msmmev or mammous SHOOT
AND ROOT FORMATION 0N LEAF-PETmLE
cumNGs OF mom x HIEMALIS
FOTSCH cv. ‘APHRODiTE mow

Thesis for the Degree of M. S.
MlCHiGAN STATE UNWERSiTY
EDWARD PAUL MEKKELSEN
1975

 

   

‘ T_'__-.—————__—fi._—.._

ABSTRACT
HISTOLOGY OF ADVENTITIOUS SHOOT AND ROOT FORMATION

ON LEAF-PETIOLE CUTTINGS OF
BEGONIA X HIEMALIS FOTSCH CV. 'APHRODITE PEACH'

BY

Edward P. Mikkelsen

A histological investigation was undertaken to determine
the origin of adventitious shoots and roots on leaf-petiole
cuttings of the Rieger begonia, g, x hiemalis Fotsch cv.
'Aphrodite Peach'. The main objective was to ascertain
whether shoots originated from single or multiple epidermal
cells of the leaf petiole. Thus, the origin of non-chimeric
plants from mutation induction might be properly interpreted.

‘ Observations made with a light microscope of 10 micron
sections of leaf petioles at various stages of shoot and root
development showed that the epidermal and subepidermal paren-
chymal cells gave rise to a callus near the cut end of the
petiole approximately two weeks after excision from the
stock plant. Roots originated within this callus and also
within parenchymal cells of the petiole three to four weeks
after excision. Five to six weeks after excision, nodular
protrusionslwhich gave rise to adventitious shoots, developed
at the surface of the callus. On the basis of these observa-

tions, it was not possible to determine whether the cells

Edward P. Mikkelsen

of the entire new shoots were the derivatives of single or
multiple epidermal cells of the leaf petiole.

The histological information suggests that adventitious
shoots may originate from more than one epidermal cell of
the petiole; therefore, some chimeric plants from irradiated
leaf-petiole cuttings would be expected. However, primarily
non-chimeric plants have been obtained to date with such
mutation breeding programs. The factors, aside from chance,
which may be involved in ensuring non-chimeric adventitious
plants above the soil line are l) certain epidermal cells of
the petiole are "chosen" to develop into adventitious shoots,
and dipontic selection is only mildly occurring before ad-
ventitious shoot formation, 2) diplontic selection is rigor-
ously occurring before shoot formation, and 3) diplontic
selection is occurring after shoot formation and is causing
the elimination of all but one cell type in each of the
histogenic layers. The implications of these factors in a
mutation breeding program are as follows: if certain cells
are "chosen" to develop into adventitious shoots, and if
diplontic selection plays a minor role, then a wide mutation
spectrum, including semi-lethals, would be expected at
relatively low irradiation doses; however, if dipontic selec-
tion plays a major role in determining which cells develop
into adventitious shoots, then semi—lethals would be expected

only at relatively high irradiation doses.

Edward P. Mikkelsen

Although there is no indisputable evidence for only one
epidermal cell of the leaf petiole and its derivatives being
involved in adventitious shoot formation, the fact remains
that the majority of mutated adventitious shoots are non-
chimeric. Therefore, irradiation of plant parts that give
rise to adventitious shoots is still the best in vizg_method

of mutation breeding of vegetatively propagated crops.

HISTOLOGY OF ADVENTITIOUS SHOOT AND ROOT FORMATION
ON LEAF-PETIOLE CUTTINGS OF

BEGONIA X HIEMALIS FOTSCH CV. 'APHRODITE PEACH'

BY
Edward Paul Mikkelsen

A THESIS

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

MASTER OF SCIENCE

Department of Horticulture

1976

To Lynn, Darcy, and Many

ii

ACKNOWLEDGEMENTS

I would like to thank Dr. K. C. Sink for his
guidance and assistance during the research and
preparation of this thesis. I would also like to
thank the other members of my committee, Dr. L. W.
Mericle and Dr. A. A. De Hertogh, for their assis-
tance. I extend my gratitude to Dr. H. P.
Rasmussen, Dr. J. Doorenbos, Dr. C. Broertjes, and
especially to my father, Mr. J. C. Mikkelsen, for
their valuable assistance and discussions.

