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THES TIS.

INTERNAL STRUCTURE OF SOME APPLE VARIETIES.

Submitted by -

James Godkin.

 

To the Horticultural Department of
the Michigan Agricultural College

as a part requirement for the M. S.
Degree.

June - 1917.
THES'S

 
6 462

FOREWORD.

Acknowledgements are due to Mr. W. C. Dutton,
Assistant Horticulturist of the Experiment
Station, and to Associate Professor R. deZeeuw
of the Botany Department, for invaluable as-
sistance rendered in the photographic and

microphotographic work.
J. G.

96425
INTERNAL STRUCTURE OF SOME APPLE VARIETIES.

OBJECT.
A compsrison of core-lines and cells from different

regions of the apple.

MATERIAL.

The apples for the work were obtained from the fol-
lowing state agricultural stations: Storrs, Connecticut;
Perdue, Indiana; Urbana, Illinois; Lexington, Kentucky;
Amherst, Massachusetts; Wooster, Ohio; Corvallis, Oregon;
State College, Pennsylvania; Blacksburg, Virginia; Durham,
New Hampshire; Geneva, New York; Pullman, Washington;

Morgantown, West Virginia.

METHODS.
The method of cutting the fruit for ordinary photo-
graphic work was similar to that employed by Professor
E. J. Kraus of the Oregon Station with but few exceptions.
A simple scheme or plan for cutting the fruit was devised

as shown in the accompanying illustrations No. 1 and No. 2

Illustration No. l.

 
wha

Illustration No. 2.

 

Mature fruit was used and cross sections studied exclusive-
ly. In making the cut it has been found better to have it
anterior to the center of the core, since in this way you
can gradually work up to the center of the core, and it is
in the latter regions that the greater possibility for study
offers itself. After making the first cut just above the
center in most cases it was found advisable to remove the
seeds with a needle or some other like instrument. In this
work a needle of a dissecting set was used. In this way the
carpels remained intact, i. e. were not so apt to be broken
down. After the seeds had been removed, a uniform slice
1/16" - 1/8" thick was cut off. These sections or slices
were cut by means of a sharp medium-sized cheese-knife, and
with a quick drawing out of the knife (see illustration

No. 2). Care was taken not to cut the sections too thin, in

such cases, since they do not show as detailed a vascular
-5-
system as do those cut the proper thickness. As soon as
the sections were cut they were labeled by means of small
tags on which, such data as were deemed necessary, were
written and in pencil. These tags were fastened to the
sections by strings forced thru the edge of the sections
and near the circumference with the dissecting needle al-
ready described.

After this had been done the sections were placed in
the following per cents of grain alcohol and left for the
indicated lengths of time:

508... - +. 2 woe 4 days.

- 6 1

2

10%. 2. 2. 2 + + 2 ee 2e § "
85%... 2. 2 + 2 ee 4
4

95% . 2. 6 2 eo we ew ee Be 6 "

Absolute. ......2-+4 "
Between each Change of alcohol and before adding another, the
alcohol of the preceding grade, i. 6. per cent, was drained
off as thoroughly as possible and the sections then pressed
lightly against filter paper, but as Professor Kraus suggests
in his bulletin on "Variation of Internal Structure of Apple
Varieties” they should not be allowed to become dry. After
the sections had been thoroughly dehydrated they were removed
from the absolute alcohol and placed in xylol and left for
a period of two or three days. No definite rule can be laid
down as to the proper length of time for each of the dif-
ferent steps described above in the dehydration and clearing
process, as this depends in a large extent upon the variety
being treated. For some varieties, as for example, Fameuse,

the xylol and absolute alcohol may be omitted and the sections
=6<
placed directly into a clearer made up of equal parts of
turpentine and carbolic acid (crystals). In the latter
case it is better to place a glass plate over the sections
in the carbolic acid clearer as this prevents any shrivel-
ling or shrinking of the tissue which might otherwise occur
since the sections in this manner are held in a flat position
in the receptacle. On the other hand certain varieties, as
the Ben Davis and Northern Spy, would not clear up in this
clearer but had to be, in some cases, placed a second time
in absolute alcohol and xylol in order that they should be
properly cleared. After the sections had been properly
cleared they were photographed as shown in Illustration
No. 3. | ? ae

 

Illustration No. 3.

