Isolation, description, inheritance, associated traits and possible uses of three barley (Hordeum vulgare
L.) starch mutants
by Tae Young Chung
A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in
Philosphy in Crop and Soil Science
Montana State University
© Copyright by Tae Young Chung (1982)
Abstract:
Two barley pericarp starch mutants that retained starch in the pericarp layer at maturity and one barley
endosperm mutant, designated "fractured" starch in which the endosperm consisted of angular, and
what appeared to be fractured starch granules, were identified, the inheritance determined, and the
characters associated with these mutants , and uses related to the human consumption determined. All
the mutants were inherited as single recessive genes and the fractured starch mutant expressed xenia.
The symbols per 1and per 2 for the pericarp starch mutant 1 (Pernubet I) and 2 (Pernubet 2) genes and
fra for fractured starch gene (Franubet) were assigned. The linkage relationship between starch mutants
and translocation breakpoints were determined using the translocation homozygote lethal stocks and
the per 1, per 2 and fra genes were located in chromosomes 1, 7 and 4, respectively. Small seed size
was in common for all three mutants. Fewer kernels per spike, high tillering ability, high lysine content
and low ß-glucan viscosity were associated with the fractured starch mutant. Most of the viscous
substances appeared to be synthesized near physiological maturity of the kernel. The waxy endosperm
genotype had significantly higher ß-glucan viscosity and fractured starch mutant had significantly
lower than their parent Nubet isotype. The flour yield of Franubet was much greater than that of Nubet
and this appeared to be associated with lower ß-glucan viscosity. The kernels of waxy endosperm and
hulless genotypes pearled slower than that of their parent isolines, though the pearling indexes were
effected by the sample size charged, moisture content of the kernel and pearling times. For the
comparisons of genotype differences at the optimum pearling index a method of adjusting pearling
index with the ash content was developed. No pronounced differences were observed between Nubet
and starch mutant diets on the gain per day, feed consumption, or digestible energy in a rat feeding trial
and energy balance study. ISOLATION, DESCRIPTION, INHERITANCE, ASSOCIATED
TRAITS AND POSSIBLE USES OF THREE BARLEY
Qlordeum vulgare L.) STARCH MUTANTS
by
TAE YOUNG CHUNG
A thesis submitted in partial fulfillment
o f the requirements for the degree
of
DOCTOR OF PHILOSOPHY
in
Crop and Soil Science
Approved:
Chairman, Examining Committee
Head, M yor Department
Graduate Dean
MONTANA STATE UNIVERSITY
Bozeman, Montana
March, 1982
ACKNOWLEDGMENTS
I express my deepest appreciation and thanks to the following people.
Professor R. F. Eslick for his support, encouragement, and guidance while serving as
my major professor, and for imparting his professional philosophies to the Korean barley
breeding program. Dr. C. F. McGuire for unselfishly giving his time and energy in support
o f my thesis and for providing the laboratory equipment. Dr. R. L. Ditterline, Dr. G. A.
Taylor, Dr. C. W. Newman and Dr. L. R. Erickson for their professionalism, contributions
to these studies and for serving on my graduate committee.
My fellow barley graduate students, Mr. D. R. Biggerstaff, Mr. P. L. Bruckner, Mrs.
C. Fastnaught, Dr. G. J. Fox, Mr. D. Hadley and Dr. V. W. Small for their friendship and
assistance through the past three years. My fellow barley breeders, Dr. Y. S. Ham, Dr. E. S.
Lee, Mr. D. H. Chung and other members working at the Barley and Wheat Research Insti­
tute in Korea for supporting my personal work. Dr. R. T. Ramage and Dr. W. McProud for
encouragement, guidance and friendship.
My father, Hae Taek, my mother, Jong Soon and my brothers for love, encourage­
ment and support throughout my life. Hyo Hwan, my wife, for her love, sacrifice, encour­
agement and typing.
TABLE OF CONTENTS
Zee
VITA............. ................. .........................................................................................................
ii
ACKNOWLEDGMENT............................................................ ..............................................
iii
TABLE OF CONTENTS........... .................................................. ...................................
iv
LIST OF TABLES..................... ................... ........................................................................
vi
LIST OF FIGURES................................ .......................... ....................................................
ix
ABSTRACT .......................................................... ............... ............................
xi
Chapter
1
INTRODUCTION....................................................................................................
I
2
LITERATURE REVIEW...................
2
3
MATERIALS AND METHODS..........................
Inheritances and Linkages of Starch M u tan ts.................................................
Description o f M utants...............................................................................
AUelism of M utants.......................................................
Inheritance of M utants....................................................................
Linkage..................................................
Characters Associated with Starch M utants.......................................
Yield and Yield Components.........................................................................
Proximate Analysis and Amino Acid C o n ten ts........... ...............................
P-Glucan Viscosity, Amylose Content and
Brabender Viscoamylogram..............................................
Water Absorption o f Grain and Pearled G rain........... ................................
Use o f Starch M utants.........................................................................................
MUling...............................................................................
Factors Affecting Pearling Index and Pearling Rate........... ........................
Rat Feeding Trial ...................................................................................
27
27
27
28
29
29
32
32
33
35
36
37
37
38
40
#-
4
RESULT..............................................
Inheritances and Linkages o f Starch M u tan ts................................................
Description o f M utants.........................................................
42
42
42
V
Page
Allelism of M utants..............................................................................
Inheritance o f M utants...........................................................
Linkage.............................................................................................................
Characters Associated with Starch M utants....................................................
Yield and Yield Components........................................................................
Proximate Analysis and Amino Acid C o n ten ts...................... ...................
(J-Glucan Viscosity, Amylose Content and
Brabender Viscoamylogram................................................
Water Absorption o f Grain and Pearled G rain.............................................
Use o f Starch M utants.........................................................................................
Milling.........................................................
Factors Affecting Pearling Index and Pearling Rate............. .....................
Rat Feeding T r ia l.........................................................................
42
48
50
56
56
60
66
72
76
76
89
103
5
DISCUSSION ......... ..................................................................
Inheritances and Linkages of Starch M u tan ts....................................................106
Characters Associated with Starch M utants................................................... 112
Use o f Starch Mutants........................
116
6
SUMMARY AND CONCLUSIONS......................
LITERATURE CITED ...................................................................
124
1
vi
LIST OF TABLES
Table
Page
3-1. Description of translocations utilized to establish hulless
tester stocks o f translocation homozygote lethals.......................................... ........
31
3- 2. Growing conditions o f barley starch mutants used for analysis.........................
34
4- 1. Sources of barley starch mutants, gene symbols and mutation
r a te s ..............................................................................................................................
43
4-2. Phenotypic appearance of a F 1 diallel cross among pericarp
starch mutants in b a rle y .............................................................................................
47
4-3. Segregation ratios of barley starch mutants phenotypes........................................
49
4-4. Linkage recombinations between translocation breakpoints
involving chromosome I and naked and short awn gene from
F a segregations of barley translocation homozygote lethals ..................................
SI
4-5. F 1 segregations of pericarp starch m utant I crossed with
translocation homozygote lethals and their linkage
recombination values...............................................................................
52
4-6. F 1 segregations o f pericarp starch mutant 2 crossed with
translocation homozygote lethals and their linkage
recombination values..................................................................
54
4-7. F 1 segregations o f fractured starch m utant crossed with
translocation homozygote lethals and their linkage
recombination values........... ........................................................................
55
4-8. Average yield and yield components o f barley starch mutants
tested in 3 environments with 4 replications............................................................
57
4-9. Analysis o f variances for yield trials o f barley starch mutants
tested in 3 environments with 4 replications............................................................
58
4-10. Proximate analysis of grains from barley starch mutants
grown in several environments (Dry m atter basis)...................................................
61
vii
Table
Page
4-11. Average amino acid contents o f grain o f fractured starch
m utant (Franubet) compared to Nubet, from samples grown
in 4 environments........................................................................................................
67
4-12. Amylose content, gelatinization temperature and amylograph
paste viscosity data o f Buhler milled flours prepared from
barley starch m u ta n ts..................................................................................................
71
4-13. Average water absorbed by grain and pearled grain o f barley
starch mutants grown in several environments for different
steep times at room tem perature................................................................................
74
4-14. Analyses o f variance o f the tempering moisture effects on
the Buhler milled fractions for barley starch m u tan ts............................................
78
4-15. Protein, ash and starch content and 0-glucan viscosity of
milled fractions from barley starch m u ta n ts............................................................
83
4-16. Phenotypic (P)1genotypic (G) and environment (E)
correlation coefficients among determinations on grain
and flour samples from barley starch mutants grown in
several environments...........................................................................
95
4-17. Analysis of variance of pearling index o f Betzes isotypes
(hulless vs. covered) for different sample sizes with 2
replications..........................
92
4-18. Regression and correlation coefficients between pearling
index and pearling time for sample sizes o f Betzes and N ubet...............................
93
4-19. Analysis of variance for 2 minutes pearling index as affected
by % moisture in grain o f Betzes isotypes (hulless vs. covered,
waxy vs. normal endosperm )...........................
94
4-20. Genotype comparisons o f grain weight and pearling indexes
for the isogenic pairs of Compana, Betzes and Titan grown
in the different environm ents...............................................................................
95
4-21. Correlation coefficients between kernel weight and pearling
index within the isotypes o f Compana, Betzes and T ita n ........... ..........................
96
viii
Table
Page
4-22. Analysis of variance o f pearling index for successive pearling
time increments on Betzes isotypes (hulless vs. covered,
waxy vs. normal endosperm)................................................. ...................................
97
4-23. Proximate analysis of barley starch mutants balanced diets
adjusted to 14.5 percent protein..............................................................................
104
4-24. Average gain/day, feed consumption, feed efficiency and
protein efficiency ratio for rats fed 14.5% isoprotein diets
containing barley starch mutants..............................................................................
104
4-25. Average energy consumption, energy digested and percent
digestible energy data from rats fed 14.5% isoprotein diets
containing starch m utants.........................................................................................
105
ix
LIST OF FIGURES
Figure
Page
4-1. Grain appearance o f barley pericarp starch mutants and
photomicrographs o f outside layers o f the seed, stained
with iodine so lu tio n ....................................................................................................
44
4-2. Photographs of cross sections o f the grain o f Franubet and
Nubet on a light table and photomicrographs o f their starch
granules..........................................................................................
46
4-3. Graphic representation o f yield and yield component
interactions o f barley starch mutants with the
environments.................................................................................................................
59
4-4. Changes o f starch content in the pearled offal fractions o f
barley starch m u ta n ts..........................
62
4-5. Changes o f ash content in the pearled offal fractions of
barley starch m u ta n ts..................................................................................................
64
4-6. Changes o f protein content in the pearled offal fractions o f
barley starch m u ta n ts........................
65
4-7. Mean 0-glucan viscosity o f grain and pearled offal fractions
o f barley starch mutants grown in several environments........... .....................
68
4-8. Changes of 0-glucan viscosity as the grain o f Nubet and
Franubet developed......................................................................................................
70
4-9. Brabender amylograms o f Buhler milled flours obtained
from barley starch mutants, with and without HgCl3 added..................................
73
4-10. Relationship o f water absorption to steeping time of the
grain and pearled grain o f barley starch mutants, mean o f
several environments.....................................................
75
4-11. Tempering moisture effects to Buhler milled fractions
(Average o f Pemubet I, Franubet and Nubet with 2
replications)................................................................................................................
77
X
Figure
Page
4-12. Ash and protein content o f Buhler milled fractions as
effected by tempering moisture (Average o f Pemubet I ,
Franubet and N u bet)............................................................................ ......................
80
4-13. Comparisons of average percent recovery and Buhler
milled fractions for barley starch mutants grown in
several environments......... ................................................ ..................... ............... ....
81
4-14. Relationships of grain protein determined by UDY and
Kjeldahl method to flour yield among barley starch
mutants grown in several environments.................... ................................................
87
4-15. Relationship o f grain 0-glucan viscosity to flour yield
among barley starch mutants grown in several environments................................
88
4-16. Relationship o f pearling index to sample size and pearling
time determined on the hulless and covered isotypes of
Betzes (Average o f 2 replications).................................
90
4-17. Relationship o f pearling index determined at 2 minutes to
the percent moisture in grain o f Betzes isotypes.................. ........................... . . .
4-18. Relationship o f pearling index to iso types (hulless vs. covered,
and waxy vs. normal endosperm) and pearling times in
Betzes barley......... ..................................
91
98
4-19. Relationship o f pearling index to ash content o f pearled
grain o f Betzes isotypes (hulless vs. covered, and waxy vs.
normal endosperm)...................................................................................................... 100
4-20. Comparisons o f linear relationship between pearling index
and pearling time among barley starch mutants grown in
several environments...................................................................................................
101
4-21. Relationship o f ash content to pearling index o f barley starch
mutants grown in several environments...................................; .............................. 102
xi
ABSTRACT
Two barley pericarp starch mutants that retained starch in the pericarp layer at
maturity and one barley endosperm m utant, designated “fractured” starch in which the
endosperm consisted o f angular, and what appeared to be fractured starch granules, were
identified, the inheritance determined, and the characters associated with these mutants
and uses related to the human consumption determined. All the mutants were inherited as
single recessive genes and the fractured starch m utant expressed xenia. The symbols per I
and per 2 for the pericarp starch m utant I (Pemubet I) and 2 (Pemubet 2) genes and/ra
for fractured starch gene (Franubet) were assigned. The linkage relationship between starch
mutants and translocation breakpoints were determined using the translocation homozy­
gote lethal stocks and the per I, per 2 and fra genes were located in chromosomes 1 , 7 and
4, respectively.
i
Small seed size was in common for all three mutants. Fewer kernels per spike, high
tillering ability, high lysine content and low 0-glucan viscosity were associated with the
fractured starch mutant. Most o f the viscous substances appeared to be synthesized near
physiological maturity o f the kernel. The waxy endosperm genotype had significantly
higher 0-glucan viscosity and fractured starch m utant had significantly lower than their
parent Nubet isotype.
The flour yield o f Franubet was much greater than that o f Nubet and this appeared
to be associated with lower 0-glucan viscosity.
The kernels o f waxy endosperm and hulless genotypes pearled slower than that of
their parent isolines, though the pearling indexes were effected by the sample size charged,
moisture content o f the kernel and pearling times. For the comparisons o f genotype differ­
ences at the optimum pearling index a method of adjusting pearling index with the ash
content was developed.
No pronounced differences were observed between Nubet and starch m utant diets
on the gain per day, feed consumption, or digestible energy in a rat feeding trial and energy
balance study.
,
Chapter I
INTRODUCTION
Alternative uses o f barley include industrial utilization, food, feed and malting.
Researchers at Montana State University are working to improve the cash return from bar­
ley production. The objectives o f this project are to increase the usage o f barley and barley
products for human consumption with quality acceptable to the consumer.
Korea has traditionally used barley as a food, but the consumption o f barley has
been declining drastically. This reduction in barley consumption is due to recent substantial
gains in rice production and consumption. The reason for this replacement in Korean diets
is that the rice grain is considered to be more palatable, attractive, and convenient than
pearled barley.
The purpose o f this study was an attem pt to find a barley m utant which would be
more like rice. The characters related to the industrial, food and feed uses, and inheritance
of new induced barley mutants were investigated.
Although this study did not develop a barley with acceptable cooking quality for the
Korean consumer these results may contribute to finding a barley more nearly like rice.
Chapter 2
LITERATURE REVIEW
Chemical Mutagenesis
Induced mutations are a suitable breeding method when new or specific breeding ob­
jectives are to be realized. It is a method which enlarges the genetic variability o f a crop
and may produce new, suitable forms necessary for attaining distinct breeding objectives.
It has become one o f several valuable tools in varietal improvement o f barley and is becom­
ing increasingly important (Scholz, 1971). Its importance is demonstrated, especially in
barley, by the release of new, improved commercial varieties. According to Sigurbjoemsson
(1975), 36 barley varieties were released between 1962 and 1975. AU these varieties were
by direct mutation or from crossing a m utant with other cultivars or lines.
A number of chemical mutagens are available to induce point mutations. O f these
agents only a few are reafly useful for inducing mutations in cultivated plants, and most o f
these are alkylating agents. Nilan (1964) stated that diethyl sulfate was one o f the most
important mutagens for barley. With appropriate treatments, diethyl sulfate induced negli­
gible frequencies of chromosome aberration, very little physiological iiyury and a higher
proportion o f viridis than albino chlorophyU seedling mutants. They suggested that muta­
gen concentration, treatment duration, pH, temperature during treatment, oxygen supply,
seed-size and caryopsis type are the important factors influencing the deleterious side
effects and mutation rates. Nilan also reported that type o f treatm ent is more important
than concentration in reducing deleterious side effects and increasing the mutagenic effec­
tiveness. Soaking barley in 0.01M diethyl sulfate solution for 6 hours was recommended.
3
Nilan et al. (1963) also suggested a buffered solution at pH 7.0, a temperature of
IOC during treatment, and adequate oxygen supply to induce higher mutation frequen­
cies with less physiological iiy'ury. Small seeds and hulless seed treated for the same length
o f time as large seeds or hulled seeds showed more physiological damage and significantly
higher mutation frequencies.
Heiner (1963) found that the ethyl group o f diethyl sulfate preferentially reacted
with guanine of DNA to induce mutations in barley.
Translocation
Since barley translocations were found by Smith (1941), they have been used as a
tool for special cytological studies and offer enormous potential for plant breeders to
engage in chromosome engineering (Ramage, 1963).
Bumham (1962) described a method using translocations to test for the indepen­
dence o f barley linkage groups. Kramer et al. (1954), using translocation-gene linkages and
F t meiotic configurations of translocation intercrosses, concluded that two linkage groups
(linkage groups III and VII) were carried by the same chromosome (chromosome I). The
Fourth American Barley Research Workers Conference adopted a system o f designating the
chromosomes and the linkage groups. In this system, Arabic numbers I through 7 are used
to designate chromosomes and linkage groups. Extensive genetic data have been compiled
using this system and published at regular intervals (Nilan, 1964; Robertson, 1967; Robert­
son, 1971).
Barley translocations have been used by a number o f workers to assign new mutants
to specific chromosomes and to test linkages. Homozygous translocations have little or no
4
'
effect on the phenotype o f barley though they may alter linkage groups and chromosome
morphology (Burnham, 1962). Heterozygous translocations are phenotypically recognized
by semisterility as a result o f aborted spores. The aborted spores are caused by deficiencies
and duplications o f a chromosome segment resulting from adjacent disjunction (Ramage,
1963).
Pollen and ovule abortion were higher than 50% in heterzygous translocations of
com (Burnham, 1962). Barley translocations, however, averaged about 25% in the ovule
and 29% in pollen. Abortion was mainly due to an excess o f alternate disjunctions. When
crossing-over in the interstitial segments is followed by alternate disjunction, these crossedover chromatids in spores are deficient in genetic information and abort. An important
consequence o f this excess alternate disjunction in barley is that very few recombinants
are recovered from genes occurring between the centromeres and breakpoints o f trans­
locations (Ramage, 1963).
Hanson (1952), as cited by Burnham (1962), reported the pattern o f crossing-over
reduction in the interstitial segment using linkage data obtained with three known markers
(K, Lg3, b l) in barley chromosome 4 and three translocations (T2-4a, T3-4a, T4-5a)
involving this chromosome.
Ramage (1966) also reported that 13 translocations involving chromosome 2 were
tested against the male sterility gene, msg 2. The range o f recombination values were 0 to
2 units in the interstitial segment. Therefore, the pattern o f crossing-over reduction in the
interstitial segment furnished the position o f the centromere and the linkage data provided
the distance between the centromere and markers (msg 2).
5
When an individual heterozygote for a translocation and for a gene pair is selfed,
four phenotypic classes can be recognized in the F 1 generation. These classes are, semisterile dominant and recessive, and fertile dominant and recessive. Either of the semisterile
classes on fertile classes provides two measurements o f linkage information. The semisterilerecessive zygotes arise from the union o f a recombinant and a nonrecombinant gamete.
Normal-recessive and homozygous translocation recessive zygotes arise from the union of
two nonrecombinant or two recombinant gametes. Therefore, the recessive classes give
more information about linkage per individual than the dominant classes (Tuleen, 1971).
If homozygous translocation recessive and normal recessive classes can be determined, the
amount o f recombination information will be maximized. Root-tip squashes or a test cross
method were suggested to determine the homozygous translocations (Burnham, 1962;
Tuleen, 1971).
Since a translocation involves an exchange o f pieces between two non-homologous
chromosomes, the linkage obtained is evidence for the location o f assigned gene actions
in either or both chromosomes. Therefore, linkage information may provide ‘pseudo­
linkage’ between genes and one o f the chromosomes involved in the translocation. To
determine which of these possibilities is true, a test must be made with an additional trans­
location involving a break-point at nearly the same locus in one o f the chromosomes or the
other breakpoint in this additional translocation being in% different chromosome (Bumham and Cartledge, 1939).
All possible combinations (21) o f translocated chromosomes have been isolated. The
recommended designations o f translocations along with the break-position, and recombi­
6
nation values o f genes and breakpoints in heterozygous translocations have been sum­
marized (Ramage et al., 1961; Ramage and Suneson, 1961; Hagberg et al., 1978).
A translocation tester set involving seven barley chromosomes has been used exten­
sively to identify the various gene locations and as a source o f marker genes.
Genes conditioning shrunken endosperm (Jarvi and Eslick, 1975; Ullrich and Eslick,
1978), male sterile (Eslick et al., 1972), seedling lethal (Rahman and Eslick, 1975), erectoid (Bockelman and Eslick, 1977), scald resistance (Bockelman et al., 1978) and high
amylose gene (Ullrich and Eslick, 1978) have been identified along with associated linkage
groups. The balanced male sterile-translocation system with dominant preflowering selec­
tive genes was proposed to produce the female stocks in commercial hybrid seed produc­
tion. Eslick (1971) suggested that translocations created the necessary linkages which is
one o f the problems in previous hybrid systems.
The utilization of translocation homozygote lethals were initially proposed by Eslick
(1972) as a useful breeding tool. The possible advantages o f these translocation homozy­
gote lethals are (I) maintaining recessive male steriles and albino genes or other lethals in
a heterozygous stock without roguing homozygous dominant plants, (2) developing
balanced male sterile stocks, (3) increasing precision o f linkage studies and eliminating the
root-tip squash method o r test cross for identifying homozygous translocations, (4) trans­
ferring desirable genes to a recurrent parent without d is k in g characteristics which may
require expensive o r tedious laboratory work (Biggerstaff and Eslick, 1978).
Kernel Structure and Mutants
The barley grain o f covered cultivars consists o f hull, pericarp, integuments, endo­
sperm and embryo. As the barley ripens the hull and pericarp becomes firmly attached to
7
the wall of the kernel. The structure and adherence o f hull are important characteristics for
germinating grain in the malting process. The adhering hull protects the seedling from
mechanical damage, and restricts excessive seedling growth without affecting the desirable
enzymic degradation (Pomeranz and Bechtel, 1978).
According to Reid and Wiebe (1979), naked caryopsis (hulless kernels) are fre­
quently found as a result o f natural mutation, especially in primitive mountainous areas
where barley is used for human food. Nilan (1964), in his genetic review o f barley, noted
that naked caryopsis n is controlled by a single recessive gene. It is located on the long arm
o f chromosome I.
Eslick et al. (1972), from the three point linkage tests, indicated that the n gene is
located between I k i (short-awn) and msg 10 (male-sterile) and the recombination values
between msg 10 and n, n and I k it and msg 10 and I k i were 7.2, 7.9, and 14.7 units,
respectively. Since translocation data indicated that the msg 10 locus is located very near
the centromere region, the n gene is probably located 7.2 units from the centromere in the
long arm o f chromosome I.
Isogenic analysis o f the covered and naked caryopsis isotypes developed with differ­
ent varietal backgrounds revealed that the average yield reduction in a wide range o f envi­
ronments by naked caryopsis was 12%. It was concluded that the kernel weight and yield
reductions of naked barley are probably proportional t ^ t h e weight of the hulls (8-16%
of the kernel weight) (Eslick, 1979). Since the hulls o f naked barley are easily separated
from the caryopsis during the threshing process, the proportion o f the seed components
changes accordingly. The protein, starch, and fat concentration o f the seed increase due to
the reduction in the crude fiber represented by the hulls (Newman et al., 1968).
