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ABSTRACT
THE ULTRASTRUCTURE AND MICROANATOMY
OF THE ROOT TIP OF PISUM SATIVUM

by Gordon C. Spink

A descriptive study of the first millimeter of the
seedling root tip of Pisum sativum var. Alaska was under—
taken. Although the pea root has no distinct areas, it
was sub-divided into seven regions for analysis: quies—
cent center, cortex, central cylinder, epidermis, colum-
ella, tip-cells, and epidermis-root—cap—complex.

The tissue was fixed, dehydrated and embedded
according to standard electron microscopy techniques.
Thin sections (400 to 1,000 Angstroms) were observed in
the electron microsc0pe and thick sections (% to 10 mi-
crons) were observed in the phase microscope. Electron
microscope sections were related to the complete root
Picture. Autoradiography of paraffin sections of tri—
tiated thymidine treated cells were made to observe the
quiescent center, as described by Clowes (1961).

l

 

 

THE ULTRASTRUCTURE AND MICROANATOMY

OF THE ROOT TIP OF PISUM SATIVUM
By

Gordon C.'Spink

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1966

 

 

ACKNOWLEDGEMENTS

I wish to sincerely thank Dr. G. B. Wilson for his
guidance and help; Mrs. June P. Mack for her technical as—
sistance and c00peration; Dr. Lee V. Leak, formerly of
Michigan State University and now at Massachusetts General
Hospital, Boston, for his help and advice with electron

microscopy; and my wife, Jane, for her understanding.

ii

 

TABLE OF CONTENTS

Page
ACKNOWLEDGEMENTS. . . . . . . . . . . . . . . . . . . ii
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . iv

LIST OF TABLES. . . . . . . . . . . . . . . . . . . . vi

INTRODUCTION. . . . . . . . . . . . . . . . . . . . . 1
DEFINITIONS . . . . . . . . . . . . . . . . . . . . . 3
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . 6
MATERIALS AND METHODS . . . . . . . . . . . . . . . . 9
OBSERVATIONS. . . . . . . . . . . . . . . . . . . . . 13
DISCUSSION. . . . . . . . . . . . . . . . . . . . . . 35

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . 46

FIGURES . . . . . . . . . . . . . . . . . . . . . . . 47

BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . . 88

 

LIST OF FIGURES

Figure

l.--Phase microscope montage photograph of the first
millimeter of root tip . . . . . . . . . . .

2.--Quiescent center . . . . . . . . . . . . . . .
3.--Quiescent center . . . . . . . . . . . . . . .
4.--Quiescent center . . . . . . . . . . . . . . .

5.-—Cross-sectional montage of cortex and central
cylinder . . . . . . . . . . . . . . . . .

6.--Cortex cells . . . . . . . . . . . . . . . . .
7.--Central cylinder cells . . . . . . . . . . . .

8.--Cross—sectional montage of central cylinder
cells. . . . . . . . . . . .

9.--Epidermal cells. . . . . . . . . . . . . . . .
lO.--Epidermal cells. . . . . . . . . . . . . . . . .
ll.--Cross-sectional montage of epidermal cells
12.-—Columella cells. . . . . . . . . . . . . . .
l3.——Columella cells. . . . . . . . . . . . . . .
l4.--Cross-sectional montage at 400 microns from tip.

l5.--Tip—cells. . . . . . . . . . . . . .

iv

 

Page

48
50
52

54

56
58

60

62
64
66
68
70
72
74

76

 

 

 

 

'LiSt of Figures--continued.

 

Figure Page
l6.--Epidermal-root-cap-complex . . . . . . . . . . . 78
l7.--Golgi bodies . . . . . . . . . . . . . . . . . . 80
l8.—-Lipoidal material. . . . . . . . . . . . . . . . 82
l9.--Rough-end0plasmic reticulum and microtubules . . 84
20.——Autoradiograph showing quiescent center. . . . . 86

v

 

 

 

LIST OF TABLES

QUIESCENT CENTER DATA SHEET

 

 

NUMBER OF ORGANELLES PER SQUARE MICRON OF

CYTOPLASM . . . . . . . .

CORTEX DATA SHEET . . . . .

CENTRAL CYLINDER DATA SHEET

EPIDERMIS DATA SHEET. . . .

COLUMELLA DATA SHEET. . . .

vi

Page

28

29
30
31
32

33

 

7 _

m—n ——

 

 

 

 

 

THE ULTRASTRUCTURE AND MICROANATOMY
OF THE ROOT TIP OF PISUM SATIVUM

INTRODUCTION

The root apical meristem, being a convenient tissue

I

for experimental study, is often used in cytological, mor-
phological and physiological investigations. The meristem-
atic area contains a large number of mitotically active
undifferentiated cells which contain few if any vacuoles

and lack secondary cell walls. The cells are easy to obtain
and relatively easy to handle and can be treated simply with
chemicals and metabolic inhibitors (Wolff, 1964).

The root apical meristem of Pisum sativum var. Alaska
has been used for many years in the Cytology Laboratory of
the Biology Research Center, Michigan State University, for
the study of mitosis, the mitotic cycle, and as a tissue for
testing cytological effects of pesticides and herbicides. A
fairly clear picture has been obtained concerning cell kin-
etics and chromosome structure of the meristematic area of
the root apex in the light microscope.

l

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

THE ULTRASTRUCTURE AND MICROANATOMY

OF THE ROOT TIP OF PISUM SATIVUM

 

INTRODUCTION

The root apical meristem, being a convenient tissue
for experimental study, is often used in cytological, mor-
phological and physiological investigations. The meristem-
atic area contains a large number of mitotically active
undifferentiated cells which contain few if any vacuoles
and lack secondary cell walls. The cells are easy to obtain
and relatively easy to handle and can be treated simply with
chemicals and metabolic inhibitors (Wolff, 1964).

The root apical meristem of Pisum sativum var. Alaska

 

has been used for many years in the Cytology Laboratory of
the Biology Research Center, Michigan State University, for
the study of mitosis, the mitotic cycle, and as a tissue for
testing cytological effects of pesticides and herbicides. A
fairly clear picture has been obtained concerning cell kin-
etics and chromosome structure of the meristematic area of
the root apex in the light microscope.

l

 

 

 

 

 

This investigation was undertaken to extend knowledge
obtained at the light microscope level to the electron micro-
scope level and to study the microanatomy and ultrastructure
of cells of the first millimeter of the primary root of

Pisum seedling.

 

 

 

 

 

 

" I
. alum..- I P
. .

 

 

 

DEFINITIONS

 

ACROPETAL ........... Proceeding from the base (the region of
the shoot-root connection at the ground

level) toward the apex.
BASIPETAL ........... Proceeding from the apex toward the base.

LIPOIDAL MATERIAL...Material is considered lipoidal if it re-
acts to certain fixatives as reported in
the classical electron microscope litera—
ture, and no attempt is made to specify

the type.

MERISTEM ............ TXmeristem is, by definition, a group of
plant cells in a state of active division
and growth. These cells are not homoge-
neous since a meristem may be subdivided
into regions that differ anatomically or
in cellular activity, which indicates
that differentiation has already progressed
to a considerable extent in these embryo-
like cells." (Miksche, 1964)

3

 

 

 

 

 

‘NQ‘ l‘J'H-i . "

 

MICROANATOMY .......... The anatomical details visible in the

electron microscope.

MICROTUBULES .......... Tubular structures found primarily at
the periphery of the cell, having an

outside diameter of 230—270 Angstroms.

NORMAL ROOT ........... A root grown under the standard condi-
tions, not chemically treated or caused
in any manner to be different; it is

used as the control for comparison.

QUIESCENT CENTER ...... Located at the junction of the columella
cells and the central cylinder region;
it is the zone that should contain the
initial cells according to the classi—
cal histogen theory; it is described
as an area in which cells seldom or
never divide, roughly hemispherical in
shape which may change shape and size
with time, and is surrounded by actively
dividing cells, the initials. (Clowes,

1961)

 

 

 

 

ROOT APICAL MERISTEM..Used in a broad sense, it is various

lengths of root proximal to the apex.

(Esau, 1965)

ROOTCAP ............... Is commonly regarded as a structure
that protects the root meristem and
assists the root in the penetration
of the soil during its growth.

(Esau, 1965) In Pisum this is more

 

a descriptive term than an anatomical

term.

ULTRASTRUCTURE ........ Is the resolution and portrayal of
cytological units in the electron

microscope. (Frey—Wyssling, 1965)

PROTOMERISTEM ......... The least determined part of the
meristem and includes the initials
and their most recent derivatives.

(Esau, 1965)

 

 

LITERATURE REVIEW

The anatomy of the root using the optical microscope
has been adequately covered by Stocking (1956) and Esau
(1965). Esau (1965) has updated her earlier work with the
limited use of electron microsc0pe observations and photo-
micrographs.