For the photo-micrographic work the following plan
of proceedure was followed: Small blocks of apple 1/8"
square were cut from the Ben Davis apple and in duplicate
from the following regions: (a) primary vascular bundle

region; (b) cortical region; (c) cambial region; and (a)
=-T~
pith region. The apples used were from the following
states: Connecticut, Indiana, Illinois, Oregon, Kentucky,
Massachusetts, Pennsylvania, West Virginia, Virginia, and
Michigan.
After the cutting had been made the section (in
Guplicate) were placed in small cheese cloth sacks with
the necessary data for identification on them. It was
found better not to use ink or pencil as these are very
apt to be washed off in the process of clearing, especially
is this true of ink. A better and safer scheme was to
punch small holes in the tags used above, 6. g. 80 many
holes indicating a certain region from which the section
had been taken. These cheese-cloth bags were next tied
up with thread and dropped into the following fixing solu-
tion and left for a period of twenty-four hours.
Chromic acid - - - - - -8 grams.
Chrome-Acetic Fixer ( Glacial acetic acid- - .5 c.c.
Water (distilled)- - 09.0 c.c.
The above process was to kill and fix the material in place
after killing. The material was next removed from the fix-
ing fluid and placed into a washing trough with running water
and allowed it to wash for another twenty-four hour period.
After washing, the material was placed in the fol-
lowing grades or per cents of grain alcohol and left for the
indicated lengths of time. |

25% 2. 2 6 se ew ww ew we wh hw heh whLc Lm 2 Ours.
BSG 2 ee ew ew tw tw ww . 1 = 2 hours.
50% 2. 2 6 we ew ew et . 1-2 hors.
70% 2 2 2 eo ew ew we ww we wh eh wl ew Bm BD GOYS.
-~8-
The object of leaving them for a longer period in the 70%
alcohol was to bring about a hardening of the apple tissue.
Next the following grades of grain alcohol were used in the
dehydration process:
85% . . . « © © © © «© © «WS hours.
95% 2. 2... +s. ss. .8 hours or over night.
Absolute alcohol. .. . .2 = 6 hours.
At the end of the period, the absolute alcohol was changed
and the material left for the same length of time before
clearing. The material was next gradually brought into
pure xylol as follows:
Absolute alcohol, 2 parts, and xylol, 1 part - 2 hrs.
" " 1 " " " 2 " - 2 "
Pure xylol - - - - - - - -2 "
At the end of the final period, the old xylol was
poured off and fresh xylol poured over the material.
After the above operation, a block of paraffin
1/2" X 1/2" xX 3/4" was taken and 1/3 of it added to the
xylol, one section in duplicate being placed ina vial.
The material was then set aside for a period of 24 hours at
room temperature, and then a second 1/3 of a paraffin block
added to each vial and the material set on top of the paraf-
fin bath, see Illustration No. 4 (on following page). At
the end of this period the remainder of a block of paraffin
was added to each vial and the material placed in the paraf-
fin bath, see Illustration No. 4. After a period of 24 hours
the corks of the vials were removed. After removing the

corks the vials were left for a period of 4 - 6 days in
-9e-

Illustration No. 4.

 

the bath. This is to allow the xylol to evaporate from
the paraffin.

The blocks of paraffin used were made as follows:
Papers measuring 13" X 2" were molded over little wooden
blocks 4" X $" X 2". When the xylol had evaporated suf-
ficiently from the vials, the material was embedded and
the degree of success met with in this operation was or should
be determined largely by the previous infiltration.

With warm needles from dissecting set, the material
was arranged quickly and accurately in the paper boats,
made as previously described. After arranging the material
in the boats, a film was formed by drawing gently over the
top of the paraffin in the boats. This kept the water from
spoiling the blocks. These blocks of paraffin now contain-
ing the material were plunged into water as cold as it was

possible to make it. In this water they were left for
-10-

15 - 2O minutes.