8
It was suggested that the advantages o f hulless barley to the end user are (I ) 30%
less storage space, (2) superior by-products from processing, (3) reduced energy require­
ments for syrup or starch production, (4) 12% less weight to transport, and (5) for the
plant breeder, rapid visual selection for potentially important biochemical mutants
(Eslick, 1979).
The pericarp consists o f the epidermis, hypodermis, cross cells, and tube cells.
The remaining tissues o f the grain are seed coat nucellar tissue and endosperm (Pomeranz
and Bechtel, 1978).
During kernel maturation, large numbers o f small, spherical starch granules were
detected in the pericarp o f barley kernels shortly after anthesis. These granules were very
quickly digested by the alpha-amylase present in the pericarp and 15 days after anthesis
no pericarp starch remained in the kernels. It was suggested that the function o f the alphaamylase is to hydrolyse the pericarp starch to provide energy for the growing kernel
(MacGregor et al., 1972).
To facilitate a study o f various tissues in small grains a staining procedure was devel­
oped for use in conjunction with a Strong-Scott barley pearler (Scheming and Rooney,
1979). May-Grunwald solution (0.5 methylene blue and 0.5% eosin-Y in methanol) stained
the germ, pericarp, and starchy endosperm o f sorghum blue, green, and pink, respectively.
The method was suggested for use in studying pearled graiS
The aleurone layer consists o f large rectangular, heavy walled starch free cells. Botanically the aleurone is the outer layer o f the endosperm (Pomeranz and Bechtel, 1978). It
has been suggested that the aleurone layer is a specialized secreting tissue situated at the
periphery o f the starch endosperm in seeds. During germination, extensive degradation of
9
the aleurone cell wall under the action o f gibberellic acid stimulation supports the enzyme
releasing function (Taiz and Jones, 1970). The proportion o f aleurone was estimated to be
6.9% o f whole grain in covered barley and to contain 21.5% o f the protein (Novacek et al.,
1966).
Endosperm Starch and Mutants
Starch consists o f two distinct molecular forms o f polymerized glucose molecules,
amylose; a linear homopolysaccharide, and amylopectin; a branched homopolysaccharide.
Amylose is composed o f alpha-D-glucose units with alpha-1, 4 linkage and the amylopectin
is composed o f alpha-D-glucose units linked in straight chains by alpha-1, 4 with branch
points being alpha-1, 6 linkages. A typical cereal starch consists o f 75% amylopectin and
25% amylose (Banks and Greenwood, 1975).
Goering et al. (1957) found that the amylose content of starch in 30 samples o f
Compana barley range from 19 to 25% on a dry basis. In 44 different varieties o f barley,
amylose content varied from 13 to 24% o f the total starch, illustrating that inherent differ­
ences exist in amylose to amylopectin ratios.
Scanning electron microscopy has revealed principally two sizes large round to oval
types over 25/im in size and small starch granules about 5 pm, both embedded in a matrix
o f reserve protein in barley endosperm (Pomeranz, 1974).
Since the cereal starch granule is formed within the plastid which controls shape and
structure, most cereal starch granules are single spherulitic structures. However, one plastid
may give rise to a multiplicity o f nuclei, and subsequently compound granules are formed
(Banks and Greenwood, 1975). The starch granules o f rice and wrinkled-seeded peas are
10
compound and fairly angular as illustrated by electron microscopic photographs. Those
starch granules are complex and exhibit a central cavity (Banks and Greenwood, 1975;
Juliano et al., 1975).
Williams and Duffus (1977) showed the development o f the amyloplast in the
barley endosperm. A t two days after anthesis amyloplasts could be seen in the endosperm,
each containing many small starch granules. After this period, some enlarge to become fullsized large granules. By 14 days after anthesis two populations of amyloplast, large and
small, appeared, the large being about 11 jim across and the smaller 3 or 4 pm across.
A wide varietal range in the ratio o f small to large granules from a minimum o f
5.5:1 to a maximum o f 37:1 on a number basis were reported. Small granules accounted
for 6.2 to 30.6% of the total starch weight (Goering et al., 1973b). No substantial differ­
ences were found between large and small starch granules separated from mature barley for
% protein, % fat, swelling power, iodine affinity and Brabender cooking viscosity curves
indicating that small granules observed in mature barley endosperm are a second discrete
population o f starch granules and not immature granules (Goering and DeHaas, 1974).
The texture o f cooked rice is determined largely by the amylose/amylopectin ratio
o f the starch (Juliano et al., 1964). According to Juliano ( 1979), in a review on rice quality,
two attributes of cooked rice commonly measured are tenderness (softness) and cohesive­
ness (stickiness). These are inversely related with amylodt content, especially hot water
insoluble amylose content. Amylose content o f milled rice varied from 9 to 33% among
the selected samples (550 samples collected from 18 counties). Japonica types, which are
acceptable in China, Korea and Japan, showed low to medium amylose content (9-15,
15-20%, respectively).
11
Waxy barley endosperm contains very little (0-3%) amylose and almost 100% amylopectin in the endosperm starches (Goering et al., 1973a). This character can be identified
by an iodine reaction on the endosperm and pollen starch grains. Iodine reacts with normal
starch producing a deep blue color while the reaction with the waxy starch produces a
reddish-brown color (Haus, 1975).
Nilan (1964) summarized the genetic studies on the waxy endosperm character. The
waxy endosperm gene, wx, is a simply inherited recessive, and normal allele is completely
dominant in Wx wx wx and Wx Wx wx endosperm and expresses xenia for the trait. This
gene belongs to the chromosome I linkage group and is located on the short arm approxi­
mately 50 recombination units from the centromere. The waxy gene produces no notice­
able gross differences in crop appearance except that it imparts an opaque hue to the grain
in contrast to the more vitreous appearance o f normal grain (Eriksson, 1969).
The waxy endosperm gene was introduced into established barley cultivars because
of the observation that waxy starch is more readily modified by enzymes and chemicals
than normal starch (Goering et al., 1973b). Low pasting temperature and easy hydrolysis
o f waxy barley allows complete conversion without conventional cooking temperatures
and times. By using waxy barley, high maltose syrups (maltose contents were 58 to 66%)
were produced in the laboratory and pilot plant (Goering et al., 1980). Waxy endosperm
has also been reported in rice, com, and sorghum. Amylodt content and starch properties
o f waxy rice, com, and sorghum are similar to the waxy barley (Medcalf, 1973). Waxy
rice is high in cohesiveness and very sticky because o f high amylopectin content (Juliano
e t a l , 1965).
I
12
High amylose endosperm was reported in Glacier barley by Merritt (1967). The
amylose content in the endosperm of this m utant was 44% compared to 24% in the
original parent. The m utant has been designated as ac 38 and was inherited as a simple
recessive. This m utant gene exhibited a dosage effect and also expressed xenia. The gene
was assigned to chromosome 2 as determined by the trisomic analysis method in Betzes
background (Ullrich and Eslick, 1978). Scanning electron micrographs revealed that high
amylose starch granules o f barley formed irregular shapes and were significantly smaller
than normal starch granules (Banks and Greenwood, 1975). Two high amylose genes
(amylose content 60-70% of starch) were also reported in com. The starch developed in
high amylose com has peculiarly shaped granules. Long bulbous granules were found
clumped together and surrounded by other types o f granules. High amylose starch has
an extremely high gelatinization temperature and is exceedingly resistant to the action
o f digestive enzymes (Sandstedt, 1965).
Because o f the linear nature of amylose, the high amylose grains are o f commercial
interest. The amylose has a number o f potential uses which a branched polymer does not.
Linear polymers are particularly suited for film and coating applications. Edible films
which are only slightly permeable to gases could be used as containers for foodstuffs
and present no waste disposal problems (Medcalf, 1973).
Reid and Wiebe (1979) referred to a kernel type iq barley in which the starch was
replaced by a sugary liquid. As the seed matured it collapsed and the collapsed seed failed
to germinate. Stocks o f this m utant could be maintained as heterozygotes which expressed
xenia.
13
Sugary endosperm genes (su I and su 2) high in sucrose were also reported in com.
These sugary endosperms stored less starch. Instead, they contained four to ten fold more
sugar than normal endosperm types. The additive effects in sucrose content o f su I su 2
genotype were exceedingly small and were largely aggregated into compound granules
which are somewhat analogous to wrinkled pea starch (Sandstedt, 1965).
Gene interactions o f waxy, high amylose, and sugary endosperm have been studied
for starch properties and structure in com. The high amylose gene (ae) and the waxy (wx)
were shown to be completely epistatic to high amylose (du) and sugary (su I, su 2) in amy­
lose content. The ae wx genotype had 15% amylose in the endosperm starch (Kramer et al.,
1956).
The complexity o f the starch granules is increased under the influence o f two or
more homozygous recessive genes. The combination o f high amylose (ae) and the sugary
(su I) genes produces a mixture o f many kinds o f granules. Some were made up o f many
exceedingly small blue staining particles (some o f which were birefringent) embedded in a
red staining and nonbirefringent matrix (Sandstedt, 1965).
Protein Synthesis and Mutants
As a percentage o f the tissue, protein is highest in the embryo, next highest in the
bran, and lowest in the endosperm. Protein stored in the endosperm is inversely related to
-
!
carbohydrate content. High protein content in grain is therefore at the expense o f stored
starch (Canvin, 1976).
14
Sixty-five generations o f continuous selection o f com for high and low crude protein
extended the upper level to 25% and the lower level to 4%. Yield capacity and seed size,
however, were depressed in high protein selections (Dudley and Lambert, 1969).
Johnson et al. (1969) suggested that a single genotype o f wheat could produce grain
varying from as low as 8% protein to as high as 18% depending on the environment in
which it is grown. The environment affects protein content o f grain to a greater extent
than the genetic system and these two effects are difficult to separate (Johnson et al., 1969).
Barley cultivars responded differently in grain protein content to increasing levels of
nitrogen application. Malting barley cultivars showed a remarkable increase in protein con­
tent, however, very little change occurred in Hiproly and C L 4362 (McGuire et al., 1979).
The endosperm protein consists o f high amounts o f glutamic acid and proline while
glycine, valine, and arginine are much lower in the endosperm compared to embryo and
aleurone protein. The protein in the germ and aleurone cells contain considerably more o f
the essential amino acids, arginine, histidine, lysine, methionine, threonine, and valine
(Munck, 1972).
Since the opaque-2 endosperms in com which had a different amino acid pattern
and 69% more lysine than the normal com , intensive investigations have been done to find
high lysine mutants in various crops (Mertz, 1976).
The high-lysine barley, Hiproly, o f Ethiopian origin, isolated from the world barley
collection (Munck et al., 1970) and the mutant Ris^ 1508, produced by chemical mutation
(Ingverson, 1975), contain significantly higher lysine (Hiproly, 30% and Ristf 1508, 45%)
than their parents. The mode of inheritance o f the high lysine trait o f Hiproly and Ristf
1508 were determined to be a single recessive (Munck et al., 1970; Doll, 1973).
15
From the study o f classical Osbome protein fractions in these mutants during the
grain filling period, it was found that high lysine in Ris^ 1508 derived from Bomi is due to
the depression of hordein synthesis. The albumin fraction in normal barley is synthesized
early in grain development, whereas hordein, glutelin and globulin are synthesized from I O
days after fertilization until maturity. The high lysine mutants, on the other hand, showed
a remarkable depression of hordein and globulin synthesis with increasing albumin
(Cameron-Mills et al., 1979).
Jarvi and Eslick (1975) and Ullrich (1978) identified six and eight shrunken endo­
sperm lines and found that the mutants contained high lysine. The high lysine traits are
controlled by single recessive genes and some o f them expressed xenia. AU of the mutants
including Hiproly and Ris^ 1508 showed lower grain yield and test weight because of
shrunken endosperms. Linkage or pleiotropy between high lysine and shrunken endosperm
are assumed because the association appears to be quite general in barley.
g-Glucan Viscosity and Brabender Viscosity
Gums extracted from raw barley and malt with warm water have presented problems
in the brewing industry by causing increased viscosity and filtration problems in the sweet
wort. In the finished beer the remaining high molecular weight substances originating from
barley gums influence quality. They raise the viscosity and improve the foam stability of
beer. Gums from barley, malt, and wort were hydrolysized to glucose, arabinose, and
xylose with acid (Djurtoff, 1958). Scott (1972) found that most o f the specific viscosity
o f worts was contributed by 0-glucan. The viscosity o f the worts due to other components
remained constant among varieties.
16
P-Glucan is a general name for all non-cellulose compounds o f two or more glucose
molecules linked together in the 0-configuration (Bourne and Pierce, 1972). The barley
0-glucans are essentially linear molecules containing both 0 -1 ,4 and 0-1,3 glucosidic link­
age which are randomly arranged (Preece and MacKenzie, 1952; Igarashi and Sakurai, 1965).
Most methods o f 0-glucan extraction methods are based on precipitation from
aqueous extracts (Bourne and Pierce, 1972). If only the hot water soluble gums are
extracted, the 0-glucan estimates tend to be low. Estimates increase if both water soluble
and insoluble fraction are extracted using strong acid or basic solutions (Forrest and
Wainwright, 1977).
For breeding purposes, a rapid viscosity method for estimating 0-glucan has been
developed (Greenberg and Whitmore, 1974; Morgan and Gothard, 1977; Morgan, 1977).
The amount o f 0-glucan is not measured directly, but is estimated by the amount o f vis­
cosity produced by an extract. Greenberg (1974) found that extract viscosity was closely
related to actual 0-glucan content in a logarithmic fashion, with a correlation coefficient
o f 0.89.
0-Glucan is believed to be situated mostly in the endosperm as a part of the cell
walls and material surrounding the starch granules (Bourne and Pierce, 1972). It was sug­
gested that the 0-glucans were contained in the barley aleurone cell walls (Taiz and Jones,
1970), however, McNeil and Albersheim (1975) found that the aleurone cell walls are
composed o f arabinoxylan and cellulose. Fulcher et al. (1977) suggested that the main
0-glucan deposition is at the sub-aleurone cell walls that are immediately adjacent to
the aleurone layer. Fluorescent microscopic photographs illustrated considerably more
sub-aleurone cell wall than the remainder o f the endosperm and these sub-aleurone cell
17
walls contained extensive deposition o f aniline-blue positive material indicating 0-glucan
deposition (Fulcher et al., 1977; Wood and Fulcher, 1978). Gohl et al. (1977) showed
the distribution o f the viscosity within the matured barley kernel using pearled fractions.
The layer between the bran and the center of the kernel revealed the highest viscosity.
The pentosan and hemicellulose content also influence viscosity (Bourne and
Pierce, 1972). The materials extracted by a 4% aqueous sodium hydroxide solution from
cereal grains after removal o f starch and gum, are hemicelluloses. Two types o f hemicel­
lulose, a husk type and an endosperm type were obtained. The husk type was found to
have a low viscosity and the endosperm type was high in viscosity.
Current thinking is that gums and hemicellulose are all derived from the same basic
source and pure extraction is difficult (Forrest and Wainwright, 1977).
Varietal differences in /!-glucan concentration was reviewed by Bourne and Pierce
(1972). Hot and dry growing conditions increase the glucan concentration and wet grow­
ing conditions appear to favor low viscosity. Varietal differences, however, remain in the
same order.
In an isogenic study on barley viscosity, using caryopsis type, waxy endosperm and
short awn genes and their normal counterparts, Fox (1981) detected a significant differ­
ence in extract viscosity among genotypes. Naked, short awn and waxy recessive genotypes
were higher in viscosity than their parents. Three recessive double recombination types
were higher in viscosity indicating additive gene action among those genotypes for g-glucan
viscosity o f the grain.
Intensive studies on the Brabender cooking viscosities have been done with barley
flours and starches to evaluate the starch and flour characteristics. Goering et al. (1970)
18
observed some genetic differences for cooking viscosity among starches from 12 barley
genotypes. In general, the hulless varieties have a higher paste viscosity than the covered
types. Some varietal differences were also determined. Starch from Nupana showed the
highest viscosity and Titan the lowest. Paste gelatinization temperatures were similar
among non-waxy cultivars.
Starch from the waxy genotypes has been compared to starch from normal geno­
types (Goering et al., 1973a). Waxy starches showed higher pasting peak at about a 20 C
lower temperature, had more granule instability and very little setback on cooling com­
pared to their normal starch counterparts. Small and large starch granules isolated from
3 different barley isogenics including high amylose, waxy and high lysine isotypes with
their derived lines were investigated for starch properties (Goering et al., 1975). No sig­
nificant difference of the Brabender cooking curves between small and large starch gran­
ules was found. These results indicated a similar stability for small starch granules com­
pared to large granules during cooking.
Cooking viscosity curves o f barley flours were similar to the starch curves for geno­
type differences. Maximum viscosities, however, are relatively lower than starch viscosities
(Goering et al., 1980).
Brabender amylographs have been used to determine gelatinization temperature on
milled rice flour and rice starch and as a means to index eating quality o f rice. Correlation
studies indicated that peak viscosity and setback characteristics on the amylogram were
related to palatability, stickiness and amylose content among the Japonica and Indica
types. The preferred rice in Japan has high peak viscosity, low temperature to attain peak
viscosity and greater breakdown (setback). The variation o f gelatinization temperatures of
19
the acceptable rice varieties has been shown to be from 55 C-79 C with 65-68 C as the
preferred range (Suzuki, 1979).
Water Absorption of Starch Mutants
According to Pratt (1964), water uptake by wheat flour is influenced by the protein
and starch content in the flour with only minor influences caused by the other constitu­
ents, such as dextrins, pentosans and cellulose. Sorum (1977) reported the alkaline water
retention capacities (AWRC) of barley flour. High water retention capacity was found in
barley flours compared to wheat flours.
The swelling properties o f starch are due to the contribution o f the hydroxylated
nature o f the D-glucose molecules, the large molecular size o f the constituent polymers,
and the granular form itself (Medcalf, 1973). Swelling power of waxy starch granules
extracted from the waxy isogenic pairs o f Compana and Oderbrucker were compared to
the normal starch granules. Waxy starch showed higher swelling power than starch from
the normal isotypes (Goering et al., 1973a).
An excellent review on the water absorption of barley grain was reported by
Brookes et al. (1976). Positive correlation between starch content and velocity o f water
uptake and an inverse relationship between nitrogen content and imbibition rate was
reported. The surface layers o f the barley kernel are the principle barriers to water entry.
The pericarp and testa are the major organs for regulating water entry. Variation o f temper­
ature during imbibition influences the rate o f water uptake by the barley seed. All barleys,
however, have a similar temperature coefficient o f imbibition.
20
Briggs (1978) reported smaller kernels take up moisture faster and to a higher final
level than larger kernels. Genotype differences for the water uptake were observed using
Compana iso types (Bruckner, 1981). Naked and waxy isotypes were found to take up
water faster than normal iso types in a 48 hour, 20 C, unaerated steep.
Barley Milling
The purpose of modem flour milling in wheat is to separate the bran and germ from
the endosperm, and to reduce the size of the endosperm particles through a series of mill­
ing steps. Bran and germ are deleterious in baking performance and may be less digestible.
Flour extraction is defined as the proportion of flour by weight, obtained by milling
a known quantity o f wheat. It has been used as an index o f the overall efficiency o f a flour
milling enterprise. Since the ash content at a given flour extraction varies within a narrow
range, ash % has been used as an indicator o f flour mill performance. As extraction
increases, ash content o f the flour increases. Percent ash in the flour reflects the efficiency
o f separation o f bran from endosperm (Pomeranz, 1971).
Pomeranz et al. (1971) described a method for milling barley to increase the flour
extraction up to 65 percent. The barley was tempered for 30 minutes with 0.5% water
added before milling, red dog and shorts were reground on an alpine mill and sifted
through a 100 xx sieve, and the throughs, called tailings flour, were mixed with patent flour.
McGuire (1979) developed the Buhler milling process for barley changing several
steps o f the wheat milling method to extract suitable flour from covered and naked barley.
Barley was tempered to 13% moisture for 30 minutes before milling and the 145 mesh
flour sieves were replaced with 88 mesh sieves. Shorts were returned to first break rolls
21
for a second passage through the mill. The feed rate for whole grain to first break was
about 75 g/min.
Cheigh et al. (1975) tempered naked barley for 48 hours to a moisture level o f
13.5%. An addition o f 0.5% more water prior to flour milling increased the milling effi­
ciency compared to 30 minutes tempering to a 14% moisture level.
Sorum (1977) reported that the dry milling of barley produced higher average
flour extraction without increasing significantly the ash content in the flour over milling
tempered barley. Because o f the soft woolly nature o f the barley endosperm, too long and
high moisture tempering tend to produce crushed or flaked barley at the first break roll
(McGuire, 1979).
Flour extraction o f barley varied between 50 and 74% depending on the milling
method applied and upon the samples used (Cheigh et al., 1975; Sorum, 1977). McGuire
(1979) found an average extraction o f 65% from Steptoe and a high extraction o f 72.2%
from Betzes.
Cheigh et al. (1975) indicated that use o f hulless barley cultivars resulted in approxi­
mately 10% greater flour extraction than that from covered barley cultivars, without
increasing ash content in the flour.
Ash content in the barley flour from milling experiments ranged from 1.23 to 1.98%
on a dry basis. Positive relations between whole grain and flour protein (r=0.763**), and
whole grain and flour ash (r=0.426**) indicated that the flour characteristics reflected the
variability o f the grain among cultivars (McGuire, 1979).
22
The correlation between the ash content o f the flour and crude fiber and Agtron
color values indicated that carbohydrate content in the flour increased as ash contents
decreased (Pomeranz et al., 1971).
Barley Pearling
Pot and pearl barley are manufactured by gradually removing the hull and outer
portion o f the barley kernel for human food. To produce the highest quality demanded by
the consumer, pot and pearl barley must be pearled to remove the coloring m atter asso­
ciated with the bran layers, and the endosperm must be as white as possible. A common
type o f pearling machine usually consists o f a cylindrical millstone and a perforated cylin­
der similar to the Strong-Scott barley pearler.
The Strong-Scott barley pearler has been used to identify the color o f the aleurone
layer and to determine the endosperm texture in grading barley for malting purposes. The
Strong-Scott pearler has been proposed as a means of determining kernel hardness in wheat
also (Taylor et al., 1939).
Various factors thought to affect pearling were investigated for kernel hardness o f
wheat (McCluggage, 1943; Kramer and Albrecht, 1948). It was found that the amount of
pearled material was linearly related to pearling time and cultivar differences. Soft wheat
pearled faster than hard wheat and as pearling time increased the amount o f pearlings
increased in both soft and hard wheat. The relationship between pearling time and pearling
index was not linear, although curvilinearity was not very pronounced in hard wheat and
only for extreme pearling times (McCluggage, 1943; Kramer and Albrecht, 1948). Various
speeds o f the pearling stone were examined for pearling intensity. The faster speeds pearled
23
barley faster. Essentially the same amount o f pearlings secured in given varieties with 3 dif­
ferent speeds o f the pearling stone could be obtained by adjusting the pearling time. No
significant interactions were detected between speed of the pearling stone, pearling time
and kernel hardness (McCluggage, 1943).
The amount of sample in the pearling machine affected significantly the amount o f
pearlings obtained in a given time. Differences between varieties for any given sample size
were essentially the same (McCluggage, 1943; Kramer and Albrecht, 1948). It was also
found that the amount o f pearlings was inversely related to the moisture content of wheat
(Kramer and Albrecht, 1948).
Using a commercial barley process, LeQerc and Garby (1920) indicated that the
recovery o f pearled barley and ash content decreased linearly as pearling progressed over
time. The edible product, or pot barley, obtained from the third operation, was almost
entirely free from the aleurone layer. Seventy-one percent o f the covered barleys was
recovered with a 1.44% ash content. The fourth and fifth pearling operation produced
pot barleys that were not only whiter, but were practically free from all bran material.
Fifty-eight percent and 47% o f pearled barley recovered showed 1.18 and 1.0% ash content
(LeQerc and Garby, 1920). Sixty-five percent recovery for pot barley and 35% recovery
for pearled barley are generally accepted amounts in the commercial pearling process
used in the United States (Geddes, 1951).
