evidence against the hypothesis that
inheritance of hypocotyl elongation in Navy beans (Phaseolus
vulgaris) can be explained according to the duplicate recessive
epistasis theory. Snyder and David (1957) report that such
a case occurs in the transmission of purple color in flowers
of the common yellow daisy and in other plant stocks, where
single flowers depend upon the simultaneous presence of two
different dominant genes, the absence of either or both resulting
in double flowers. These authors also mention that in human
beings, deaf—mutism appears to be inherited in the same manner
but that, nevertheless, duplicate epistasis is more common in
plants than in animals.

The above conclusion is a slight modification of that
reached by Lamprecht (1948) in a report following a study on
the inheritance of the slender-type of Phaseolus vulgaris by
crossing dwarf and tall varieties of beans. He remarked that
slender is caused by recessivity in two genes for the length
of the internodes: Cry, La, each in dominant condition,

preventing the length growth of the internodes.

SECTION III

54

55

DEVELOPMENTAL STUDIES

Materials and Methods

This experiment was intended to determine whether the
difference in hypocotyl elongation between Sanilac and 211-V
is due to difference in cell lengths, to difference in cell
number, or to both. At the end of the spring, 1960, Foundation
seeds of both varieties were sown at the Plant Science Green-
house of Michigan State University. Twenty-two days after
germination, four seedlings of each group of plants were
selected and their hypocotyl region cut into four equal parts.
Pieces one centimeter long were taken from each one of these
parts. After fixation and embedding in paraffin, these sections
were sliced to a 10 micron thickness with a microtome, mounted
onto slides, and finally triple-stained. For each variety,

twelve longitudinal-section slides were prepared by this method.

Results

Upon observation under a compound microscope equipped
with a micrometer, the cortical region appeared to be more
uniform in the arrangement of the cell layers and to offer
more ease in the measurement of the cells than the tissues
inward. From each slide, twenty-four cells of the first three
cortical layers beneath the epidermis were measured with a
precision of one micron under a 125 X magnification. These
twenty-four observations were grouped into two sets of twelve

measurements each and the mean value of each set recorded.

56
The results are reported in Table 17 and Plate IV (A, B)
illustrate typical cell expansion in both varieties. Under
the above mentioned magnification, the average hypocotyl
cortical cell length of Sanilac was found to be 11.33 microns
and that of 211-V to be 10.92 microns. In Table 18 an
analysis of variance carried out for the obtained data shows
no significant difference between these two means which
represent relative rather than absolute values of the average

cell length for the region considered in both varieties.

Discussion of Experimental Results

As the long hypocotyl variety 211-V seems to develop
cells even slightly shorter than those of the short hypocotyl
type Sanilac, it becomes mandatory that the hypocotylary axis
of the former contain more cells than that of the latter, at
least for the tissue under consideration. Tentatively an
explanation of this increase in cell division rate in 211-V
as compared to Sanilac may take into account the possibility
of phytohormonal influences. Various growth promoting substances
have been extracted from higher plants. Among many instances,
lately, in 1958 Radley ( 20 ) reported the presence of 0.4
‘pg (microgram) of Gibberellic Acid (GA) in an extract from
250 gr of tall peas. She also obtained gibberellin-like
substances in dwarf bean seed (Phaseolus vulgaris). The same
year Macmillan and Suter identified gibberellic substances

from immature runner bean seeds as Gibberellin A (13). A

57

TABLE 17. Experiment III. Cortical cell lengths of Sanilac
and 211-V.*

 

 

 

 

 

 

 

Sanilac 211-V

Plant No. l 2 3 4 l 2 3 4

Section 1 12 9 9 12 9 13 8 l3

13 12 11 ll 10 12 9 13

2 14 12 ll 9 14 ll 8 12

15 11 13 9 13 ll 9 l4

3 11 ll 12 10 10 10 9 12

10 ll 14 10 12 10 8 12

Average 12.50 11.00 11.67 10.17 11.33 11.17 8.50 12.67

Grand Average 11.33 10.92

 

*Lengths expressed in microns with a 125x magnification.

TABLE 18. Experiment III. Analysis of variance for cortical
cell lengths of Sanilac and 211—V.

 

 

 

Source of Var. D.F. S.S. M.Sq. F
Total 47 151.25
V 1 2.08 2.08 .56 N.S.
P 6 21.58 3.60 .96 N.S.
S 2 6.50 3.25 .87 N.S.
V x S 2 .67 .34 .09 N.S.
P x S 12 30.67 2.60 .67 N.S.
Error 24 89.75 3.74

 

V: Variety P: Plant S: Section
N.S.: Not significant at the 5% level.