After removing from the water the blocks were dried
and cemented with hot paraffin to blocks of wood 4" x 3" x 8",
The paraffin was allowed to thoroughly harden on the blocks
before the work of cutting was begun. After the blocks had
hardened sufficiently they were trimmed down to the required
size, taking care to have the upper and lower edges exactly
parallel with each other and with the edge of the knife.

The blocks containing the vascular systems were cut
15 microns in thickness, the blocks containing the rest of
the material were cut both 15 microns and 20 microns in thick-
ness, best results being obtained with the material contain-
ing the pith cells by cutting it 20 microns in thickness.
A Bausch and Lomb simple rotary microtone and a special
Torry razor were used to cut the material in the paraffin
blocks.

After the material had been cut in paraffin ribbons
it was arranged in a cardboard box or tray, a very thin
film of albumen fixative was rubbed evenly over perfectly
clean slides. After this, the pieces of ribbon were ar-
ranged on slides so that cover-glasses used would easily
cover them. Next the sections were floated on water and
warmed gently until all the wrinkles had been smoothed out.
This was accomplished by placing the slides on top of the
paraffin bath for about a half to one minute or more. After
this operation the slides were set aside for about 24 hours,
or until perfectly dry. When the slides were thoroughly dry,

the sections were covered with xylol from a dropper bottle,
~ll-
and not allowed to run-off for about one-half minute, then
it was drained off and the operation repeated. The xylol
was next rinsed off the sections with absolute alcohol, and
this operation followed by rinsings with 95% alcohol and then
70% alcohol.

The slides were next run down from paraffin to 70%
alcohol and then transferred to 50% alcoholic safranin stain
and left for a period of 24 hours. After this operation the
slides were dehydrated by flooding successively for one-half
minute each with 70%, 95% and absolute alcohols. Then they
were cleared in xylol for about one minute. The xylol on
the under side and upper side as near to the sections as
possible was wiped off with a clean cloth, and then the
sections were mounted in balsam. When the balsam had harden-
ed sufficiently the sections were photographed with photo-
micrograph as shown in Illustration No. 5.(on following page).

RESULTS.

From a study of sections of fruit and photographs, the
internal structure for the same variety of apple, but grown
in different regions of the country, was in the main nearly
identical. This was true regardless of the size or form of

the fruit studied.

CELL MEASUREMENTS.

Average Size of Cells.
(Microns )

Ind. Ben Davis -~ P. V. B. cross-section 18.75 X 22.5

Mich. Ben Davis- P. V. B. " " 16.875 X 18.75
Va. Ben Davis - P. V. B. " " 17.25 x 22.5
-12-

 

Illustration No. 5.

Photomicroscophic Apparatus.

(Cut taken from E. Leitz Company's Catalog)
~13- Average Size of Cells
Microns )
W. Va.e Ben Davis - P. V. B. cross-section 15.00 X 16.875

Pa. Ben Davis - Pp. V. B. " " 16.875 X 20.625
Conn. Ben Davis - P. V. B. " " 19.50 X 23.25
Tll. Ben Davis - P. V. B. " " 15.75 xX 18.75
Ore. Ben Davis -~ P. V. B. " " 135.50 X 18.00
Ky. Ben Davis -~ P. V. B. " " 18.00 X 19.50
Mass. Ben Davis - P. V. B. " " 15.75 xX 15.75
Ky- Ben Davis - Co. cross section 71.25 X 90.00
Mich. Ben Davis - Co. " " 101.25 X 121.875
Conn. Ben Davis - Co. " " 78.75 X 101.25
Ore. Ben Davis ~- Co. " " 84.375 X 95.625
Penn. Ben Davis - Co. " " 90.00 X 108.75
Va. Ben Davis - Co. " " 88.125 X 131.25
Ill. Ben Davis =< Co. " " 116.25 XxX 138.75
W. Va. Ben Davis - Co. " " 118.125 X 150.00
Ind. Ben Davis ~=- Co. " " 112.50 xX 142.50
Mich. Ben Davis - Ca. cross section 91.875 X 140.625
Penn. Ben Davis ~- Ca. " " 65.625 X 84.375
Va. Ben Davis - Ca. " " 103.125 X 168.75
Mass. Ben Davis ~- Ca. " " 76.875 X 129.375
Conn. Ben Davis - Ca. " " 88.125 X 151.875
Ore. Ben Davis - Ca. " " 67.50 X 121.875
Ind. Ben Davis - Ca. " " 99.375 X 159.375
Ill. Ben Davis - Ca. " " 75.00 X 106.875
Ky. Ben Davis - Ca. " " 73.125 X 148.125
Penn. Ben Davis - P. cross section 88.125 X 241.875

