The Reserve Polysaccharides of Various Corn Genotypes

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P U R D U E U N IV E R S IT Y

THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION

BY

ENTITLED

William Dvonch

THE RESERVE POLYSACCHARIDES OF VARIOUS

_________________ CORN GENOTYPES________________________

COMPLIES WITH THE UNIVERSITY REGULATIONS ON GRADUATION THESES

AND IS APPROVED BY ME AS FULFILLING THIS PART OF THE REQUIREMENTS

FOR THE DEGREE OF

Doctor of Philosophy

P r o f e s s o r IN C h a r g e

H

eap of

School

TO THE LIBRARIAN:----•SS» THIS THESIS IS NOT TO BE REGARDED AS CONFIDENTIAL.

GRAD. SCHOOL FORM 9—3 . 4 9 —1M

or

of

T h e s is

D epa r tm en t

THE RESERVE POLYSACCHARIDES OF VARIOUS CORN GENOTYPES

A Thesis Submitted to the Faculty of

Purdue University

by

William Dvonch

In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy June, 1950

ProQuest N um ber: 27714147

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.

uest ProQuest 27714147 Published by ProQuest LLC (2019). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

ACKNOWLEDGMENTS

The writer wishes to express his appreciation to Dr. Roy L. Whistler for suggesting and directing this work.

He

is indebted to Dr. H. H. Kramer and Mr. Gerald Dunn of the Department of Agronomy for the supply of genetic mater­ ial, Dr. H. J. Yearian of the Department of Physics for the X-ray diffraction patterns, and Mr. L. C. Shenberger and Mr. Lee House for the photomicrographs.

He

is particularly indebted to Elsie Mihelich Armstrong for much of the analytical work.

TABLE OF CONTENTS

ABSTRACT

...............................................

INTRODUCTION............................................. MATERIALS AND ANALYTICAL METHODS........................ Production of the Genetic Material................... Isolation of Starch........ .......................... Analytical Methods.

............ .............. .

Determination of water-soluble polysac­ ....................... charides and starch. Micro-Kjeldahl protein determination............. Iodine sorption.................................. Gelatinization temperature of the starch granules............................. ......... X-ray diffraction patterns of the starch granules.......................... ............ RESULTS AND DISCUSSION.................................. Reproducibility of Gene Action...... ................ Gene Dosage Effects.................................. The Starch Fraction......................... ....... NATURE OF THE WATER-SOLUBLE POLYSACCHARIDES............ Experimental....... .................................. Isolation of the water-soluble polysaccharides... Purification of Fraction 1 .................... Preparation of ^ - a m y l a s e ................. ....... Preparation of limit dextrins.................... Periodate end-group assay........................ Determination of color with iodine........... Formation of cupric chloride patterns...........

Table of Contents (Gont. ) Page Discussion.............

46

NATURE OF THE AMYLOSE AND AMYLOPECTIN COMPONENTS OF THE STARCH.........................................

49

Experimental...............

50

Fractionation procedure..........................

50

Determination of viscosities......

51

Results and Discussion................... CONCLUSIONS

........................................

BIBLIOGRAPHY.................... VITA

52 57 61

LISTS OF TABLES AND FIGURES List of Tables Table

Page

1.

Reproducibility of Iodine Sorption Values.........

2.

The Genetic Constitution, Endosperm Description,

13

and Quantities of Polysaccharides in the Endosperm of Various Genotypes.................. 3*

15

Comparison of the Quantities of Polysaccharides in Non-Isogenic Endosperm Types.................

4.

19

The Quantities of Polysaccharides in Various Genotypes Involving suamsu......................

5*

21

The Quantities of Polysaccharides in Various Genotypes Involving Su suam ............

6.

21

The Quantities of Polysaccharides in Various Genotypes Involving Su su.......................

7-

22

The Endosperm Description and Quantities of Polysaccharides in the Endosperm of Various Homozygous Genotypes.................

8.

24

Microscopic Appearance and X-ray Diffraction Data for the Starches of Various Homozygous Genotypes

9*

..........

27

Prominent Rings in the Diffraction Patterns of Various Starches............

10.

Amounts of Starch not Dissolved upon Autoclaving 3 Hours at 124°.

11.

34

........

35

End-Group Assay of Limit Dextrins by Periodate Oxidation......

.

43

Table 12.

Page

Properties of Fraction 1 and 2 from Golden Bantam Sweet Corn...............................

13*

Yields of Fraction 1 and 2 from Golden Bantam Sweet Corn.......

14.

15*

45

46

Properties of the Amylose and Amylopectin Isolated from Various Starches..................

53

Corrected Per Cents of Amylose in Various Starches.

56

List of Figures Figure 1.

The Relationship Between Per Cent Starch and Per Cent Amylose Content of the Starch in the Endosperm of Various Homozygous Genotypes.......

25

2 . Starch Granules from American Maize Co. Corn Starch (normal).

X 600...........

28

3* Starch Granules Typical for Golden Bantam Sweet Corn, no. 1 8 , 9 > 19 » 21, and 30 Starches. X 600.......... 4.

29

Starch Granules Typical for no. 24, 27, and 29 Starches.

X 600...............

30

5* Starch Granules from no. 35 (supersugary) Starch, X 600............................................

31

6 . Starch Granules from no. 36 (waxy) Starch. X 600...