The factors affecting cooking quality o f boiled barley in Korea appeared to depend
upon whiteness and water absorption o f pearled grain. The Korean consumers preferred
the white pearled grain. Pearled barley that absorbed more water cooked easier than that
which absorbed less water. The whiteness o f pearled barley was linearly related to pearling
time and inversely related to pearling index within limited cultivars (Ryu, 1979).
Barley Nutritive Value
The primary energy source for monogas trie animals is supplied by the cereals. Al­
though barley is similar in chemical composition to com and wheat, its feed value has been
considered to be inferior. As barley contains more crude fiber than com and wheat, the
hulls o f barley may be responsible for a dilution effect of nutrients in the grain.
The inhibitory effects o f barley hulls on nutrient digestion, absorption, and util­
ization o f barley in diets were reported by Larson and Oldfield (1960). A remarkable
depression o f weigh gains caused by reduced digestibility o f the energy supplying compo­
nents was found in pig feeding trials. This observation was supported by Gill et al. (1966).
Effects o f the hull on pig performance was studied by Newman et al. (1978) using
hulless and covered iso types. Gains o f pigs fed hulless Compana were superior to those fed
Compana barley. Glacier barley was equal to hulless Glacier. From these results, they sug­
gested that differences may exist in the nutritive value o f hulless barley that are not asso­
ciated with lower crude fiber content.
-
Using four Betzes iso types, differing in length of awn and caryopsis covering, differ­
ences in nutritional quality traits were tested. The length o f awn was associated with
0-glucan differences in the grain. The presence o f the hull appeared to have a minor influ­
ence on rat performance. True protein digestibility and digestible energy in covered barley
was lower than that o f hulless barley in rat feeding trials. The presence o f hulls increased
feed consumption and produced poor feed conversion in pigs, bu t the rate o f gain was not
25
changed by the presence o f the hull (Truscott, 1980). In chick diets Andersson et al.
(1961) found hulless and covered barleys to be quite similar.
Viscous barleys caused a problem termed “ sticky droppings” in poultry, which leads
to poor growth and production. This problem can be overcome by water-treatment or the
addition o f /3-glucanase to barley. Gohl (1977) discovered that water treatment did not sig­
nificantly affect dry matter, crude protein digestibility or biological value. Water treatment
o f barley increased the gains of body weight for rats over those o f untreated barley. Starch
disappearance in the gastrointestinal tract differed little between treated and untreated
barley.
Available energy in grains and purified starch from barleys o f different amylose con­
tent were evaluated in rat and pig feeding trials (Calvert, 1975; Newman, 1979). The waxy
gene had no effect on the nutritional value o f barley starch, whereas the high amylose
starch from high amylose Glacier appeared to be lower in nutritional value than starch
from Glacier, Compana and Wapana. These results are supported by the performance of
swine fed normal and waxy starch types in com and sorghum (Jensen et al., 1973; Cohen
and Tanksley, 1973).
The digestible energy (DB), which is determined by subtracting gross energy o f the
feces from the gross energy o f the barley, has been used to estimate the available dietary
energy. Digestible energy determined by the use o f a bomb calorimeter is probably the
least precise measure o f a feeds energy value to an animal. This is also one o f the easiest
methods available to determine digestible energy (Maynard et al., 1979).
The apparent digestibility coefficients o f energy o f diets varied from 79 to 89% in
barley diets and those o f starch are about 95%. The hulless barley diets showed signifi­
26
cantly higher digestible energy than covered barley diets in metabolic trials of rats
(Vermorel and Keller, 1967; Gobi, 1977;Truscott, 1980).
Chapter 3
MATERIALS AND METHODS
Inheritances and Linkages of Starch Mutants
Description of Mutants
Pericarp starch mutants were obtained from the M2 seeds o f barley cultivar, Nubet,
treated with 0 .01M o f diethyl sulfate. Seed was treated with diethyl sulfate in the spring of
1978 by the method recommended by Nilan (1964) and planted at Bozeman, Montana.
Bulked seed, harvested in 1978, was soaked in 1% iodine (IKI) solution for 5 minutes and
the treated seeds rinsed with tap water. Dark and light blue stained seeds were selected.
Each o f 10 seeds were increased at the experimental field o f Arizona State University in
Mesa, Arizona. Four plants o f dark blue and 5 plants of light blue stained seed were har­
vested and the phenotypic appearances o f Ms seeds were observed. Three plants, Per 16,
Per 17, and Per 18, stained dark blue and 5 plants. Per 28, Per 30, Per 31, Per 32, and
Per 33, stained light blue with no segregation on the head. The remaining dark blue m utant
plant, Per 19, segregated light and dark blue within the spike and Per 30 and Per 33, light
blue mutants had thin kernels. These three lines were excluded from the study.
The fractured starch m utant was selected from the same material as the pericarp
mutants. A low percentage o f opaque seeds occurred among the normally vitreous seeds.
These opaque seeds were selected, grown, threshed as individual plants, and the seed
examined. It had been previously observed that waxy endosperm were opaque. The true
breeding plants with opaque seed were stained with iodine. The waxy endosperm mutants
stained reddish brown, the endosperm o f other opaque mutants stained dark blue as did
the fractured starch mutant. The fractured starch m utant, characterized by the angular
starch granules, was identified later by microscopic observation. Small pieces o f the endo­
28
sperm starch were placed on a glass slide with a few drops o f water and a cover glass
applied. The m utant was easily distinguished from the round starch granules of the normal
endosperm with the 16 X 10 or 100 X 10 power magnifications. The three lines o f the
fractured starch m utant used in this study were provided by Eslick.
Allelism o f Mutants
During the summer o f 1979, each o f these mutants and Nubet were planted at the
Agricultural Experiment Station, Bozeman, in a 3 m single row, spaced 30 cm apart. A
diallel cross among the 8 lines o f pericarp starch mutants was made to determine allelism.
Two or three spikes o f each line for a cross were emasculated by hand before the anthers
turned yellow and covered with glassine paper bags to protect from foreign pollen. After
three or four days, the emasculated heads were pollinated with male pollen from selected
parents. To identify crosses and parents, each was marked with a proper identification tag.
Crosses between the starch mutants and Nubet and Nupana were also made using the
method described above. In November 1979, 10 seeds o f each cross were planted at the
Mesa experimental field o f Arizona State University and 4 plants o f each cross were har­
vested separately. F i plants (F 1 seeds) o f the 8 parent diallel cross among pericarp starch
mutants were tested for phenotypic appearances with the iodine solution to determine
allelism. A fter each m utant was confirmed as a member o f a m utant group, i.e., allelic, the
mutant stocks maintaining each o f the three lines were designated Pemubet I , dark blue
staining m utant, Pemubet 2, light blue staining m utant, and Franubet, fractured starch
mutant.
29
Inheritance o f Mutants
In the summer of 1980, four families o f the segregating F 3 populations from the
individual F 1 plants o f each cross harvested in Mesa, Arizona, were grown at the Agricul­
tural Experiment Station, Bozeman. Whenever possible, 120 seeds o f each family were
planted in a row 30 cm apart and 12 m long. For the F 3 segregations o f pericarp starch
mutants, the individual F 3 plants were harvested and the phenotypic appearances deter­
mined with the iodine solution.
After the heterogeneity X3 o f each family was tested against the total segregation
ratio o f each cross, the data obtained from each family were added for the segregation
ratio o f a cross.
Linkage
During the summer of 1979, translocation homozygote lethals developed by Bigger*
staff (1981) involving all possible combinations o f the seven barley chromosomes were
crossed to Shonubet (short awn-Nubet) to establish a hulless tester set o f translocation
homozygote lethals. Information on translocations utilized are presented in Table 3-1. In
November 1979, 10 seeds o f each cross with the translocations were planted at the Mesa
experimental field of Arizona State University and 4 semisterile plants of a cross, when
■
I
possible, were harvested. The segregating F 3 populations were grown at Bozeman in 1980.
Whenever possible, 120 seeds o f four families were planted in rows 30 cm apart, 12 m long.
A t heading time the semisterile, short awn plants in the F 3 populations o f trans­
location crosses were tagged and crossed with the starch mutants. Since the short awn gene
is tightly linked with the hulless gene, the homozygous hulless, short-awn heterozygote
30
translocations were the expected parents. To determine the recombination o f the trans­
location breakpoints involving chromosome I with the short awn and naked genes, F 1
phenotypic segregation ratios o f these populations were classified
The crossed seed between the starch mutants and the hulless translocations were
harvested with the parents. The male parents, presumed to be hulless translocation hetero­
zygotes were confirmed at maturity. The F t plants o f these crosses were increased in the
greenhouse. Sixteen seeds o f each cross, a total o f 46 combinations, between the 3 starch
mutants and hulless translocation homozygote lethals, were planted in 3 benches. Due to
the physiological sterility caused by inadequate control o f greenhouse conditions, geneti­
cally semisterile plants were not easily identified and very few seeds were harvested from
some plants. The available seed harvested from the individual plants was planted in a 3 m
row in the field at the Huntley Agricultural Experimental Station in May 1981. Since the
F 1 seeds increased in the greenhouse were too few to establish recombination, the remain­
ing available crossed seeds were planted again in the greenhouse in February 1981. All the
semisterile plants were harvested and planted in the Bozeman field in June o f 1981.
During the summer o f 1981, the semisterile and fertile plants o f a cross were sepa­
rated, and the plants threshed individually. F 1 segregation ratios o f the pericarp starch
mutants crossed with translocation homozygote lethals were obtained from the material
planted in the Bozeman field. F 1 segregation ratios o f the fractured starch m utant were
obtained from the population grown at Huntley and Bozeman. Ten to 12 seeds, harvested
from a fertile plant, were observed under the light microscope to determine the phenotype
for fractured starch. The results obtained from fertile classes from Bozeman and Huntley
data were combined. To obtain the linkage recombination, the maximum-likelihood
31
Table 3-1. Description of translocations utilized to establish hulless tester stocks o f trans­
location homozygote lethals.
Translocation
Designation
Cultivar
Tl-3e
Tl-Ae
Tl-Sf
Tl-6a
Bonus
Bonus
Bonus
Mars
I
L
S
S
L
S
L
Sat
Tl-Se
Bonus
L
S?
Tl-Sj
Bonus
L
Sat
Tl-7c
Mars
S?
Sat
Tl-71
Tl-Tk
T2-3a
T2-3c
T2-4a
T2-4d
T2-5a '
T2-5e
T3-4b
T3-4d
TA-Se
Bonus, ert a
Bonus, ert a
Gull
Bonus
Mars
Bonus
Bonus
Bonus
Mars
Bonus
Bonus
L
I
S?
S?
Sat
L
S
L
TA-7b
TS-Sb
T5-7g
TS-Tc
Bonus
Bonus
Bonus
Bonus
Breakpoints*
.
-
-
S?
L?
L
S?
-
-
-
S
-
-
L
L
S
L
L
S
Sat
L
L
S
Authority
Persson (1969)
Persson (1969)
Ramage et al, (1961)
Ramage et al. (1961),
Tuleen (1974)
Persson (1969),
Nilan (1964)
Persson (1969),
Ramage (1971)
Nilan (1964, Ramage and
Suneson (1961)
Ramage (1971)
Persson (1969)
Kasha and Burnham (1965)
Kasha and Burnham (1965)
Ramage et al. (1961)
Ramage et al, (1961)
Nilan (1964)
Persson (1969)
Ramage et al. (1961)
Ramage et al. (1961)
Hagbert et al. (1975)
Tuleen (1974)
Ramage et al. (1961),
Ramage and Suneson (1961)
S - short arm; L ■ long arm; Sat - satellite; ? * probably In that
arm; - * breakpoint not determined.
* ; Interchange chromosome with lower number listed first, higher
number listed second.
F
32
method modified by Eslick (personal communication, 1981) for application to trans­
location homozygote lethals was used.
Characters Associated with Starch Mutants
Yield and Yield Components 123
Three starch mutants and their parent, Nubet, were evaluated for yield and yield
components in 3 environments (Bozeman-1980, Bozeman-19 8 1, and Huntley-1981). The
yield trials were planted in randomized complete blocks with four replications in bordered
four rows, 3 m long plots. The seeding rate was I g per 30 cm and the center 2 rows were
harvested. The harvest area was 1.8 ma per plot.
In yield trials at Bozeman in 1980, three lines of Pemubet I (Per 16, Per 17 and Per
18), two lines of Pemubet 2 (Per 28 and Per 31) and 3 lines of Franubet (Opa-1, Opa-2
and Opa-3) were tested and data obtained from each line were averaged for each starch
m utant in each block.
In yield trials grown at Bozeman and Huntley in 1981, the starch m utant stocks con­
sisting o f a mixture of the 3 lines o f each m utant. The data were analyzed by a factorial
arrangement using the environments and isotypes as major effects.
Measurements o f yield and yield components were conducted as follows:
1. Yield, measured in grain weight per harvested area, was converted to kg per hectare.
2. Number o f kernel per spike was obtained by counting the number o f grains from 20
spikes collected at random from the border rows o f the yield trials.
3. Kernel weight (mg/kemel) was obtained by weighing the counted seed from the 20 col­
lected spikes.
33
4. No. o f spikes per m1 was calculated by the following formula: No. of spikes/m2 =
Grain weight o f 1.8 m2 (g)/Grain weight (g) X No. o f kernel per spike X 1.8.
Proximate Analysis and Amino Acid Contents
The grain samples of starch mutants used in proximate analysis were grown under
the conditions presented in Table 3-2. Seeding rates at all the environments was I g per
30 cm in rows spaced 30 cm apart. Wanubet was not grown in the Bozeman-1980 nursery
and Pemubet 2 was not increased at Mesa-1981. Special care was taken to clean the seed
and to equalize the moisture content o f grain. The samples were stored and air dried in the
seed room before analysis. Whenever moisture content varied more than 2 percent, all the
samples were dried in an oven at 50 C for 12 hours. Grain for chemical analysis was ground
with a UDY Cyclone mill fitted with a 1.0 mm screen. Paired T-tests between Nubet and
starch mutants were used to compare averaged data.
The fractions of pearled offals analyzed for chemical components were obtained by
the collection o f grits during the pearling experiments. Three to six samples o f each starch
m utant grown in the different environments were pearled in sequence. A fter finishing 3-6
samples with a predetermined pearling time, the grits were collected from the pearler.
Therefore each pearled offal represented the average o f 3-6 environments.
Chemical analysis determinations were performed by the following procedures:
1. Protein content by UDY method: Used the AACC method (1962) and facilities in the
Cereal Laboratory at Montana State University.
2. Proximate analysis including protein by Kjeldahl method, ash, ether extract, and crude
fiber content were determined at the Animal Nutrition Center, Animal and Range
Table 3-2. Growing conditions o f barley starch m utants used for analysis.
Environment
number
Year
Location
Planting
date
Experiment
Previous
crop
Mu tant
not grown
I
1980
Bozeman, Mt. .
May I
Yield trial
Alfalfa
Wanubet
2
1980
Bozeman, Mt.
May 30
Increase
Fallow
Wanubet
3
1980
Bozeman, Mt.
May 9
Increase
Fallow
Wanubet
4
1980-1981
Mesa, Arizona
Nov.
Increase
-
5
1980-1981
Chandler, Arz.
Nov.
Increase
-
Huntley, Mt.
May
Yield trial
6 .
1981
Fallow
Pernubet
35
Science Department of Montana State University. AOAC standard methods (1970)
were used for these analyses.
3. Starch content: The enzymatic procedure described by Banks and Greenwood (1971)
was used to hydrolyze the starch and the glucose content was estimated using glucooxidase.
4. Amino acid analysis: Four pairs o f samples o f Franubet and Nubet grown at Bozeman,
in 1980, were analyzed. The growing conditions o f three pairs, environment numbers I,
2 and 3, are described in Table 3-2, and the other pair of samples was grown in the same
field condition but planted late (July 3, 1980). Amino acid analysis o f the above
samples was performed using a Bechman 120 C automatic analyzer (Spackman et al.,
1958) and was conducted by A A A. Laboratories: 6206, 89th Avenue, Southeast,
Mercer Island, Washington, USA.
'
'
/KSlucan Viscosity, Amylose Content and Brabender Viscoamylogram I.
I.
f -Glucan viscosity: Viscosity was determined using a method described by Fox
(1981). The resultant viscosity is termed “j$-glucan viscosity.” The grain samples used were
grown under the conditions described in Table 3-2. Pearled offal samples analyzed were
reground in a UDY Cyclone Mill using a 1.0 mm screen.
For the analysis o f 0-glucan viscosity in developing grain o f Franubet and Nubet,
samples were harvested intermittently from the border rows o f the Bozeman-1980 yield
trial. Since anthesis o f Franubet was 3 days later than that o f Nubet, the first four samples
of Franubet were obtained 3 days later than Nubet to synchronize the days from anthesis.
After 25 days from anthesis, the samples were harvest on the same day. Harvested plants
36
were dried in the greenhouse and the sample preparations were the same as the grain
samples.
2. Amylose content: Amylose content o f flour samples was determined by the
potentiometric titration of iodine affinity. Only two flour samples o f each starch mutant,
grown at Bozeman in 1980, environment numbers I and 2 as described in Table 3-2, were
analyzed. Amylose content and Brabender viscoamylogram were conducted in the Cereal
Chemistry Laboratory o f Montana State University.
3. Brabender viscoamylogram: Three flour samples o f each starch m utant, grown
under environment numbers I , 2 and 3 (Table 3-2), were prepared on the Buhler test mill.
The milling method used is explained in the next section. The Brabender viscoamylograms
were conducted at the 11% level o f paste (dry matter basis) by the method described by
Smith (1964). Each sample was run with and without the addition o f 200 mg HgCl, which
is a potent inhibitor o f the alpha-amylase enzyme system. Paste viscosity peaks and the
drops after holding at 92.5 C, and gelatinization temperature were obtained from the Brabender viscosity curves and three graphs o f each m utant were averaged to draw the com­
posite curves presented.
Water Absorption o f Grain and Pearled Grain
Seed from starch mutants grown in several environments described in Table 3-2 was
placed in a small round tube designed to drain the water through the bottom of the tube.
Ten grams o f each sample were soaked for 10 seconds in the water and drained for 30
seconds on tissue paper. The weight o f the 10 second soaked sample was determined with
the container for the initial sample weight. The increments o f weight due to water absorp­
tion by the grain were measured after 3, 6, 12, 24 and 48 hours o f steep time at room
37
temperature, approximately 21 C. Surface water on the sample was drained for 30 seconds
before weighing. Moisture percentages were calculated on a IO g sample basis. Data were
statistically analyzed using the paired T-test between Nubet and starch mutants at each
steep time.
For the sample o f pearled grain, 50 g o f each m utant was pearled for 2.25 minutes
and for an additional 30 seconds for Wanubet and 15 seconds for Franubet.
Use o f Starch Mutants
Milling
Barley starch mutants were milled with the Buhler test mill. To determine the ef­
fects of tempering moisture on the milling barley, the samples from environment number 2
of Pemubet I and Nubet and a sample from environment number 3 o f Franubet explained
in Table 3-2 were tempered to 3 levels o f moisture. About I kg of seed was cleaned and
tempered for 30 minutes to a 11, 13 and 15% moisture level before milling. Since the
moisture content o f samples showed near 9%, about 20 ml, 40 ml and 60 ml o f water were
added to each sample to get the assigned tempering moistures. The mill feeding rates were
about 80-120 g per minute adjusting the vibration nub at 28-30. Total grain weight before
milling and the weight o f the milled fractions were used to determine the percent
recovery. The suction system o f the Buhler test mill was disconnected and a cloth bag was
• attached to collect the red dog flour. The rest o f milling procedure was that described by
McGuire (1979) including the sieve changes and tailing process. Shorts were returned to
1st reduction rolls for a second passage through the mill called tailing process and the
throughs are referred to tailed flour. The red dog flour collected in the cloth bag was
remilled same as the tailings. Each milling stream collected separately.
38
The experimental design was random factorial arrangement with 2 replications.
The methods o f chemical analysis used for this study were the same as those o f the pre­
vious method. Only one sample o f a milled fraction was analyzed.
To compare the milling properties and flour yields o f starch mutants, grain o f the
starch mutants grown in several environments (Table 3-2) were milled. Samples were
tempered for 30 minutes adding 15 ml o f water before milling. The suction system was
connected and the percent recovery o f each sample was determined. The percentage o f
milled fractions were determined on the basis o f recovery o f all streams. Three kinds o f
flour, collected from break roll, reduction roll, and tailed flour, were combined and con­
sidered as total flour. The chemical analysis o f the milled fractions was conducted by the
methods explained above. Paired T-tests were applied for the comparison between Nubet
and the starch m utant means.
Factors Affecting Pearling Index and Pearling Rate
Five separate experiments were conducted to determine the pearling index and
pearling rate. A Strong-Scott barley pearler, model 38, equipped with 10-mesh wire screen
was used in these experiments. The term pearling index refers to the percent of initial
grain weight recovered as pearled grain.
Pearling index = Pearled grain weight X 100/Initial grain weight
Either the slope (byx) by linear regression between pearling time and pearling index
or the differences of pearling index for I minute are referred to as the pearling rate. After
the sample size experiment was conducted, 50 g samples were used in the remaining
experiments.
39
Experiment I : Sample size effect on pearling index.
Betzes and Nubet grain grown at Bozeman, in 1975, were used with 2 replications.
Samples of 25, 50 and 100 g were pearled for 15 second intervals from 0.5 and 3 minutes.
A factorial arrangement with a complete random design was used for statistical analysis.
Experiment 2: Tempering moisture effects on pearling index.
Four Betzes iso type, Betzes, Nubet, Wabet and Wanubet grown at Huntley, in 1981,
were pearled for 2 minutes. Grain samples were dried in the oven for 50 C for 12 hours.
Moisture contents of samples ranged from 7.2 to 7.6 percent. Water was added to obtain
the assigned moisture levels at the end o f 2 hours before the samples were pearled. The
amount o f water added was determined on the basis o f the initial moisture and assigned
moisture levels. A factorial arrangement with a complete random design was applied for
the statistical analysis. The factors used were caryopsis shape, waxy endosperm, normal
shape and endosperm and moisture levels.
Experiment 3: Genotype comparison o f pearling index.
Pairs of iso types o f Compana, Titan and Betzes were pearled. The waxy iso types of
Compana, Titan and Betzes grown in 22, 16 and 18 different environments, respectively,
were compared for pearling index to their normal iso types. Brachytic isotype o f Betzes
I
grown in 29 environments and hulless iso type o f Betzes grown in 10 environments were
compared to their normal isotypes. AU o f these samples were provided from stocks on
hand. The paired T-test was used for the comparisons o f the I and 2 minute pearling
index for the waxy isotypes. Pearling rate was determined by the difference between the
low and high pearling index and adjusted to a per minute basis.
40
Experiment 4: Pearling rate differences o f Betzes isotypes and the relationship o f pearling
index to ash content o f pearled grain.
The purpose of this experiment was to determine the pearling rate differences
among the layers of grain and to determine a method for comparing the genotype differ­
ences o f pearling index at the same pearling rate. Grain samples o f waxy and hulless iso­
types of Betzes grown in Bozeman, 1979, and Huntley, 1981, were pearled for successive
time intervals up to 3 minutes. The time intervals of the first minute were 15 seconds and
the remaining intervals were 30 seconds. The pearled grains collected at different pearling
times were weighed to determine the pearling index and were analyzed for ash content.
The factorial arrangement o f three factors including pearling time, waxy ness and caryopsis
types was used with 2 field replications o f a completely random design.
Experiment 5 : Pearling rates o f starch mutants.
The seed samples of starch mutants grown in the several environments previously
described were pearled with successive time intervals as for experiment 4. The linear
regression coefficients between pearling index and pearling time were compared for Nubet
and the starch mutants.
Rat Feeding Trial
To evaluate the energy and nutritive value o f the starch mutants, a rat feeding trial
and an energy balance trial were conducted in the Animal Nutrition Center, Animal and
Range Science Department of Montana State University.