 

iii

TABLE OF CONTENTS

Page

INTRODUCTION......................................... 1
LITERATURE REVIEW.................................... 5
MATERIALS AND METHODS................................ 9
RESULTS AND CONCLUSIONS.............................. 11
DISCUSSION........................................... 25
SUMMARY.............................................. 33

BIBLIOGWHYOOOOOO'COOOOOCOOOOOOOOOOOOOOO0.0.0.000... 35

iv

LIST OF FIGURES

FIGURE Page
1. Initial stages of callus formation on Rieger
begonia leaf-petiole cuttings.................. l3

2. Root initiation and development. rp--root
primordium000000000OOOOOOOOOOOOOOOOOOOO00...... 15

3. Advanced root development. c--callus;
p--parenchymal tissue; pt--petiole trace;
ar”‘adventitious root.coo00000000000000.0000... 18
4. First stages of shoot formation................ 20

5. Advanced stages of shoot formation. 1--leaf
primorditlm00OOOOOOOOOOOOOOOOOOOOOOO0.0.0.000... 22

6. Summary view of shoot initiation and an
advanced adventitious shoot. s--shoot initial. 25

INTRODUCTION

Cultivars of Rieger begonia, Begonia x hiemalis Fotsch,
are triploid (Arends, 1970) and sterile. While the exact
origin of the Rieger begonias is unknown, it is believed that
they are the result of crosses between tetraploid g, x tuber:

hybrida (4x=56) with diploid g, socotrana (2x=28) (Doorenbos,

 

1973). Rieger begonias have 27 long chromosomes from

g. x tuberhybrida and 14 short chromosomes from g, socotrana

 

and hence, are 3x-1=4l (Arends, 1970). Because the Rieger
begonias are sterile, new varieties cannot be produced by
standard hybridization among themselves; either new crosses

between E, x tuberhybrida and g, socotrana must be made,

 

 

natural sports must be selected, or desirable mutations must
be artificially induced. Since most varieties of Rieger
begonias can regenerate adventitious shoots and roots from
leaf-petiole cuttings, mutation breeding by subjecting leaf-
petiole cuttings to mutagenic agents has been an effective
means of producing new varieties (Doorenbos and Karper,
1975; Mikkelsen, Ryan, and Constantin, 1975).

As Broertjes (1968) has pointed out, the most effective
use of artificial mutation induction is in propagation

systems where an adventitious shoot originates entirely from

a single cell. In this regenerative process, and theoreti-
cally only under this condition, the majority of mutated
adventitious Shoots will be non—chimeric. If more than one
cell is involved in the development of an adventitious shoot,
and if a single cell of that group is mutated, then that
portion of the Shoot arising from that mutated cell will be
genotypically different from the rest of the shoot--a
chimeric condition. Of course, more than one cell in that
group of cells giving rise to the adventitious shoot can be
mutated; however, the probability of any two of them being
mutated exactly the same is extremely low.

Many mutations resulting in chimeras are never detected.
For example, if a mutation is a change in flower color, and
if the mutated sector is a flower, then the mutation will be
observed; but if that mutated sector is a leaf, then it will
not be observed. If a mutated sector is detected, then that
portion of the plant must be propagated asexually. If this
new plant is still chimeric, the portion of the plant with
the desired phenotype must again be asexually propagated.
This process of selection and asexual propagation must be
repeated until an entire plant of the desired phenotype is
produced. Also, if a number of cells is involved in the
formation of an adventitious shoot, there is the possibility
of competition among these cells with respect to involvement
in the formation of the shoot apical meristem. Cells

incapable of dividing will not contribute to any growing

portion of the plant; whereas, those cells which are divid-
ing relatively slowly could be displaced by faster divid-
ing cells. This type of cell growth is termed diplontic
selection. Because of these three disadvantages of chimer-
ism (mutations which are not detected, the necessity of a
few generations of asexual propagation to "true-up" the
detected mutations, and diplontic selection), the efficiency
of mutation breeding increases as the number of cells that
are involved in the formation of an adventitious shoot at
the time of mutation induction decreases. With fewer cells,
the mutated sectors encompass a larger proportion of the
plant and are more easily detected, and "trued-up". Also,
there is less diplontic selection because fewer cells are
involved.