32

7.

Starch Granules from no. 38 (sugary waxy) Starch. X 600............................................

33

THE RESERVE POLYSACCHARIDES OF VARIOUS CORN GENOTYPES

AN ABSTRACT The reserve polysaccharides of corn endosperm are known to be affected by four sets of alleles, (Du d u ) , and Qtfx v^).

(Su suainsu), (Su^su^) .

Certain combinations of these genes

were studied for their effect on the amount of water-soluble polysaccharides and starch and the amylose content of the starch.

Previous work by Cameron with the gene pairs suamsu

and Du du was verified.

Similar results were found for the

pairs Su^suy, Du d u : Su su, SuySUv: Su su. Du d u .

A rise

in amylose content of the starch was always accompanied by a drop in the amount of starch and an increase in the amount of water-soluble polysaccharides.

The total polysaccharides

were always less than the total in normal corn endosperm whenever amylose content was increased in the starch. The effect of the sugary gene su was most marked in that it always caused a large increase in the amount of watersoluble polysaccharides in every background.

This was evi­

dent in sugary waxy (su wx) endosperm also; however, amylose was still absent. The starch from the triple recessive su su^du had an amylose content of 63^.

Cameron1s su du type gave a similar

value and was found in similar amounts. Comparison of identical combinations of the genes studied in non-isogenic lines grown in a uniform environment showed

il

a wide variability in the endosperm composition.

It is sug­

gested that unknown genes are responsible. The nature of the starch grain was markedly affected by genic change as evidenced by changes in microscopic ap­ pearance, X-ray diffraction pattern, and gelatini zation be­ havior. The water-soluble polysaccharides were studied and were found to be highly branched amylopectins in contradiction to findings of previous workers. In the homozygous genotypes studied, the amylose fraction of the starch was found to be lower in molecular weight than normal corn starch amylose and variable in iodine sorption. As the method of calculating amylose content in starch depends upon the value assumed for 11pure11 amylose, it is suggested that a value of 190 ng. iodine per g. of amylose rather than 200 mg. be used in future work. The amylopectin fraction was normal except in the su du and suamsuxdu starch type.

In these two amylopectins, the

degree of branching was the same as in the water-soluble polysaccharides. Cameron has suggested a scheme for the genic control of the phosphorylase system for starch synthesis Involving the gene pairs suamsu and Du du. not in agreement with it.

The data presented here are

It is suggested that if any endo­

sperm characteristic is desired, such as high amylose content, selection will need to be resorted to, particularly if more genes involved in the carbohydrate interrelationships are found.

THE RESERVE POLYSACCHARIDES OF VARIOUS CORN GrENOTYFES

INTRODUCTION The recent researches of Beadle, Tatum, and associates in the genic control of amino acid and vitamin syntheses in Neurosoora have stimulated work on the physiological bases of gene action in the higher plants and animals.

Of interest

in this connection, both from a theoretical and practical point of view, are the recent studies directed towards the elucidation of the genetic bases for the reserve polysac­ charides in corn and particularly for the linear fraction, amylose. A starch consisting solely of amylose would be of •Interest Industrially as a linear high polymer with properties similar to those of cellulose.

A breeding program to develop

such a starch Is being carried out at Purdue University by cooperation between the departments of Agricultural Chemistry and Agronomy.

To arrive at this goal, an understanding of

the genes involved, their effect, and their interaction is necessary.

Also, since the normal processes In the corn

endosperm can be fundamentally deranged by genetic change, it seemed necessary to determine if any changes had occurred in the starch, amylose, or amylopectin (the branched chain fraction) and to determine the nature of the water-soluble polysaccharides formed In large quantities in many genotypes. These polysaccharides had been assigned a role in starch

2

synthesis by Cameron (8 )* At present, there are 4 sets of alleles known to affect the endosperm carbohydrate.

The recessive gene waxy (wx),

first described by Collins (10), is responsible for the formation of starch consisting entirely of amylopectin.

The

gene derives its name from the dull, waxy appearance it im­ parts to corn endosperm.

Brimhall et al.(5) found another

allele, wxa , at this gene locus that when homozygous gives an endosperm with a small amount of amylose. A set of multiple alleles (Su suamsu) is responsible for starchy, pseudostarchy, and sugary endosperm.

The two

higher alleles both give smooth opaque kernals /Mangelsdorf (27)_/ while su kernals are translucent and wrinkled.

They

are also characterized by the presence of a large amount of water-soluble polysaccharides /Culpepper and Magoon (12)_/. Cameron studied the interrelationships between the two gene pairs suam3u. Du du (dull) in the 16 possible geno­ types and found 65% amylose in the starch isolated from the true-breeding su du genotype.

However, this genotype had

very little starch present and a great deal of water-soluble polysaccharides in the endosperm. Horovitz et al. (19) described the gene sugary -x (su%) and its interaction with su to increase the per cent of sugars in mature seeds, but Kramer and Whistler (24), in part of the breeding program at Purdue University, found that it increases amylose content also.

They determined

amylose content in certain combinations of the 4 recessive

3

genesÎ sugary, dull> sugary - x , and waxy, but found no amylose content higher than that In the su du genotype. The work presented here is concerned principally with the continuation of the work of Kramer and Whistler represented in the genetic material produced in 1949. MATERIALS AND ANALYTICAL METHODS Production of Genetic Material H.