In the feeding trial, eight female weanling rats (mean weight 70 g) stratified accord­
ing to initial body weight were individually fed balanced diets o f starch mutants with a
41
caesin diet check for four weeks. Barleys used in rat feeding trials were grown in Bozeman
in 1980 (environment 2 in Table 3-2). Diets were prepared with com starch, com oil,
vitamins, minerals and each barley m utant to contain 14.5% protein. Protein contents of
the m utant grain was diluted to the assigned protein content by adding com starch and 1%
of com oil and supplements consisting o f vitamins and minerals were added to each diet.
The proximate analysis of the balanced diets are presented in Table 4-23. Sufficient
water and diet were supplied free choice. The rats were fed for 28 days and were weighed
weekly. Feed consumption and feed efficient (feed consumed/gain) were calculated at the
completion o f the experiment. Protein efficiency ratios (PER) were determined by units of
gain/protein consumed.
To determine the digestible energy o f the starch m utant diets used in the feeding
trial, female growing rats were placed on a 9 day metabolism trial (5 days for adjustment
and 4 days for collection). Sixteen rats were stratified by initial body weight and four rats
with an average 9 1 g were assigned to each diet.
Each rat was fed 10 grams o f diet per day with water free choice. The calculation of
digestible energy for each rat was according to the following formulas:
1. Determine fecal weight on the dry m atter basis.
2. Total energy loss in feces = Fecal DM X Cal/g o f feces.
3. Total gross energy consumed = g o f feed consumed X Cal/g o f feed.
4. Digestible energy coefficient = Gross energy - Fecal energy/Gross energy X 100.
The amount o f energy in feed and fecal material was determined by a Parr Adiabatic
oxygen bomb calorimeter.
Chapter 4
RESULTS
Inheritance and Linkages of Starch Mutants
Description of Mutants
Pericarp starch mutants, Pem ubet I and Pemubet 2, were found after examining ap­
proximately 2,500 and 1,700 Ma seeds, respectively (Table 4-1). Appearance of mutants is
shown in Figure 4-1. Pemubet I stained dark blue (black in the picture) and Pemubet 2
stained light blue (gray in the picture) on the surface of grain with iodine solution. The im­
mature grains o f either mutant and Nubet tended to be green in grain harvested 2 to 3
weeks after anthesis. The greenish color gradually disappeared as the grain matured. Both
Pemubet 2 and Nubet retained the green color when the immature grains were stained with
iodine solution.
Pernubet I seed stained as early as 12 days after anthesis without interference o f the
chlorophyll color and the intensity o f blue tended to become darker as the grain approached
maturity. The stained layers o f the pericarp starch mutants are illustrated in the photo­
micrographs (Fig. 4-1). The layers between the epicarp and aleurone were darker in the
mutants due to positive starch stain (IKI) whereas the large rectangular heavy walled cells
o f the aluerone were free of iodine staining, and thus starch.
The pericarps were separated from the endosperm without the green layers by peel­
ing the seed coat o f the immatured grain at 2 1 days after anthesis. The samples were stained
with iodine. In the samples from Pemubet I , large numbers o f small spherical starch gran­
ules were detected in the epicarp under the microscope. The large starch granules were not
present in Pemubet 2. Instead, the light blue staining was scattered in the pericarp cells.
Since green pigments originating from chloroplasts are located in the cross cell layers.
Table 4-1. Sources o f barley starch m utants, gene symbols and m utation rates.
Mutant
Parent
Mutation
rate
per I
Nubet
1/2,500
B79
B79
B79
B79
per
per
per
per
16
17
18
19*
Pernubet 2
per 2
Nubet
1/1,700
B79
B79
B79
B79
B79
per
per
per
per
per
28
30*
31
32*
33
Franubet
fra
Nubet
Name of stock
Gene symbol
Pericarp
starch I
Pernubet I
Pericarp
starch 2
Fractured
starch
* : Excluded from the study
Previous designation
of mutants
B79 opq I
B79 opq 2
B79 opq 3
44
NUBET
IMMMMt M
12
18
21
25
30
Days a f t e r a n t h e s is
35 I
PERNUBET ! ( P 2M E2LO
IMMMMMt
12
18
21
25
30
Days a f t e r a n t h e s is
35
PERNUBET 2
P«r 2)
HtMHMMt
12 • 18
21
25
30
Days a f t e r a n th e s is
35
Figure 4-1. Grain appearance of barley pericarp starch mutants and photomicrographs of
outside layers of the seed, stained with iodine solution.
-» indicates the starch stained.
45
starch deposition in this m utant appeared to start at the layer between epicarp and hypodermis and proceed inward.
Appearance o f the fractured starch m utant, named Franubet, is shown in Figure 4-2.
When viewed on the light table, the seed cross section shows an opaque endosperm (darker
in picture) similar to waxy endosperm. The endosperm o f Nubet appeared relatively vitre­
ous with clear strips o f cell wall. In contrast, Franubet endosperm was shown to have a
white hue with indistinct cell walls in the center o f the endosperm. The difference between
Wanubet (Waxy endosperm isogene) and Franubet was shown after treatment with iodine
solution. Waxy endosperm developed a reddish brown color right after iodine treatment.
In contrast, the Franubet endosperm revealed the blue staining of a nonwaxy endosperm.
The vitreous ring around the kernel near the aleurone layer was also a good indicator o f
the Franubet genotype. Most starch granules o f Franubet were angular and irregular in
shape and size while the starch granules o f Nubet were round and regular. Most o f the
granules o f Franubet were smaller than the large granules o f Nubet. Some o f the Franubet
granules were larger than the large Nubet granules. These extra-large granules seemed to
consist of many small granules.
The irregular and angular shapes o f the starch granules o f Franubet were observed
as early as 9 days after anthesis with a few normal granules. Most o f the round starch was
cracked or fractured and formed a center cavity when the grain matured.
Allelism of Mutants
Allelism was detected among the 8 pericarp mutants (Table 4-2) selected for study.
F 1 seeds (F t plants) o f an 8 parent diallele cross among lines o f pericarp starch mutants
were treated with iodine solution. F 1 seeds crossed between Per 16, Per 17 and Per 18
46
Franubet
Nubet
Figure 4-2. Photographs of cross sections o f the grain of Franubet and Nubet on a light
table and photomicrographs of their starch granules.
Table 4-2. Phenotypic appearances o f a Fi diallel cross among pericarp starch mutants in barley.
Male mutant no.
Female
mutant no.
per 16
per 17
per 18
per 28
per 30
per 31
per 32
per 33
per -16
-
W
per 17
B
-
per 18
B
B
-
per 28
W
W
W
-
per 30
W
W
W
L
-
per 31
W
W
W
L
L
-
per 32
W
W
W
L
L
L
-
L
per 33
W
W
W
L
L
L
L
-
W
B : Blue stained by Iodine solution.
L : Light blue stained by Iodine solution.
W : Not stained by iodine solution.
W
-
-
L
L
L
48
(Pemubet I) stained blue and F 1 seeds crossed between Per 2 8 ,3 0 ,3 1 ,3 2 and 33 (Pemubet 2) stained light blue. Crosses with Per 16, Per 17, and Per 18 to Per 2 8 ,3 0 ,3 1 ,3 2 and
33 did not stain. No differences were found in reciprocal crosses.
Inheritance o f Mutants
F 2 monohybrid ratios for starch m utants were obtained from crosses o f mutants
with Nubet and Nupana (Table 4-3). F 3 segregation ratio of Pemubet I agreed with a 3:1
ratio at the 95% probability level. No difference between Nubet and Nupana crosses indi­
cated the inheritance of Pemubet I was not changed by the genotype background.
F 1 segregation ratio of Pemubet 2 did not fit a 3:1, 15:1, or 13:3 ratio. F 1 segre­
gation of the crosses between Pemubet I and Pemubet 2 showed the white, light blue and
blue class. No additive gene appearance was noted in the segregation (Table 4-3). F 1 segre­
gation did not fit 9:3:4 or 45:3:16 ratio also indicating the Pemubet 2 gene involved other
factors affecting its inheritance.
Crossed seeds o f reciprocal crosses between Franubet and Nubet were observed
under the microscope to verify the suspected xenia effect o f the fractured starch gene.
Seed from both crosses had the round granule characteristic of Nubet. This result revealed
that normal starch granules were dominate to fractured starch granules in the endosperm
genotypes o f Fra fra fra and Fra Fra fra and the gene expressed xenia. F 1 segregation ratios
(F 1 seeds) o f Franubet crosses fit a 3:1 ratio for starch granule type with over 95% prob­
ability regardless o f genotype background. Backcross F 1 seed segregation also showed an
agreement with the expected ratio o f IAa: Iaa with a 75-50% probability.
Table 4-3. Segregation ratios o f barley starch m utants phenotypes.
Cross
Female
Generation
Male
Ratio
Chi-
Total
tested
square
Segregation
A-
aa
Nubet x Pernubet I
Nupana x Pernubet I
Fy plant
Fy plant
303
311
103
104
406
415
3:1
3:1
Nubet x Pemubet 2
Fy plant
1,014
109
1,123
3:1
15:1
13:3
Nubet x Franubet
Franubet x Nubet
Nubet x Fra/Franubet
Nubet x Franubet
Franubet x Nupana
F^ seed
F^ seed
BC^ seed
Fy seed
Fy seed
25
35
71
436
104
0
0
64
148
35
25
35
135
584
139
: A-B- A-bb aa-
per I x per 2
Fy plant
808
54
5
0.030
0.001
140.09
16.67
60.29
Probability
of fit
<0.95
<0.95
>0.005
>0.005
>0.005
1:1
3:1
3:1
0.363
0.037
0.002
0.75-0.50
<0.95
<0.95
9:3: 4
45:3:16
39:9:16
41.74
157.11
4.843
>0.005
>0.005
0.10-0.05
Total
1177
A- : Dcxninant classes of starch mutants.
aa : Recessive classes of starch mutant.
A-B- : Double dominant for per I and per 2. A-bb : Per I- per 2 per 2.
aa- : Either per I per I Per 2- or per I per I per 2 per 2 respectively.
50
Linkage
Linkage was determined between translocation breakpoints and starch mutants
from F2 plant segregation ratios. F 3 segregation data for location o f the pericarp starch
mutant I gene are presented in Table 4-5. The F3 segregations were expected to fit a ratio
6 semisterile dominant:2 semi-sterile recessive:] fertile dominant: I fertile recessive when
the gene was independent from the translocation breakpoint in the crosses between trans­
location homozygote lethal stocks and the gene studied.
Linkage chi-square was calculated by subtracting the chi-square values for 3A-: Iaa
and 2 semi-sterile:I fertile ratios from the chi-square o f the 6 :2 :3 :1 ratio expected with
independence. With no crossing over the expected ratio is 2 :0 :0 :1.
The linkage chi-squares obtained from the crosses of T l-6 j, T l-7c, T2-5a and T5-6b
were significant. Since the segregations o f the T2-5a and T5-6b crosses showed an excess
of recombinants, the significant linkage chi-squares o f these crosses appeared to be due to
unusual segregation (an excess of recombinants over that expected with independence) and
were considered as independent in determining linkage.
Since linkage is indicated for the breakpoints o f two translocations, T I - 6j and T I -7c,
with the Pem ubet I locus, and the chromosome in common is chromosome I , the Pemubet I m utant is assigned to chromosome I by this method. Linkages between breakpoint
and pericarp starch I genes were 1.3 ± 0.6% in T l-6 j and 5.3 ± 2.8% in T I-7c combinations.
During the conversion o f the covered translocation tester set to hulless, the linkage
recombinations were determined between the translocation breakpoints involving chromo­
some I and the naked caryopsis and the short awn gene (Table 4-4). The crosses o f T l-4e,
T l-6e and T I -7k showed 0.5 to 0% recombinations between the breakpoints and both the
51
Table 4-4. Linkage recombinations between translocation breakpoints involving chromo­
some I and naked and short awn gene from F2 segregations of barley translo­
cation homozygote lethals.
Translocations
Recombination (7.)
No. of plants
observed
naked (n)
Breakpoints
short awn (lk2 )
Tl - 3e
273
6.7 + 1.4
10.2 + 1.8
Tl - 4e
328
0.1 + 0.7
0.1 + 0.7
Long arm
beyond Ikg
Tl - 5f
391
2.6 + 0.7
7.8 + 1.3
Short arm*
Tl - 6a
364
3.7 + 0.9
7.2 + 1.3
Short arm*
Tl - 6e
442
0
0
Long arm
beyond Ikg
Tl - 6j
338
3.5 + 0.9
7.8 + 1.4
Tl - 7c
323
5.8 + 1.2
10.8 + 1.7
Tl - 71
374
2.9 ± 0.8
9.1 + 1.4
Long arm near
centromere
Tl - 7k
332
0.2 + 0.2
0.5 + 0.3
Long arm
beyond Ikg
+ 0.4
* : Possibly in long arm near centromere.
+0.4
Short arm
Long arm near
centromere
Short arm
Table 4-5. F 2 segregations o f pericarp starch m u tan t I crossed w ith translocation hom ozygote lethals and their
linkage recom bination values.
Male in
cross
Semi-sterile
Fertile
Chi-•square
Total
Recombination
(%)
2
6:2:3:!-/
Linkage x
232
50
276.96**
49.98**
264.75**
17.95**
1.3 + 0.6
5.3 + 2.8
4
7
78
158
3.26
11.51**
0.00
0.18
independent
independent
64
46
15
9
155
165
27.32**
3.17
5.28*
3.17
independent
independent
14
13
31
25
9
8
109
91
1.48
0.56
0.04
0.02
independent
independent
44
65
14
14
43
21
23
3
124
103
27.56**
10.73**
3.92*
2.11
independent
independent
77
24
28
7
136
0.10
independent
Per I
per I
per I
Per I
T1-6J
Tl-7c
150
21
3
2
I
2
78
25
T2-3a
T2-4d
46
99
13
24
15
28
T2-5a
13-4b
49
78
27
32
T3-4d
T4-5e
55
45
T5-6b
T5-7g
T6-7c
per I
per I
3.98
a/ : 6:2:3:1 is a ratio of SSA-:SSaa:FA-:Faa expected.
*, ** : Significant at Pe OeOS and 0.01 levels, respectively.
\
53
short awn and naked caryopsis genes. Since crossing over is greatly reduced in the inter­
stitial segment o f a translocation heterozygote, the breakpoint o f these translocations was
postulated to be distal to the short awn gene. The largest recombinations were shown in
the crosses of T l-3e and T I -7c. Recombinations in the crosses ranged from 2.6 to 3.7%
with the naked caryopsis gene and from 7.2 to 9.1% with the short awn gene.
F 1 segregation and recombination data for the location o f pericarp starch m utant 2
gene are presented in Table 4-6. None o f the crosses fit the expected 6 :2 :3 :1 ratio, prob­
ably due to the abnormal segregations o f Pemubet 2 previously discussed. When the link­
age chi-square and maximum likelihood methods were applied, assuming single gene con­
trol for this phenotype, the linkage chi-squares for Tl-7c, T l-7i and T5-7g which have
chromosome 7 in common were shown to be linked and their recombinations were 26.0 ±
4.9, 34.2 ± 7.1 and 2.9 ±1.7% , respectively. The linkage chi-square o f T l-6j was also sig­
nificant, but the segregation showed an excess o f recombinants and thus considered inde­
pendent. Pericarp starch m utant 2 appears to be associated with chromosome 7. Addi­
tional information would be desirable.
■<
.
F 1 segregations for the fractured starch mutant are shown in Table 4-7. Since the
normal starch genes express xenia, the heterozygous and homozygous genotypes for
fractured starch gene were classified in F 1 plants. Chi-square values were expected to fit a
ratio lA A :2A a:laa when the fractured starch gene was independent of translocation
breakpoints. With no recombination the expected ratio is 0:0:1. The segregations o f T2-4a
and T2-4d were significantly different from the expected ratio and linkage recombinations
of T2-4a and T2-4d were 25.8 ± 8.5 and 30.8 ± 6.9%, respectively.
Table 4-6. F 2 segregations o f pericarp starch m utant 2 crossed with translocation hom ozygote lethals and their
recom bination values.
Male in
cross
Semi- sterile
Per 2
per 2
per 2
Fertile
Per 2
Total
per 2
per 2
Chi- square
6:2:3:!-/
Recombination
Linkage x2
(%)
independent
Tl-6 j
18
13
18
2
51
7.94**
6.62**
Tl-7c
74
2
21
11
108
23.41**
13.06**
26.0 + 4.9
Tl-71
42
13
10
8
73
8.87**
6.40**
34.2 + 7.1
T3-4d
65
13
43
10
131
6.86**
0.00
independent
T4-5e
90
11
25
5
131
19.58**
1.74
independent
T5-7g
58
I
2
22
83
68.27**
67.21**
2.9 ± 1.7
a/ : 6:2:3:1 is a ratio of SSA-:SSaa:FA-:Faa expected.
*, ** : Significant at p=0.05 and 0.01 levels, respectively.
Table 4-7. F2 segregations o f th e fractured starch m utant crossed w ith translocation hom ozygote lethals and
their linkage recom bination values.
Male in
cross
Tl-3e
Tl-6a
Tl-6 j
Tl-7c
T2-3a
T2-4a
T2-4d
T2-5e
T3-4b
T3-4d
T4-5e
14-Tb
T5-6b
T5-7g
T6-7c
Fertile
Total
AAA
Aaa
AAa
aaa
8
I
4
5
26
3
12
14
50
7
4
7
19
23
13
7
5
11
12
38
17
21
19
64
27
9
6
27
34
34
5
I
2
3
21
25
40
13
38
18
9
2
15
15
10
20
7
17
20
85
45
73
46
152
52
22
15
61
72
57
Chi-square for a ratio
Recombination
IAAA:2Aaa:Iaaa
(%)
2.700
1.286
1.941
1.200
1.541
24.200**
34.644**
1.435
5.684
4.730
3.000
3.933
1.328
2.000
2.438
independent
independent
independent
independent
independent
25.8 + 8.5
30.8 + 6.9
independent
independent
39.5 + 8.4
38.6 + 12.9
independent
independent
independent
independent
A, a Indicates the dominant genotype (Fra) and the recessive genotype (fra),
respectively.
*, ** : Significant at p*0.05 and 0.01 levels, respectively.
56
Independent assortment was shown for the crosses between Franubet and T2-3a and
T2-5e translocations. Chromosome 2 breakpoints thus appear to be inherited indepen­
dently of the fractured starch gene. This places the fractured starch gene on chromosome 4.
The segregation of T3-4d and T4-5e crosses also showed a linkage between the frac­
tured starch gene and translocation breakpoints. Weak linkage obtained appeared to sup­
port the gene assignment on chromosome 4 because both translocations have chromosome
4 in common and in other crosses both chromosome 3 and 5 breakpoints appear to be
independent o f the fractured starch gene. T2-4d and T4-5e breakpoints are reported in the
long arm of chromosome 4. T3-4b and T4-7b are reported in the short arm of chromosome
4. This would place the gene distal to the breakpoints in the long arm of chromosome 4.
It would also place the breakpoints of T2-4a and T3-4d in the long arm o f chromosome 4.
Additional information from crosses with genes or breakpoints in the long arm o f chromo­
some 4 would be desirable.
'
Characters Associated with Starch Mutants
Yield and Yield Components
Yield performance o f starch m utants was evaluated in replicated trials in 3 environ­
ments (Table 4-8). Pemubet I and Pemubet 2 were inferior to Nubet in yield while the
yield o f Franubet was not significantly different from the yield of Nubet. Starch mutants
had lighter kernel weight than that o f Nubet with this being the main reason for reduced
yields of Pemubet I and Pemubet 2. Number of kernels per spike in Franubet was signifi­
cantly less than that for Nubet and the number o f spikes per m3 o f Franubet was signifi­
cantly higher than Nubet.
t
57
Table 4-8. Average yield and yield components o f barley starch mutants tested in 3 envi­
ronments with 4 replications.
Mutants
Yield
kg/ha
No. of kernels
per spike
Kernel wt.
mg
No. of spikes
per
Nubet
3,174
23.7
40.4
334
Pemubet I
2,587*
24.2
35.9*
302
Pernubet 2
2,745*
23.1
36.2*
332
Franubet
2,975
20.1*
33.3*
451*
Glonubet
2,975
22.4*
36.6*
367
LSD (5%)
259
0.66
1.03
35.6
* : Significantly differs from Nubet at pe0.05 level.
The analysis o f variance for yield trials grown in 3 environments (Table 4-9) revealed
significant interactions between genotypes and environments for the yield components.
Interactions for yields, however, were not significant. These results could be explained by
the compensating effects o f the yield components. Yield and yield component inter­
actions are presented in Figure 4-3. The number o f spikes per m1 in Franubet and the
number of kernels per spike in Pemubet I and Pemubet 2 appeared to be major compo­
nents compensating to maintain the yield in the high yielding conditions such as Huntley1981 and Bozeman-19 8 1. More increments o f these yield components than for other
mutants apparently resulted in the yield component interactions. These extra increments
of yield components were accompanied by excessive reduction o f the other yield com­
ponents. In Franubet, the increased number o f spikes per ma was associated with a
reduction o f kernel weight. The lower numbers o f spikes per unit area o f Pemubet I and
Table 4-9. Analysis o f variances fo r yield trials o f barley starch m utants tested in 3 environm ents w ith 4 replications.
Mean squares
Source
No. of kernels - Kernel wt.
per spike
No. of spikes/mz
df
Yield
Environments (A)
2
4,851,804**
152.08**
Error a
9
109,774
1.45
3.06
2,630
Genotypes (B)
4
623,919**
30.15**
77.62**
39,197**
AxB
8
181,076
6.34**
5.42*
5,925**
36
97,795
0.64
1.56
1,840
Main plot
58.20**
63,187**
Sub plot
Error b
Total
59
*, ** : Significant at p-0.05 and 0.01 levels, respectively.
No. of kernels per spike
No. of spikes per m*
Figure 4-3. Graphic representation o f yield and yield component interactions o f barley starch mutants with the
environments.
60
2 were followed by higher numbers o f seed per spike. In this way, the yield differences
at each o f the environments and the order o f mutant yields among environments were sim­
ilar to each other and thus resulted in the no significant interaction for yield.
Proximate Analysis and Amino Acid Contents
Proximate analysis of the grain o f the starch mutants grown in several environments
is shown in Table 4-10. Three to six samples were compared by paired T-test. Protein con­
tents determined by the UDY method were significantly higher for Franubet than Nubet.
Protein contents determined by the Kjeldahl method were not different for Nubet and
Franubet.
Starch contents of Pemubet I were significantly lower than that of Nubet and the
remaining mutants had the same starch content as that o f Nubet. Ether extract (lipids),
crude fiber and ash contents o f the m utants were not significantly different from those
o f Nubet.
Starch contents were determined on the pearled offal fractions collected from the
abraded grits o f the grains from the pearling machine (Fig. 4-4). The 0-5% fraction o f
pearled offal consisted principally of germ, pericarp while the 15-20% fraction o f pearled
offal apparently consisted mostly o f aleurone layer and endosperm starch. As expected,
the starch contents increased as pearled offal fractions were collected from the outside o f
the kernel toward the center. The fractions free from outside layers had about 70% starch.
The starch contents of these pearled offal fractions not only reflected genotype differences,
but overlapping fractions due to the sample preparation (pearling) also influenced the
results.
Table 4-10. Proxim ate analysis o f grains from barley starch m utants grown in several environm ents (Dry m atter
basis).
Mutants
Measurements
Nubet
Environment
6
Pemubet I
6
Pemubet 2
5
Franubet
6
Wanubet
3
% protein (UDY)
Difference
14.84
15.04
(+0.20)
15.81
(40.45)
16.11*
(+1.27)
13.46
(40.17)
% protein (KJeldahl)
Difference
14.34
14.71
(+0.37)
16.13
(+1.31)
15.14
(40.80)
11.88
(-0.29)
% starch
Difference
59.7
57.9**
(-1.8)
56.5
(-3.2)
59.8
(+0.1)
57.4
(-1.9)
% ether extract
Difference
2.55
2.61
(40.06)
2.51
(-0.05)
2.90
(40.35)
2.95
(40.42)
% crude fiber
Difference
0.38
0.32
(-0.06)
0.34
(-0.04)
0.39
(40.01)
0.43
(+0.10)
% ash
Difference
1.86
1.94
(40.08)
1.80
(-0.08)
1.97
(+0.11)
1.96
(40.05)
The numbers in the parentheses indicate average differences from Nubet.