Mich. Ben Davis - P. t" " 78.75 X 225.00
~14-
Average Size of Cells

Va. Ben Davis - P. cross section ag.1o9t ter Rs) ans
Conn. Ben Davis - P. " " 93.75 X 271.875
Mass. Ben Davis - P. " " 88.125 X 225.00

Ind. Ben Davis ~- P. " " 84.8758 XK 234.375

Abreviations Defined.
P. V. Be «~~ «© © « - Primary Vascular System.
CO. « « « «© « « « eCortical Region.
Case 6 « «© « « + + eCambial Region.
Po s+ + © © © © © « «Pith Region.

Owing to a disintegration of cell structure from some
of the apple material it was impossible to obtain photo-
micrographs of all of the regions intended. This disintegra-
tion seemed to effect the pith cells worst of all and the
vascular cells least of all. This may have been due to the
large size of the pith cells, having a greater tendency to
disintegrate, and the small vascular cells with a tendency
to be of a woody texture remained intact for a longer period
of time. Better results may have been obtained from apples
in an earlier stage of maturity. Each region studied pre-
sented its peculiar type cell, the vascular cell heing al-
most round and the smallest of all, the cortical being round
to spherical and much larger than the vascular-system cells.
The cambial cells were much like the cortical cells in size
and shape but had a greater tendency toward the spherical or
elliptical. The pith cells were the largest of any of the
other cells being oblong in shape and 2% - 3 or more times as

long as they were broad. The cells for each region of the
~15~-

apple studied seemed to be quite uniform thruout in size and

shape for the same variety, but from different regions of the

country. |
LITERATURE CITED.

Kraus, E. J.
1916.
Variation of Internal Structure of Apple Varieties.
Oregon Agricultural Experiment Station.
Station Bulletin No. 135.

deZeeuw, R.
Laboratory Guide for Botanical Microtechnique.
Michigan Agricultural College.
Department of Botany.

DESCRIPTION OF PLATES.

Plate I.
Fig. 1. Illinois Ben Davis.
Fig. 2. Pennsylvania Ben Davis.
Fig. 3. Virginia Ben Davis.
Fig. 4. Kentucky Ben Davis.

Plate II.
Fig. 1. Connecticut Ben Davis.
Fig. 2. Indiana Ben Davis.
Fig. 3. Oregon Ben Davis.
Fig. 4. West Virginia Ben Davis.

Plate III.
Fig. 1. Massachusetts Ben Davis.
Fig. 2. Michigan Ben Davis.
Fig. De Oregon York.
Fig. 4. Virginia York.

Plate IV.
Fig. 1. Kentucky York.
Fig. 2. Ohio York.
Fig. 3. Massachusetts York.
Fig. 4. Pennsylvania York.

Plate V.
Fig. 1. Washington York.
Fig. 2. Washington York.
Fig. 3. Indiana York.
Fig. 4. Massachusetts York.

Plate VI.
Fig. 1. Virginia Rome.
Fig. 2. New Hampshire Rome.
Fig. 3. New York Rome.
Fig. 4. Kentucky Rome.
Plate VII.
Fig. l.
Fig. 2.
Fig. 3.
Fig. 4.

Plate VIII.
Fig. l.
Fig. 2.
Fig. 3.
Fig. 4.

Plate IX.
Fig. l.
Fig. 2.
Fig. 3.
Fig. 4.

Plate X.
Fig. l.
Fig. 2.
Fig. 3.
Fig. 4.