H. Kramer, Department of Agronomy, Purdue University,

was responsible for the breeding plan for the production of the genetic material studied here*

The breeding work was

carried out by Mr. Gerald Dunn, graduate assistant in the Department of Agronomy, during 1948 and 1949•

It is mostly

Dr. Kramer* s description of the source of the genotypes that follows. The sample no. below in Table 2 are the Department of Agricultural Chemistry no. for the 1949 samples; the cul­ ture no. are the Department of Agronomy no. for the 1948 samples.

The accession no. are those of the Department of

Agronomy. No. 1-8 , 11-14, and 17-29$

In 1947 a cross was made

between a plant homozygous for su^ (acc. 132 from R. L. Cushing; suo is considered identical to suv) and a plant homozygous for su du (acc. 178 from J. W. Cameron). plants were self pollinated in 1948.

Phenotypic separation

of kernels in the resulting ears resulted in four classes* normal, opaque, dull, and wrinkled.

In 1949» plants from

4

■th.© normal, opaque $ and dull classes were self pollinated and used as males with plants of a homozygous suamdu line (acc* 177 from J. W. Oameron).

Plants from the wrinkled

class were selfed and used as males on plants of 2 homo­ zygous tester lines, one of which was the suamdu line, while the other was an su-y line (acc. 288 from 0. E* Nelson) •

By

classifying the selfed ears and the corresponding crossed ears from the tester plants, it was possible to identify the 8 homozygous genotypes of the 3 factor pairs Su s u , Suxsux » and Du du. No. 9» 10, 15, 1 6 , 30, 31:

In 1947 a cross was made

between a plant homozygous for sux (acc. 132) and one homo­ zygous for suamdu (acc. 177)• in 1948.

plants were self pollinated

Classification of kernels on one ear resulted in

normal kernels, dull kernels, and kernels which appeared similar in the degree of wrinkling to the suamdu parent. Kernels from the latter 2 classes were planted, and plants were self-pollinated and used as males on the tester lines. No* 32:

A homozygous suamdu line (acc* 113 from R. L*

Cushing) was maintained by self pollination. No. 33:

Cameron’s sua% u line was crossed with Cushing's

suamdu line In 1948.

Analysis of the F% seed and of each

parent of a similar cross gave similar values.

In 1948,

Fi plants from the 1948 cross were self pollinated. No. 34:

A homozygous sur line (acc. 288 from 0. E.

Nelson) was maintained by self pollination. No. 35:

Cameron's su du line was maintained by self

5

pollination* No. 36:

A homozygous wx line (acc. 136 from A. M.

Brunson) was maintained by self pollination. No. 37:

A white endosperm equivalent of Inbred Ml4

(acc. 137 from A. M. Brunson) was maintained by self pollina­ tion. No. 38:

A su wx line was derived from a cross between

a sugary line (acc. 129 from Cornell University) and a waxy line (acc. 136) made in 1946 and was maintained by self pollination. Culture 4:

A homozygous suamdu line (acc. 134 from

Cornell University) was maintained by self pollination. Culture 79:

A homozygous su b u y line (acc. 315 from

0. E. Nelson) was maintained by self pollination. The ears of these various genotypes were allowed to mature fully in the field and were stored in the air-dry condition until used.

In many instances, only a small amount

of kernels was available for analyses, limiting the analysis for water-soluble polysaccharides and starch to one deter­ mination and not providing enough starch for fractionation of some of the homozygous genotypes. Isolation of Starch Granular endosperm starch was Isolated by a modified version of a method published by Brimhall et al. (5).

Fifty

g. of corn was soaked in distilled water until the pericarp readily peeled off (about 10-30 minutes). pericarp were removed.

The germ and

If too much difficulty was encountered

6

in removing ilie pericarp, it was left on.

The endosperm

tissue was air-dried overnight and ground in a small Wiley mill to pass the 40 mesh sieve*

The ground material was

placed in a 250 ml. centrifuge bottle and treated twice with 73% ethanol for 2 hours.

In this and succeeding operations,

the various extractions were carried out with stirring at room temperature » and separation of the extracts was made by centrifugation in the International centrifuge*

The

tissue was then treated with a 0 .2% solution of sulfur di­ oxide (about 33 ml. of commercial 6% reagent per liter of solution) overnight.

The sulfur dioxide solution was re­

placed with water, and the mixture blended in a Waring Blendor for a few minutes.

The slurry was passed through

a coarse bolting cloth, and the protein and fiber were re­ suspended to wash out the remaining starch.

The slurry was

treated as before, and the material not passing through the bolting cloth was discarded.

The remaining starch and pro­

tein was treated with 0.06 IT sodium hydroxide solution for 2 hours.

The mixture was then treated with water, neutralized

to the phenophthalein end point with dilute hydrochloric acid, transferred to 50 ml. plastic centrifuge tubes, and centri­ fuged in a small angle-head centrifuge.

The glutinous layer

which formed was scraped off, and the starch re suspended and cleaned until no glutinous layer formed.

The starch suspen­

sion was then passed through a No. 25 standard silk bolting cloth to remove any small particles of fiber.

The clean

starch was transferred to an extraction thimble and extracted

7

for 2 days with 85^ methanol in a Soxhlet apparatus.