58

.Axoav >IHHN
mo sofiuumm ahuooomhfi
m CH UGOEQOHO>OO HHGU

.m mmDOHm

 

.Axoav omenomm
mo coauomm H>uOUOm>£
m CH ucwfimoam>oo HHOO

.5 mmDOHm

 

59
leaf growth substance, with the same Rf value in isopropano—
ammonia as GA, was found in the acid fraction of the extracts
from dwarf bean primary leaves and colyledons. Besides the
Gibberellins, Indoleacetic acid (IAA) and other auxins
naturally occurring in plants have been recognized to promote
growth of stems and coleoptiles. Kinetin, another growth—
regulating substance, has not been reported as occurring
naturally in plant tissues.

As to the specific effects of the various organic
growth substances that have been tested so far, contradictory
opinions have been expressed. To remain within the scope of this
study, only a few of the recent papers published on the subject
have been reviewed. Preston and Hepton ( 19 ) reached the
conclusion that the major growth-regulating effects of most
auxins is exercised on the cell wall and that Indoleacetic
acid produces a marked increase both in elastic and plastic
extensibility on living turgid parenchyma cells short or
elongate. The silence of these authors on the effects of IAA
upon cell division may suggest that either these effects are
nil or insignificant.

Guthridge and Thompson ( 24 ) concluded from measurements
of cell and petiole lengths in strawberry that cell division
and elongation were both induced by GA. Previous evidence,
brought up by Greulach and Haesloop ( 7 ), also suggested
that stems elongate under the influence of Gibberellin more

from cell division than from increase in cell size of dwarf

60
bean internodes. This last point seems to be in very good
accordance with the observations recorded in the present
investigation.

Although action of gibberellin is seen to depend in
some extent on external conditions, the current interpretation
in phytohormonal studies is toward gibberellin-like rather

than auxin-like growth activity.

Summary and Conclusions

Cortical cells of hypocotyl sections of Sanilac and
211-V were measured with a compound microscope equipped with
a micrometer for comparison of cell lengths and cell number
in the hypocotylary axis of these two varieties of Navy beans.
No significant difference was noticed in the mean cell length
of the seedlings of both the short and the elongate types of
plants for the tissue considered. It was therefore concluded
that more cells must be present in the cortex strands of 211-V
to account for its longer hypocotyl region as compared to that
of Sanilac- Reviewing recent works on phytohormonal influences
in higher plants with special reference to Phaseolus vulqaris,
it is suggested that gibberellic or gibberellin-like substances
could be assumed to be responsible for the increase growth
rate in the long hypocotyl variety. It is hoped that further
studies on phytohormones and their relation to growth and
development are successful in opening additional avenues of

information regarding this subject.

61

GENERAL SUMMARY AND CONCLUSIONS

To investigate the basis of the differential hypocotyl
elongation of two commercially grown varieties of Navy beans,
Sanilac and 211-V, environmental, genetic, and developmental
studies were undertaken.

Following application of four combinations of day
length and light intensity treatments, it has been observed
that although differences in mean hypocotyl length between the
short and the elongate types of plants were most pronounced
under long day, high light intensity conditions, environmental
influences were not solely or primarily responsible for the
difference in hypocotyl elongation between Sanilac and 211-V.
Intrinsic factors have been assumed to chiefly control growth
patterns of the seedlings of these two varieties of beans.

Upon study of the mode of inheritance of the short
hypocotyl trait in Sanilac and its counterpart, the long hypocotyl
character in 2ll-V, by means of crosses and back crosses up to
the second generation level, no significant evidence was found
to rule out the hypothesis that transmission of the hypocotyl
elongation trait in Navy bean (Phaseolus vulgaris) could be
explained on the basis of the duplicate recessive epistasis
hypothesis..

Cytological assays on cortical cell layers of the
hypocotylary axis of the seedlings of these two types of bean
plants reveal no appreciable difference in cell lengths of both

varieties. Such an indication implies the presence of a larger

62
number of cells in a given cortical strand of le-V than in
a corresponding strand in Sanilac. Referring to recent works
on phytohormonal activity in higher plants, particularly in
the species Phaseolus vulqaris, it has been assumed that
gibberellic or gibberellin-like substancesmight be taken into
account in a tentative explanation of the increased cell
division rate in 211-V over Sanilac.

The results from the genetic and developmental studies
considered together, suggest the following alternative
hypotheses:

1. All bean varieties possess gibberellin-like substances
which tend to promote hypocotyl elongation, but most varieties
like Sanilac also possess a genetic inhibitory system, which,
when two non—allelic genes are present in dominant condition
effectively suppress the phytohormonal growth action.