Both Doorenbos and Karper (1975) and Mikkelsen 25 El.
(1975) have produced mutations of B. x hiemalis Fotsch, by
irradiation of leaf-petiole cuttings with x-rays, gamma rays
or fast neutrons. Doorenbos and Karper (1975) and Mikkelsen
(personal communication) have reported that a vast majority
of the mutated shoots were not chimeric. From their results,
it seemed logical that the adventitious shoots originated
from single epidermal cells of the leaf petiole; however,
there is no histological evidence reported to date to sup-
port or refute this assumption. Therefore, it was the

purpose of this histological_investigation of B. x hiemalis

Fotsch cv. 'Aphrodite Peach' to determine the origin of the
adventitious shoots and roots, and especially to determine
whether or not the shoots originated entirely from single

epidermal cells.

LITERATURE REVIEW

The literature pertaining to histological studies of
the origin of adventitious shoots and roots from leaf and
leaf-petiole cuttings in_!i!g_reveals that the process and
location of organogenesis is different amongst the various
species examined. The origin ranges from parenchymal and
epidermal cells giving rise to shoots and parenchymal cells
giving rise to roots, to epidermal cells giving rise to both
roots and Shoots. Lilium bulb scales (Walker, 1940) and

Peperomia leaves (Harris and Hart, 1964) regenerate both

 

shoots and roots endogenously. With Sedum leaf-petiole

cuttings (Yarbrough, 1936), Saintpaulia leaf-petiole cuttings

 

(Naylor and Johnson, 1937), and Begonia rex leaves (Hartsema,

 

1926), shoots develop from epidermal cells, and roots
develOp from endogenous, parenchymal cells. Likewise, with

Kalanchoe (Broertjes and Leffring, 1972), Streptocarpus

 

 

(Broertjes, 1968), and Achimenes (Broertjes, 1968), the
shoots develop from "single" epidermal cells. With Crassula
(McVeigh, 1938), the shoots and roots develop from cells of
epidermal origin. A detailed description of the development

of shoots and roots of Lilium, Sedum, Saintpaulia, and

 

 

Crassula are given below.

 

In Lilium candidum and L. longiflorum, the shoots and

 

roots of the detached bulb scale both originated from
parenchymal cells (Walker, 1940). A few days after the bulb
scale was removed from the bulb and planted, callus tissue
formed over the cut surface. The cells at the cut edge had
collapsed and the adjacent parenchymatous cells were
embryonic with periclinal divisions. Later, divisions were
along no particular plane. The actively dividing cells were
distinguished by their small size, dense cytoplasm, and
darkly stained nuclei. Rapid divisions of these parenchymal
cells and the adjacent epidermal cells led to the develop-
ment of the meristematic tissue of the bulblet primordia.
A small bulblet appeared about two weeks after planting.
After the development of a few leaf primordia, roots de-
veloped from callus near the traces of the bulb scale just
below and lateral to the bulblet primordia. Root and shoot
development continued independently until parenchymal cells
between them became meristematic and differentiated into
vascular tissue connecting the root and Shoot. There were
no vascular connections between primordia and bulb scale
traces.

The development of adventitious shoots and roots in

Sedum strahlii followed a different sequence of events

 

(Yarbrough, 1936). The cells at the cut surface of the

petiole collapsed within a few hours after being excised

from the plant. After approximately 30 to 48 hours callus
arose from parenchymal cells of the petiole. About five
days after excision, the callus had formed a large pad
resulting from all the living parenchymal cells of the
petiole having undergone divisions. At the same time, root
primordia appeared within the interior of the callus. Shoot
primordia appeared on the lateral surface of the callus
within 10 to 12 days and were composed of both epidermal

and subepidermal parenchymal cells of the callus tissue.

Saintpaulia ionantha represents a third mode of

 

adventitious shoot and root formation (Naylor and Johnson,
1937). A few days after excision of the leaf from the plant,
a callus tissue developed over the cut end of the petiole.
Ten days to two weeks later, the petiole swelled unevenly
around the wounded surface. Root initials derived from thin
walled parenchymal cells between the vascular traces pushed
out through the epidermis or down through the wound callus.
There was no evidence that any of the roots originated
directly from the wound callus. Redifferentiation of paren-
chymal cells between root primordia and petiole traces into
vascular tissue served to connect the root primordia to the
nearest petiole trace. Six to seven weeks after excision,
shoot primordia began to form from the epidermis, one or two
millimeters from the cut end. The first division of the

epidermal cells was anticlinal. Further divisions were

randomly oriented. Again, redifferentiation of parenchymal
cells into vascular tissue connected the shoot primordia
with the nearest petiole trace. Each shoot was reportedly
derived from a single epidermal cell, although adjacent
epidermal and subepidermal cells of the petiole contributed
to its final formation.