The

starch was removed from the thimble, air-dried, and ground to pass a 60 mesh screen. This procedure was sufficient, in most cases, to yield a clean, bright starch, low in protein and fiber.

However,

with the variety of genetic material used, difficulty was encountered in some instances in obtaining a satisfactory starch.

In these difficult cases, further manipulations

were resorted to to obtain a good starch.

These included

re-extractions of the starch with 75% ethanol and 0.06 N sodium hydroxide solution and repetition of the procedure given above from this point.

Sometimes it was helpful to

isolate the glutinous layer while the mixture was in the alkaline suspension. The yields of starch obtained were very variable, rang­ ing from 4-62%, based on endosperm tissue, or 3-54% based on the whole corn.

The recoveries of starch were also very

variable, 26% to about 100%.

This range reflects the dif­

ficulty in obtaining starch from some of the samples. Analytical Methods All data reported here are on a dry weight basis.

Mois

tures were determined for all samples by drying in vacuo at 100° overnight.

Sample weights given in the analytical

procedures are air-dry weights. Determination of water-soluble polysaccharides and starch.

To obtain data comparable to Oameron1s data, his

method of analysis for water-soluble polysaccharides and

8

starch was used, as these values are subject to the method used.

His method, modified in some manipulative details,

is as follows:

The embryos were removed from 10.0 g. of

corn, and the remaining tissue was weighed.

It was considered

endosperm, even though the pericarp was present.

After the

endosperm was ground to pass the 40 mesh sieve in a small Wiley mill, moisture samples were taken, and the rest of the tissue was weighed into an extraction thimble.

After the

sample was extracted with 80$ ethanol in a Soxhlet apparatus overnight, it was dried in vacuo over calcium chloride, and transferred to a 250 ml. centrifuge bottle.

Sixty ml. of

10 per cent trichloracetic acid solution was added, and the mixture was stirred for 0.5 hour.

The suspension was separated

in an International centrifuge, and the residue twice re­ extracted.

The combined extracts were precipitated by the

addition of 560 ml. of ethanol.

The mixture was let stand

0.5 hour and the precipitate collected in a 250 ml. centri­ fuge bottle.

It was washed twice with ethanol, dried while

in the bottle in vacuo over calcium chloride, transferred to a weighing bottle, and dried overnight in a vacuum oven at 100°. The residue was washed 3 times with water, dried at 50°, weighed, and reground to pass 40 mesh.

Duplicate

1.00 g. samples were taken for starch determinations, and moisture samples were taken. Starch was determined by the method of Brimhall and Hixon (4).

The 1.00 g. sample was heated in 100 ml. ammonium

9

persulfate solution (0.3 g. per 100 mlj for 45 minutes in a boiling water bath.

The cooled mixture was transferred to

a 50 ml. centrifuge tube and centrifuged.

The supernatant

liquid was decanted into a weighed Gooch crucible fitted with a disc of filter paper and a layer of Filter-Gel, the residue was washed with water and transferred to the Gooch crucible.

After it was washed several times, the residue

was dried at 100° overnight.

The difference between sample

and residue represents starch. The reproducibility of these results is good consider­ ing the nature of the experimental material.

Duplicate

analyses have been performed on several samples and typical results are as follows (the value for water-soluble poly­ saccharides is given first; all values expressed as per cent of mature endosperm tissue): 37.8 , 14.3.

culture 79 - 38.9 , 13*9 ;

No. 36 - 4.7, 71*8 ; 3*7, 72.3-

For further analyses, two ways to improve and facilitate the analysis suggested themselves during the course of the work.

One suggestion is to regrind the residue left after

the 80% alcohol extraction through the 60 mesh sieve in the Wiley mill in order to insure complete extraction of the water-soluble polysaccharides in the next step.

Oameron

states that 95% of this material is removed, but it is be­ lieved that extraction is not that complete in the most sugary type of tissues.

The second suggestion is to not

determine moisture values on the ground corn or the residue for starch determination as no significant difference in the

10

values obtained is found if a dry substance value of 0.91 is taken for both of these materials.

Of the 76 values

from the 38 corns, 0.91 is the modal value, occurring 47 times, and it is also the average value.

If this is

done, one must be certain that the corn used is mature and that the residue for starch determination is properly airequilibrated by letting it stand overnight. Mlcro-K.leldahl protein determination.

The A.O.A.C*

method (26) was modified to give the following procedure: Samples of 0.200 g. of starch were weighed into 25 ml. micro-Kjeldahl flasks, and 0.5 g* potassium sulfate-copper sulfate mixture (2% in copper sulfate pentahydrate) and 2 ml. concentrated sulfuric acid were added.

The mixture was

digested until clear (1.5 to 2.0 hours), cooled; one drop of ethanol was added; and the mixture digested again until clear. After the sample was cooled, it was transferred to a Kirk distilling apparatus (21), using not more than 10 ml. of water in the process.

Ten ml. of 45^ sodium hydroxide

solution was added, and the ammonia was distilled into a 50 ml. Erlenmeyer flask containing 2 ml. of 4 per cent boric acid solution by boiling the solution for 7 minutes, the last 2 minutes with the receiving flask lowered to flush the delivery tube.