Whaley, Mollenhauer, and Leech (1960), in a review
article, described the components and ultrastructure of the
apical root meristematic cells of various plants. While
different types of cells from various plants were discussed.
Whaley gt 3;. used their own photographs of the meristematic
cells of rootcap of fig; gays for illustration. Leech, Mol-
lenhauer, and Whaley (1963) in a later related work, dealt
with certain changes in the cellular characteristics which
become apparent in the development of particular cell types
basipetal to the initial area. They followed this develop-
ment in part for 10 centimeters from the initial area.
Clowes and Juniper (1964) studied the fine structure of the
quiescent center and its neighboring tissue in gga and

6

 

 

 

 

attempted to compare the undifferentiated, non-meristematic

cells of the quiescent center with those of meristematic,
non-differentiated cells of the meristem and those of the
differentiated, non—meristematic cells of the cap. Clowes
(1961, 1964) has studied the root apical meristem with par—
ticular interest in the quiescent center, and published

photographs of tritiated thymidine treated Pisum roots show-

 

ing this quiescent center. No data, however, as to the age
or length of roots or conditions of growth were given.

While limited investigations are being pursued on
the cellular anatomy of meristems, much information has
accumulated on cellular organelles of plant material. Frey-
Wyssling and Mfihlethaler (1965) have thoroughly discussed
ultrastructural plant cytology and the relationship to molec-
ular biology. Porter and Machado (1960) studied the Allium
root tip extensively with particular interest in the endo-
plasmic reticulum. Golgi bodies and their possible functions
have been investigated by Whaley's group at the University
of Texas (Whaley gt a1. 1960; Mollenhauer, Whaley, and Leech,
1961; Mollenhauer, 1965). Most recently, Ledbetter and
Porter (1963, 1964) have reported a new component of plant

cells, microtubules. Exclusive of microtubules, the cellular

 

 

 

 

Components of plant cells have been thoroughly reviewed in

The Cell, vol. II (Brachet, 1961) and The Cell, vol. VI (Bra-

 

chet, 1964).

The previous anatomical studies on Pisum sativum have
been the investigations with the optical microscope of Popham
(1955), Reeve (1948), and Neumann (1939) in which they at-
tempted to classify the anatomical boundaries and levels of
tissue differentiation. There is still disagreement on the
interpretation of their results and also on the development
of the anatomical organization. Torrey (1955) has done a

thorough descriptive study of the Pisum root tip with com-

 

parison of the intact to the excised tip. Although particu—
larly interested in the development of vascular patterns,

Torrey's study of the pea root anatomy was quite extensive.

 

 

 

 

MATERIALS AND METHODS

The observations in this study were made on the first
millimeter of the root tip of Pisum sativum, var. Alaska.
Ferry Morse and Vaughn Seed Companies, whose seeds were used,
guaranteed that the peas possessed relative genetic homogene-
ity, were disease—free and had not been treated with herbi—
cides or insecticides.

The peas were soaked for 6 hours in glass distilled
water (pH 5.5 - 5.7) at 22.50 C; rolled in paper toweling
and placed vertically in beakers containing water. Wax
paper was placed around the rolled toweling to prevent dry-
ing of the peas by evaporation.

After 36 hours of germination at 22.50 C, the peas,
the roots of which were 1-2 centimeters in length, were sus-
pended on waxed, one-quarter inch wire mesh over dishes which
contained 350 milliliters of full strength modified Hoaglund
nutrient (balanced salt) solution. To allow for acclimatiza—
tion, the peas remained in the nutrient for 4 hours. The
nutrient solution, pH 5.5 - 5.7, was constantly aerated by

9

 

 

 

lO

filtered air. The Hoaglund solution consists of the follow-

ing in grams per liter:

Calcium nitrate Ca(NO3)2 7.6
Ammonium nitrate NH4NO3 10.32
Magnesium sulfate MgSO4°7 H20 14.4
Potassium monobasic phosphate KH2P04 10.68
Potassium dibasic phosphate KZHPO4 .56

 

Seedlings were grown in the aerated nutrient solution
in a constant temperature water bath at 22.50 C in a 22.50 C
constant temperature room. Samples were taken at various
times from 0 hour to 120 hours.

Root tips 1 to 2 millimeters in length were harvested
with a clean sharp razor blade and placed immediately into
the fixative. Various fixatives were used: 5% unbuffered

potassium permanganate (KMnO4), 2.5% KMnO buffered at pH 7.5

4

with veronal acetate, 2.5% KMnO4 buffered at pH 7.2 with

Sorenson’s buffer, 6.25% glutaraldehyde and post-fixed in

2.5% KMnO buffered to pH 7.2 with Sorenson's buffer, and

4
6.25%.glutaraldehyde and post-fixed in 1% osmic acid (0504)
buffered in Sorensen's at pH 7.2. Duration of fixation, all

at room temperature, was 1% to 2 hours and that of post-

fixation 1 hour. The material was dehydrated in one hour

 

 

 

 

 

 

11

in a series of alcohols; then embedded in Epon 812 according
to Luft (1961).

Sections were obtained with glass and diamond du Pont
knives on a Leitz ultramicrotome and a Porter-Blum ultramicro-
tome MT-2. For the best tissue orientation, flat embedded
root tips were placed in a vise-type holder on the MT-2.
Thick sections of % to 10 microns were viewed unstained and
photographed on a Wild phase contrast M-20 microscope on 35
millimeter film using various filters. Naked 400 mesh and
parlodion-carboned grids of 100 and 200 mesh were used. Some
sections were stained with Reynold's lead citrate (1963),
uranyl acetate (Pease, 1964), or double stained with a com-
bination of both (Pease, 1964). Grids were observed on a
Philips EM-100B and photographed on 35 millimeter Kodak Fine
Grain Positive film.

Seedlings needed for the autoradiographic studies
were suspended over dishes containing tritiated thymidine
(New England Nuclear) at a concentration of 2 microcuries
per milliliter of nutrient solution. Samples were taken
from 0 to 120 hours and fixed in Pienaar's fixative (1955).
which consists of a 6:3:2 mixture of absolute methanol,

chloroform, and propionic acid; placed in a vacuum for 10

 

 

 

 

11

in a series of alcohols; then embedded in Epon 812 according
to Luft (1961).

Sections were obtained with glass and diamond du Pont
knives on a Leitz ultramicrotome and a Porter-Blum ultramicro-
tome MT—2. For the best tissue orientation, flat embedded
root tips were placed in a vise-type holder on the MT—Z.
Thick sections of % to 10 microns were viewed unstained and
photographed on a Wild phase contrast M—20 microscope on 35
millimeter film using various filters. Naked 400 mesh and
parlodion-carboned grids of 100 and 200 mesh were used. Some
sections were stained with Reynold's lead citrate (1963),
uranyl acetate (Pease, 1964), or double stained with a com—
bination of both (Pease, 1964). Grids were observed on a
Philips EM—lOOB and photographed on 35 millimeter Kodak Fine
Grain Positive film.

Seedlings needed for the autoradiographic studies
were suspended over dishes containing tritiated thymidine
(New England Nuclear) at a concentration of 2 microcuries
per milliliter of nutrient solution. Samples were taken
from 0 to 120 hours and fixed in Pienaar's fixative (1955),
which consists of a 6:3:2 mixture of absolute methanol,

chloroform, and propionic acid; placed in a vacuum for 10

 

 

 

 

 

 

 

12

minutes; and stored in the refrigerator until embedded in
paraffin. Paraffin blocks were sectioned 8 microns thick

on an A—O Spencer microtome and sections were mounted on
albumin coated glass slides. Paraffin was removed by xylol
and slides containing the radioactive material were then
hydrated to water at which time they were coated with Kodak
liquid emulsion NTB-3 according to Prescott (1964). At
varying times from one to three weeks, slides were developed
to determine the progress of exposure (Prescott, 1964). After
development, slides were then stained in leucobasic fuchsin
Schiff reagent (Lillie, 1951) and mounted permanently in

diaphane mounting medium.

 

 

 

 

 

 

 

 

OBSERVATIONS

For convenience in describing the first millimeter

of the root apical meristem of Pisum sativum, the meristem

was subdivided into seven areas (Figure 1). It should be

remembered, however, that in the pea root tip there are no

discrete areas with well defined limits, and that various

types of transitional cells are found between these seven

areas. The seven areas to be discussed are as follows:

quiescent center, cortex.

central cylinder, epidermis,

columella, tip cells, and epidermis—root-cap—complex.