The development of adventitious shoots and roots in

Crassula multicava represents another means of regeneration

 

(McVeigh, 1938). Epidermal cells of the petiole began to
divide within two days after excision from the plant. The
first divisions were periclinal, later they were also anti-
clinal. The activity was not confined to a few cells; most
of the cells around the entire petiole near the wounded
surface divided. Groups of cells projected above the sur-
rounding cells because of their more rapid divisions. New
plants arose from these projected groups of cells. Initially
roots were formed, and then vascular connections were made
to the petiole traces by differentiation of parenchymal
cells. Subsequently, the first pair of leaf primordia were
formed. This represents a case where both roots and shoots

arose from cells of epidermal origin.

MATERIALS AND METHODS

The cultivar of Rieger begonia chosen for this experi-
ment was 'Aphrodite Peach', which is a product of mutation
breeding of 'Aphrodite Rose' shoot tip cuttings. Aside
from a different flower form and color, 'Peach' is capable
of producing an average of about 60 adventitious shoots per
leaf-petiole cutting under the optimal conditions of winter
and about 20 under the suboptimal conditions of summer;
whereas, 'Rose' produces about 10 adventitious shoots per
petiole cutting in the winter and almost none in the summer
(unpublished data).

Leaf-petiole cuttings at various stages of root and
shoot development were obtained as needed from Mikkelsen's
Inc., Ashtabula, Ohio, where they were grown under normal
production methods as follows. Leaf-petiole cuttings were
taken when the leaf petioles were about 1 to 1.25 cm long.
The cuttings were planted in 5.5 cm. plastic pots contain-
ing a mixture of 45% peat, 45% perlite, and 10% soil, and
the pH adjusted to 5.5. After being thoroughly "watered in",
they were placed in the "rooting area" where the temperature
was 22°C and the light intensity about 1200 ft-ca. They

were kept moist by hand misting with a hose. After 4 weeks,

10

they were transferred to the "Shooting area" where the
temperature was maintained as close to 18°C as possible.
The light intensity was increased to 6000-7000 ft-ca.

Upon receiving the developing cuttings they were rinsed
in tap water to remove the soil. Any excess roots were cut
off, the leaf and the upper part of the petioles were
trimmed off, and the remaining pieces of petioles with the
developing roots and shoots were dehydrated, stained and
mounted using a modification of a technique described by
Sass (1971). The basic procedure was as follows. The
pieces were fixed in FAA for at least 24 hrs. Then they
were dehydrated by soaking for 24 hrs. in each of six solu-
tions of an n-butyl alcohol dehydration series, infiltrated,
imbedded, and cast in "Paraplast'. Ten micron sections were
microtomed, mounted on glass slides with Weaver's solution
and dried on a hot plate at 55°C. The sections were then
stained with safranin-fast green double staining. Usually,
the slides were in safranin for 12 to 24 hrs. and in fast
green for 2 to 5 mins. The slides were made permanent with
'Permount' and then were examined with a microscope.
Photographs of selected areas were taken using fine grain

'Panatomic X' film.

RESULTS AND CONCLUSIONS

Microsc0pic observations made on serial sections of
B. x hiemalis Fotsch cv. 'Aphrodite Peach' leaf-petiole
cuttings at sequential times of development revealed the
following sequence of events in the development of adventi—
tious shoots and roots. At the time of excision, the paren—
chyma cells at the cut end of the petiole dehydrated and
collapsed (Figure 1A). Within two weeks, the epidermal
and subepidermal parenchymal cells at the petiole base had
divided periclinally and anticlinally to form a small callus
area (Figures 1B and 1C). This callus usually surrounded
the entire portion of the petiole base; however, if the
epidermis was not intact at some area of the petiole due to
injury, callus formation by epidermal or subepidermal cells
did not occur where the epidermis was injured (Figure 1D).
Three or four weeks after excision, root primordia became
evident as clusters or groups of cells below the surface of
the callus divided rapidly, eventually becoming organized
as root meristems (Figures 2A and 2B). The root primordia
subsequently grew laterally through the callus and emerged
at the surface of the petiole (Figure 2C). Roots were also

initiated from internal parenchymal cells. These were

11

12

Figure 1. Initial stages of callus formation on Rieger

begonia leaf—petiole cuttings.