Two drops of bromcresol green-methyl red

indicator solution (20 ml. of 0.1% solution of methyl red in ethanol added to 100 ml. of 0.1% solution of bromcresol green in ethanol) were added, and the solution was titrated with standard 0.01 N hydrochloric acid solution (standardized

11

gravi metrically ) to tlie colorless end point»

A blank was run»

It is important that the distilling apparatus is steamed out before u s e , and that the pH of the boric acid solution is adjusted to the end point of the indicator by appropriate addition of 0.01 N base. Protein was calculated as N x 6.25*

A value of 0.9

was taken for the dry substance of the starches as small deviations from this value would not affect the usefulness of the results. Iodine sorption.

The iodine sorption of the starches

and amylose was determined by the modified potentiometrie titration method of Bates et, al. (1, 42).

For starches, a

sample of 0.1100 g. was weighed into a 50 ml. Erlenmeyer flask and 25*00 ml. of 0.5 N potassium hydroxide solution was added.

The flask was flooded with nitrogen, stoppered,

and stored at 5°*

The solution was carefully swirled several

times after a few hours and once a day until completely free from undispersed material. is reached in 2 days.

Usually, complete dissolution

Any sedimentation of the solution can

be ignored. The sample was then warmed to room temperature, and 5*00 ml. was pipetted into a 250 ml. beaker.

Five ml. of

0*5 N potassium hydroxide solution and 70 ml. of water were added, and the solution was made neutral to methyl orange with 0.500 N hydrochloric acid solution.

Ten ml. of 0.5 N

potassium iodide solution was added, and the sample was titrated potentiometrically with 0.001 N iodine solution.

12

0,05 N In potassium iodide and chloride.

The electrode

system consisted of a Leeds and Northrup no, 1199-31-A saturated calomel half-cell and a bright platinum electrode, and the potentials were determined by a Leeds and Northrup Type K potentiometer.

After each addition of iodine solution,

the system was let come to equilibrium for 2-3 minutes.

A

blank curve was obtained in the same manner, Amylose samples were dispersed in 1 N potassium hydrox­ ide solution; the addition of the 5-00 ml. of 0.5 N potassium hydroxide solution was omitted, and 75 ml. of water was used. The point of inflection in the curve potential vs. ml. of iodine solution added was taken as the end point of the titration.

The ml. of iodine solution for the blank at this

same potential was subtracted from the value for the sample, and the iodine sorption value of the sample calculated as mg. iodine sorbed per g. starch. As no previous work has been done at this laboratory on the reproducibility and precision of this method, a sample of corn amylose, once-recrystallized, was designated as a standard and analyzed by 3 workers.

To check the re­

producibility of samples of starch isolated by different workers, 2 samples of Golden Bantam sweet corn starch were prepared and analyzed. shown in Table 1.

The results of the analyses are

It whould be pointed out that each value

was obtained by a complete independent analysis.

13

Table 1 Reproducibility of Iodine Sorption Values. Elsie Mihelich Armstrong

William Dvonch

Carolyn Hillis Johnson

Aver­ age

Dev­ iation

Standard corn amylose, 6 56 (1)

19.22# 19.36 19.29

18.90% 18.96 18.93

19*12%

19*11%

6/1000

Golden Bantam sweet corn starches

5.82 5.88 5.94 5.88

6.06

5.97

15/1000

For the starches reported here, the limit of error is approximately +

of the determined value•

For the amylose,

It is ± 0*3%» Gelatinization temperature of the starch granules.

The

gelatinization temperature of the starch granules was deter­ mined after the method of Reichert (36) by slowly heating an aqueous suspension of the starch in a water bath and observing the temperature at which the granules gelatinize, i.e., swell, lose their birefringence, burst. X-ray diffraction patterns of the starch granules.

All

X-ray diffraction patterns were obtained by exposing for 2 to 3 hours a 1 mm. thick sample of the wet starch to nickel filtered copper radiations at 35 K.V.P., 23 ma. and a specimento-film distance of 5*0 cm. in a flat film camera* was contained between Scotch tape.

The sample

This procedure does not

interfere with the starch pattern as only a diffuse halo is obtained with a moistened Scotch tape blank.

14

RESULTS AND DISCUSSION The analytical data obtained is shown in Table 2 with the genotype and endosperm description.

The per cent amylose

in the starches is calculated on the assumption that "pure" amylose sorbs 200 mg. of iodine per g.

It is shown later

that this procedure is not completely justified, but it serves to give comparative values.

In this connection it

should be pointed out that Cameron does not give his basis for calculating amylose content.

As the base for the method

of calculating amylose content is questionable, the values are only given to the nearest per cent.

The determination

of protein in the starches analyzed gave values from 0.23 to 0*93%»

A correction for protein content would not sig­

nificantly alter the results. Reproducibility of Gene Action The genotypes produced were not isogenic in order to insure more general application of the conclusions drawn as to their action although precision in determining gene action in a specific genotypic background was sacrificed.

It is

evident that, besides the different modifying complexes that affect the endosperm carbohydrate, the particular amounts of water-soluble polysaccharide, amylose, and amylopectin are subject to environmental modification as well.

These 2

effects are both probably operating to give the results made evident in Table 3* in which similar genotypes are grouped for comparison within a background isogenic for the genes

15

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of Various Homozygous

Genotypes.

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Table

7.