*,** : Significantly differs from Nubet at p*0,05 and 0.01 levels, respectively.
Percent s ta rc h ( a s Is b asis) (y )
62
—*
—*
0 5 10 15
I t t t
5 10 15 20
20
t
80
N ubet
:
Pernubet I :
Franubet :
30
40
0.412
0.9 9 0
0 . 3 1 6*
0.452
09 7 9
0 .9 S 7
60
Percent grain weight removed as pearled offal (x )
a/ : Slope of log 10 of starch content on log 10 of pearled
offal fractions.
b/ : Correlation coefficients between log 10 of starch content
and log 10 of pearled offal fractions.
* : Differs from Nubet at p-0.05 level.
Figure 4-4. Changes o f starch co n ten t in pearled offal fractions o f barley starch m utants.
63
Pericarp starch mutant I tended to have a higher starch content in outside fractions
than Nubet, and between outside fractions and center o f kernel were lower in starch con­
tent than Nubet. This tendency might explain the kernel weight differences. Since Pemubet I had smaller grain than Nubet, the outer layers of the kernel should represent a higher
proportion in the intermediate offal fractions. The log conversions o f the starch contents
and % pearled offal fractions were made to test the slope differences between Nubet and
the mutants. The correlation coefficients between the two traits calculated from the log
conversion data were 0.990, 0.979 and 0.987. A significant difference between the slope
o f Nubet and Pemubet I were detected.
Ash content o f the pearled offal fractions o f the starch mutants and Nubet are com­
pared in Figure 4-5. In general, the ash content of the pearled offal fractions showed an
inverse relationship to starch content. Inverse transformations o f the ash content were
made to test the slope differences. No significant differences o f slope between Nubet and
the mutants indicated a similar pattern o f ash content in the offal fractions o f Nubet and
the mutants. The offal fractions near the outside o f the kernel consisting mainly o f the
pericarp and aleurone layer ranged from 3.7% to 4.5% according to genotype. The pericarp-aleurone offal fractions o f the Pemubet I appeared to have a lower ash content than
Nubet, but the statistical analysis o f these comparisons did not support this conclusion.
The endosperm, presumably free o f aleurone and outer layers consisted o f 0.60% ash with
relatively small genotype differences.
Protein contents o f pearled offal fractions showed a pattern similar to those o f ash
content (Fig. 4-6). The outermost layer o f the kernel consisted o f more than 25% protein
and the inner fraction, free from the outside layers, approached 10% protein. No signifi-
64
•
* • *
4
O 5 10 15
I
i
i
i
5 10 15 20
20
i
30
i
30
40
Nubet
Pernubet I
Pernubet 2
Franubet
40
50
i
50
i
60
60
I
70
Percent of grain removed as pearled offal (x )
a/ : Slope of 1/ash content on pearled offal fractions,
b/ : Correlation coefficients between 1/ash content and pearled
offal fractions.
Figure 4-5. Changes o f ash co n ten t in pearled offal fractions o f barley starch m utants.
I
I
65
»" — '■#
0 - ■— A
^
Nubet
Pernubet I
Pernubet 2
Franubet
20
40
50
30
0 5 10 15
( ( t (
I
I
I
(
5 10 15 20
50
60
40
30
Percent of grain removed as pearled
60
I
70
offal ( x )
70
I
100
a/ : Slope of I/protein content on pearled offal fractions,
b/ : Correlation coefficient between I/protein content and pearled
offal fractions.
Figure 4-6. Changes o f protein content in pearled offal fractions o f barley starch m utants.
66
cant differences between the slope o f Nubet and the slope o f mutants were determined
from the transformed linear regression data. Linearity was established by dividing one by
the protein content and regressing on the pearled offal fractions.
The amino acid content o f the fractured starch m utant was compared to Nubet
utilizing paired samples grown in 4 environments. Average percent amino acid in the grain
and in the protein was calculated (Table 4-11).
Glutamic acid and proline were the dominate amino acids present in both Nubet and
Franubet. Significant differences between Franubet and Nubet were shown for isoleucine,
lysine, glutamic acid and proline on the basis o f the amount in the grain. Lysine content in
Franubet was over 18% higher than in Nubet. The increased lysine in Franubet was asso­
ciated with a decrease in glutamic acid, isoleucine and proline in the grain. Lysine content
based on the protein was 2.22% in Nubet and 2.81% in Franubet. These might be relatively
lower than usual and are suspected to be due to the high protein content in the samples
which were grown at high fertility level.
While comparing amino acids in the protein, Franubet had more alanine, aspartic
acid, glycine, leucine, lysine, threonine and tyrosine and significantly less glutamic acid
than Nubet.
g-Glucan Viscosity, Amylose Content and Brabender Viscoamylogram
P-Glucan viscosity o f grain and pearled fractions o f the starch m utants were
measured with a Brookfield viscometer on the alkali extractions (Fig. 4-7). P-Glucan vis­
cosities o f Pemubet I and Pemubet 2 were not significantly different from the viscosity o f
Nubet. The lowest P-glucan viscosity was shown by the Franubet grain determinations,
Table 4 -1 1. Average am ino acid contents o f grain o f fractured starch m utant (F ranubet) com pared to N ubet,
from samples growin in 4 environm ents.
Amino acid
As % of grain (DMB)
Fr^nybet
As % of protein (DMB)
Difference
(y-x)
Nubet
(x)
Franubet
(V)
Difference
(y-x)
40,23*
40.69
40.36**
-2.47*
40.26**
Alanine
Arginine
Aspartic acid
Glutamic acid
Glycine
0.599
0.607
0.982
5.755
0.491
0.602
0.682
0.983
4.995
0.504
40.003
40.075
40.001
-0.760**
40.013
3.33
3.36
5.45
31.91
2.73
3.56
4.06
5.81
29.44
2.98
Histidine
Isoleucine
Leucine
Lysine
Methionine
0.235
0.615
1.080
0.400
0.308
0.257
0.583
1.070
0.473
0.302
40.002
-0.032**
-0.010
40.073*
-0.006
1.30
3.42
6.01
2.22
1.71
1.53
3.44
6.35
2.81
1.78
Phenylalanine
Proline
Serine
Threonine
Tyrosine
0.953
2.400
0.828
0.597
0.474
0.879
2.140
0.804
0.593
0.492
-0.074
-0.260*
-0.024
-0.004
40.018
5.28
13.28
4.60
3.32
2.63
5.21
12.62
4.75
3.51
2.91
Valine
Taurine
0.807
0.923
0.781
0.782
-0.026
-0.141
4.48
5.14
4.61
4.61
18.000
16.900
Total
40.22
40.03
40.34*
40.59*
40.07
-0.07
-0.66
40.14
40.19**
40.28*
40.13
-0.53
-1.100**
*,** : Significantly differs from Nubet at p=0.05 and 0.01 levels, respectively
68
80 ■
- g lu c a n v is c o s ity ( c p s .)
70 ■
60 ■
50 ■
40 •
30 ■
«q 20 ■
10 ■
20
30
0 5 1015
40
50
60
70
I
i
i
i
i
i i i
i
i
100
30
5 10 15 20
50
40
70
60
Percent of grain weight removed as pearled offal
Indicates the average viscosity of grain grown
in several environments.
*, ** : Significantly differs from Nubet at p-0.05 and 0.01 levels,
respectively.
Figure 4-7. Mean 0-glucan viscosity o f grain and pearled offal fractions o f barley starch
m utants grown in several environments.
69
averaging 4.0 centerpoise units, which was significantly lower than Nubet. Wanubet had
the highest 0-glucan viscosity in grain and was almost double that of Nubet.
The area o f deposition of substances contributing to viscosity in the grain are also
illustrated in Figure 4-8. Outer layers o f pearled offal tended to have the lowest /S-glucan
viscosity regardless o f genotype. The pearled offal fractions collected nearer the center
of the kernel showed higher jJ-glucan viscosity than pearled offal fractions from the outer
layers.
The pearled offal collected as the 30 to 40% or 40 to 50% fractions had the maxi­
mum viscosity and the more central fractions of the endosperm tended to decline slightly
in viscosity. Genotype differences appeared to be greatest at maximum 0-glucan viscosities.
Synthesis of 0-glucan substance in the developing kernel is shown in Figure 4-8.
0-Glucan viscosity o f Nubet started to increase 3 weeks after anthesis and was at maximum
from 30 to 35 days after anthesis until maturity. Franubet showed the same tendency as
Nubet with much lower viscosity.
Amylose contents o f starch mutants were determined by potentiometric titration o f
iodine affinity (Table 4-12). Since the electrode had been stored in a dry condition for too
long a time, the standard curve appeared to have shifted. Therefore results obtained from
the samples are suspected to be I or 2% higher values than they should be. The amylose
content o f flour samples from starch mutants ranged from 22.0% to 24.5% in non-defatted
samples and from 27.5% to 30.5% of dry weight in defatted samples. The genotype differ­
ences were not significant.
The gelatinization temperatures o f the starch mutants were about 60 C with little
variation (Table 4-12). No significant differences among genotypes were detected.
70
N ubet
o 10
F ra n u b e t
9
13 15
18
21
harvest
Days from anthesis
Figure 4-8. Changes o f 0-glucan viscosity as the grain of Nubet and Franubet developed.
Table 4-12. Amylose content, gelatinization tem perature and am ylograph paste viscosity data o f Buhler milled
flours prepared from barley starch m utants.
Mutants
No. of
Measurements
7o
Environments
Nubet
Pernubet I
Pernubet 2
Franubet
2
2
24.5
30.5
22.0
27.5
24.5
29.0
23.5
29.0
2
60.0
60.5
(59.0)
60.0
1630
370
1650
380
(1500)
( 400)
1583
430
770
170
( 660)
( 260)
800
180
amylose
Non-defatted
Defatted
Gelatinization
temoerature ( C)
Amylograph viscosity with HgCl? (EU)
Peak
Drop at 92.5 C
3
3
Amylogranh viscosity without HgCl? (EU)
Peak
Drop at 92.5 C
3
3
( ) : Data from I sample only.
830
300
72
Amylograph paste viscosities o f flours milled from starch mutants with HgQ, and
without HgCl2 are presented in Table 4-12 and Figure 4-9. The greatest differences oc­
curred between with HgCl2 and without HgCl2 indicating alpha-amylase activities during
starch cooking. The maximum viscosity o f the Nubet flour with HgQ2 was about 1600BU
and viscosity dropped at 92.5 C to about 400BU. No consistent differences between
genotypes occurred in the curves and no significant differences were detected for maxi­
mum viscosities and viscosities dropped at 92.5 C.
Water Absorption of Grain and Pearled Grain
The percent water absorptions o f grain and pearled grain o f the starch mutants were
determined by successive increments o f steeping time and data are presented in Table 4-13
and Figure 4-10. The water absorptions plotted against the steeping times showed hyper­
bolic curves in either grain or pearled grain. The water absorptions o f pearled grain were
considerably faster than the water absorptions o f intact grain. The maximum absorptions
were reached within 12 hours in pearled grain and after 48 hours o f steeping in intact grain.
The intact grain o f Wanubet absorbed more water after 24 and 48 hours steeping
than Nubet but no significant differences between Nubet and Wanubet were found after
steeping for 12 hours or less. The pearled grain o f Wanubet had an exceptionally high
water absorption as compared to the pearled grain o f Nubet. The greatest difference oc­
curred between the pearled grain o f Nubet and Wanubet at the 12 hours steeping with little
change after 12 hours steeping. These results demonstrated that the water permeability o f
Wanubet grain appeared to be similar to that o f Nubet. The water holding capacity of
Wanubet endosperm, however, was much higher than Nubet endosperm.
73
Temperature
Temperature
70
-e—
40 *C
4 0 eC
i t .......
Pernubet I
I
IBUl
& L .
70
1800 -
1800
V iscosity
—
••
1400
II
with HflCia
1000
/
Pernubet 2
5 1400
ea
fl
• • " ♦ W i t h HflCI2
i\
- 1000 ■
w
o
o
KJ
600
600
A ,/
200
/
20
200
Without HgCla
40
J
60
20
Time I mlns.l
4 0 eC
Temperature
92.3
40 "C
Without HflCI2
40
60
Time ImInsJ
Temperature
92J
70
........ ' ■ -N ..M...... »■
1800
(BUI
3
V iscosity
Franubet
- IOOO
•
o
o
O
1400
•
>
600
200
\\
I ! ♦ —Wlt h HgCI2
l \
j I:A
#
20
Without HflCI2
40
60
Time (mlns.l
80
Figure 4-9. Brabender amylograms o f Buhler milled flours obtained from barley starch
mutants, with and w ithout HgQj added.
Table 4-13. Average w ater absorbed b y grain and pearled grain o f barley starch m utants, growin in several envi­
ronm ents, for different steep tim es a t room tem perature.
Nubet
Environment
6
Pernubet I
6
Pernubet 2
5
Franubet
6
Wanubet
3
Water absorotion of grain. %
3
6
9
12
24
48
hours
hours
hours
hours
hours
hours
13.2
22.6
30.1
35.3
48.5
59.2
14.3*
24.9**
32.8**
38.5**
52.2*
63.1
14.0
23.7
31.6
37.1
50.2
61.8
15.9*
27.1**
35.9**
41.6*
55.1*
66.3**
13.5
23.5
32.1
38.3
55.2**
70.4**
Water absorption of pearled grain. 7.
Average pearling
index
3 hours
6 hours
9 hours
12 hours
24 hours
(67.4)
(66.6)
(68.0)
31.5
53.2
59.1
62.3
63.8
39.8*
57.6*
63.4*
67.1*
68.9*
37.6
55.1
61.5
64.6
65.9
*, ** : Significantly differs
(67.7)
43.5**
60.6**
63.0
65.6
66.4
(69.5)
46.1*
69.0**
76.7**
80.6**
81.6**
from Nubet at p=0.05 and 0,01 level, respectively.
75
P e a rle d grain
$ Grain
Nubet
Pernubet I
Franubet
Wanubet
3 6 9 12
24
Steeping tim e (h o u rs)
Figure 4-10. Relationship of water absorption to steeping time o f the grain and pearled
grain o f barley starch mutants, mean o f several environments.
76
Pearled and intact grain o f Franubet absorbed more water than that o f Nubet at all
steeping times, however, no sign (leant differences from Nubet were found after 9 hours of
steeping the pearled grain.
The water absorptions o f intact and pearled grain of Pemubet I were significantly
higher than Nubet at all steeping times. The endosperm o f Franubet and Pemubet I ap­
peared to absorb water faster than the endosperm of Nubet.
No significant differences were obtained between Pemubet 2 and Nubet in either
intact or pearled grain at all steeping times.
Use o f Starch Mutants
Milling
The average effects o f tempering moisture on milling fractions o f starch mutants
(Pemubet I, Franubet and Nubet) recovered from the Buhler test mill are presented in
Figure 4-11. Analysis o f variances o f the recovery o f each fraction revealed that reduction
roll flour, tailed flour and bran were significantly different for the isotypes and the
tempering moisture levels (Table 4-14). Interaction o f the bran fraction o f isotypes with
moisture levels was significant. This indicated the moisture effected the bran recovery o f
one isoline differently than the others. The interactions o f the other milled extractions
were not significant so that only average tempering moisture effects are discussed here.
As the moisture o f the tempered barley increased, the flour yield recovered from the
reduction roll and tailed flour decreased as the bran fraction increased at the expense o f
flour yield.
77
100
■
90
■
Shorts
I
to
I
Xl
80
Bran5z
Shorts
Bran^z
Shorts
B ran 5z
■
>1
I$
I
70
&
Tailed
flour
60
f7
O
2
■n
£
50
Tailed
flour
6,
40
S/
E
g
0)
Tailed
flour
■
30
■ .
R e d u c tio n
R e d u c ti o n
ro ll flo u r
ro ll f lo u r
&
R e d u c tio n
ro ll flo u r
H
2
20
■
iI nU
B re a k
B re a k
B re a k
ro ll f l o u r
ro ll flo u r
ro ll f l o u r
11
13
15
Tempering m oisture (%)
Figure4-11. Tempering moisture effects on Buhler milled fractions (Average o f Pemubet I , Franubet and Nubet with 2 replications). The different letter super­
scripts indicate a significant difference among each milled fractions at
P=O OS level.
Table 4-14. Analyses o f variance of the tempering moisture effects on Buhler milled fractions for barley starch
mutants.
Source
DF
Mean squares
Break
flour
Reduction
flour
Tailed ■.
flour
1.96
Bran
Shorts
Genotypes (A)
2
7.46*
115.43**
Moistures (B)
2
0.12
308.28**
AxB
4
2.22
3.15
4.19
Error
9
0.99
3.36
1.72
2.26
1.55
Total
17
1.30
2.39
1.71
1.96
1.63
LSD (5%) for
moisture
29.87**
,
*, ** : Significant at p=0.05 and 0.01 levels, respectively.
89.29**
71.98**
437.86**
0.31
17.95**
5.12
79
In general, barley bran was crumbled rather than flaked and some o f the endosperm
starch remained attached. Well separated bran from the endosperm was not obtained
regardless of the genotype or tempering moisture level. The shorts appeared to be flaked
pieces from the crushed endosperm. Shorts were light and fluffy. The yield o f the shorts
was relatively consistent for all tempered moisture levels, but a considerable amount of
bran and the first shorts to come from the reduction sieves increased with increasing
moisture levels. The constant amount o f shorts obtained are suspected to be due to the
tailing process. Average flour yield, obtained by adding break roll, reduction roll and tailed
flour, were significantly lowered as tempering moisture increased.
Ash and protein contents o f the flours collected from each mill stream tended to
increase as the tempering moisture decreased and as the flour yield increased. Higher per­
centages of ash and protein in the tailed flour appeared to be an increasing source o f ash
and protein contents o f the flour (Fig. 4-12).
The milling properties and flour yields of starch mutants were evaluated by com­
parison to Nubet (Fig. 4-13). Significant differences were obtained between Nubet and
Franubet, and Nubet and Wanubet for percent recovery, yield of flour, and yield of bran.
The percent recovery and flour yield o f Franubet were higher, and those o f Wanubet
were lower than Nubet. Pemubet I and 2 were not different from Nubet.
The flour yield o f Franubet ranged from 70 to 81% and averaged 13% more than
was obtained from Nubet. The flour yield o f Wanubet was significantly less than Nubet
drastically increasing the bran fraction. The pericarp starch mutants were not significantly
different from Nubet for flour, bran and short recovery.
80
13% m oisture :
15%moisture : I'
,
Breakroll
Reduction roll
Flour
Tailed
Breakroil
Fteductionroii
Flour
Tailed
Shorts
Percent p ro te in (DMB)
Percent ash (DMB)
11% m oisture : ;*x*:*:
Milled
B ran
Shorts
fraction
Figure 4-12. Ash and protein content o f Buhler milled fractions as effected by temper­
ing moisture (Average of Pemubet I , Franubet and Nubet).
!
81
Recovery, %
90
100
90
.y.y.v
90
91
93**
m
87*
x x :::
•shw-:
XyXy
80
IlS
70
Illill
c
o
60
BH
4*
O
2
50
«
40
2
Kl
■
30
20
BNM
Itli
#
ISSBBl EBB - :
IS
IM II
0
B
Nubet
Pernubet I
/ # # #
:I
• f
Il
Pernubet 2
H:;#-
P
Franubet
Wanube1
Nubet and mutants
* : Differs from Nubet at p«0.05 level.
Figure 4-13. Comparisons o f average percent recovery and Buhler milled fractions for
barley starch mutants grown in several environments.
82
Protein, ash and starch contents o f the milled fractions recovered from starch
mutants were compared with those o f Nubet (Table 4-15). In general, the protein and ash
content o f the flours were relatively lower than the protein and ash content o f bran and
shorts. Starch content o f the flour was higher than starch content of the grain (Table
4-10). These results indicated that barley endosperms were separated, to some degree, from
the bran layer through the milling process. No differences were shown for protein and
starch content of the flour between Nubet and Wanubet, ash content o f the Wanubet flour
was higher and the protein and ash content o f bran less than that o f Nubet. The reduction
of protein and ash contents in the bran appeared to be due to a failure to separate bran
from the endosperm.
P-Glucan viscosity data of the milled fractions are presented in Table 4-15. The
viscosity of the flour was basically less than grain viscosity (Table 4-7) and viscosity of
the shorts was very high. The milled fractions of Franubet had much lower viscosities in
flour, bran and short fractions as compared to those o f Nubet. These results appear to
reflect the lower grain viscosity in the grain o f Franubet. The milled fractions of Wanubet
were expected to have a higher viscosity than the milled fractions o f Nubet. Only the
viscosity of the Ipran was higher than Nubet. Since the shorts were shown to have an
extremely high viscosity compared to flour and bran, it was suspected that the cell wall
material in the endosperm was separated from the starch and collected in the short stream
in the milling process. Low 0-glucan viscosity o f Franubet shorts compared to the Nubet
shorts indicated that the less viscous cell wall substances in the Franubet endosperm which
is supported by the indistinct cell wall observed in Franubet endosperm (Fig. 4-2).
Table 4-15. Protein, ash and starch content and 0-glucan viscosity o f milled fractions from barley starch mutants.
Nubet
No. of paired samples
Pemubet I
6
6
Pemubet 2
5
Franubet
6
Wanubet
3
Flour
Protein, % (UDY)
Ash, %
Starch, 7.
B-glucan viscosity,cps.
11.8
1.315
68.2
10.1
12.5
1.464**
66.2**
11.3
12.5
1.263
65.4*
9.8
13.5*
1.439*
65.0**
3.9**
11.6
1.628**
65.4
14.3
Bran
Protein, 7. (UDY)
Ash, %
B-glucan viscosity,cps.
17.6
2.804
18.6
16.9
2.731
31.2
18.0
2.491
29.7*
20.9**
3.515*
5.1**
12.8*
2.067*
52.8**
Shorts
Protein, 7. (UDY)
15.1
Ash, 7.
2.460
B-glucan viscosity,cps. 1,195
15.5
2.412
1,314
15.9
2.259
833
*, ** : Significantly differs
19.1**
3.537**
23**
11.7
2.306
242*
from Nubet at p=0.05 and 0.01 level, respectively.
84
Phenotypic, genotypic and environment correlations among determinations on grain
and flour samples from starch mutants are presented in Table 4-16. Twenty-six samples
representing five isotypes o f Nubet grown in several environments provided the data for
these correlations.
Strong positive relationships appeared between whole grain and flour protein, and
whole grain and flour ash. Highly significant phenotypic correlations for these traits indi­
cated the strong influences o f grain measurements on the flour quality. Environmental
correlations between grain and flour protein and grain and flour ash content were highly
significant. These environmental correlations show the strong effect o f environment on
variation among kernel traits. Highly significant positive correlations between grain pro­
tein determined by either UDY or the Kjeldahl method and flour yield indicated that
the higher the protein content of grain the higher the flour yield. This protein relation­
ship is also shown by protein in the flour. In contrast to the above, the genotypic cor­
relation between grain protein determined by the UDY method and flour yield and the
environmental correlation between grain protein determined by Kjeldahl method and
flour yield were positive and significant. The relationship o f these traits are shown in
Figure 4-14. Significant positive environmental correlation between flour yield and grain
protein determined in Kjeldahl method could be explained with this figure. Wide ranges of
grain protein within genotypes (Fig. 4-14) and no differences detected between Nubet and
starch mutants (Table 4-10) indicated that most o f the variation was due to the environ­
ments. When the higher protein samples are considered to represent the high protein envi­
ronments, the samples grown in high protein conditions showed higher flour yield than the
samples grown in low protein conditions.
Table 4-16. Phenotypic (P), genotypic (G) and environm ent (E) correlation coefficients am ong determ inations
o n grain and flour samples from barley starch m utants grown in several environm ents.
(2)
Grain protein, 7.
(UDY)
Grain protein, %
(Kjeldahl)
Grain ash, 7.
Grain starch, %
Grain B-glucan
viscosity, cps.