In the following descriptions of a typical cell

of the quiescent center, cortex, and central cylinder, it

is assumed when average measurements or quantities are given.
they are for a thin section through the center of the cell

in a median longitudinal section of the pea root. These

values should therefore be used only for comparison. The

criterion used was that the nucleus and cytOplasm be cut

equally in two. Because of the random placement of the

nucleus, vacuoles and specific areas in the cytoplasm, the

13

 

. . . . . . . , ‘1 -' i
.' ’o‘a‘u‘ .0. o '1’ :0... A'Js‘uvo‘n'i‘i'o'w' n' 1' WTQ'

 

 

 

 

 

 

 

OBSERVATIONS

For convenience in describing the first millimeter
of the root apical meristem of Pisum sativum, the meristem
was subdivided into seven areas (Figure 1). It should be
remembered, however, that in the pea root tip there are no
discrete areas with well defined limits, and that various
types of transitional cells are found between these seven
areas. The seven areas to be discussed are as follows:
quiescent center, cortex, central cylinder, epidermis,
columella, tip cells, and epidermis-root-cap-complex.

In the following descriptions of a typical cell
of the quiescent center, cortex, and central cylinder, it
is assumed when average measurements or quantities are given.
they are for a thin section through the center of the cell
in a median longitudinal section of the pea root. These
values should therefore be used only for comparison. The
criterion used was that the nucleus and cytoplasm be cut
equally in two. Because of the random placement of the
nucleus, vacuoles and specific areas in the cytOplasm, the

13

 

 

 

 

 

 

 

 

14

cells of the epidermis, columella, tip-cells, and epidermis-
root-cap-complex lacked bilateral symmetry when cut longi-
tudinally or transversely. It was, therefore, impossible

to arrive at average numbers of organelles, as was done for
the other described areas.

Table 2 shows a comparison of the number of organ-
elles per square micron of cytOplasm for the quiescent cen-
ter, cortex, and central cylinder. Data were calculated
from a thin section of material through the center of the
cell in a median longitudinal section. Of the 7 regions,
only the quiescent center, cortex, and central cylinder
could be compared in this manner for these 3 exhibit the
necessary bilateral symmetry.

The following information was obtained from observa-
tions of numerous sections of pea root seedlings. Data were
obtained directly from photographs and microscopic observa—

tion (light, phase, and electron).

Quiescent Center

Cells in the quiescent center (Figures 2, 3, and 4)

were found to be irregularly shaped though more or less

 

 

 

 

 

15

spherical, packed tightly in a random manner, and approxi-
mately 12 microns in diameter. (See Table l and 2 for more
detailed information.) Autoradiographic studies of paraffin
sections concurred with those of Clowes (1961), and demon-
strated that this area had a very low tritiated thymidine
uptake during the range of time of this study. Therefore

it can be concluded that the cells in this area were not
dividing, or at least, as a group, were dividing very slowly
during the period of exposure to the radioactive isotope.
The nucleus, centrally located, filled the majority of the
cell volume. aThe nuclear envelope showed typical pores,
areas of larger discontinuities, and very few connections

with endOplasmic reticulum (ER). CytOplasmic free ribosomes

 

and rough-ER were evident in osmium tetroxide (0504) fixed
material. Short pieces of E3 were found randomly displaced,
while longer pieces were usually found around the periphery

of the cell. Small lipoidal drgplets, usually separated,

were scattered throughout the cells. Proplastids, about 8

per thin section, were equally divided between spherical and
cylindrical shaped bodies, of which about % exhibited a bright
refractive substance, probably stored starch. Few Golgi

bodies (an average of 3 per thin sectioned cell) with

 

 

 

 

 

16

associated small vesicles were seen in this area. Most of
the mitochondria were spherical types, less than 1 micron in
diameter, and were found in lower number than in most other
areas. Although vacuoles were not prominent in this area.
five different small types were located. Plasmodesmata

connected adjacent cells.

Cortex

The cells of the cortex tissue, when cut transversely
(Figure 5), appeared oval (12x20 microns) with very conspic-
uous intercellular spaces at the junctions of surrounding
cells. The nucleus was centrally located while the vacuoles
were found at the ends of the oval. In a median longitudinal
section the cells were square (12x12 microns) to rectangular
with the longer axis cross-wise to the long axis of the root
(Figure 6). Since the longer axis of the oval shape was
arranged along the circumference, in median longitudinal
sections the smaller diameter of the cell was evident. (See
Tables 2 and 3 for more detailed information.) The 3 most
characteristic features of cells in this region were: 1)

squarish shape of cells in longitudinal sections, 2) large

 

 

 

 

17

number of organelles, and 3) very conspicuous intercellular
spaces. Another characteristic feature of cells in the cor—
tex was the great abundance of microtubules. These were
found only in glutaraldehyde-0504 fixed material. They were
also found, however, to a lesser extent in the epidermis—
root-cap—complex. Microtubules were more evident only when
the cell periphery was cut obliquely. This was due to their
complex (not yet fully understood) distribution pattern. The
nucleus occupied about l/3 of the cell volume and character-
istically showed many pores. There appeared to be some ques-
tion concerning the number and types of vacuoles. In KMnO4-
fixed material, however, there appeared to be no membrane-
limited vacuoles having grainy contents. This is further
pursued in the discussion. The cells in this region contain
what Whaley §£.§l- (1960) have termed prevacuoles and appeared
as dark homogeneous areas, less than 1 cubic micron in volume,
and were characteristically star-shaped. These also appeared
to be in association with or part of the ER. All lengths.

up to 6 microns, of EB were found randomly dispersed through-
out the cell. In cross-section many plasmodesmata between

all cells were evident. while in longitudinal section, be-

cause of the section being parallel to the intercellular

 

 

 

 

 

18

spaces, connections appeared only between cell walls perpen-
dicular to the long axis of the root. Lipoidal droplets,
usually single, were found scattered in the cells. Pr0plas-
33g§_were evenly divided between spherical shapes with a
diameter of about 2 microns and cylindrical, dumbell, and
irregularly shaped types. Cylindrical proplastids were of
the typical size, as found previously, 3x1 microns, while
many irregular and dumbell shaped types, not seen in the
quiescent center, were evident. Almost twice as many spher—
ical and cylindrical mitochondria were found as any other
type per thin sectioned cell. Very few dumbell shaped mito-
chondria were evident. gglgi bodies were more numerous as

were their vesicles.

Central Cylinder

As one proceeds basipetally from the quiescent cen—
ter to l millimeter from the tip, distinct but gradual changes
were evident. The cells elongated into regularly stacked
cylinders (Figures 5, 7, and 8) with the long axis parallel
to that of the root. It appeared that this region of the

tissue was in transition. and a stable cell type was not

 

 

«nu-yap “

   

 

19

noted. Great variations in vacuole types and cytoplasm were
noted. Cell organelles, however, remained fairly constant
for the region. (See Tables 2 and 4 for more detailed in-
formation.) It is very possible that this variation of the
cells is due to early differentiation into pre-phloem and/
or pre-xylem. Dense quantities of free cytoplasmic £399—
somes and rough-ER were evident. The ribosome content
appeared to vary between cells in this region. Again as

in the quiescent center, E5 was randomly dispersed but the
longer pieces were more apt to be located around the cell
periphery. More of the ER was 1 to 6 microns in length.
than 1 micron or less. Plasmodesmata were found in all
cells. Single lipoidal droplets, % micron in diameter,
were randomly scattered, while clumps were very rare. Be-
cause of the increased cell size, the nucleus filled about
25% of the cell, and exhibited the usual pores, holes, and
ER—connections. Vacuoles could best be described as in a
transition from the membrane-limited textured interior type
found in the quiescent center, to the highly sculptured or
clear area vacuoles found at 1000 microns from the tip.
Very few dumbbell shaped proplastids were seen, while 3

times more spherical types than cylindrical ones were

 

   

 

 

 

20

present. Nearly all of the mitochondria were 3/4 to 1 micron
diameter spheres, while a very few long dumbells were found.
Typical appearing Golgi bodies were evident. each with ap—

proximately 5 vesicles, % micron in diameter.

Epidermis

The epidermal cells (Figures 9, 10, and 11) to be
described were located at 1000 microns from the tip and
were oval cylinders with their long axis the same as that
of the root. (See Table 5 for more detailed information.)
Approximately 90% of the cell was composed of a vacuole.
which may or may not be limited with a membrane, whose
contents may be grainy and/or fibrous. As a result of the
large vacuolar area, the dense cytoplasm and nucleus were
located at the periphery. Depending on the manner in which
the cell was sectioned, either dense cytoplasm or vacuolar
area or some of both were found. Few free ribosomes were
evident in the cytoplasm and, as a result, roughfiER was
clearly seen. Long single strands of weaving ER (up to 10
microns) along with many short pieces were found at the per—

iphery of the cytoplasm. Up to 10 loosely stacked individual

 

 

21

strands of ER were also found localized in this area. Nu-
merous Golgi bodies with many vesicles were seen, and these
vesicles appeared to be engaged in the process of deposit-
ing their contents outside the plasma membrane. These
vesicles appeared to move toward the plasma membrane, be-
come fused to it, then burst Open giving the plasma membrane
a slightly sca110ped appearance. Lipoidal droplets were
absent as were plasmodesmata. Proplastids were both spher-
ical and cylindrical and filled with many starch grains.
Mitochondria were equally divided between spherical and
cylindrical bodies. Epidermal cells were separated from
each other and from the interior cell layer by a prominent
cell wall, 2 to 5 microns thick. A granular substance ex-
tended about 4 microns into the mucilaginous material that

covered the root tip.