Longitudinal section (LS) of a petiole 2 days
post-excision (PE) showing the collapsed cells
at the cut end. 120X.

Cross section (CS) of a petiole 12 days PE show—
ing the first periclinal divisions of the
epidermal cells. 335x.

LS of a petiole 2 wks. PE showing periclinal and
randomly oriented planes of division of epidermal
and subepidermal cells. 30X.

External view at the cut end of a petiole 9 wks.
PE showing the absence of callus and shoots where
the epidermis was not intact. 12X.

 

 

Figure

14

Figure 2. Root initiation and development. rp--root
primordium.

A. LS of a root primordium 4 wks. PE. 33SX.

B. LS of a more advanced root primordium 5 wks. PE.
335x.

C. LS of a root initial emerging at the callus
surface 4 wks. PE. 120X.

 

15

 

16

observed in the basal portion of petioles and were present
even when the entire lower portion of the epidermis was
injured and there was no epidermal callus (Figure 3A).
Vascular strands between the roots and petiole traces were
formed by redifferentiation of callus and parenchymal cells
into vascular tissue (Figure 3B).

Shoot formation began 5 to 6 weeks after removal of the
leaf-petiole cutting from the stock plant. Cells at the
surface of the callus divided anticlinally forming a number
of small daughter cells, and sometimes the callus cells
directly below the surface also divided (Figure 4). This
rapid division was not confined to a single cell and its
derivatives but involved a number of cells and their deriv-
atives. A few of these derivative cells, out of many,
formed a nodular protrusion above the surface of the callus
(Figures 5A and 5B). The size of the protrusion increased
by cell division (Figure 5C), and some of the cells of the
protrusion eventually organized into a shoot meristem. This
meristem continued growing to differentiate leaves and
lateral buds (Figure 5D). Also, cells of the protrusion
and cells adjacent to it differentiated into bud scales and
other supportive tissue of the base of the newly formed
shoot (Figure 5E). Concurrent with the development of the
protrusion, cells below the protrusion began to organize as
vascular tissue to connect the new shoot vascular system

with the leaf—petiole vascular system (Figure 5F).

17

Figure 3. Advanced root development. c——callus;
p--parenchymal tissue; pt--petiole trace;
ar--adventitious root.

A. External view 5 wks. PE showing a root initiated
from a region lacking epidermis and callus. 15X.

B. LS of an adventitious root and CS of the leaf
petiole showing vascular tissue connecting the
adventitious root with a petiole trace. 32X.

18

 

Figure 3

19

Figure 4. First stages of shoot formation.

A. CS of a petiole 5 wks. PE showing divisions of
epidermal and subepidermal cells of the callus.
480x.

B. CS of another petiole 5 wks. PE, same as A.
480x.

20

 

Figure 4

21

Figure 5. Advanced stages of shoot formation. 1--leaf

A.

primordium.

LS of a slight protrusion 6 wks. PE showing anti-
clinal divisions. 335x.

LS of other protrusions above the callus 6 wks.
PE. 480x.

LS of a more advanced protrusion 6 wks. PE.
335x.

Oblique section of an advanced initial 6 wks. PE
showing a leaf primordium. The seemingly narrow
connection to the callus is an artifact due to
angle of the slice. 335x.

LS of a bud scale and a shoot primordium arising
from the callus 6 wks. PE. 33SX.

LS of a petiole 6 wks. PE showing the redifferen-
tiation of callus cells below the surface to form
vascular tissue. 335x.

22

 

Figure 5

23

Figure 6A shows the formation of a callus with cells of a
portion of the callus beginning to form a shoot initial.
Figure GB shows an advanced adventitious shoot.

In conclusion, the epidermal and subepidermal cells of
the basal portion of the petiole of a Rieger begonia leaf-
petiole cutting form a callus. Roots form from cells of
the internal portion of the callus and also from parenchymal
cells of the petiole. Shoots form from cells on the surface
of the enlarging callus. Since from the time of callus
formation to the time an organized shoot appears many cells
have divided numerous times, it is virtually impossible to
determine from the histological observations whether the
cells of the entire new Shoot are the derivatives of a
single epidermal cell of the petiole or are the derivatives

of a number of epidermal cells.