Plate XI.
Fig. l.
Fig. 2.
Fig. 3.
Fig. 4.

Plate XII.
Fig. l.
Fig. 2.
Fig. 3.
Fig. 4.

Plate XIII.
Fig. l.
Fig. 2.
Fig. 3.
Fig. 4.

Plate XIV.
Fig. l.
Fig. 2.
Fig. 3.
Fig. 4.

Plate XV.
Fig. l.
Fig. 2.
Fig. 3.
Fig. 4.

Plate XVI.
Fig. l.
Fig. 2.

~16-

Washington Grimes.
Illinois Grimes.
Kentucky Grimes.
Oregon Grimes.

Massachusetts Grimes.
Indiana Grimes.
Oregon Maiden Blush.
Washington Rome.

Massachusetts Maiden Blush.
Michigan Maiden Blush.
New York Maiden Blush.
New Hampshire McIntosh.

New Hampshire McIntosh.
Michigan McIntosh.
New York McIntosh.
Massachusetts McIntosh.

Washington McIntosh.
Massachusetts Tolman.
New Hampshire Tolman.
New York Tolman.

Virginia Tolman.
Washington Jonathan.
Virginia Jonathan.
Oregon Jonathan.

Jonathan.
Jonathan.

New Hampshire
Massachusetts
Ohio Baldwin.

Massachusetts Baldwin.

Massachusetts Winter Banana.
Ohio Winter Banana.

Oregon Winter Banana.

New York Winter Banana.

New York Baldwin.

New Hampshire Baldwin.

Pennsylvania Rhode Island Greening.
Connecticut Rhode Island Greening.

West Virginia Hubbardston.
Massachusetts Hubbardston.
-17-
Fig. 3. Ohio Hubbardston.

Plate XVII.
Indiana Ben Davis - cross-section primary vascular
bundle - 150X.

Plate XVIII.
Virginia Ben Davis - cross-section primary vascular
buncle - 150X.

Plate XIX.
Kentucky Ben Davis - cross-section primary vascular
bundle - 150X.

Plate XX.
Michigan Ben Davis - cross-section primary vascular
bundle - 150xX.

Plate XXI.
Illinois Ben Davis - cross-section primary vascular
bundle - 150X.

Plate XXII.
Pennsylvania Ben Davis - cross-section primary vascular
bundle - 150X. |

Plate XXIII.
Connecticut Ben Davis - cross-section primary vascular
bundle - 150X.

Plate XXIV. |
Oregon Ben Davis - cross-section primary vascular
buncle - 150X.

Plate RXV.
Massachusetts Ben Davis - cross-section primary vas-
cular bundle - 150X.

Plate XXVI.
West Virginia Ben Davis-cross-section primary vascular

Plate XXVII.
Virginia Ben Davis - longitudinal section primary vas-
cular bundle - 150X.

Plate XXVIII.
Illinois Ben Davis .- longitudinal section primary vas-
cular bundle - 150X.

Plate XXIX.
Indiana Ben Davis - longitudinal section primary vas-
cular bundle - 150X.

Plate XXX.
Connecticut Ben Davis - longitudinal section primary
vascular bundle - 150X.
-18~

Plate XXXI.
Michigan Ben Davis - longitudinal section primary vas-
cular bundle - 150X.

Plate XXXII.
Kentucky Ben Davis - cross-section cortical region - 150X.

Plate XXXIII.
Kentucky Ben Davis
= 150X.

longitudinal section cortical region

Plate XXXIV.
Michigan Ben Davis

cross-section cortical region - 150X.

Plate XXXV.
Michigan Ben Davis
region - 150X.

longitudinal section cortical

Plate XXXVI.
Connecticut Ben Davis - cross-section cortical region

Plate XXXVII.
Oregon Ben Davis - cross-section cortical region - 150X.

Plate XXXVIII.
Oregon Ben Davis - longitudinal section cortical region
= 150X.

Plate XXXIX.
Pennsylvania Ben Davis - cross-section cortical region
- 150X.

Plate XL.
Pennsylvania Ben Davis - longitudinal section cortical
region - 150X.