Columella

The cells located in the columella exhibited very
distinctive characteristics (Figures 12, 13, and 14). (To
my knowledge these columella cells have not been previously

described in the literature using electron microscope

 

 

 

 

techniques.) The cylindrical cells formed regular columns

between the quiescent center and the tip-cells. (See Table
6 for more detailed information.) The diameter averaged 15
microns and the long axis averaged 18 microns. The plasma
membrane was slightly scalloped probably due to the vesicles
from Golgi membranes. Again as in the epidermal cells.
these do not show bilateral symmetry when cut transversely
or longitudinally. The irregularly shaped nucleus, contain-
ing pores, was located toward the basipetal end of the cell.
Large irregularly shaped spherical proplastids containing
large refractive starch grains were located in the acropetal
end of the cell (Esau, 1965). Numerous mitochondria both
spherical and cylindrical shaped were found. Lipoidal drop-
lgtg % micron in diameter were seen scattered in the cyto-
plasm in some cells, While in others they were concentrated
at the periphery of the cell. Long single strands of EB.
about 10 microns in length, along with shorter pieces, were
usually seen near the periphery. Unique to this region were
the Specific areas of great amounts of ER, usually not longer
than 4 microns, in close association with vesicles. These
vesicles, less than % micron in diameter appeared to be the

result of ER-budding. Many Golgi bodies with up to 6 cisternae

 

 

 

 

 

 

 

 

 

and numerous small vesicles were found adjacent to. but not

in this region. Also vesicles % to 1 micron were found
scattered in the cytoplasm. These vesicles may be larger
free Golgi vesicles. Plasmodesmata were evident between
cells having cell walls of less than 1 micron. Some
membrane-limited vacuoles were seen, but more prominent
were the clear areas located in the cytOplasm. (The clear
areas were considered vacuolesfor they appeared as such
in the light microscope.) The membrane—bounded vacuoles
were about 4 microns in diameter and exhibited a typical
fibrous-textured interior. Some vacuole membranes contained
many pores or small holes. Also observed were vacuoles.
single or in clusters, with very whorled membranes. In
some cells, membrane-bounded areas of less dense cytOplasm
containing scattered organelles were in association with

these irregularly whorled vacuoles.

Tip-Cells

Tip-cells (Figure 15) can best be described in terms
of differences from columella cells. There are four obvious

changes; 1) the cytOplasm is denser. 2) clear areas are no

 

"

 

 

 

 

24

longer found in the cytOplasm, 3) there is a great increase
in the clear membrane bounded vesicles % micron in diameter,
and 4) these vesicles appear to be moving from the interior
of the cytoplasm to the plasma membrane and discharging their
contents outside of the plasma membrane, thus giving the
typical highly scalloped cell shapes found throughout this
area. This scalloping rapidly progresses acropetally from
the slightly sca110ped columella cells to the deeply scal-
lOped tip-cells. Highly sca110ped cells were evident 3 to

4 cells basipetal to the tip. As the scalloping progresses.
the cytoplasm is equally reduced, and that which is left

is more dense. The substance deposited by the vesicles.
between the cell wall and the plasma membrane, was of dif-
ferent texture than the still evident original cell wall
(middle lamella). Even with the most scalloped cells, it
was possible to see the original shape of the cell and this

corresponded to the size and shape of the columella cells.

Epidermis—Root—CaprComplex

The epidermis-root-cap-complex (Figure 16) is that

part of the root tip apex that is basipetal to cells we can

 

 

 

 

 

25

designate as typical epidermal cells (with smooth plasma
membranes) and extending inward until columella cells or
cortical cells are reached and down to the apex to the
typical tip-cells. Thus in 3 dimensions, it is a cone

with a hole at the vertex. The epidermal-root-cap cells
can best be described by comparing them to the typical
epidermal type, columella cells, and tip-cells. Cytoplasmic
free ribosomes and rough—ER similar to that of columella
cells were seen. In longitudinal section they usually
appeared lenticular and circular in cross-section. Their
long axis approximately paralleled that of the tip and the
size was approximately 30 by 12 microns. They have large
membrane bounded vacuoles, whose contents are of granular
texture, and may also have small whorled membrane inclusions
in or near the large vacuoles or isolated. The vacuoles
may also be of the sculptured type. Some microtubules were
apparent in the glutaraldehyde-0504 fixed material. A wide
variance in organelle contents and distribution was noted;
however, the large number of Golgi bodies and vesicles that
were present in the columella and tip-cells were not found.

Also the distribution of nuclei and plastids was not found

to be as regular. The plasma membrane is slightly scalloped

 

 

 

26

but never to the extent of that of the tip—cells. The most
characteristic features are their lenticular shape in longi—
tudinal section and prominent large membrane bounded vacuoles.
The closer they are to the columella and tip—cells, the more
they resemble them; the closer they are to epidermal cells,
the more they look like them. Their lenticular shape is a
practical shape for it lends itself to packing in the area.

as the tip curves toward the point.

In the light of the above observations, it is evident
that for any cell classification or identification, it is
necessary to consider both the gross cell morphology and the
ultrastructure of the protoplasm. With the electron micro-
scope it is impossible to classify a cell by studying only
the organelles or only the gross shape of the cell. However
when both are coordinated, it is possible to classify and
identify a cell, even with the artificial divisions that
must be used.

These artificial divisions become very evident when
one attempts to draw in boundaries on a photograph of a med-
ian longitudinal section of the root apex. It is all a
series of gradations. To go from any one point to another

in this area, it is necessary to proceed through a gradual

 

 

 

 

 

27

change of cell types. To go from the outer epidermis to the
central cylinder, one must traverse the following cell types:
outer-epidermis, inner—epidermis, epidermal-like cortex.
cells looking more like cortex, cortex, central-cylinder-
1ooking cortex cells, and finally central cylinder cells.
And one may also go through variations by starting in cen-
tral cylinder cells and proceeding to the tip-cells. This
even gradation into all areas may be due to the lack of an
apical initial cell (or cells), which would form definite
layers and regions (Clowes, 1961). In a photograph of the
pea seedling root, at all stages of develOpment, even from
early embryogenesis (Reeve, 1948), it is impossible to draw
definite boundaries for the apical meristem. It is possible.
however, to draw in definite regions and layers only a short

distance more basipetal than 1 millimeter from the tip.

 

 

 

28

 

TABLE 1
QUIESCENT CENTER DATA SHEET
a_== r w
CELL ARRANGWT ....... . ....... . . . . . . . . . . . . . .irregularly packed
CELL SHAPE. . . . . . . . ........................... irregularly shaped spheres
CELL DIMENSIONS ......... . . . . . . . .............. 12xl2xl2 microns
CELL VOLUME. . . . . . . ....................... .about 900 cubic microns
RIBOSOMES. . . . . . . ........... . ................. yesM
ENDOPLASMIC RETICULUM.(ER)
Rough ....... . . . .............. yes**
Av # of pieces***
1 micron or less ......... 11 (3-17)*
1-7 microns .............. 7 (2—20)
Distribution pattern ......... long pieces usually around cell periphery
NUCLEUS
Location. . . . . . . . . . . ...... . . . . . . . . ........ centrally located
$ of cell filled. . . . . . . . ................. more than 50$
Nuclear envelope ............. . ......... ..pores and larger holes
Nuclear envelope -KR connections . . . . . . . . . .few
LIPOIDAL
Single--av. # ........ . .- ...... lh (Lt-21)
--av. diameter ....... . . 1'; micron
Clumps--av. #. ............... 3 (04+)
--av. size. . ........... fxl micron
PROPLASTIDS
Spherical--av. #.......... ............... 1+ (l-8)
--av. diameter . . . . .............. 1 micron
Cylindricalm-av. #. . . . . . . ....... . . . ...... 3 (0-6)
--av. size. . . ..... . ......... . .3xl micron
Dumbell--av. #m... ................... ..1 (0-5)
--av. size.. ....... ....... ...2§xl micron
Reflective material ........... . .......... f; of all proplastids
GOLGI
Av. # ..... 3(0-6)
Av. size ..................... 3/14 micron long
Vesicles .......... . .......... 3 per Golgi, 1/10 micron diameter
JMITOCHDNDRIA
Spherical--av. # ............. . ........... 15 (It-28)
--av. diameter... ..... ..........3/h micron
Cylindrical--av. #. . ........... . ......... 2 (0-6)
--av. size . . . . ................ 2x1 micron
Dumbbellnav. # .......................... less than 1
--av. size.... ..... ..............2xlmicron

PLASMDDESMATA. . . . . . . ..... . . . . . . . .15 connecting all adjacent cells

 

 

 

 

 

 

29

Table 1--Quiescent Center Data Sheet (continued)

VACUOLES
Type ..... Membrane-limited
Shape....spherical cylindrical
Contents.grainy grainy
Av..#....2 (0-6) 2 (0-5)

Av. size.l micron diameter 3x1 micron (fixl to 3x5)

COMMUTS: other types rarely found, 1) membrane-limited with clear
contents, 2) star-shaped opened-ER, 3) swollen-RR, 1%)
clear areas in cytoplasm which may or may not have scat-
tered organelles.