The Endosperm

Description

and Quantities

of Polysaccharides

43

00 02

PER CENT

AMYLOSE

25

gO

p• i --------- i ” ■ "i i— 1---------ri -----

70

"

60

© oOSU s u oDU u O - SU SUX DU

50 40

20 10 0

11

-

SU001 SU0” 1DU O su sux °S U SUX

Q O x

©su Osu 30

i 1

SUamSUx DU °° OSUx DU °SU X DU

-

i i------- ri ....... i 1

-

DU DUo

su am SUam o Q0G NORMAL

-

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© su WX wx q SU oWX ©WX i l 1 1 1 1 . l_j_____ i_____ i_____ i_____ i_____ |_____ i _____ 1 i ______i _____ _____ _ ____ 1_ __ 0 10 20 30 40 50 60 70 80 90 -

PER CENT STARCH Fig. 1.

The Relationship Between Per Gent Starch and Per Gent Amylose Content of the Starch in the Endo­ sperm of Various Homozygous Genotypes.

26

The Starch Fraction The starches from lines of the 12 homozygous genotypes were examined microscopically and by X-ray diffraction. data is summarized in Table 8.

The-

The 5 typical starch forms

are shown in Figures 2-7 with their appearance in polarized light whenever birefringence was exhibited.

The new X-ray

patterns found are shown with the data of Bear and French (2) for the A and B patterns of starch in Table 9There Is obviously no explanation for the characteristics of the granules on the basis of the genes studied here; for, as an example, in the case of Golden Bantam sweet corn starch and no. 24 starch the granules are markedly different in spite of both being sugary genotypes. The same conclusion must be made for the differences in X-ray diffraction patterns.

The B pattern of starch,

which was thought to be characteristic for root starches, has also been found in a starch containing 60-70$ amylose from wrinkled seeded peas by Hilbert and MacMasters (T f )• However, it obviously is not correlated with high amylose content. Hilbert and MacMasters found that the gelatinization behavior of high amylose pea starch was unusual in that it was not completely gelatinized even after heating 1 hour at 120°, for it was only partially swollen and still showed birefringence•

This behavior is duplicated by the starches

of the genotypes studied here.

For even after heating 3

hours by auto slaving at 124° for the fractionation procedure

27

Table 8.

Microscopic Appearance and X-ray Diffraction Data

for the Starches of Various Homozygous Genotypes. Ho.

Genotype

Microscopic appearance

American normal Maize Go.

Simple biréfringent granules. Fig. 2.

Golden Bantam

Biréfringent compound granules with few simple ones. Fig. 3.

su

24

18

X-ray diffraction pattern A

Mostly small biréfringent simple granules. Fig. 4. du

Fig. 3-

A

9

suamdu

Fig. 3.

B

35

su du

Some compound, mostly simple granules with many misshapen. Little birefringence. Fig. 5 .

19

sux

Fig. 3 . More compound granules.

27

su sux

Fig. 4. Little bi re fringence.

21

Fig. 3-

A

30

suxdu suam suxdu

Fig. 3.

B

29

su suxdu

Fig. 4. Little birefringence.

36

wx

Fig. 6.

38

su wx

Fig. 7-

11su du" pattern

B "su SUx" pattern

no pattern

28

o

Fig- 2*

Starch Granules from American Maize Co. Corn Starch (normal) •

X 600

29

cy

Fig. 3 .

Starch. Granules Typical for Golden Bantam Sweet Corn, no. 18, 9» 1 9 » 21, and 30 Starches.

X 600

30

Fig. 4*

Starch Granules Typical for no. 24, 27, and 29 Starches.

X 600

31

l ^ 0o t

I ,

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Fig. 5 .

:

Q

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Starch Granules from no. 35 (supersugary) Starch, X 600

32

Fig. 6.

Starch Granules from no. 36 (waxy) Starch X 600

33

Fig. 7

Starch Granules from no. 38 (sugary waxy) Starch. X 600

34

Table 9*

Prominent Rings in the Diffraction Patterns of

Various Starches. Normal corn starch A pattern^) a. &

Normal potato starch su du pattern B pattern^) a, £ a. £

su sux pattern

a, S

•

8.9 7-88

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5-92

S

5.92

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S

5.22

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5.22

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4.54

M

F

4.72

F

4.56

F

8 M M

4.40 4.04 3.70 3.42

VF MB MB M

Bear and French

VF

(2).

discussed later, incomplete gelatinization occurred as evidenced by the recovery of undissolved material. It is seen that the amount of undissolved material shown in Table 10 parallels amylose content*

The material

was starch as evidenced by the fact that protein tests were always negative and iodine sorption of one of the recovered samples was identical with that of the starch used. The gelatinization temperature that is used for characterizing starches is rather meaningless for the starches studied here.

Incomplete gelatinization occurs, as

seen above, and, also, loss of anisotropy which is used as

35

Table 10.

Amounts of Starch not Dissolved upon Autoclaving

3 Hours at 124°. No.

Starch type

American Maize Go.

normal

25

0

24

sugary

31

2

18

dull

34

2

34

sugary-x

37

7

21

suxdu

39

4

32

amylaceous sugary

44

7

30

suamsuxdu

53

24

25,26,35^

supersugary

58

31

Amylose %

Per cent not dissolved

(£) See Tables 14 and 15*

one criterion of gelatinization does not occur simultaneously with swelling and breaking up of granule structure.