Flour yield, %
Flour protein, %
(UDY)
Flour ash, %
(I) P
G
E
(2) P
G
E
P
(3)
G
E
(4) P
G
E
(5) P
G
E
(6) P
G
E
(7) P
G
E
(8) P
G
E
(3)
0.876** 0.112
0.917* -0.187
0.867** -0.093
-0.373
-0.289
-0.340
'
(4)
-0.245
0.129
-0.354
-0.156
-0.136
-0.164
-0.006
0.388
-0.109
(5)
(6)
-0.531** 0.512**
-0.801
0.879*
-0.458*
0.345
0.581**
-0.449*
0.678
-0.572
0.633**
-0.417
-0.233
0.037
-0.180
0.124
0.232
-0.639**
0.332
-0.196
-0.776
0.633
0.160
0.279
-0.799**
-0.988**
-0.213
(7)
(8)
0.832**
0.826
0.841**
0.847**
0.570
0.945**
-0.141
0.379
-0.239
-0.050
0.267
-0.117
-0.404*
-0.792
-0.340
0.569**
0.843
0.673**
-0.067
-0.564
0.243
-0.240
-0.761
0.165
0.701**
0.863
0.712**
0.028
-0.014
0.057
-0.231
-0.327
-0.043
-0.253
-0.375
-0.006
0.558**
0.327
0.231
Number of samples are phenotypic-26, genotypic-5 and environment-21, respectively
*, ** : Significant at p=0.05 and 0.01 level, respectively.
86
Significant genotypic correlation between flour yield and grain protein determined
by UDY method were suspected to be due to the altered protein associated with Franubet.
Franubet showed higher flour yield than Nubet (Figs. 4-13 and 4-14) and higher UDYprotein than KJeldahl protein (Table 4-10). These two traits associated in Franubet may
be the reason for the significant genetic correlation.
Negative phenotypic correlations between grain protein determined by both
methods and /J-glucan were significant. The higher the protein content the lower the
0-glucan viscosity of the grain. The environmental variation o f protein appeared to provide
the major contribution to the significant correlation. A significant environmental corre­
lation between UDY protein and 0-gIucan viscosity o f the grain supported this statement.
Correlations between starch content and the other determinations were not signifi­
cant. These results indicated that the starch content o f grain was not related to the pro­
tein content of grain or flour yield.
Strong negative relationships were obtained between /J-glucan viscosity and flour
yield (Table 4-16, Fig. 4-15). The higher the 0-glucan content o f the grain, the lower the
flour yield. Both traits showed random variation among environments within isotypes
(Fig. 4-15), but highly significant genotypic correlation indicated that most o f the vari­
ations of these traits were due to the isotype differences. As shown in Table 4-10, Figures
4-13 and 4-14, Franubet and Wanubet were distinguished from the other isotypes.
Significant phenotypic correlation between flour yield and flour protein indicated
the higher the flour yield was the higher the flour protein. The flour yield from the high
protein grain may have included more by the outside layers which had a high protein
content.
87
Flour yield (% ),(Y )
80 -
9 =12.4+3.26 X
r» 0.531
*
*
70 •
60 50 -
Nubet
•
Pernubet I *
Pemubet 2 ♦
Franubet
*
Wanubet
•
■
40
12
14
16
Grain protein by UDY (%),(x)
Flour yield ( % ) ,(¥ )
Ys 19.5 + 2.89 X
r . 0.581
18
*
*
o
Nubet
•
Pemubet I *
Pemubet 2 ♦
Franubet
*
Wanubet
•
12
14
16
Grain protein byKjeldahl(% ) ,( X)
18
Figure 4-14. Relationships o f grain protein determined by UDY and Kjeldahl method to
flour yield among barley starch mutants grown in several environments.
88
Flour yield ( % ) ,( Y)
Nubet
•
Pernubet I *
Pernubet 2 ♦
Franubet *
Wanubet
•
: 73.9+ 0.87 *
r« 0.799
10
20
30
Grain B glucan viscosity(cps.),(x)
40
Figure 4-15. Relationship of grain 0-glucan viscosity to flour yield among barley starch
mutants grown in several environments.
89
Factors Affecting Pearling Index and Pearling Rate
To determine the factors affecting the pearling index, sample size, moisture effects
and isotypes were investigated with Betzes, Compana and Titan isolines. The pearling
index data for sample sizes are presented in Figure 4-16. As can be seen from this figure
the pearling indexes for a given pearling time were dependent upon sample size. Analysis
of variance of this experiment (Table 4-17) verified these differences. The interactions
between iso type and sample size, isotype and pearling time, and sample size and pearling
time were significant, but these variances were relatively small compared to the variances
of the main effects. The linear relationship o f pearling index to pearling time among dif­
ferent sample sizes for the two isotypes are presented in Table 4-18. The differences of
slopes were significantly different within the same sample sizes. It is interesting th at the
covered isotype pearled more rapidly than the hulless isotype.
With the very high correlation coefficients observed for all sample sizes, it would
appear that all three sample sizes are equally accurate. Fifty gram samples were pearled
in subsequent tests. Barley is usually commercially pearled to 60 to 75 of the original
weight.
The relationship o f moisture content o f the grain to pearling index was tested using
4 Betzes iso types, each grown in 2 environments (Fig. 4-17). Moisture levels ranged from
10 to 30%. The pearling indexes increased as moisture levels increased for the fixed pearl­
ing time of 2 minutes. The waxy endosperm isotypes in both covered and hulless had
higher pearling indexes than their counterpart normal isotypes at each moisture level and
hulless iso types also showed higher pearling indexes than covered isotypes.
;>r ■
90
OO 90
BO
70
60
50
40
30
20
10
Pearling time ImInsJ
Relationship o f pearling index to sample size and pearling time determined
on the hulless and covered isotypes o f Betzes (Average o f 2 replications).
91
90*-
—•
■e
-O
O**Me*«0
10
14
18
22
Nubet
Betzes
Wanubet
Wabet
30
Percent m o is tu re In grain
Figure 4-17. Relationship o f pearling index determined at 2 minutes to the percent
moisture in grain o f Betzes isotypes.
92
Table 4-17. Analysis o f variance o f pearling index o f Betzes isotypes (hulless vs. covered)
for different sample sizes with 2 replications.
DF
Source
Mean square
F
Isotype
(A)
I
315.36
195.80**
Sample size
(B)
2
5972.87
3708.51**
Pearling time (C)
10
2520.72
1565.09**
AxB
2
10.93
6.89**
AxC
10
3.33
BxC
20
131.06
A x B x C
20
1.11
Error
66
1.61
Total
131
2.07*
81.38**
0.69
*, ** : Significant at p=0.05 and 0.01 levels, respectively.
The analysis o f variance (Table 4-19) o f the moisture effects indicated significant
interactions between moisture effect and isotype. Hulless types showed slower increases of
the pearling index at the low level o f moisture and increasing sharply at the higher
moisture levels. The covered types showed rapid increases with increasing moisture at the
low levels o f moisture and slower increases at the higher levels o f moisture. The possible
explanation o f these results was a difference in speed o f water absorption between covered
and hulless isotypes for 2 hour steep period used. When comparing genotypes these results
suggest that the comparisons should be made with grain o f the moisture level commonly
present for commercial pearling; i.e., air dry.
93
Table 4-18. Regression and correlation coefficients between pearling index and pearling
time for sample sizes of Betzes and Nubet.
Slope
Correlation
coefficient
25g
-23.79“
-0.999**
Il
50g
-15.74-
-0.999**
II
IOOg
- 1 1 .1 0 -
-0.999**
Isotype
Betzes
Sample size
Nubet
25g
-26.19-
-0.999**
it
50g
b
-15.98-
-0.999**
Il
IOOg
-12.06-
-0.999**
** : Significant at p=0.01 level.
The different letter superscripts indicate a significant difference
within isolines at p-0.01 level.
Pearling indexes and pearling rates o f isotypes were compared to determine the
effect of certain genes (Table 4-20). Significant differences of pearling index were present
for each comparison. Pearling indexes o f the waxy endosperm and hulless isotypes were
higher than those o f the normal parents (isotypes) and brachytic iso types had a lower
pearling index than the normal isotype.
The pearling rates were determined by the differences o f pearling indexes deter­
mined at two pearling times. Pearling rates o f waxy isotypes were significantly lower than
those o f normal parents and pearling rates o f brachytic and hulless isotypes were no t sig­
nificantly different from the normal isotypes.
94
Table 4-19. Analysis o f variance o f 2 minutes pearling index as affected by % moisture in
grain o f Betzes iso types (hulless vs. covered, waxy vs. normal endosperm).
DF
Source
Mean square
F
Endosperm type (A)
I
210.45
434.98**
Hull type
(B)
I
56.10
115.95**
Moisture level (C)
4
164.44
339.88**
AxB
I
7.03
14.53**
4
0.06
0.12
BxC
4
8.27
17.10**
A x B x C
4
1.98
4.09*
Error
21
0.48
Total
39
AxC
?
*, ** : Significant at p=0.05 and 0.01 levels, respectively.
Correlation coefficients between kernel weight and pearling index within each iso­
type are presented in Table 4-21. The variations o f the kernel weight within each isotype
were due to environment.
Wabet (waxy Betzes) and the derived Betzes, Watan (waxy Titan) and brachytic
Betzes showed significant positive correlations and Wapana (waxy Compana) and the
derived Compana showed strong negative correlations.
Since the samples with lower kernel weights consisted o f much small grain, and
since many of the small grains were found in the pearled offal box during the pearling
Table 4-20. Genotype comparisons of grain weight and pearling indexes for the isogenic pairs o f Compana,
Betzes and Titan grown in different environments.
Genotype
Compana
Betzes
Titan
Betzes
Betzes
Pearling index
Pearling
rate per
min.. 7.
Isotype Environments Kernel wt.
No.
mg
I mins.
1.5 mins.
Normal
Waxy
22
22
50.28
48.29**
64.9
67.4**
-
Normal
Waxy
18
18
37.79
36.04**
68.8
69.7
Normal
Waxy
16
16
34.29
33.76
70.2
71.6*
Normal
Brachytic
29
29
38.60
30.24**
71.7
68.0**
62.6
58.4**
-
18.1
19.2
Covered
Hulless
10
10
39.57
35.33**
71.9
76.2**
63.2
66.7**
•
17.4
19.0
*
*
-
2 mins.
42.7
49.2**
22.3
18.4**
48.6
53.1**
20.2
16.5**
47.0
56.6**
23.2 ?
15.0**
•
*, ** : Significantly differs from normal genotypes at p=0.05 and 0.01 levels,
respectively.
96
Table 4-21. Correlation coefficients between kernel weight and pearling index within the
isotypes o f Compana, Betzes and Titan.
Environments
No.
Correlation coefficients
r
Normal
Waxy
22
22
-0.606**
—0.640**
Normal
.Waxy
18
18
0.646**
0.904**
Titan
Normal
Waxy
16
16
0.325
0.741**
Betzes
Normal
Brachytic
29
29
0.062
0.727**
Covered
Hulless
10
10
Genotype
Isotype
Compana
Betzes
.
Betzes
-0.503
-0.322
*, ** : Significant at p=0.05 and 0.01 levels.
operation, the positive correlations for Titan and Betzes isotypes could be due to the
shriveled grain.
The negative correlation indicated that the larger grain pearled faster than smaller
grain in Compana isotypes. Most o f the pearled grain of Compana and Wapana contained
half kernels. Many broken pieces o f kernels were found in pearled offal fractions. The sig­
nificant negative correlations o f these isotypes appear to be because the larger kernels
broke easier than smaller kernels.
Pearling index of a sample for successive time increments provides information on
the action o f the pearler on the various layers of the barley kernel. Two isogenic pairs of
97
waxy endosperm and caryopsis covering isotypes Betzes grown in 2 environments were
pearled in successive increments for 3 minutes.
The relationships o f pearling index to pearling time and iso type are presented in
Figure 4-18 and Table 4-22. The pearling indexes for the first 15 seconds show sharp dif­
ferences between covered and hulless iso types and most o f the pearled grain was free
from the hulls. After the first 15 seconds, the grain was pearled at a constant rate as pearl­
ing time increased. The correlation coefficients were r=0.999 and pearling rates per minute
ranged from 13.7 to 17.1%. The pearling rates per minute for normal endosperm isotypes
were significantly faster than those o f waxy endosperm isotypes with no difference in
rate of pearling between covered and hulless isotypes.
Table 4-22. Analysis o f variance o f pearling index for successive pearling time increments
on Betzes isotypes (hulless vs. covered, waxy vs. normal endosperm).
DF
Mean square
Endosperm type (A)
I
537.1
278**
Caryopsis type (B)
I
245.6
127**
Pearling time
7
1885.0
976**
AxB
I
13.1
AxC
7
0.8
BxC
7
17.6
A x B x C
7
0.6
32
1.9
Source
Error
(C)
F
6.8*
0.4
9.1*
0.3
*, ** : Significant at p=-0.05 and 0.01 levels, respectively.
98
100
JK
X
o>
n
c
0)
C
I
Nubet
Betzes
Wanubet
Wabet
as
1.0
0.999
1.5
20
2.5
3.0
P earlin g tim e Imins.) lx)
The different letter super sc ipts indicate a significant difference
among isotypes at p=0.05 level.
Figure 4-18. Relationship o f pearling index to isotypes (hulless vs. covered, and waxy vs.
normal endosperm) and pearling times in Betzes barley.
99
Since the pearling index and pearling rate were linearly dependent upon the pearl­
ing time and endosperm characteristics, the ash content was used to equalize the pearling
index for a comparison of genotypes. In Figure 4-19 the relationship o f pearling index to
ash content for the different isotypes is shown. The ash contents o f the grain, with suc­
cessive layers removed, were probably the same for the waxy endosperm isotypes and their
normal isotypes. The plotted lines of Betzes and Wabet for ash content and pearling index
are probably the same line, as are the Nubet and Wanubet plot. Since these pairs o f iso­
types were expected to have similar ash distribution in the endosperm the lines obtained
from ash content and pearling index represent the pearling rates freed o f the pearling time.
Accordingly the relationships o f pearling indexes o f waxy iso types were not different from
the normal endosperm isotypes when pearling time is determined by ash content. The
relationships o f covered iso types were much different from the relationship o f hulless iso­
types. Since the hull o f the covered isotypes consisted o f material with a high ash content
these differences are regarded as the effect o f hulls on ash content.
Pearling index and ash content o f pearled grain o f starch mutants were compared to
those o f Nubet (Fig. 4-20). The pearling rates of Pemubet I and Pemubet 2 were not sig­
nificantly different from Nubet and their pearling rates per minute were 18 to 17%. Pearl­
ing rate per minute o f Franubet and Wanubet was significantly different from that of
Nubet. Franubet pearled slower than Nubet and faster than Wanubet.
Relationships o f pearling index to ash contents are presented in Figure 4-21. In
general, ash content was lower as the pearling index decreased, and when the pearled grain
was free from the outside layers, the decreasing rates o f ash content were stabilized. It
would appear necessary to remove more o f the outer layers o f the Pemubet mutants to
100
2 .5 1-
/-S 1.5
o 1.0
—• Nubet
■f Betzes
4# Waaubet
■it Wabet
Rearing index
Figure 4-19. Relationship o f pearling index to ash content o f pearled grain o f Betzes iso­
types (hulless vs. covered, and waxy vs. normal endosperm).
101
o
a 60
Nubet
Pernubet I
Pernubet 2
Franubet
Wanubet
as
10
15
2.0
2.5
P earlin g tim e lm ins.|,(x|
3.0
*,** : Significantly differs from Nubet at p=0.05 and 0.01 levels,
respectively.
Figure 4-20. Comparisons o f linear relationship between pearling index and pearling
time among barley starch mutants grown in several environments.
102
2.0
A sh c o n te n t (%)
1.5
1.0
0.5
e — — • Nubet
Pemubet I
-<^***"***l£- Pemubet 2
Franubet
‘
I ____________ I ____________ I ____________ a____________I ____________i ____________ I
100
90
80
70
P e a rlin g
60
50
In d e x
40
I
remaining
Figure 4-21. Relationship o f ash content to pearling index o f barley starch mutants
grown in several environments.
103
obtain pearled barley with a 1% ash content than would be necessary with Nubet or the
other mutants.
Rat Feeding Trial
Results obtained from the feeding trials o f weanling female rats fed barley starch
mutant rations are presented in Table 4-24. Differences were noted in respect to rat gain
per day and feed consumption between the casein diet and barley diets. A slightly lower
rate o f growth was shown for the Pemubet I diet as compared to rats fed the Nubet
diet, but no pronounced differences were observed among the other diets. The feed to gain
ratios indicating conversion ranged from 0.228 to 0.246 but the differences were nonsig­
nificant. These results indicated that the energy-nutrition o f starch mutants was similar to
Nubet.
The protein efficiency ratios (PER) also exhibited no significant differences among
iso types. Since these results were obtained with 14.5% protein diets (Table 4-23), no dif­
ferences in the protein efficiency ratios were expected. These data indicated the protein
conversion to body weight.
Energy consumption and digestible energy were estimated and results are presented
in Table 4-25. The Franubet diet showed slightly lower digestible energy than the other
iso types but the differences were not significant.
104
Table 4-23. Proximate analysis o f barley starch mutants balanced diets adjusted to 14.5
percent protein.
Protein
7.
Fat— ^
%
Nubet
14.8
1.6
Pernubet I
14.6
Pernubet 2
Ration
Fiber
%
Ash
7.
NFE-/
7.
H 2O
%
1.3
2.3
73.2
6.8
1.6
1.5
2.6
72.6
7.1
14.6
1.3
1.2
2.3
73.6
7.0
Franubet
14.4
1.8
1.7
2.7
73.1
6.3
Casein
15.0
2.1
0.1
0.8
75.3
6.7
.
a/ : Ether extract.
b/ : Nitrogen-free extract.
Table 4-24. Average gain/day, feed consumption, feed efficiency and protein efficiency
ratio for rats fed 14.5% isoprotein diets containing barley starch mutants.
Ration
Nubet
Pernubet I
Pernubet 2
Franubet
Casein
LSD (57.)
No. of
rats fed
8
8
8
8
8
Gain/day
g
4.25
4.00*
4.18
4.11
3.64*
0.19
Feed consumed Gain/feed
g/4 weeks
ratio
501
492
514
492
416*
38.4
0.237
0.226
0.228
0.234
0.246
NS
a/ : Protein efficiency ratio.
* : Significantly differs from Nubet at p~0.05 level.
NS : Non-significant.
PER-/
1.60
1.55
1.56
1.62
1.63
NS
105
Table 4-25. Average energy consumption, energy digested and percent digestible energy
data from rats fed 14.5% iso protein diets containing barley starch mutants.
Ration
No. of
rats fed
Energy consumed
Kcal/day
Energy digested Digestible
Kcal/day
energy, %
Nubet
4
39.9
33.4
84.1
Pemubet I
4
39.7
33.7
83.4
Pemubet 2
4
39.6
32.8
84.3
Franubet
4
40.6
33.5
81.6
NS
NS
NS
LSD (5%)
NS : Non-significant.
Chapter 5
DISCUSSION
Inheritances and Linkages o f Starch Mutants
MacGregor et al. (1972) observed changes of starch storage in the pericarp layers o f
barley. Large numbers o f small starch granule are present in the pericarps during the first
10 days after anthesis but the granules are quickly degraded by alpha-amylase. No starch
granules were detected in the pericarp layers 16 days after anthesis within the limited
number o f cultivars examined.
Pericarp starch mutants found in this study appeared to deposit starch in the peri­
carp 12 days after anthesis. The reasons for starch deposition are not clearly understood
but the flour samples milled from pericarp mutants exhibited the same amount o f alphaamylase activity as the flour sample o f Nubet used in the amylogram tests. Preliminary
observations o f alpha-amylase activity determined by blue plate agar assay also indicated
no differences between Nubet and the pericarp mutants. Deposition o f starch in the peri­
carp was probably not due to a deficiency o f alpha-amylase.
As mentioned by Blakely et a l (1979) the testa cells originating from the integu­
ments were collapsed and so closely adhered to the pericarp layer during grain develop­
ment that the pericarp and testa layer could not be identified with microscopic observa­
tion. No significant differences of morphological cell structure and thickness o f bran were
found between Nubet and pericarp starch mutants. The pericarp starch mutants did not
appear to have thin or missing bran layers.
107
Blakely et al. (1979) reported sorghum grain mutants with no testa layer. This
report suggested the possibility o f a structural mutant o f barley with the bran layer miss­
ing. This m utant was identified by measurement o f the thickness o f the pericarp-aleurone
junction under the light microscope but quicker methods are required to select efficiently
from a large population. The staining method with iodine or May-Grunwald solution was
considered to be a good method for selecting these mutants, but the chemical compo­
sition o f the cells interfered with the selection o f structural mutants.
F2 segregations for starch in the pericarp obtained from the crosses between Pernubet I and Nubet and Nupana fit to a 3:1 ratio. A single recessive gene controlled this
character. No xenia effect was observed. The seed o f the F2 plants showed the pheno­
types o f the parents. No segregation was obtained within the seed from a single plant.
From a three point linkage study, the location o f hulless caryopsis, n and short awn
gene, Zk2 were determined by Eslick et al. (1972). The n and I k 2 genes were both located
on the long arm of chromosome I and their recombinations from the centromere were
14.7% for short awn and 7.2% for the hulless caryopsis gene.
The recombination values obtained from the crosses o f translocations involving
chromosome I with n and I k 1 were generally lower than recombination values reported
from the crosses between male sterile 10 (ms 10) and n and Zk2. No recombination geno­
types between either n and I k 1 and the breakpoint o f chromosome I in the T l-4e, T l-7k
crosses were obtained. These results confirmed the recombinations reported by Persson
i
(1969) which showed the tight linkages between T l-4e and n, and T l-6e and T l-7k and
dense ear (ari-d) located near the n gene. These translocations appeared to be exchanged
in the chromosome I arm distal to I k 1. The breakpoints o f Tl-6j and T l-7i occur on
108
chromosome I near the centromere and T l-3e, Tl-Sf and Tl-6a exchanged the short arm
o f chromosome I appeared which agrees with previous results summarized in Table 3-1.
Since the linkage values between pericarp starch m utant I (per I) and the trans­
location breakpoints in Tl-6j and T l -7c crosses were 1.3% ± 0.6 and 5.3% t 2.8, respec­
tively, and the linkage chi-squares were significant, the per I gene is assigned to chromo­
some I. Though the linkage chi-squares o f T2-5a and T5-6b crosses were also significant,
excess recombinants in both crosses over that expected with independence suggest that
these segregations should be considered as independent in determining linkage.
From the recombinations obtained from T1-6J and T l-7c crosses with per / , the
gene location o f per I was estimated. This gene appeared to be located on the long arm o f
chromosome I at a point between the centromere and
it
The location o f translocation
breakpoints involving chromosome I and per I, based on data presented in this paper,
were diagrammed as following.
Chromosome I
Tl-Se
T l-S f
T l-6a
T l-7c
*
*
*
*
short arm
Tl-6j
T l-7i
centromere
Nr
T M e **
T l-6e • •
T l-7k • •
per I
h ------- M
13%
n
Ik 1
long arm
5.3% ^
Kr
8%
K14%
*, Breakpoint location considered to be somewhere in short arm, **, or distal to I k 1 in
long arm.
109
F1 segregation o f pericarp starch m utant 2 (per 2) was 1019 Per 2 — 109 per 2
per 2. The nearest expected segregation ratios are 3:1, 15:1 and 13:3. The segregation
obtained did not fit any o f these ratios.
F2 segregation o f this gene obtained from the crosses with translocations showed
considerable variation. Three segregations fitted a 3:1 ratio and the other three segre­
gations were similar to that o f the parental crosses.
These results indicated other factors were affecting the inheritance o f this gene.
Biggerstaff and Eslick (1978) reported unusual segregations from the progeny o f the
translocations treated with diethyl sulfate. Gamete lethal selective genes were assumed to
be the most likely cause o f the unusual segregations. One possible explanation for the dis­
agreement o f per 2 with the expected ratio might also be gamete lethal gene involvement.