NOTE: Av. # is given per thin sectioned cell, median longitudinal section.
*Range

**080h fixation
***KMn0h fixation

 

 

 

 

 

 

 

 

 

TABLE 2
NW 01" ORGANELLES Pm SQUARE MICRON 0F CYTOPLASM
Organelles Quiescent Cortex Central
Center Cylinder
(63 sq, microns)* (105 sq. microns)* (231 sq. microns)*

m--1:icron

or less .17 (.05-.27) .21 (.03-.53) .03 (.01-.07)

1-7 microns .ll (.03-.32) .25 (.11-.hh) .06 (.0h- .1)
LIPCIDAL

Single .22 (.06- 33) .16 (.07-.3l) .02 (0-.0h)

Clumps .05 (0-.06) .00 .00
PROPLASTIDS

Spherical .06 (.02-.13) .07 (0-.l6) .03 (.01-.0h)

Cylindrical .05 (0-.1) .05 (.02-.12) .01 (0~.02)

Dumbbell .02 (0-.08) .01 .00
GOIGI .05 (0-.l) .06 (.01-.1) .02 (0-.0h)
LMITOCEDNDRIA

Spherical .2h (.06-.hh) .26 (.0h-.h7) .09 (.07-.l)

Cylindrical .03 (0—.1) .06 (.02-.13) .01 (.00—.02)

Dumbbell . 00 . 00 . 00

 

*The average square microns of cytoplasm per cell, taken from electron
micrographs of sections Of’material 600-1000 Angstroms thick.

 

 

 

 

30

Tfilflli3
CORTEX DATA SHEET

CELL SIZE, SHAPE, AND ARRANGEMENT (cross section)...ova1, 12x20 microns,
with large intercellular spaces between cells; long axis of oval-
shaped cell is arranged along the circumference Of the root.

CELL SIZE, SHAPE, AND ARRANGEMENT (median longitudinal section)...square
to rectangular, longer axis perpendicular to long axis Of root; av.

size 12x12 microns; due to arrangement of oval-shaped cell, we see

short axis of oval in median longitudinal section.
mvoLmoOOOOOOO

O. ..... 00....

. ......... ....about 2&10 cubic microns
RIHOSOMES................. ................... .cytoplasm very dense due
to heavy concentration**
MICROTUBULES.................. ................ yes**
ENDOPLASMICIRETICULUM.(ER)
Rough ....... ....,........yes**

Av. # of pieces***
1 micron or less.....22 ( 3-56)*

1-7 microns.. ..... ...26 (12-h6)
Distribution pattern ..... none
NUCLEUS
Location .............. . ................... central
5 of cell filled........... ............ ...30$
Nuclear envelope (NE)... ............... ...many pores
NE-ER connections..... .................... none
LIPOIDAL
Single--sv.:f...... ...... 17 (7-33)
--av. diameter ..... é-micron
C1umps--av.:f............very few
--av. size.. ..... ..fixl micron
PROPLASTIDS
Spherica1--av. # ...... ...... ......... .....7 (0-17)
--av. diameter..... .............. 2 micron
Cylindrical--av. #.. ........ ..............5 (2-13)
--av. size ..................... 3x1 (1x2 to 1x6 to 2x5)
Dumbbelluav.# ..... .. ..... l
(& Irregular)- size range... .............. (1x7 to 2x3 to 1x3)
GOLGI
Av. # ........... ....6 (1-10)
Av. size. ................ 2 micron in length
vesicles.................Av. 8 per Golgi, fi-micron diameter
MITOCHDNDRIA

Spherical--avu.fi..........................27 (h-50)

--av. diameter ............... ....1 micron or less
Cylindrical--av.:f....... ................. 6 (2-lh)
--av. size ..................... 2x1 (511% to 1x8)
D1nnbbell--av. #. . . . . ...................... very few
PLASMUDESMATA

25 connections to all cells but not across intercellular spaces

I

i
v
Q.

.'

f
'
I
.
.-
'3
'J
i"
a
.4.
...-
1'"

 

 

 

 

 

 

31

Tam-9 3--Cortex Data Sheet (centinued)

VACUOLES
Type.. ..... prevacuolar areas
Shape......irregular star-shaped, maybe in association with ER
Contents...dark, homogeneous
Av. f......either none or 6 to 10
Av. size...less than 1 cubic micron
COMMENTS: No membrane limited vacuoles in xnnoi; some in anu

NOTES: Av. # is given per thin sectioned cell, median longitudinal section.

*Range
**080h fixation
”MO 1, fixation

 

TABLE h

CENTRAL CYLINDER DATA SHEET

 

lel to root long axis

CELL DIMENSIONS. ........ .....11x3O microns
CELL VOLUME ....... . ......... .about 2850 cubic microns
RIBOSOMES ..... . .............. dense cytoplasm due to free ribosomes**
ENDOPLASMIC RETICULUM (ER)
Rough.................. ......... .....yes**
Av. # of pieces***
1 micron or less ............ ..... 8 (2-20)*
1-7 microns ...................... 15 (10-23)
Distribution pattern ......... . ....... long pieces usually at cell
periphery
NUCLEUS
Location ................. centrally located

$ of cell filled.........25$
Nuclear envelope (NE)....pores and larger holes
NEnER connections........yes

LIPOIDAL ‘
Single--av. #....... ...................... h (0-9)
--av. diameter ................... ...é-micron
Clumps--av. f... .......................... very rarely found
--av. size............. ............. fi-micron
PBOPLASTIDS
Spherical--av.-# ..... ....7 (2-10)

--av. diameter..l%-micron
Cylindrica1--ava-{.......2 (0-5)
--av. size....1x3 microns
Dumbbell--av. #..........1ess than 1
--av. size ..... ..3xlfi-micron

 

 

 

 

 

32

Table huCentral Cylinder Data Sheet (continued)

GOIGI
Av.:#....................h (O-lO)
Av. size......... ........ 1 micron
vesicles......... ........ 5 per Golgi, fi-micron diameter
MITOCHDNDRIA
Spherical--Av.#...... ..... ...........22 (16-28)
--Av. diameter... ...... .....3/h to 1 micron
Cylindrical--Av.A#u......... ....... ..3 (1-5)
--Av. size................3xl micron
Dumbbell-~Av..i........... ........ ...less than 1
--Av. size.......... ......... 2x%-micron (spherical ends 1
micron in diameter, connect-
ing rod é-micron in width,
total length may be 5 microns)
PLASMDDESMATA.... ........ ....30 connecting all adjacent cells
VACUOLES
Spherical, cylindrical, and dumbbell shapes with and without distinct
limiting membranes.
Opened ER connected to spherical vacuoles
Sculptured vacuoles
ER in association with sculptured vacuoles
COMMENTS: Appear to be in transition from.membrane-limited textured
interior type of quiescent center to the sculptured and
clear areas evident at 1000 microns from.tip which appear
to be the typical vacuoles for the region.

NOTE: Av.-# is given per thin sectioned cell, median longitudinal section.

 

*Range
**OsOh fixation
***KMnOh fixation

 

TABLE 5

. DIDERMIS DATA 31mm
M m
CELL SHAPE AND ARRANGEMENT...oval cylinders with long axis parallel to

that of the root
CELL DIMENSIONS (cross-section)..........Oval 15x10 microns
CELL DIMENSIONS.(median longitudinal section)....l5x50 microns
CELL VOLUME..................about 5660 cubic microns
RIBOSOMES....................few**
ENDOPLASMIC RETICULUM (n)
Rough....... ......... ....very evident due to few free ribosomes **
Distribution pattern.....long single strands (up to 10 microns)
of weaving ER along with.many short pieces found at periphery
of cell. Up to 10 loosely stacked strands of ER also found at
periphery.

 

 

33

Cable 5--Epidermis Data‘Sheet (continued)

NUCLEUS
Location.... ....... . .....
i of cell filled .........
LIPOIDAL......... ........... .
PROPLASTIDS
Spherical-1}.............

--av. diameter..

Cylindrical-sf...........

--av. size....

Dumbbell-sf. ....... ......