For

example, culture 4 starch, amylose content 42%, lost anis­ otropy at 65-70° (normal starch gelatinizes in this range), but most granules were not swollen.

At 80° about half the

granules had the characteristic gelatinized appearance, but after heating for 2 hours at 120°, 13% were still not dis­ solved. In addition, since many of the starches have small granules that are not biréfringent, it is difficult to ob­ serve what changes do occur.

36

NATURE OF THE WATER-SOLUBLE POLYSAGOHARIDES The water-soluble polysaccharides of sweet corn, par­ ticularly Golden Bantam, have been examined by different workers who arrived at somewhat different conclusions. These workers separated the water-extractable material into two fractions on the basis of solubility in 6?^ acetic acid.

The more soluble fraction which stains red with iodine

was considered a plant glycogen, and the name "phytoglycogen'* was suggested for it by Sumner and Somers (39)*

The more

insoluble fraction which stains blue with iodine was con­ sidered as starch by Morris and Morris (32) and Hassid and MoGready (16) while Sumner and Somers considered it as a polysaccharide distinct from starch and suggested the name "glycoamylose" for it.

On observing the iodine-stained

glycoamylose fraction microscopically, Gameron found both blue and red staining particles and, therefore, considered the fraction impure.

In view of this fact and since the

amylose-like nature of the glycoamylose has not been established, the total water-soluble polysaccharide fraction was re-examined. It appeared doubtful that glycoamylose is amylose-like as it has been our experience that amylose degraded to the extent that it stains red with iodine is not readily water-soluble. Experimental Isolation of the water-soluble polysaccharides.

Although

polysaccharides were isolated from open pollinated Golden Bantam, open pollinated Country Gentleman, Inbred line Purdue

37

39A sweet corn, and culture 7 9 » complete experimental results are given only for one of these, namely, G-olden Bantam, The isolation procedure is based on that of Sumner and Somers and of Hassid and McOready.

Ninety-five per cent

ethanol was used in all precipitations. Air-dried mature corn was finely ground and 500 g. was extracted for 30 minutes at 15-20° with 1500 ml. of 10^ trichloroacetic acid solution.

The solids were separated

by centrifugation and re-extracted with 750 ml. of trichloro­ acetic acid solution.

Both extracts were combined and clari­

fied by passage through a supercentrifuge at 40,000 r.p.m. Two volumes of ethanol were slowly added with rapid stirring; the precipitate was allowed to settle, and the supernatant liquid was decanted.

The precipitate was alternately washed

with ethanol in a Waring Blendor and filtered until free of acid. ide.

The precipitate was dried in vacuo over calcium chlor­ The yield was 26%.

On extraction with hot 85% methanol

(38), 0.7% of fatty substance was removed.

This quantity

corresponds to that which can be removed under similar con­ ditions from commercial corn starch. The polysaccharide mixture was further separated into 2 fractions.

A 100 g. portion was mixed with 1000 ml. of

water, heated on a steam-bath for several minutes, and then autoc laved 2 hours at 120°.

On cooling, the solution was

passed through a supercentrifuge to remove precipitated protein (2.7%)•

Two volumes of glacial acetic acid were

added, and the mixture was held overnight at 5°.

The super-

38

natarrt solution containing phytoglycogen (Fraction 2) was decanted.

The precipitated glycoamylose (Fraction 1) was

washed with ethanol in a Waring Blendor, filtered off, dissolved in 500 ml. of water, adjusted to pH 6.5 with sodium hydroxide, and precipitated by the addition of 1*5 volumes of ethanol.

The precipitate was washed with several portions

of ethanol in a Waring Blendor and dried in vacuo over cal­ cium chloride.

The yield was 82 % of the soluble polysac­

charide mixture. Fraction 2 was isolated from the supernatant solution by precipitation with 1.5 volumes of ethanol.

It was then

treated In the same manner as Fraction 1 except that 2 volumes of ethanol were used in the final precipitation.

The yield

was 15% of the soluble polysaccharide mixture. Based on the original sweet corn. Fraction 1 was ob­ tained in 21% yield and Fraction 2 in 4% yield. Similarly, the yield of water-soluble polysaccharides from culture 79 corn was 38%.

The polysaccharide mixture

contained 0.5% of fatty substances, 2.3% protein, and 61% of Fraction 1 and 36% of Fraction 2.

Based on the original

corn. Fraction 1 was obtained in 23% yield and Fraction 2 in 14% yield. In the separation of the two fractions, storage over­ night at 5° is not important since identical results are obtained if the precipitate is worked up immediately. Purification of Fraction 1.

In the solid state or in

solutions of about 1% or more concentration. Fraction 1 is

39

colored blue by Iodine,

The blue-staining portion is removed

by adsorbtion on cotton (34-).

Thus, if a 1$ solution of the

carbohydrate is passed through a column of previously washed U.S*P. absorbent cotton, the effluent is colored lavender with iodine, while the blue-staining material is bound near the top of the column.

Its position can be located by ex­

truding the column and streaking with iodine solution. 3^ of the fraction is adsorbed by the cotton. indicated by passing 400 ml. of a

About

This was

solution of Fraction 1

through a cotton column 34 mm. by 200 mm.