Plate XLI.
Virginia Ben Davis

cross-section.cortical region - 150X.

Plate XLII.
Virginia Ben Davis
region - 150X.

longitudinal section cortical

Plate XLIII.
Illinois Ben Davis

cross-section cortical region - 150X.

Plate XLIV.
Illinois Ben Davis
region - 150X.

longitudinal section cortical

Plate XLV. .
Indiana Ben Davis - longitudinal section cortical region
- 150X.
- 19.

Plate XLVI.
Michigan Ben Davis - cross-section cambial region - 150X.

Plate XLVII.

Pennsylvania Ben Davis - cross-section cambial region

Plate XLVIII.
Pennsylvania Ben Davis - longitudinal section cambial
region - 150X.

Plate XLIX.
Virginia Ben Cavis - longitudinal section cambial region
- 150X.

Plate L.
Massachusetts Ben Davis ~ cross-section cambial region
- 150X e

Plate LI. |
Massachusetts Ben Davis - longitudinal section cambial
region - 150X.

Plate LII.
Connecticut Ben Davis - cross-section cambial region

Plate LIII.
Indiana Ben Davis - cross-section cambial region = 150X.

Plate LIV.
Indiana Ben Davis - longitudinal section cambial region

Plate LV.
Kentucky Ben Davis
- 150X e

longitudinal section cambial region

Plate LVI.

Illinois Ben Davis cross-section cambial region - 150X.

Plate LVII.
Illinois Ben Davis
- 150X.

longitudinal section cambial region

Plate LVIII.
Michigan Ben Davis.
- 150X e

longitudinal section pith region

Plate LIX.
Virginia Ben Davis
~ 150X.

longitudinal section pith region

Plate LX.
Indiana Ben Davis ~- longitudinal section pith region
- 150X.
 

 

 

 

 

 

Fig. 3. Fig. 4.
 

 

 

Fig. 4.
 

 

 
PG A DB TIES

 

Fig. 2.

Fig. 1.

 

 

 

Fig. 4.

Fig. 3.
 
PLATE V.

 

 

 

Fig. &.

Fig. l.

 

 

 

Fig. 4.

Fig. 3.
PLATE VI.

 

Fig. 3. Fig. 4.
PLATS Wil.

 

 
PLATE VIII.

 

 

 

 

 

Fig. 3. Fig. 4.
PGA oD BE.

 

 

 

Fig. l. Fig. 2.

 

 

 
PLATE X.

 

 

Fig. l. Fig. 2.

 

 

 

 
 

PLATE XI.

 

 

 

 

Fig. V 5 Fig. 2.

 

 

 

 

Fig. 3.
 

PLAT B AIT,
 

Fig. Se

 

XIII.

 
 

 

 

—
PLATE XV.

 

 

 
 

 

 
PLA TE XVIT.

 
PLATE XVIII.

 
PLATE XIX.

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PLATE XX.

 
PLATE XXI.

 
PLATE XXII.

 
PLATE XXIII.

 
PLATE XXIV.

 
PLATE XXV.

 
PLATE XXVI.

 
PLATE XXVII.

 
PLATE XXVIII.

 
PLATE XXIX.

 
PLATE XXX.

 
PLATE XXXI.

 
PLATE XXXII.

 
PLAT. KXKITI.

 
PLATE XXXIV.

 
PLATE XXXV.

 
PLATE XXXVI.

 
PLATE XXXVII.

 
PLATE XXXVIII.

 
PLATE XXXIX.

 
PL
A
TE
Xx
L

 
PLATE XLI.

 

 
PLATE XLII.

 
PLATE XLIII.

 
PLAT E XLIV:

 
PLATE XLV.

 
PLATE XLVI.

 
PLATE XLVII.

 

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PLATE XLVIII.

 
 

PLATE

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XLIX.

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PLATE UL.

 
PLATE LI.

 
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PLATE ULIII.

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PLATE LV.

 
PLATE LVI.

 

 

 

 
PLATE. LVI.

 
PD AT EB OLVITT:

 
PLAT ©. bE,

 
PLATE Lx.

 
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