Reflective material......
GOLGI

compressed between vacuole and cell membrane
at periphery of cell.

very'small

..... .......none evident

about é-of all present

2 microns

about é'of all present

3xlé-micron

none

filled with many large bodies

Av. #....... .............. . ......... ....numerous

Av. size.. .............................. lé-micron

Vesicles...................... .......... many, fi-micron in diameter
MITOCHONDRIA

Spherical-1} ..... ........ab0ut é of all present

—-av. diameter..§-micron
Cylindrical-~i . ..... ....about g-or all present
-—av. size....éxl micron

Dumbbell--av. f- ...... ....none
PLASMODESMATA...............................none
VACUOLES

Type .................... ..may or may not be limited with a membrane

Shape....................irregular

Contents ..... ... ..... ....grainy and/or fibrous

Ava f........... ..... ....usually only 1

Av. size ................. fills 90$ of cell
NOTE: Av. #-is given per thin sectioned cell, median longitudinal section.

**anh fixation

 

TABLE 6

COLUMELLA DATA SHEET
m
CELL SHAPE AND ARRANGEMENT...regu1ar cylindrical columns (in cross sec-

CELL DIMENSIONS ......... .....

CELL VOLUME......

RIBOSOMES ....................

tion, oval; in a median longitudinal sec-
tion, squarish)

cylinder diameter 15 microns; long axis 18
microns

about 3180 cubic microns

few**

 

 

34

Table 6--Columella Data Sheet (continued)

ENDOPLASMIC RETICULUM (ER)
Rough....................very evident due to fewer free ribosomes**
Distribution pattern.....(l) Great.nnmbers of ER (not longer than
A microns) in close association with vesicles, found in definite
regions. (2) Long single strands of ER, up to 10 microns, with
shorter pieces were usually found near the periphery.

NUCLEUS
Location..... ...... ................toward basipetal end of cell,
shape very irregular
$ of cell filled..... ....... .......small
Nuclear envelope (NE)... ....... ....numerous pores but no large holes
NE-ER connections.. .............. ..none
LIPOIDAL

Single-fl}................scattered pieces, some localized at periphery
--av. diameter.....$ micron

PROPLASTIDS
Distribution pattern..... ....... ...irregularly shaped spherical pro-
plastids located near acropetal
end of cell
Size...................... ...... ...up to 6 microns
Reflective material....... ....... ..many large starch bodies
GOLGI

Av. #....................many, up to 6 cisternae
Av. size.................2 microns in length

vesicles..... ..... . ..... .numerous small vesicles
MITOCHONDRIA
Spherical-—av. #3..... ...... . ...... numerous

--av. diameter............1 micron
Cylindrical--av.-#.................numerous
--av. size..............2xl micron (5x2 to 5x1)
PLASMODESMATA...... ...... ....between cells having cell walls less than 1
micron thick
VACUOLES
Type . . . . . . . . . . . .membrane limited vacuoles (membrane may have many
pores or small holes)
Shape.... ...... .may be highly irregular or whorled
Contents........fibrous textured, some may have less dense cytoplasm
with scattered organelles
Av. #. . ....... . .few
Av. size........h microns diameter
COMMENTS: More prominent were cytoplasmic clear areas with.no mem-
brane limited boundary.
CYTOPLASM............large clear areas, no ribosomes evident
............regions containing both short pieces of ER and
many vesicles
VESICLES
: to 1 micron in diameter, found scattered in cytoplasm
micron diameter in definite regions with short pieces of ER

NOTE: Av. # is given per thin sectioned wall, median longitudinal section.
**030h fixation

 

 

 

34

Table 6--Columella Data Sheet (continued)

ENDOPLASMIC RETICULUM (ER)
Rough.. .... ...... ..very'evident due to fewer free ribosomes**
Distribution pattern..... (1) Great.numbers of ER (not longer than
4 microns) in close association with vesicles, found in definite
regions. (2) Long single strands of ER, up to 10 microns, with
shorter pieces were usually found near the periphery.

NUCLEUS
Location.... ....... . ........... ....toward basipetal end of cell,
shape very irregular
5 of cell filled..... ........... ...small
Nuclear envelope (NE). ......... ....numerous pores but no large holes
NE-ER connections.. ...... ..........none
LIPOIDAL
Single-a}... ...... .. ..... scattered pieces, some localized at periphery
--av. diameter.....§ micron
PROPLASTIDS
Distribution pattern..... .......... irregularly shaped spherical pro-
plastids located near acropetal
end of cell
Size... ....... . . .............. . .up to 6 microns
Reflective material. ...... ...... ..many large starch.bodies
GOLGI
Av. 5.... ................ many, up to 6 cisternae
Av. size.................2 microns in length
vesicles..... ............ numerous small vesicles
MITOCHDNDRIA
Spherical--av. #...... ...... . ...... numerous

--av. diameter............l micron
Cylindrical--av.-#.................numerous
--av. size.......,......2xl micron ($x2 to 5x1)
PLASMODESMATA... ..... ........between cells having cell walls less than 1
micron thick

VACUOLES
Type...... ..... .membrane limited vacuoles (membrane may have many
pores or small holes)
Shape.... ....... may be highly irregular or whorled
Contents ....... .fibrous textured, some may have less dense cytoplasm
with scattered organelles
Av. #” ..... ....few

Av. size........h microns diameter
COMMENTS: More prominent were cytoplasmic clear areas with.no mem-
brane limited boundary.
CYTOPLASM............large clear areas, no ribosomes evident
..... ....... regions containing both short pieces of ER and
many vesicles

VESICLES

‘ to 1 micron in diameter, found scattered in cytoplasm

micron diameter in definite regions with short pieces of ER

NOTE: Ava # is given per thin sectioned wall, median longitudinal section.
**°90h fixation '

 

 

 

 

DISCUSSION

The cellular organization of the root tip of Pisum
has been well established with the use of the light micro-
scope (POpham, 1955). The foregoing electron microscope
observations substantiate the descriptions based on light
microscope studies and provide further information on the
organization as well as the ultrastructure and microanatomy.

The interpretation of the pattern of organization,
termed the Open or Pisum type, has been modified only
slightly since first reported by Janczewski (1874). This
Open type is characterized by the lack of distinct organi-
zational patterns and the apparent continuation of the cell
column from the tip into the central cylinder cells. This
characteristic pattern is also clearly apparent at the elec-
tron micrOSCOpe level. As has been reported earlier, there
are no distinct cell or organelle changes detectable by
electron microscopic observation. From any one point to
any other point, one must go through a transition of cellu-
lar and organelle changes. DeSpite this, however, it is
still possible in a general way to subdivide the meristem—

atic segment into seven areas.

35

 

 

36

This open or Pisum type of organization is only one
of many forms. Another type, which has a closed or very
distinct pattern, is found in agg. Because of this and the
ease of identification of areas, it is often used for study.
Whaley, Mollenhauer, and Leech, Cell Research Institute,
University of Texas, have made extensive electron microscope
studies using ggg and, up to the present, their publications
are the most complete in this field. Thus, our finding
should be compared to theirs.

Since the work of Whaley §£_gl. was the first and
still is the most complete published investigation on the
root meristem using the electron microscope, it is often
used as the standard for plant cells. Unfortunately, it is
often forgotten that their results and micrographs are based
only on Egg and thus the impression, either implied or stated.
is given in the general literature that this may be typical
for plants. Until further comparative studies are made
this is an unjustified assumption. Pisum and Vicia are
certainly different from Egg and many other variations may
yet be found.

It is unfortunate also that the earlier work of

Whaley is most often quoted. This is a general review of

 

 

 

 

 

37

the meristematic region, more concerned with organelles than
cell types, and does not consider the characteristics of
discrete regions. The published micrographs certainly do
not indicate the diversity of cell types. The micrograph
used as the typical cell of this region is termed a "trans—
verse section through a meristematic rootcap cell" (Whaley
.EE._l-v 1960) and in later works which use this as a refer-
ence, it is implied and inferred that this is a typical
meristematic root tip cell. Because of the great diversity
in the apical root meristem found in our work on Pisum, I
do not believe one can show a typical meristematic root tip
cell. Fortunately, a more complete study of cell types found
in Egg was later published (Leech, Mollenhauer, and Whaley,
1963) and showed some of the diversity.of cells found within
10 centimeters of the tip. However they still did not show
all the cell types found or the cellular relationships. If
one needs to show a typical meristematic root tip cell, the
definition of meristem should be made very clear and very
'rigid.

While no criticism of the work of Whaley, Mollenhauer
and Leech is implied, the generalizations from the work.

either implied or stated. are a little disturbing as this

 

 

 

indicates a uniformity which does not exist. Because the

organization in root meristems is so diverse and the fact
that £23 has such a distinctive type, I do not believe that
it should be implied or considered as typical of plants.
ggg has a well organized and clearly evident meristem with
a distinct rootcap. It also has a distinct quiescent center
that may be easily outlined on a photOgraph of a median lon-
gitudinal section. Other plants have equally distinct, but
differently organized meristems. On the other hand, plants
like Vicia, Pisum, and others, have no distinct regional
boundaries (Clowes, 1961).