After the column

was washed with 300 ml. of water, the washings and filtrate were combined, concentrated, and the solids content determined by evaporation of 10 ml. portions oh Filter-Oel (9).

The

recovery was 97%Larger amounts of purified Fraction 1 were prepared by passing 300 ml. of 10^ solution through a column of cotton 40 mm. by 600 mm. , washing with 600 ml. water, concentrating the combined solutions in vacuo, and precipitating with ethanol. Preparation of ft -amylase.

The procedure used at the

Northern Regional Laboratory was followed.

Eight hundred g.

of Vigo soft red winter wheat, 1948 crop, was finely ground. Dry Ice was added to the wheat while it was being ground to prevent overheating.

The wheat was extracted with 4 liters

of 50% ethanol (by volume) in a stainless steel beaker fitted with a stainless steel stirrer.

After 1.5 hours, the wheat

was separated by means of tho basket head of the Inter-

40

national centrifuge. 3 hours.

The centrifugate was cooled to 0° for

The precipitated protein was centrifuged off in

the Sharpies supercentrifuge, and the centrifugate was raised to 80% ethanol ("by volume).

The solution was cooled to 0°

for 4 hours, and the supernatant liquid was decanted off the precipitated enzyme and supercentrifuged.

The two precipi­

tates were washed with cold absolute ethanol until they be­ came friable and could be filtered on a Buchner funnel.

The

enzyme precipitate was resuspended in fresh ethanol, filtered, and dried in vacuo over calcium chloride.

In a typical

experiment, 13 g. of enzyme powder was obtained. per g. was 121 Kneen-Sandstedt

The strength

-amylase units (22).

A

solution of the enzyme was made by extracting with water in portions (800 ml. water for 12 g. enzyme powder). solution was filtered, and any

This

-amylase present was In­

activated by lowering the pH to 3*0 at 30° for 2 hours (23)* The solution was preserved with toluene and thymol and kept at 5°. Preparation of limit dextrine.

Limit dextrine were

prepared essentially according to the procedure of Hodge et al. (18). In a typica.1 experiment, 20 g . purified Fraction 1 was dissolved in water, made up to 200 ml. volume, and 20 ml. enzyme solution was added.

A few drops of toluene were

added to the solution, and the flask was Immersed in a bath maintained at 36° and shaken at intervals.

Aliquots were

taken for reducing sugar determinations periodically.

The

41

enzyme brought about a 52$ conversion of white potato amylopectin; Hodge et al. report 54$.

For Fractions 1 and 2,

the limits of hydrolysis were 47$ and 45$, respectively, and were reached in 2 days; whereupon, they remained constant for 4 days even when fresh enzyme solution was added each day. The limit dextrins were precipitated from the hydrolyzates by adding ethanol to 60$ concentration by volume.

The

precipitate was redissolved in 10-12 parts of water and pre­ cipitated by addition to 9 volumes of ethanol in the Waring \

Blendor.

The dextrin was filtered off, re-dissolved, pre­

cipitated, and washed in ethanol as before.

Ethanol was

employed for precipitation and washing since the limit dex­ trins were found to be soluble in the 50$ ethanol used in the isolation of amylopectin limit dextrins.

Even so, the

limit dextrin of Fraction 2 was partially soluble as evi­ denced by the fact that only 56$ was recovered.

On the other

hand, 92$ of the limit dextrin of Fraction 1 was recovered. Periodate end-Rrouo assay.

The method of Hirst et al.

(6, 1*4) was used without change as it seems to work well with àmylopectin-1ike materials. Samples of 0.2 or 0.4 g. were weighed into three 500 ml. glass-stoppered Pyrex bottles, and 100 ml. of potassium chloride solution containing 5 g. of salt per 100 ml. water was added.

Twenty ml. of 0.3 M sodium periodate solution and

20 ml. of water were then added.

The reaction was carried

out in the dark at 15-20° with shaking, and 25*00 ml* aliquots

42

were taken out at 2 , 4, and 6 days, one set of aliquots from each bottle.

The formic acid present was determined by

addition of 5 drops of ethylene glycol to react with excess periodate and titration with 0.01 N barium hydroxide to the methyl red end point.

The end-group assay value is the

moles glucose anhydride residue per mole formic acid pro­ duced.

The values are considered reproducible to * 1 glu­

cose residue. Results obtained on the limit dextrin of the total water-soluble fraction of sweet corn and the limit dextrin of white potato amylopectin are shown in Table 11.

These

show that the formic acid production levels off satisfac­ torily and that the end-group value obtained is not affected much by sample size. The amount of periodate consumed was also determined by an adaption of Jackson1s procedure (20).

The iodate present

in the sample used for formic acid analysis was determined by adding 5 ml. of 0.5 H hydrochloric acid, 1 ml. of 20 % potassium iodide solution, and titrating the liberated iodine rapidly with standard 0.1 N.sodium thiosulfate solution.

The

periodate present in a 25*00 ml. sample was determined as follows :

The sample was neutralized with 20 ml. saturated

sodium bicarbonate solution and 25*00 ml. of 0.1 N standard sodium arsenite solution and 1 ml. of 20^ potassium iodide solution were added.

After 10-15 minutes, the excess arsenite

was back-titrated with standard 0.1 N iodine solution.

The

difference between the amounts of periodate and iodate found

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