24

Figure 6. Summary View of shoot initiation and an advanced
adventitious shoot. s-—shoot initial.

A. LS of a petiole 6 wks. PE showing callus with
the initiation of a protrusion to form a shoot.
120X.

B. LS of an advanced adventitious shoot 6 wks. PE.
120X.

 

 

25

 

DISCUSSION

Histological observations of leaf-petiole sections of
'Aphrodite Peach' suggest that adventitious shoots arise
from a number of cells which may or may not be the deriva-
tives of one epidermal cell of the petiole. Considering
that 1) an epidermal cell divides numerous times in the
formation of the callus, 2) that the cells of the callus
surface divide several times before a protrusion develops,
and 3) that only some of the cells of the protrusion even-
tually form the shoot apical meristem, it is possible by
chance alone for the shoot apical meristem to be organized
from cells, all of which are the derivatives of the same
epidermal cell of the petiole. However, there appears to
be no a priori reason why the shoot apical meristem could
not be organized from cells which are the derivatives of
several different epidermal cells.

The fact that the irradiation induced mutation work on
leaf—petiole cuttings of B. x hiemalis Fotsch by Doorenbos
and Karper (1975) and Mikkelsen g2.§l. (1975; personal com-
munication), have produced a low percentage of chimeric
plants is evidence that the shoot apical meristem, at the

time that the shoot emerges above the soil level, is

26

27

composed of an identical, or homogeneous, phenotype.
However, it is not evidence that the entire adventitious
shoot originated from a single epidermal cell of the petiole,
nor is it evidence that the cells of the protrusion which
organized into the shoot apical meristem were the derivatives
of a single epidermal cell. The high percentage of non-
chimeric plants only provides evidence that the shoot apical
meristem is composed of a phenotypically homogeneous group
of cells at the time that the shoot emerges above the soil
line.

Naylor and Johnson (1937) originally reported that a

single epidermal cell in Saintpaulia gives rise to the

 

entire shoot except for the vascular strands which connect
the vascular system of the shoot with that of the petiole.
The most logical reason for the high percentage of non-
chimeric plants which were obtained during radiation induced
mutation breeding was that the entire plant came from one
cell; therefore, the shoot apical meristem would obviously
be genetically homogeneous (Sparrow, Sparrow, and Schairer,
1960; Broertjes, 1968). Actually, it seems that the pub-
lished histological observations of both Naylor and Johnson
(1937) and Broertjes (1968) do not indisputably support the
hypothesis that the derivatives of only one epidermal cell
are involved in the formation of the shoot apical meristem.

The high percentage of non-chimeric mutants supports that

28

hypothesis, but does not prove it. As mentioned earlier,
Walker (1940) has reported that a number of cells, both
epidermal and parenchymal, are involved in the production
of adventitious shoots in Lilium, but Broertjes (1972; per-
sonal communication) has stated that mutation breeding of
lily bulb scales immediately after scaling produced mainly
solid mutants as determined by flower color. Therefore,
a high percentage of non-chimeric mutations is not suffi-
cient evidence to show that adventitious shoot formation
involves only a single cell. More recently Broertjes (per-
sonal communication) has stated that a number of epidermal
cells are dividing at the time of adventitious shoot forma-
tion in Saintpaulia. Therefore, the fact that a number of
cell types may be involved in the formation of an adventi-
tious shoot in 'Aphrodite Peach' and concomitantly there is
a high percentage of non-chimeric induced mutations is not
an isolated occurrence. The question which arises is what
processes are involved during adventitious shoot formation
and subsequent growth of the shoot which usually insures a
homogeneous group of cells in the shoot apical meristem at
the time of shoot emergence above the soil level and subse—
quent flowering. There are a number of possible answers to
this question, and one or all of them may be correct.
Although more than one epidermal cell of the petiole

divides at the time of shoot formation, it seems likely that

29

one cell out of a group will divide first. As Broertjes
(personal communication) states it:

Many epidermal cells divide and form a number of meri—
stems. In Saintpaulia, the number of epidermal cells
per meristem is approx. 100-200. One cell, however,
is the first one and this probably becomes the center
of this group and grows out into a series of vegeta—
tive daughter cells. On top of this meristem one or

a few cells then form the apex of the adventitious
shoot and because of the restricted number of cells
involved in it, the chance is very large that they are
genetically identical, thereby forming a genetically
homogeneous plantlet. The correct definition conse-
quently is: the apex of an adventitious shoot ulti—
mately originates from a single epidermal cell.