Nevertheless, even with the great difference in
organization between 2g; and Piggm, electron micrographs
do show similarities in cytoplasmic organelles (Whaley g5
g1., 1960) and in most cell types (Leech §£__l., 1963). No
specific organelle comparison is possible with ggg, since
the data were analyzed in a different manner. Whaley E;
El: (1960) reported generally on the organelles of the
whole root tip and Clowes and Juniper (1964) are concerned
only with organelle content in the root axis. Although

electron micrographs of Zea and Pisum are generally com-

parable, they do differ in specific regions. The micrograph

 

 

 

39

that Whaley g; _l. (1960) used as the representative of the
meristem, the "transverse section through a meristematic
rootcap cell," would certainly not be a representative cell
for Pisum. The nearest equivalent cell type in Pisum is
that of the quiescent center, not of the rootcap.

Apparently, no cells are found in Zea which are

comparable to the columella and tip-cells of Pisum. No

 

mention in the literature was found of these cell types
being studied by electron microsc0py. The regions of short
pieces of ER and small vesicles may be unique to the colum-
ella cells. The tip-cells appear to be extreme cases of
the result of action of active Golgi vesicles. The cyto—
plasm is much denser as if the clear-watery-material is
being collected in the Golgi vesicles and then deposited
outside the plasma membrane.

The work on.3;§gm fully supports that of Mollenhauer.
Whaley and Leech on Golgi bodies. In agreement with them
(Figure 17). we found that larger (more active appearing)
Golgi bodies with large flattened vesicles were located in
the outer cells (epidermis, epidermis-root-cap-complex, and
tip-cells) and the smaller Golgi with smaller and fewer

vesicles were located in the interior of the root (Whaley,

 

 

 

 

4O

Kephart, and Mollenhauer, 1959; Mollenhauer, 1965). Also,
the observations reported here fully support Mollenhauer's
and Whaley's theories of Golgi vesicle involvement with

cell wall deposition. ggg cells were shown with slightly
sca110ped edges though never to the extreme of that of the
columella and tip-cells seen in giggm, Also g§g_cells re-

vert to smooth plasma membranes, while those of Pisum do

- u—

not (Mollenhauer, _p‘gl., 1961). Nevertheless, the mode
of action appears to be the same.

A feature of Pisum that was not found discussed in
the literature is the occurrence of a high concentration of
lipoidal material in the quiescent center region (Figure 18).
After 0504 fixation the material appears as a very dark,
smooth, homogeneous material, well—contained with no appar—
ent unit membrane. After KMnO4 fixation, since KMnO4 dis-
solves away some of the lipoidal material, the material may
appear as an “empty-body" with a heavy, dark, thick layer
at the periphery (Figure 18). This heavier concentration
of lipoidal material is not only evident in the electron

microsc0pe, but also very evident in the phase microscope

because of its highly refractive property.

 

41

As has been noted. different fixatives and fixation
procedures may give different results. Variation of the
concentration, duration, temperature, and pH of the basic
fixative may each give different results. Depending on the
fixative used, different buffers even at the same pH may
give different results. On the choice of fixatives, there
are 2 schools of thought: 1) Whaley and his group believe
permanganate to be the best; 2) K. R. Porter, Harvard Uni-
versity. and his followers stress the importance of glutar-

aldehyde fixation followed by post-fixation in 050

4.
For membranes and ER. KMnO4 gives very dark, sharp
images, almost like a line drawing. The ground substance

of the cytoplasm is very smooth and structureless (Mollen-
hauer, 1959). Glutaraldehyde fixation with Sorenson's
buffer (Ledbetter and Porter, 1963) followed by a post-
fixation of 0504 or KMnO4 apparently gives the most accu-
rate picture, but certainly in some cases not the sharp
images of KMnO4 alone. After glutaraldehyde fixation and
a post-fixation, ribosomes, rough-ER and microtubules are
clearly seen (Figure 19), but in some cases due to the

dense ribosomes, smooth-ER may be completely hidden and

organelles barely visible. It is felt that glutaraldehyde

 

 

 

 

42

fixation is superior, since everything seen in KMnO4 is

 

present. plus other definite components (ribosomes, rough-
ER. microtubules. plus perhaps many other things). Morpho—

logically, the basic picture obtained with KMnO and glutar-

4
aldehyde fixation followed by post—fixation of 050 or KMnO

4 4
are the same. The fixative choice depends upon what is to
be studied and emphasized in one's micrographs. Ledbetter
has done extensive work on buffers for glutaraldehyde and
we concur with his choice of Sorenson's buffer at pH 7.2.
Quite obviously fixatives need much more study in terms of
the artifacts they produce.

Buffers are also of particular importance. Different
buffers or pH's cause great variations in the occurrence and
type of vacuoles found. The differences may range from
swollen ER to a great increase in small 1 micron diameter
spheres to a few large vacuoles that nearly fill the cell.
Much care must be taken to compare regions only when they
have had the same treatment. This was painfully learned
by the author.

As previously mentioned, the vacuoles evident in

the cortex region do not present a constant picture. In

this region, it appears that different types of vacuoles

 

 

 

 

 

43

may be seen after different fixations, but the results may
not always be consistent. Perhaps under different physio-
logical conditions, different fixations create different
results. Also, and perhaps this is the most logical explan-
ation, it may be only a technical problem that is caused if
the section is not an exact median longitudinal section or
if the column of cortex cells in the root itself is twisted
or at an angle. As mentioned above, the cells in cross—
section are oval with the nucleus in the center and vacuoles
usually found at either end of the oval. Thus, if the short
axis of the oval is not exactly cut, the appearance would be
variable. This technical problem may be evident only in the
cortex cells due to their particular arrangement. It cer-
tainly will not give a different picture if central cylinder
cells are cut slightly off—center.

The work of Clowes (1959, 1961, 1964) on the quies-
cent center was also compared to our work. He demonstrated
the existence of this region in many types of plants, e.g.
ggg, Pisum, and others. Our autoradiographic photOgraphs
of paraffin sections fully reproduced his results (Figure
20). Clowes and Juniper (1964) published an excellent

electron micrograph montage of the quiescent center and

 

 

44

the surrounding cells in ggg on which they were able to draw
in the distinct boundaries of this region. Again as in the
case of the ggg work by Whaley §£__l,, incorrect generaliza-
tions have been made. It is sometimes assumed by persons

not completely familiar with the work of Clowes or with
patterns of organization in roots, that a distinct quiescent
center can be drawn on photographs of all plants. Although
this region can not be clearly seen structurally in Pisum,

it is equally evident in Pisum and ggg after autoradiographic
procedures.

In conclusion, it is clear that the first millimeter
of the Pisum root is a complex tissue containing a great
variety of cell types. It is in fact a highly specialized
region, the function of which is the production of new cells
at a controlled rate. It also is in this region that the
vascular pattern is determined (Torrey, 1955).

Using this work as a base-line, comparative studies
of either induced or random changes may now be pursued. At
present, the induced changes caused by treatment with 5-
amino uracil (an analog of thymine) are being studied in
our laboratory. Also a complete series of electron micro—

scope autoradiographic experiements are planned to further

 

 

 

45

study the quiescent center. Considerable knowledge could

also be gained by observing antimitotic and herbicidal ac—
tion on the meristematic region. Until the present study

of the normal root tip was completed, no real comparative

studies could be attempted since there was no base-line

for a standard.

 

 

SUMMARY

A descriptive study of the ultrastructure and microanat-
omy of the first millimeter of the root tip of Pisum has

been presented.

Since the pea root tip has no distinct boundaries, it
was necessary to sub-divide the root tip into 7 regions:
quiescent center, cortex, central cylinder, epidermis,

columella. tip—cells. and epidermis-root-cap-complex.

Each area was investigated and data collected.

The areas studied in the electron microsc0pe were shown
in relationship to a phase contrast photograph montage

of a median longitudinal section of a root tip.

Comparison of the ultrastructure and microanatomy was
made between the work on Pisum of this study and the

published work on Zea.

46

 

 

 

 

FIGURES

48

...... I

Figure l.-—Phase microscope montage photograph of the

first millimeter of root tip.
A phase microscope montage of the first millimeter

of the pea root in a median longitudinal section. Specimen
is a % micron thick section of material fixed by usual elec—
tron microscope techniques and embedded in Epon and sectioned
by a diamond knife on an ultramicrotome. Overlay shows the
7 regions: 1) quiescent center; 2) cortex; 3) central
cylinder; 4) epidermis; 5) columella; 6) tip-cells; 7)
epidermis—root—cap-complex. Glutaraldehyde-KMnO fixation.

4

Approximately X 450.

 

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50

Figure 2.——Quiescent center.

An enlargement of Figure 1, showing 0
(circled on overlay). Note the concentratior
oidal material (L). Arrow, pointing to root
root axis. Glutaraldehyde—0504 fixation. A;

2.800.

 

 

 

 

 

 

 

52

Figure 3.--Quiescent center.