Since the F1 segregation o f per 2 in combinations with translocations were backcrossed
twice with the parental genotypes, the chances o f an association o f the gamete selective
gene with per 2 were reduced if the per 2 gene inheritance was influenced by a gamete
selective gene. F1 segregations obtained from crosses o f the parental genotypes backcrossed only once would have a greater chance o f having a gamete selective gene present.
The linkage chi-squares for the crosses between T l -7c, T l-7i and T5-7g and peri­
carp starch m utant 2 (per 2 ) were significant and their recombinations were 26.0 i 4.9,
34.2 ± 7.1 and 2.9 ± 1.7%, respectively, when the gene was assumed to be a single re­
cessive gene. All these translocations have chromosome 7 in common.
no
The breakpoints o f T l -7c, T l-7i and T5-7g were identified by Nilan (1964),
Ramage (1971) and Tuleen (1974). The satellite o f chromosome 7 (short arm) was inter­
changed in T l -7c and T l-7i and long arm interchanges were reported for T5-7g. Therefore
the per 2 gene appeared to be located on the long arm of chromosome 7. The linkage chisquare o f T1-6J was also significant, bu t an excess o f recombinants in this cross was con­
sidered as independent inheritance.
The starch granules of Franubet were angular and irregular in shape and size. These
shapes formed during the early stages o f grain filling. Some starch granules were much
longer than those o f Nubet. These extra Iyge granules, however, appeared to be com­
pounded o f many small granules. A few round granules were found in this m utant but
most o f them were fractured or cracked and showed a center cavity when samples were
obtained from mature grain. The starch granules o f Franubet resemble to some degree the
starch granules o f sugary endosperm in com but the starch content o f Franubet endo­
sperm was very high. The starch granules o f rice are similar to the starch granules o f this
m utant with regard to the angular shape, but quite different in size and regularity.
Vitrious endosperms are considered to be due to the high protein content o f the
endosperm in wheat. This character has been used as a selection criterion for hard wheat
(Pomeranz, 1971). A vitrious ring around the Franubet endosperm was postulated to have
a high protein content (Eslick, 1981, Personal Communication) but further studies are
necessary to prove this assumption.
The F 2 and backcross segregation o f crosses o f the fractured starch m utant fitted a
ratio o f 3:1 and I : I , respectively. The crossed seeds were all normal starch phenotype.
Ill
These results indicated a single gene inheritance with xenia. The genotype o f Fra (Normal
starch genotype) appeared to be dominant over the genotype o f fra (fractured starch
genotype) in either the heterozygous genotypes o f endosperm (Fra fra fra or Fra Fra fra)
and no dossage effect was observed in these endosperms.
The recombinations obtained using translocation homozygote lethals and the frac­
tured starch gene revealed the location o f this gene.
Ramage et al. (1961), Nilan (1964) and Persson (1969) reported the breakpoints o f
translocations involving chromosome 4. T2-4d and T4-5e are exchanged in the long arm
.
i
'
1
o f chromosome 4 and the short arm exchanges of this chromosome are reported to be T3T3-4b and T4-7b. The recombination values obtained from the crosses o f T2-4a, T2-4d,
T3-4d and T4-5e appeared to place the fractured starch gene (fra) distal to those break­
points and over 50 map units distance from the centromere o f the long arm. The indepen­
dent assortments o f T 3 ^ b and T4-7b also appeared to support this assumption. The posi­
tion o f translocation breakpoints and th e /ro gene obtained are diagrammed as following.
Chromosome 4
T3-4d
T4-5e
T3-4b*
T4-7b*
T2-4d
long arm
K -----26%
Short arm
centromere
K
K-
fra
T2-4a
—I— --------1---------
31%
over 50%
*, Breakpoint location considered to somewhere in short arm.
—
4-
>1
112
Characters Associated with Starch Mutants
Side effects associated with starch mutants were observed. The significant reduc­
tion o f the grain size o f Pemubet I appeared to cause a lower yield than Nubet. The
starch content o f grain was also lower in Pemubet I than Nubet, although the most starch
granules were deposited in the pericarp layer of Pemubet I . The lower seed weight o f
Pemubet I was suspected to be due to inferior deposition o f starch in the endosperm and
a genetic association between the pericarp starch gene and grain size. The roll o f the peri­
carp starch in the accumulation o f starch in the endosperm is not known.
The number o f kernels per spike and kernel weight o f Franubet were significantly
smaller than those o f Nubet but the number o f spikes per m1 o f Franubet were much
higher than Nubet. No significant difference in yield was found between Franubet and
Nubet. This appeared to be due to the compensating effects o f yield components. In addi­
tion to the differences o f yield components as compared to Nubet, the shorter height and
later heading were constantly associated with Franubet in the several environments.
No significant differences were obtained between Nubet and starch mutants for
chemical composition determined by proximate analysis o f the grain except for the
UDY-protein content o f Franubet and the starch content o f Pemubet I .
Protein content o f grain is influenced to a great extent by growing conditions.
Environmental effects on grain protein were reported by Canvin (1976), Johnson e t al.
(1969) and McGuire et al. (1979). It is generally accepted that the genetic variations o f
protein content are much smaller than the variations due to environment. Good genetic
gains for protein content were reported with 65 generations o f continuous selection for
113
high protein in com. Yielding capacity and seed size were lower for high protein selections
(Dudley and Lambert, 1969).
Since the UDY method determining protein content is related to the amount o f
NH3 ions in the sample, the results o f the protein analysis determined by the two methods
suggested a difference in amino acid content o f the proteins. An amino acid analysis re­
vealed significant increases o f lysine and decreases o f isoiuciene, glutamic acid and proline
in Franubet.
High lysine barley has been studied by many breeders (Munck et al., 1970; Ingverson et al., 1975; Jarvi and Eslick, 1975; Ullrich and Eslick, 1978). Most o f the mutants
are associated with shrunken endosperms causing lower grain yield.
Franubet had a plump (non-shnmken) kernel with high lysine and this m utant
should be considered as a new source o f high lysine cultivars.
According to Palmer (1979), the main objective of the malting process is to encour­
age the development o f flavor compounds and to promote enzymatic hydrolysis o f the
protein and carbohydrate reserves of the grain. The synthesis o f enzymes such as alphaamylase, endo-0-glucanase and endo-protease is of primary importance in malting.
Fincher (1975) has shown that the cell walls o f endosperm contain 70-75% 0-glucan
and 20-25% arabinoxylan. Because o f the chemical nature o f these 0-glucans and the
function o f cell walls, which is entrapping the starch and protein reserves, the degradation
o f the starchy endosperm is prevented until the cell walls o f the endosperm are trans­
formed. Significant degradation o f 0-glucan occurs by the action o f f-glucanase during
malting and in consequence, endosperm starch and protein are exposed to enzymes that
convert to sugars and amino acids (Palmer, 1979).
114
Since Franubet showed a significantly lower 0-glucan viscosity indicating low fl-glucan, problems related to 0-glucan in malting would be expected to be reduced when this
m utant was used for malting. Only a short period o f germination may be required for
developing alpha-amylase and protease. More rapid modification o f the endosperm might
be expected o f this genotype. Some relationship was suspected between low viscosity and
less visual cell wall material in Franubet (Fig. 4-2, Fig. 4-7). Further investigation will be
required to determine this.
An elevated 0-glucan viscosity o f the waxy genotype was in agreement with results
reported by Fox (19 8 1). The genetic relationship o f viscosity to waxy and fractured starch
endosperms remains to be defined but different mechanisms for control o f viscosity are
suspected.
The 0-glucan viscosity curves o f developing endosperms showed that Nubet endo­
sperm started to increase in viscosity 21 days after an thesis and maximum viscosity was
reached at physiological maturity. This result appeared to agree with a previous study
reported by Coles (1979). According to his suggestion, starch accumulation is nearly com­
plete when the moisture content o f the endosperm declines to 40%, whereas the accumu­
lation o f 0-glucan and hemicellulose occurs near physiological maturity.
Contrasting results are reported on the storage site o f the viscous substance in the
kernel. Taiz and Jones (1970) suggested that 0-glucans were contained in the aleurone cell
walls, but McNeil and Albersheim (1975) found the aleurone cell wall consisted o f arabinoxylan and cellulose. Fulcher et aL (1977) suggested that the main deposition o f
0-glucans are at the sub-aleurone cell walls but results showed by Gohl et al. (1977) indi­
cated the highest deposition o f 0-glucans were near the center o f the endosperm.
115
The results o f viscosity obtained from the fractions o f pearled offal appeared to be
similar to the results reported by Gohl et al. (1977). The outside layer including germ,
pericarp, testa and aleurone layer had lower viscosity than the center o f the endosperm
and gradually increased from outside to inside. This indicated that the cell walls originating
from the bran and aleurone layers contributed very little viscosity to the grain though
most of the cellulose and hemicellulose are located in these layers. The genotype differ­
ences for 0-blucan viscosity o f the pearled grain increased as the outside layers o f the
grain were removed.
No differences were shown for amylose content, gelatinization temperature, paste
viscosity and amylogram curves between Nubet and the starch mutants. These results
indicated that the starch properties were not altered in the mutants. O f particular note is
that the structural changes o f starch granules in the endosperm o f Franubet did not influ­
ence the amylose content or amylogram properties.
Miller et al. (1973) suggested that the major contribution to the viscosity o f
cooked starch paste is the molecules extruded from the granules during cooking. Fox
(1981) reported the relationship o f Brabender peak pasting viscosity to 0-glucan viscosity
and alpha-amylase activity. A strong negative correlation existed between log 10 Brabender peak pasting viscosity and log 10 alpha-amylase activity. A positive correlation
was found between log 10 Brabender peak pasting viscosity and log 10 0-glucan viscosity
within 12 samples o f waxy isotypes o f Betzes, Compana and Titan. The differences of
Brabender peak pasting viscosity between samples treated with HgQ2 and without
HgCl2 suggested the influences o f alpha-amylase on the past viscosities. Effect o f low
116
0-glucan viscosity in the Franubet flour was not evident in the amylograph viscosity.
Genotype differences are suspected to be the reason for the differing results.
Fast imbibition rate in pearled grains appeared to support the role o f surface layers
o f the barley kernel in moderating water entry as reported by Brookes et al. (1976).
The significant differences o f water absorption between Nubet, Franubet and
Pemubet I found in either whole grain or pearled grain indicated fast water uptakes by
the endosperms o f those mutants. Since kernel sizes o f the starch mutants were signifi­
cantly smaller than those o f Nubet, these results appeared to be similar to the results
obtained by Briggs (1978). Pearled grain o f Wanubet absorbed more water at a significant­
ly faster rate compared to the pearled grain o f Nubet. The water absorption o f Wanubet
seed appeared to be regulated by surface layers. Since waxy endosperms are basically dif­
ferent in amylose content and viscous substance from normal endosperm, these character­
istics appear to be related to water holding capacity.
The variations obtained suggest the water absorption rate for a given steep time and
the maximum water absorption are variable among barley cultivars and these differences
may be at the maximum when surface layers are removed.
Use o f Starch Mutants
Tempering is generally practiced in wheat milling for toughening bran and changing
the physical characteristics o f the endosperm before milling. The milling efficiency is usu­
ally improved with proper tempering.
Moisture effects o f tempered barley were compared to the recovery o f milled
fractions. As moisture level in tempered barley increased, the flour yield recovered from
117
the reduction roll was decreased and the bran was increased. Positive effects o f tempering
were reported by Cheigh et al. (1975). Barley was tempered for 2 to 48 hours and moisture
was adjusted to 14 percent.
Sorum (1977) reported th at dry milling of barley produced higher flour extraction
without increasing ash content. The basic tendency o f these results supports the results o f
Sorum (1977). No sign o f improvement on the separation between endosperm and the
bran was noted with differing tempering levels. More recovery o f bran appeared to be due
to the endosperm attachment on the bran when barley was tempered to a higher moisture
level.
A considerable amount o f a barley sample was lost during milling with Buhler test
mill, especially when the crushed barley sample “ plugged” the milling stream. This loss
was mainly due to the suction system on the Buhler test mill. The “plugging” problem
appeared to be one o f the major problems when milling barley with this mill. The feed
rate was adjusted so as to minimize this problem. When the “ plugging” problem was
reduced by using the correct feed rate the recovery o f milled product was maximized.
This highest recovery of total of milling fractions in Franubet suggests good milling
properties in this iso type while the lowest recovery for Wanubet suggest poor milling pro­
perties. The tempering, feeding rate, and endosperm characteristics were suspected to cre­
ate the “ plugging” problem but an evaluation method for this problem was not established
in this study.
Average flour yield o f Franubet was increased 13% over N ubet and Wanubet aver­
aged 16% less flour yield than N ubet. No differences were found between pericarp starch
118
mutants and Nubet. Flour yields ranged from 42% to 75% among genotypes and the dif­
ferences from the highest to the lowest within genotypes was 10 to 14 percent. This vari­
ation indicated that the genotype differences on the flour yield appeared to be greater than
variation due to environments including the variation originating from the milling process.
The effectiveness of milling barley is characterized by a decrease in flour ash and a
change in ash distribution in the milling fractions. The Franubet flours consisted o f signi­
ficantly higher contents o f ash and protein and a lower starch content than those of
Nubet. Forty-four percent o f the total ash was found in the flour of Nubet and 58% of
the ash was found in the Franubet flour. Since the outside layers of the kernel consisted
of material high in ash and protein and low in starch content more contamination by
the outside layers was suspected to be present in Franubet flour.
The 0-glucan viscosity o f the flour was less than grain viscosity and 0-glucan vis­
cosity o f shorts was very much higher than flour viscosity. Most o f the viscous substances
which caused the differences between grain and flour 0-glucan viscosity appeared to be
collected in the shorts fraction. Since the viscous substance was distributed in the endo­
sperm along with starch granules, the collection o f these substances in the shorts sug­
gested the separation o f starch granules from the viscous substances. Significant differ­
ences were shown for the 0-glucan viscosity between the milled fractions o f Franubet and
Wanubet as compared to Nubet. The differences in 0-glucan viscosity between Franubet
and Nubet appeared to reflect the isotype differences for the grain characteristics. The
viscosity o f the Wanubet fractions did not seem to be associated with changes in the grain
characteristics.
119
These results suggest the flour characteristics may be not only influenced by the
grain characteristics, but also associated with the % flour recovered.
Significant positive correlations between whole grain protein and flour protein, and
grain ash and flour ash appeared to confirm the results reported by McGuire (1979) indi­
cating that the strong influence o f grain quality on flour characteristics. Significant posi­
tive correlations between flour yield and flour protein, and protein and flour ash were ob­
tained. These results may be due to a failure to separate flour from the oustide layers o f
the kernel. The positive relationship with a strong correlation between grain protein and
flour yield indicated the higher the protein content of the grain the higher the flour yield.
The significant environmental correlation between grain protein (determined by Kjeldahl
method) and flour yield suggests that the genetic variations o f protein content among
starch mutants are much smaller than variations due to environment. However, significant
genotypic correlation between grain protein determined by UDY method and flour yield
appeared to confirm the different amino acid distribution in Franubet compared to Nubet.
The inverse relationship with a strong correlation between 0-glucan viscosity and
flour yield suggested that the highly viscous substances in the grain were the major factors
reducing flour yield. The significant genotypic correlation indicated that most o f the vari­
ations for this correlation were due to the isotype differences.
Factors affecting the pearling index in barley were sample size, moisture content,
pearling time and isotype differences. Effects o f sample size, moisture content and pearl­
ing time to pearling index showed basic tendencies similar to those reported by Taylor
et al. (1939), McQuggage (1943) and Kramer and Albrecht (1948).
120
Errors associated with the Strong Scott pearler were relatively small and the great­
est variances appeared to be due to the sample differences. Peariing time was suspected to
be the major factor causing variation in pearling index. The strong linear relationship of
pearling index to pearling time indicated that pearling properties could be represented by
a comparison o f the slope (byx) obtained from two indexes o f one sample pearled for
two different lengths of time.
Significant differences o f pearling index and pearling slopes (byx) were found be­
tween waxy and normal endosperm genotypes indicating isotype variation. Higher pearl­
ing indexes at a given pearling time and slower pearling rates were shown for waxy endo­
sperm isotypes compared to their normal isotypes. The pearling rate is an indication o f
kernel hardness when comparing hard and soft types o f wheat (Taylor et al., 1939;
McGuggage, 1943; and Kramer and Albrecht, 1948).
In general, the harder the grain, the less the material removed in pearling. These
results suggested that the grain o f waxy endosperm was a harder grain than the normal
isotypes. By the “ bite” test waxy endosperm types have been observed to be less hard
than normal endosperm types. Usually opaqueness in wheat is associated with low protein
and softness. Waxy endosperm barleys are opaque. Hardness tested by other methods is
needed to confirm the results observed here. It is possible that high viscosity is associated
with a slower pearling rate.
In the comparisons o f normal and bachytic or hulless caryopsis isotypes in Betzes,
isotype differences were found for pearling index but no differences were noted in pearl­
ing slopes between normal and m utant isotypes. These results suggested that pearling rates
were similar for isotypes when the endosperm types were the same. The pearling index at
121
a given time was different between isotypes but this difference is probably related to
kernel shape or size.
The significant positive correlation between grain weight and pearling index in the
Betzes and Titan genotypes revealed that grain size o f a sample affects the pearling index.
Small kernels or broken kernels in a sample sifted out into the pearled offal and resulted
in a reduced pearling index when determined for a constant pearling time.
The negative correlation o f these traits for Compana and Wapana indicated that the
large grains pearled faster. Broken kernels o f the pearls in these cultivars probably caused
these results.
Chung et al. (1974) reported that the resistant forces o f the large grain at the initi­
ation o f pearling were greater than those o f small grain. The broken kernels observed may
be partially due to the initial resistance of large kernels. The strong linear relationship
between pearling index and pearling time also indicated that the differences in pearling
rates among grain layers, if they exist in kernels, were not measurable with this machine.
Probably the pericarp and aleurone layers were too thin to be detected with this machine.
Since the pearling rate was directly dependent upon the pearling time and endosperm
properties, the comparisons o f optimum pearling rates among genotypes were difficult and
other measurements were required to equalize the pearling rates. Ash content o f pearled
grain was observed for this purpose and an acceptable relationship was found between ash
content and pearling rate. Pearling rates among genotypes are comparable only if the ash
contents o f genotypes are similar and if the distributions o f ash among layers o f tissues
are uniform.
122
One percent o f ash content o f the pearled grain was assumed to be an acceptable
pearl for human consumption. The pearling indexes at 1% ash for Betzes and Wabet were
68 and 66% and those o f Nubet and Wanubet were 78 and 77%, respectively. Pearling
rates adjusted to one percent ash content o f the pearls and converted to pearling indexes,
67% for the covered, and 77% for the naked corresponded well with the conventional
pearling indexes o f naked and covered barley used in Korea. The variation o f ash content
due to the genotype and environment should be considered in determining the grain
ash content but further information regarding this point was not obtained.
Pearling rates o f starch mutants were compared to that o f Nubet. Franubet pearled
slower than Nubet and faster than Wanubet and Pemubet I and Pemubet 2 showed no dif­
ferences from Nubet in pearling rates. Pearling indexes determined at I percent ash content
were relatively higher for Nubet and Franubet than Pemubet I and Pemubet 2 but these
differences were not considered to be due to the ash content associated with the isotypes.
Differences o f growth performance o f rats fed the four starch m utant diets ad­
justed to 14.5% isoprotein levels were not significant. This was true for a rat feeding trial
and an energy balance trial. The results indicate the energy value o f the starch mutants
are basically that o f Nubet.
Newman (Personal communication, 1980) suggested that the evaluation o f energy
values in cereals is quite difficult to measure in a rat feeding trial. Variations due to the
genotype o f the rats might be a reason and the physiological energy balance within the ani­
mal body and the energy trade o ff between protein and carbohydrate may be other reasons.
123
Since Franubet has significantly less viscous material, though to be mainly 0-glucan,
a greater growth performance o f chicks in a feed trial may be expected with this barley in
the diet.
Chapter 6
SUMMARY AND CONCLUSIONS
Two pericarp starch mutants (Pemubet I and Pemubet 2) accumulated starch in the
pericarp layer and one fractured starch mutant (Franubet) in which the endosperm con­
sisted of angular o r fractured starch granules were identified and their inheritances were
investigated. Characters associated with these genes and their use related to the human
consumption were studied.
The pericarp layers o f the grain were mutated by the diethylesulfate and the pheno­
typic appearances o f these mutants identified by the treatment with iodine solution (blue
and light blue staining on the surface o f the grain) expressed the homozygous recessive
genotypes o f the plants. Pericarp starch m utant I was inherited as single gene and assigned
the symbol per I. Pericarp starch mutant 2 (Pemubet 2) symbolized per 2 appeared to
inherited also as single recessive gene, however, unusual F1 segregations occurred in some
crosses with parents or translocations.
The linkage studies using translocation homozygote lethal stocks indicate that per I
was located on the long arm o f chromosome I near the centromere and per 2 was placed
on the long arm o f the chromosome 7. Probable gene order from the centromere o f
chromosome I was Tl-7i-Tl-6j-per J-K-Ik1.
Changes o f ash and starch distribution were associated with starch deposition in the
pericarp layer o f Pemubet I . Smaller seed size appeared to be associated with both peri­
carp starch mutants.
The additional starch accumulations in the pericarp layer did not influence the
chemical composition, such as protein, ash, ether extract, crude fiber, amylose content
125
and 0-glucan viscosity o f the grain. No significant advantages were found for pearling, mill­
ing and energy or nutritive value of these grains.
The fractured starch m utant gene (Franubet) was inherited as single recessive gene
expressing xenia and was assigned the symbol fra. Seeds of this m utant appeared as opaque
on a light table and the endosperm consists o f angular, irregular, and compound starch
granules which are easily distinguished from the round, regular granules o f normal Nubet
starch. This gene was assigned to chromosome 4, distal in the long ami, by linkage with
translocation breakpoints.
In addition to the reduced seed size, fewer kernels per spike, shorter culm length,
and higher tillering ability, lower /3-glucan viscosity, faster water absorption, and higher
lysine content of the grain were associated with this mutant. P-Glucan viscosity o f the
Nubet grain increased from 3 weeks after anthesis to physiological grain maturity and
most of the viscous substances appeared to be stored in the endosperm. The highest
0-glucan viscosity was obtained for the waxy endosperm isotype and the lowest for the
fractured starch mutant. These results indicated that different genetic mechanisms for the
P-glucan viscosity existed among the mutants.
No differences for chemical components of the grain, such as starch, protein, ash,
crude fiber, ether extract and amylose content were found between Franubet and N ubet.
No significant changes o f the viscoamylograph curves were attributable to the starch
granule changes in Franubet.
126
Increasing moisture level of tempered grain appeared to cause flour yield reductions
and created “ plugging” problems in the Buhler milling process. No significant improvement
o f the separation o f brain and endosperm was observed when the tempering moisture levels
were altered.
The variation of the flour yield due to the genotype differences was significantly
greater than the variation due to the environment including the errors associated with the
milling process. The highest flour yield was obtained for Franubet and the lowest flour
yield was from the grain of Wanubet. The 0-glucan viscosity of the grain appeared to be
related to the flour yield and a strong negative correlation (r= -0.799**) was obtained
between 0-glucan viscosity o f the grain and percent flour yield obtained from Nubet and
its starch mutants. The high flour extraction from Franubet appeared to elevate the ash
and protein content in the flour probably by contamination with outside layers.
The pearling index of barley grain was largely dependent upon the sample size
charged, moisture content o f grain and pearling time assigned. Genotype differences for
pearling rates could be determined by pearling for 2 minutes or more lengths o f time with
the same sample size and moisture level. The grain o f waxy endosperm and hulless geno­
types pearled slower than their normal counterparts. The pearling rates o f kernel layers
were not distinguishable with the successive increments of pearling time and it was difficult
to determine the optimum pearling index for the pot barley for Korean consumption with
a Strong-Scott barley pearler.
For the comparisons o f genotype differences o f pearling index, the pearling can be
equalized by taking the pearling index at the same amount o f ash content in the pearled
grain. Ash contents due to different genotypic background should be considered before
127
applying this method. The pearling index at 1% ash content in the pearled grain appeared
to be acceptable for incorporation in the Korean diet and for comparing genotype differ­
ences. Pearling indexes of Pemubet I and Pemubet 2 appeared to be lower than those o f
Nubet and Franubet at the 1% ash level.