2. Only those varieties of beans with recessive alleles
in the homozygous state at both the postulated loci involved
are able to produce the gibberellin-like material and no

direct inhibiting system is present.

10.

11.

12.

13.

63

L I TE RATURE C I TED

Adams, M. W. Notes on 211-V. Personal communication.

Andersen, A. L. Dry bean production in the Eastern States.
USDA Farmers' Bulletin No. 2083, 1955.

Down, E. E., and Andersen, A. L. Agronomic use of an X—ray
induced mutant. Quarterly Bulletin, Michigan
Agricultural Experiment Station, 39:378, 1956.

Emerson, R. A. A genetic study of plant height in Phaseolus
vulgaris. Nebraska Agricultural Experiment Station
Research Bulletin, No. 7, 1916.

Esau, K. Plant Anatomy. New York: John Wiley and Sons,
Inc., 1953.

 

Feucht, T. R. and Watson, D. P. The effects of Gibberellin
on internodal tissues of Phaseolus vulgaris.
Amer. Jour. Bot. 45:520—522. 1958.

Greulach, V. A. and Haesloop, J. G. The influence of
Gibberellic acid on cell division and cell elongation
in Phaseolus vulgaris. Amer. Jour. Bot. 45:566-570,
1958.

Highkin, H. R. Temperature-induced variability in peas.
Amer. Jour. Bot. 45:626-631. 1958.

Humphries, E. C. and Wheeler, A. W. The effects of Kinetin,
Gibberellic acid, and light on cell expansion and cell
division in leaf disks of dwarf beans (Phaseolus
vulgaris). Jour. Exp. Bot. 11:81—85. 1960.

Klein, W. H., et al. Photocontrol of growth and pigment
synthesis in the bean seedling as related to irradiance
and wavelength. Amer. Jour. Bot. 44:15-19. 1957.

Kooiman, H. N. Monograph on the genetics of Phaseolus
(g, vulgaris and g, multiflorus). Bibliographica
genetica. Vol. VIII. 1931.

 

Lamprecht, H. The inheritance of the slender type of Phaseolus
vulgaris and some other results. Agri. Hortique
Genetica. 5:72-84. 1948.

Macmillan, J. and Suter, P. J. The occurence of Gibberellin
A, in higher plants: Isolation from the seed of
Runner Bean (Phaseolus multiflorus). Naturwissenschaften,
45:46-47, 1958.

 

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

64

Makarem, S. A. Studies with some agronomic characters in
Navy beans. Thesis for the degree of M.S., Michigan
State University, 1952.

Mendel, G. J. Experiments in plant hybridization. Classic
Papers in Genetics, by J. A. Peters. New York:
Prentice—Hall, Inc., 1959.

Meyer, B. S. and Anderson, D. B. Plant Physiology. 2nd
edition. New York: D. Van Nostrand Company, Inc.,
1956.

Norton, J. B. Inheritance of habit in the common bean.
Amer. Nat., Vol. 49. 1915.

Orman, A. C. Field beans for canning. Agr. Gaz. New S.
Wales, 51:538, 1940.

Preston, R. D. and Hepton, J. The effects of Indoleacetic
acid on cell wall extensibility in Avena coleptiles.
Symp. Soc. Exp. Bot., 6:320, 1952.

Radley, M. The distribution of substances similar to
Gibberellic acid in higher plants. Ann. Bot., 22:
297—307, 1958.

Sax, K. Quantitative inheritance in Phaseolus. Journ. Agr.
Res. Vol. 33, 1926. '

Snyder, L. H. and David, P. R. The Principles of Heredity,
5th edition. Boston: D. C. Heath and Company, 1957.

Tisdale, S. L. and Nelson, W. L. Soil Fertility and Fertili-
zers. lst edition. New York: The Macmillan Company,
1956.

Thompson, P. A. and Guttridge, C. G. The effects of Gibberellic
acid on the initiation of flowers and runners in the
strawberry. Nature, 183:264, 1959.

Viglierchio, D. R. and Went, F. W. Plant growth under con-
trolled conditions. Amer. Jour. Bot., 44:449-453,
1957.

Wade, B. L. Breeding and Improvement of Peas and Beans.
USDA Yearbook of Agriculture, 251—282, 1937.

Zeeuw de, D. and Leopold, A. C. The prevention of auxin
responses by ultraviolet light. Amer. Jour. Bot.,
44:225—228. 1957.

MICHIG STATE UNIVER ITY LIBRARIES

Ilol IIJJIIIIl Illl ll

3 75 4439

l If)”
3

1293