An electron micrograph of cells in the quiescent
center following Glutaraldehyde—KMnO4 fixation. Note the
nucleolus (NU), nuclear envelope pores (P), chromatin ma-
terial (C), mitochondria (different shapes) (M), proplas—
tids (different shapes) (PP), plasmodesmata (PD), cell
wall (W), lipoidal material (L), endoplasmic reticulum
(ER), and Golgi (G). Arrow, pointing to root apex, indi—
cates root axis. Median longitudinal section. Approxi—

mately X 6,000.

 

 

 

 

 

 

 

 

 

 

 

 

 

54

Figure 4.--Quiescent center.

Cells of the quiescent center after
Note the differences from Figure 3. New cel
formed between daughter cells (X). Nuclear
not yet completed after division. Arrow, pc
apex, indicates root axis. Median longitudi

Two and one-half percent KMnO pH 7.5 with

4]

tate. Approximately X 6,000.

 

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qu—qv- I -—- “.-.f' ‘—'_1l-l

56

Figure 5.——Cross-sectional montage of cortex and central
cylinder.

 

A cross-sectional montage of a portion of root at
1000 microns from the tip, showing cortex (COR) and central
cylinder cells (CC). Note the intercellular spaces (IS).
Fixation in 2.5% KMnO4, pH 7.5 with veronal acetate buffer.
Approximately X 2,300. '

 

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58

Figure 6.——Cortex cells.

A section showing cortex cells. Note the large
intercellular spaces (IS). Arrow, pointing to root apex
indicates root axis. Median longitudinal section. Glu-

taraldehyde-KMnO4 fixation. Approximately X 6,000.

 

59

 

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60

Figure 7.--Centra1 cylinder cells.

Portions of central cylinder cells. Note the
double nucleolus (NU), in a polyploid cell, and the pore
(P) in the nuclear envelopes which are evident because c
the oblique cut of the nuclei. Arrow, pointing to the
root apex. indicates root axis. Glutaraldehyde-KMnO4

fixation. Median longitudinal section. Approximately

X 6,000.

 

 

62

Figure 8.--Cross-sectional montage of central cylinns
cells.
A cross-sectional montage of a portion of root .I
1000 microns from the tip. showing central cylinder cel.s
(CC). Note the sculptured vacuoles (V) and nuclei (N).

Fixation in 2.5% KMnO4, pH 7.5 with veronal acetate buf.:

Approximately X 2,300.

 

    

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64

Figure 9.——Epiderma1 cells.

Portions of epidermal cells. Note the endoplasmic
reticulum (ER) localized at periphery, clear cytoplasmic
areas (X), heavy concentration of starch in proplastids
(PP), Golgi (G) and Golgi vesicles (GV), and highly scal-i
loped plasma membrane (PM). Arrows at bottom of photOgraph
indicate portions of 2 cells located at the periphery of
the root. Median longitudinal section. Arrow, pointing
to root apex, indicates the root axis. Glutaraldehyde-KMnO

4
fixation. Approximately X 6,000.

 

 

 

66

Figure lO.—-Epiderma1 cells.

Portions of 2 epidermal cells showing the central
large vacuoles and cytoplasm at the periphery of the cells.
Note the grainy mucilaginous covering of the root (X).
Median longitudinal section. Arrow, pointing to root apex,
indicates root axis. Glutaraldehyde-KMnO fixation. Ap-

4
proximately X 6,000.

 

 

.tral

cells.

apex.

67

 

 

 

 

" ' .“ -w'mm“" ...—.—

 

 

 

68

 

 

Figure ll.--Cross-sectiona1 montage of epidermal cells.

A cross-sectional montage of a portion of root at
1000 microns showing epidermal (EPI) and sub-epidermal cells.
Note the smooth plasma membrane (PM) of the outer epidermal
cells. Fixation in 2.5% KMnO4, pH 7.5 with veronal acetate

buffer. Approximately X 2.300.

 

 

 

 
 
 

 
   

...-..o-nI'l.s‘.tn°"“‘

- 'I
uni- .—--. . .—-#—- I.

 

 

 

70

Fig. 12.—-Colume11a cells.

Photograph showing cells of the columella. Note
the endoplasmic reticulum (ER) - vesicle (V) region and
placement of the nucleus (N) and proplastids (PP). Median
longitudinal sectiOn. Arrow, pointing to root apex, indi—
cates root axis. Glutaraldehyde-KMnO4 fixation. Approxi—
mately X 6.000.

 

71

lots

ind

m
.l
d

e
1

indi'

 

roxi—

 

 

 

 

72

Figure 13.-—Columella cells.

Portion of a columella cell located closer to the
root apex than those in Figure 12. Note the placement of
the nucleus (N) and proplastids (PP), endOplasmic reticulum
(ER) - vesicle (V) region, Golgi (G), and cytoplasmic clear
area with scattered organelles (X). Median longitudinal
section. Arrow. pointing to root apex, indicates root axis.

Glutaraldehyde-KMnO4 fixation. Approximately X 6.000.

 

 

 

 

 

 

 

 

 

74

Figure l4.--Cross-sectional montage at 400 microns
from tip.
A cross-sectional montage of a portion of root
at 400 microns from the tip. Note the highly scallOped
outer cells (X) and the gradations to the columella cells
(COL) containing lipoidal material (L) and endoplasmic
reticulum (ER) localized at the periphery in some cells.

Fixation in 2.5% KMnO pH 7.5 with veronal acetate

4!
buffer. Approximately X 1,170.

 

 

 

 

>0t
)ped

cells

115-

 

 

 

 

->--"-' “LwW$ 7"

 

 

 

 

76

Figure 15.—-Tip-cells.

Portions of highly scalloped tip—cells. Note
great numbers of Golgi bodies (G) and Golgi vesicles (GV),
deposited Golgi vesicle contents (X), and former cell
shape indicated by original cell wall (W). For higher
magnification, and details of the Golgi bodies, see
Figure 17 A. Median longitudinal section. Arrow, point-
ing to root apex, indicates root axis. Glutaraldehyde-

KMnO4 fixation. Approximately X 6,000.

 

77

 

 

 

 

‘7 5"“ 'UW.V" x7“. W?mm ”.Iew9.lf‘_ffi. ”—4

 

 

 

78

Figure l6.--Epidermal-root-cap—complex.

Portions of cells showing the variation of types
found in the epidermis-root-cap-complex. The cell in
photograph A.is located near the columella cells and the
cell in photograph §_is near the epidermis and tip-cells.
Both exhibit the lenticular shape. Arrow, pointing to
root apex, indicates root axis. Median longitudinal sec-
tion. Glutaraldehyde-KMnO fixation. Approximately X

4
6.000.

 

 

 

 

80

Figure l7.--Golgi bodies.

This plate shows two types of Golgi bodies found
in the pea. Photograph A has a Golgi body (G) with larger
vesicles (GV) found in cells near the periphery of the
root. Photograph B shows a Golgi body (G) with smaller
vesicles (GV) found in cells in the interior of the root.
Note the endoplasmic reticulum (ER), plasma membrane (PM)
being more scalloped in A, mitochondria (M), vacuoles (V),

and cell wall (W). Fixation 2.5% KMnO4. pH 7.5 with veronal

acetate. Approximately X 38,500.

 

ound

larger

 

 

 

 

 

 

'4 “E v

82

Figure l8.--Lipoidal material.

This plate shows lipoidal material (L). A, Lipoidal

drOplet (L) after Glutaraldehyde-0504 fixation. Approxi-

mately X 42,300. g, Lipoidal droplet (L) after KMnO4 fix-

ation. Approximately X 42,300. 9, Showing high concentra-

tion of lipoidal material (L) found in the quiescent center.

Glutaraldehyde—0504 fixation. Approximately X 5,000.

 

 

3.7,,- ‘y— .“v, .1; ....— , . - G‘-

84

Figure 19.-—Rough-endoplasmic reticulum and microtubules.

This plate shows rough-endOplasmic reticulum and
microtubules. A, Shows rough-endoplasmic reticulum (RER)
and free ribosomes (R). Glutaraldehyde-0504 fixation.
Approximately X 72,000. “B. Shows microtubules (MT) at
the periphery of the cell. Note plasma membrane (PM).
cell wall (W), and dense free cytOplasmic ribosomes (R).
Glutaraldehyde-0504 fixation. Approximately X 72,000.

g, Cross-sections of 3 microtubules (MT) at the periphery
of a cell. Glutaraldehyde-0504 fixation. Approximately
(X 72,000.

 

 

85

 

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earl. .rr

 

 

“51'

 

 

 

 

.— .wmfl‘fim ”.WWJWT . . , r . , .4, ._ U —..m'—“r7' ;4 .-.. .. 4.

 

86

Figure 20.--Autoradiograph showing quiescent center.

An autoradiographic paraffin section of root tip
grown for 18 hours in nutrient solution and tritiated
thymidine. Note the quiescent center (QC) with low up-
take of tritiated thymidine into the nuclei (N). Approx-

imately X 700.

II.

 

 

87

 

 

 

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*"fi'm
.. _ ' .....- _

 

 

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