The rat feeding trial and energy balance studies were conducted to evaluate the
nutritional value o f the starch mutants. No significant differences were found between
Nubet and starch mutant rations for gain per day, feed consumption, or percent digestible
energy.
The two pericarp starch mutants were shown to have no advantage in accomplishing
the objectives o f this study, however, the Pemubet I gene may be a good marker for
genetic studies.
Franubet m utant was shown to be lower in 0-glucan viscosity, higher in lysine con­
tent of the grain, to imbibe water more rapidly and mill easier with higher flour yield
than Nubet. The angular starch and fewer cell walls in Franubet endosperm may contribute
to finding a barley that tastes like rice.
LITERATURE CITED
LITERATURE CITED
AACC. 1962. American Association o f Cereal Chemists. Cereal Laboratory Methods (7th
ed.). The Association, St. Paul, Minn.
Andersson, J. O., D. C. Dobson, and R. K. Wagstaff. 1961. Studies on the value o f hulless
barley in chick diets and means o f increasing this value. Poultry Sci. 40: 1571-1584.
AOAC. 1970. Official Methods o f Analysis (I Ith ed.). Association o f Official Agricultural
Chemists, Washington, D. C.
Banks, W. and C.T. Greenwood. 1971. The characterization of starch and its components.
Part 4. The specific estimation o f glucose using glucose oxidase. Starke 23: 222-228.
Banks, W. and C. T. Greenwood, (ed.). 1975. Starch and its components. John Wiley and
Sons, New York. 342 p .
Biggerstaff, D. R. 1981. Combining cytogenetics and mutagenesis to recover unusual mu­
tants at specific loci in barley (Hordeum Vulgare L.). MS. Thesis, Montana State
Univ. p.p. 24 typed.
Biggerstaff, D. R. and R. F. Eslick. 1978. Combining cytogenetics and mutagenesis to
recover unusual mutants at specific loci. Barley Newsletter 22:13.
Blakely, M. E., L. W. Rooney, R. D. Sullin, and F. R. Miller. 1979. Microscopy o f the
pericarp and the testa of different genotypes o f sorghum. Crop Sci. 19: 837-842.
Bockelman, H. E. and R. F. Eslick. 1977. Absence of crossing-over o f ert-c in crosses
involving T2-3a and additional information on the linkage o f uz and msg 5. Barley
Genetics Newsl. 7: 15-16.
Bockelman, H. E., E. L. Sharp, and R. F. Eslick. 1978. Observed recombination between
T3 translocation stocks and scald resistance loci. Barley Genetics Newsletter 8:
17-20.
Bourne, D. T. and J. S. Pierce. 1972. 0-glucan and g-glucanase. Tech. Q. Master Brew.
Assoc. Am. 9: 151-157.
Briggs, D. E. (ed.). 1978. Barley. Chapman and Hall, London, p. 291.
130
Brookes, P. A., D. A. Lovett, and I. C. MacWilliam. 1976. The steeping o f barley. A re­
view o f the metabolic consequences o f water uptake, and their practical impli­
cations. I. Inst. Brew. 82: 14.
Bruckner, P. L. 1981. Effect o f the waxy endosperm gene on germination, agronomic,
and malt quality characteristics in barley (Hordeum Vulgare L.). MS. Thesis in
Crop and Soils Science, Montana State University, p.p. 127 typed.
Burnham, C. R. 1962. Interchanges, p. 66-116. In Discussions in cytogenetics. Burgess
Publishing Co., Minneapolis.
Burnham, C. R. and J. L Cartledge. 1939. Linkage relations between smut resistance and
semisterility in maize. Agr. Jour. 31: 924-933.
Calvert, C. C. 1975. Nutritional implications o f amylose-amylopectin ratio in barley
cultivars for rats and swine. MS. Thesis, Montana State Univ. p.p. 74 typed.
Cameron-Mills, V., A. Brandt, and J. Ingverson. 1979. The molecular biology of barley
storage protein synthesis, p. 339-364. In Inglett G. E. and L. Munck (ed.) Cereals
for Food and Beverages. Academic Press, New York.
Canvin, D. T. 1976. Interrelationships between carbohydrate and nitrogen metabolism,
p. 172-191. In Genetic Improvement o f Seed Proteins. Proc. o f a Workshop. Nat.
Res. Council, Washington, D. C.
Cheigh, H. S., H. E. Snyder, and T. W. Kwan. 1975. Rheological and milling characteris­
tics o f naked and covered barley varieties. Korean J. Food Sci. Technol. 7: 85-90.
(English)
Chung, T. C., S. J. Clark, J. C. Lindholm, C. A. Watson, and R. J. McGinty. 1974. The
pearlograph technique for measuring wheat hardness. Unpublished.
Cohen, R. S. and D. Tanksley, Jr. 1973. Energy and protein digestibility o f sorghum
grains with different endosperm textures and starch types by growing-finishing
swine. J. Anim. Sci. 3 7 :931.
Coles, G. 1979. Relationship o f mixed-link 0-glucan accumulation to accumulation of free
sugars and other glucans in the developing barley endosperm. Carlsberg Res.
Commun. 44: 439-453.
131
Djurtoff, R. 1958. Non-starchy polysaccharides (barley gums) in barley, malt and beer.
The Brewers Digest 33: 38-42.
Doll, H. 1973. Inheritance o f the high-lysine character of a barley mutant. Hereditas 74:
293-294.
Dudley, J. W. and R. J. Lambert. 1969. Genetic variability after 65 generations of selection
in Illinois high oil, low oil, high protein, and low protein strains o f Zea mays L.
Crop ScL 9: 179-181.
Eriksson, G. 1969. The waxy character. Hereditas 63: 180-204.
Eslick, R. F. 1971. Balanced male steriles and dominant pre-flowering selective genes for
use in hybrid seed production. Barley Genetics II: 292-297. Proc. Second Int.
Barley Genetic Symp., Washington State Univ., Pullman, Wa.
Eslick, R. F. 1972. Barley genetics-Rolls Royce, Model T, Chrysler, or Ford. Barley News­
letter 25: 69-70.
Eslick, R. F. 1979. Barley breeding for quality at Montana State Univ. p. 2-25. In Pro­
ceedings of Joint Barley Utilization Seminar. Korean Science and Engineering
Foundation and United State National Science Foundation, Suweon, Korea.
Eslick, R. F. 1981. Personal communication. Montana State Univ.
Eslick, R. F., E. A. Hockett, and G. D. Kushnak. 1972. Recombination values o f four
genes on chromosome I. Barley Genetics Newsletter 2: 123-124.
Fincher, G. B. 1975. Morphology and chemical composition o f barley endosperm cell
walls. J. Inst. Brew. 81: 116-122.
Forrest, I. S. and T. Wainwright. 1977. Differentiation between desirable and trouble­
some 0-glucans. p. 401-413. In Proc. 16th European Brewing Congress. Elsevier,
Amsterdam.
Fox, G. J. 1981. The effect o f the waxy endosperm, short awn, and hulless seed genes
upon biochemical and physiological seed characteristics important in barley (/Zordeum Vulgare L.) utilization. Ph D. Thesis, Montana State Univ. p.p. 214 typed.
132
Fulcher, R. G., G. Setterfield, M. E. McCully, and P. J. Wood. 1977. Observations on the
aleurone layer. II. Fluorescence microscopy of the aleurone-sub-aleurone junction
with emphasis on possible B-1, 3-glucan deposits in barley. Aust. J. Plant Physiol.
4:917-928.
Geddes1 W. F. 1951. Technology o f cereal grains, p. 2018-2090. In Jacobs, M. B. (ed ).
Chemistry and Technology o f Food and Food Products. Interscience, New York.
Gill, D. R., J. E. Oldfield, and D. C. England. 1966. Comparative values o f hulless barley,
regular barley, com and wheat for growing pigs. J. Anim. Sci. 25: 34-36.
Goering1 K. J. and B. DeHaas. 1974. A comparison o f the properties o f large-and smallgranule starch isolated from several isogenic lines of barley. Cereal Chem. 51: 573578.
Goering, K. J., B. W. DeHaas1 D. W. Chapman, R. F. Eslick, and R. E. Gramera. 1980.
New process for production o f ultral high maltose syrup from special genetically
derived barley. Starke 32: 349-352.
Goering, K. J., R. F. Eslick, and B. W. DeHaas. 1970. Barley starch. IV. A study o f the
cooking viscosity curves o f twelve barley genotypes. Cereal Chem. 47: 592-596.
Goering, K. J., R. F. Eslick, and B. W. DeHaas. 1973a. Barley starch. V. A comparison o f
the properties o f waxy Compana barley starch with the starches o f its parents.
Cereal Chem. 50: 322-328.
Goering, K. J., R. F. Eslick, and C. A. Ryan, Jr. 1957. Some effects o f environment and
variety on the amylose content o f barley. Cereal Chem. 34: 437-443.
Goering, K. J., D. H. Fritts, and R. F. Eslick. 1973b. A study of starch granule size
and distribution in 29 barley varieties. Starke 25: 297-302.
Goering, K. J., L. L Jackson, and B. W. DeHaas. 1975. Effect o f some nonstarch com­
ponents in com and barley starch granules on the viscosity of heated starch-water
suspensions. Cereal Chem. 52: 493-500.
Gohl, B. 1977. Influence o f water treatment o f barley on the digestion process in rats.
Z. Turphysical Turemaehr Futtermillelkd 39: 57-67.
133
Gohl, B., K. Larsson, M. Nilsson, 0 . Thoander, and S. Thomke. 1977. Distribution o f car­
bohydrates in early harvested barley grain. Z. Turphysical Turennaehr Futtermittelkd39: 1-15.
Greenberg, D. C. 1974,0-glucan and extract viscosity. J. Inst. Brew. 80: 435.
Greenberg, D. C. and E. T. Whitmore. 1974. A rapid method for estimating the viscosity
o f barley extracts. J. Inst. Brew. 80: 31-33.
Hagberg, G., L Lehman, and P. Hagberg. 1975. Segmental interchanges in barley: I. Trans­
locations involving chromosomes 5 and 6. Hereditas 80: 73-82.
Hagberg, A., L. Lehmann, and P. Hagberg. 1978. Segmental interchanges in barley.
II. Translocations involving chromosomes 6 and 7. Z. Pflanzenztichg. 81: 89-110.
Hanson, W. D. 1952. An interpretation o f the observed amount o f recombination in inter­
change heterzygotes in barley. Genetics. 37: 90-100.
Haus, T. E. 1975. Description o f genetic stocks: Waxy endosperm (wx). Barley Genetics
N ew sletters: 97.
Heiner, R. E. 1963. The action of the chemical mutagen, diethyl sulfate, on barley. Ph D.
Thesis, Washington State Univ. p.p. 103 typed.
Igarashi, O. and Sakurai, Y. 1965. Studies on the non-starch polysaccharides o f the endo­
sperm of naked barley. Agric. Biol. Chem. 29: 678.
Ingverson, J. 1975. Structure and composition of protein bodies from wild-type and highlysine barley endosperm. Hereditas 81: 69-76.
Jarvi, A. J. and R. F. Eslick. 1975. Shrunken endosperm mutants in barley. Crop Sci. 15:
363-366.
Jensen, A. H., D. H. Baker, P. B. Lynch, and B. G. Harmon. 1973. Using acid-preserved,
high-moisture corns and waxy corns in diets for swine. 111. Pork Industry Day, Co­
operative Ext. Ser., Univ. o f 111. AS- 655a.
Johnson, V. A., P. J. Mattern, D. A. Whited, and J. W. Schmidt. 1969. Breeding for high
protein content and quality in wheat, p. 29-40. In Panel Meeting on New Ap­
proaches to Breeding for Improved Plant Protein. By the joint FAO/IAEA o f
Atomic Energy in Food and Agr.
134
Juliano, B. O. 1979. The chemical basis o f rice grain quality, p. 69-1 W .In Proceedings of
the Workshop on Chemical Aspects o f Rice Grain Quality. IRRI, LosBanos1Philipines.
Juliano, B. O., G. B. Cagampang, and E. P. Navasero. 1975. Grain quality. Genetic evalu­
ation and utilization program. IRRI Ann. report for 1975: 83-90.
Juliano, B. O., G. B. Cagampang, L. J. Cruz, and R. G. Santiago. 1964. Some physico­
chemical properties of rice in Southeast Asia. Cereal Chem. 41: 275-286.
Juliano, B. O., L. U. Onate, and A. M. del Mundo. 1965. Relation of starch composition,
protein content and gelatinization temperature to cooking and eating quality of
milled rice. Food Technol. 19: 1006-1011.
Kasha, K. J. and C. R. Burnham. 1965. The location o f interchange breakpoints in barley.
I. Linkage studies and map orientation. Can. J. Genet. Cytol. 7: 62-77.
Kramer, H. IL and H. R. Albrecht. 1948. The adaptation to small samples o f the pearling
test for kernel hardness in wheat. J. Amer. Soc. Agron. 40: 422-431.
Kramer, H. H., P. L Pfahler, and R. L. Whistler. 1956. Gene interactions in Maize affect­
ing endosperm properties. Agr. Jour. 50: 207-210.
Kramer, H. H., R. Veyl, and W. D. Hanson. 1954. The association of two genetic linkage
groups in barley with one chromosome. Genetics 39: 159-168.
Larsen, L. M. and J. E. Oldfield. 1960. Improvement o f barley rations for swine. III. Ef­
fects o f fiber from barley hulls and purified cellulose in barley and corn rations. J.
Anim. Sci. 20: 440-444.
LeClerc, J. A. and C. D. Garby. 1920. Pearl barley: Its manufacture and composition. J.
Ind. Eng. Chem. 12: 451-455.
MacGregor, A. W., A. G. Gordon, W. O. S. Meredith, and L. Lacroix. 1972. Site o f alphaamylase in developing barley kernels. J. Inst. Brew. 78: 174-179.
Maynard, L. A., J. K. Loosli, H. F. Hintz, and R. G. Warner. 1979. Animal Nutrition (7th
edition) p. 602. McGraw-Hill Book Co., New York.
135
McCluggage1 M. E. 1943. Factor influencing the pearling test for kernel hardness in wheat.
Cereal Chem. 20: 686-700.
McGuire, C. F. 1979. Roller milling and quality evaluation o f barley flour, p. 89-93. In
Proceedings of Joint Barley Utilization Seminar. Korean Science and Engineering
Foundation and United States National Science Foundation, Suweon, Korea.
McGuire, C. F., E. A. Hockett, and D. M Wesenberg. 1979. Response o f agronomic and
barley quality traits to nitrogen fertilizer. Can. J. Plant Sci. 59: 831-837.
McNeil, M. and P. Albersheim. 1975. The structure of plant cell walls. VII. Barley aleurone
cells. Plant Physiol. 55: 64-68.
Medcalf, D. G. 1973. Structure and composition of cereal components as related to their
potential industrial utilization, starch, p. 121-134. /n Pomeranz, Y. (ed.) Industrial
Uses o f Cereals. Symp. Proc. 58th Ann. Meeting AACC. St. Louis, Missouri.
Merritt, N. R. 1967. A new strain o f barley with starch o f high amylose content. J. Inst.
Brew. 73: 583-585.
Mertz, E. T. 1976. Case histories o f existing models, p. 57-70. In Genetic Improvement o f
Seed Proteins. Proc. o f a Workshop. Nat. Res. Council, Washington, D C.
Miller, B. S., R. I. Derby, and H. B. Trimbo. 1973. A pictorial explanation for the increase
in viscosity o f a heated wheat starch-water suspension. Cereal Chem. 50: 271-280.
Morgan, A. H. 1977. The relationship between barley extract viscosity curves and malting
ability. Jour. Inst. Brew. 83: 231-234.
Morgan, A. H. and P. G. Gothard. 1977. A rapid, simple viscometric technique for indi­
rect estimation o f soluble 0-glucan content of raw barley. J. Inst. Brew. 83: 37-38.
Munck, L. 1972. Improvement of nutritional value in cereals. Hereditas 72: 1-128.
Munck, L., K. E. Karlsson, A. Hagberg, and B. O. Eggum. 1970. Gene for improved nutri­
tional value in barley seed protein. Science 168: 985-987.
Newman, C. W. 1979. A summary of ten years efforts in the nutritional evaluation of
barley cultivars. p. 109-136. In Proceedings of Joint Barley Utilization Seminar.
Korean Science and Engineering Foundation and United States National Science
Foundation, Suweon, Korea.
136
Newman, C. W. 1981. Personal communication. Montana State Univ.
Newman, C. W., R. F. Eslick, and J. W. Pepper. 1978. Performance o f pigs fed hulless
and covered barley supplemented with or without a bacterial diastase. Proc. West.
Sect. Amer. SocL Anim. Sci. 48: 187.
Newman, C. W., 0 . 0 . Thomas, and R. F. Eslick. 1968. Hulless barley in diets for wean­
ling pigs. Jour. Anim. Sci. 27: 981-984.
Nilan, R. A. 1964. The cytology and genetics o f barley, 1951-1962. Monographic Supple­
ment No. 3.—Research Studies 32 (I). Washington State University, Pullman, Wa.
278 pp.
Nilan, R. A., C. F. Konzak, R. E. Heiner, and E. Froese-Gertzen. 1963. Chemical muta­
genesis in barley. Barley Genetics I: 35-54. Proc. First Int. Barley Genetic Symp.,
Wageningen.
Novacek, E. J., C. F. Petersen, and A. E. Slinkard. 1966. A separation on anatomical parts
of barley from the by-products o f barley pearling. Cereal Chem. 43: 384-391.
Palmer, G. H. 1979. The morphology and physiology of malting barleys, p. 301-338. Zn
Cereals for Food and Beverages, (ed.) Inglett, G. E. and L Munck. Academic Pre.
Co., New York.
Persson, R. T. 1969. An attem pt to Fmd suitable genetic markers for dense ear loci in bar­
ley II. Hereditas 63: 1-28.
Pomeranz, Y. (ed.) 1971. Wheat: Chemistry and technology. AACC Inc., St. Paul, Minn.
Pomeranz, Y. 1974. A note on scanning electron microscopy o f low and high-protein bar­
ley malts. Cereal Chem. 5 1: 545-552.
Pomeranz, Y. and D. B. Bechtel. 1978. Structure o f cereal grains as related to end-use
properties, p. 85-104. In Pomeranz, Y. (ed.) Cereals’ 78: Better Nutrition For The
World’s Millions.
Pomeranz, Y., Ke Helen, and A. B. Ward. 1971. Composition and utilization of milled
barley products. I. Gross composition of rollermilled and air separated fractions.
Cereal Chem. 48: 47-58.
137
Pratt, Jr. D. B. 1964. Criteria o f flour quality, p. 209. In Hlynka (ed.) Wheat Chemistry
and Technology. AACC Inc., St. Paul. Minnesota.
Preece, I. A. and K. G. MacKenzie. 1952. Non-starchy polysaccharides o f cereal grains.
I. Fractionation o f the barley gums. J. Inst. Brew. 58: 353-363.
Rahman, M. M. and R. F. Eslick. 1975. Linkage o f male sterile genes with seedling lethal
genes. Barley Genetics Newsletter 5: 42.
Ramage, R. T. 1963. Chromosome aberrations and their use in genetics and breedingtranslocations. Barley Genetics I. 995-115. Proc. First Int. Barley Genetic Symp.,
Wageningen.
Ramage, R. T. 1966. Techniques for mapping barley chromosomes. Barley Newsletter
10:44-49.
Ramage, R. T. 1971, Translocations and balanced tertiary trisomics. Barley Genetics
Newsletter 1:74-80.
Ramage, R. T., C. R. Burnham, and A. Hagberg. 1961. A summary o f translocation studies
in barley. Crop Sci. 1: 277-279.
Ramage, R. T. and C. A. Suneson. 1961. Translocation-gene linkages on barley chromo­
some 7. Crop Sci. 1: 319-320.
Reid, D. A. and G. A. Wiebe. 1979. Taxonomy, botany, classification, and world col­
lection. p. 78-104. In. Barley: Origin, Botany, Culture, Winter hardiness. Genetics,
Utilization, Pests. USDA Agr. Handbook No. 338.
Robertson, D. W. 1967. Linkage studies o f various barley mutations (Hordeum species).
Crop Sci. 7: 41-42.
Robertson, D. W. 1971. Recent information of linkage and chromosome mapping, Barley
Genetics II: 220-242. Washington State Univ., Pullman, Wa.
Ryu, I. S. 1979. Grain quality o f barley for human diet. p. 94-108. In Proceedings of
Joint Barley Utilization Seminar. Korean Sci. and Eng. Foundation and United
States National Science Foundation, Suweon, Korea.
Sandstedt, R. M. 1965. Fifty years progress in starch chemistry. Cereal Sci. Today 10:
305-314.
138
Scheming, J. F. and L W. Rooney. 1979. A staining procedure to determine the extent
o f bran removal in pearled sorghum. Cereal Chem. 56: 545-548.
Scholz, F. 1971. Utilization o f induced mutation in barley. Barley Genetics II: 94-105.
Proc. Second Int. Barley Genetic Symp., Washington State Univ., Pullman, Wa.
Scott, R. W. 1972. The viscosity o f worts in relation to their content o f 0-glucan. J. Inst.
Brew. 78: 179-186.
Sigurbjoemsson, B. 1975. The improvement of barley through induced mutation. Barley
Genetics III: 84-95. Proc. Third Int. Barley Genetic Symp., Garching.
Smith, L. 1941. An inversion, a reciprocal translocation, trisomics and tetraploids in bar­
ley. J. Agr. Res. 63: 741-750.
Smith, R. J. 1964. Viscosity o f starch paste. In Methods in Carbohydrate Chemistry vol.
4. p. 114. ed. by Whistler, R. L., Academic Press, New York.
Sorum, D. L 1977. A study adapting soft wheat evaluation procedures to barley. MS.
Thesis, Montana State Univ. p.p. 79 typed.
Spackman, D. H., W. H. Stein, and S. Moore. 1958. Automatic recording apparatus for
use in chromatography of amino acid. J. Anal. Chem. 3 0 : 1190.
Suzuki, H. 1979. Amylography and alkali viscography of rice. p. 262-282. In Proceedings
o f the Workshop on Chemical Aspects o f Rice Grain Quality. IRRI, LosBanos,
Philippines.
Taiz, L. and R. L. Jones. 1970. Gibberellic acid, B I , 3-glucans and the cell walls of barley
aleurone layers. Planta 92: 73-84.
Taylor, J. W., B. B. Bayles, and C. C. Fifield. 1939. A simple measure o f kernel hardness
in wheat. J. Amer. Soc. Agron. 31: 775-784.
Truscott, D. R. 1980. A nutritional evaluation o f four Betzes barley isogenes influenced
by length of awn and presence or absence o f hulls. MS. Thesis, Montana State Univ.
p.p. 146 typed.
Tuleen, N. A. 1971. Linkage data and chromosome mapping. Barley Genetics II: 208212. Proc. Second Int. Barley Genetic Symp., Washington State Univ., Pullman, Wa.
139
Tuleen, N. A. 1974. Unpublished data in personal communication to R. T. Ramage dated
April, 1974.
Ullrich, S. E. 1978. Agronomic, biochemical and genetic characterizations and adaptation
of high lysine, shrunken endosperm mutants o f barley (JHordeum Vulgare L.).
Ph.D. Thesis, p.p. 114.
Ullrich, S. E. and R. F. Eslick. 1978. Evidence for assigning the high amylose locus,
amo I o f Glacier barley to chromosome 2. Barley Genetics Newsletter 8: 112-113.
Vermorelv M. and Keller, J. 1967. Energetic utilization by growing rate o f the principal
cereals as components o f diets balanced with respect to protein and amino acids, p.
387-396. In Energy Entabolism o f Farm Animals. Proc. o f 4th Symp., Warsaw,
Poland.
Williams, J. M. and C. M. Duffus. 1977. The development o f endosperm amyloplasts
during grain maturation in Barley. J. Inst. Brew. 84: 47-50.
Wood, P. J. and R. G. Fulcher. 1978. Interaction o f some dyes with cereal 0-glucans. Cer­
eal Chem. 55: 952-966.
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