 

Broertjes (1972) further states that the cells which form
the center of activity are somehow "chosen". Two explana~
tions are apparent in his hypothesis. First, the cell whose
derivatives will form the shoot apex are "chosen", and
second, because of the number of cell divisions and of the
restricted number of cells forming the apex, the homogeneity
of the cells in the shoot apex is a matter of chance.
Another possible explanation for the high percentage
of non—chimeric plants is that diplontic selection is taking
place at the time of shoot formation. Cells which exhibit
less physiological damage after irradiation treatment divide
faster and contribute considerably more cells to the develop-
ment of the callus. When the cells at the surface of the
callus begin to divide to form the protrusion, the most
vigorous cells divide faster and form the protrusion.

Broertjes (1972) stated that diplontic selection is working

30

to a lesser degree before and including the time of the
organization of the shoot apical meristem. The basis for
his conclusion is that he has observed dwarf mutants even at
low levels of irradiation when most cells probably were not
mutated. The non-mutated cells were possibly more vigorous
than the dwarf mutant cells; hence, the non-mutated ones
should have successfully competed against the dwarf mutants
if diplontic selection were taking place. However, there is
no a priori reason to believe dwarfism is a semi-lethal
characteristic resulting from cells which are physiological-
ly less vigorous. If dwarfism were solely the result of
less cell elongation, the cells could divide as rapidly or
could even divide more rapidly than the non—mutated cells.
The emergence of a chlorophyll deficient mutant or slower
developing mutant would be more convincing evidence that
diplontic selection was occurring to a lesser extent during
the early stages of shoot development.

Diplontic selection is an accepted theory in the growth
of a developing meristem; therefore, it is a possible ex-
planation of how the shoot apical meristem could be composed
of homogeneous cells at the time of emergence above the soil
line. If a number of cell types were involved in the
development of the organized meristem, then diplontic selec-
tion could conceivably result in elimination of all but one

cell type in each of the histogenic layers. Sometimes,

31

mixtures of cell types in the layers of the apical meristem
persist which ultimately result in variegation patterns of
the leaves, shoots, or flowers. Such mutations have indeed
been reported by Sparrow, Sparrow, and Schairer (1960) in
their work on Saintpaulia. For some reason, they did not
refer to these mutations as being chimeric.

Any or all of the above explanations may be the real
reason why a shoot apical meristem emerging above ground is
composed of a homogeneous group of cells when more than one
adjacent epidermal cell of the petiole have divided so that
by chance alone some chimeras would be expected. Cells
could be "chosen" by some unknown factor, selected against
only mildly before and at the time of meristem formation,
and then if more than one cell type did occur in any of the
histogenic layers of a meristem, all but one type in each
layer would be eliminated by diplontic selection. As with
most experimental observations, the combined information
obtained from this histological study and from the irradia—
tion mutation breeding projects of Doorenbos and Karper
(1975) and Mikkelsen gt_al. (1975) are open to a number of
possible explanations. What are the implications of these
explanations for practical mutation breeding? If cells are
indeed "chosen", and diplontic selection is less strict
during the initial stages of development, then under rela-

tively low irradiation doses a wide mutation spectrum,

32

including semi-lethals, would be expected. If diplontic
selection is occurring during the early stages of shoot
development as it does after shoot meristem formation, then.
a narrower mutation spectrum, excluding semi-lethals, would
be expected. If the semi-lethals are desired, then they
might be produced by subjecting the petioles to a high dose
of irradiation so that most cells are damaged to the extent
that they cannot divide, thus allowing the lesser damaged
semi-lethal cells to develop into shoots. Regardless of the
explanation for observing few chimeras, the fact is that
few chimeras are produced by irradiating leaf-petiole
cuttings; therefore, irradiation of plant parts that will
give rise to adventitious shoots is a very efficient means
of inducing desirable mutations in asexually propagated

higher plants.

SUMMARY

Epidermal and subepidermal cells of the leaf petiole
formed a callus at the basal portion of the petiole of
Rieger begonia cv. 'Aphrodite Peach' leaf-petiole cuttings.
Roots arose from cells of the internal portion of the callus
and also from parenchymal cells of the petiole. Shoots
formed from cells on the surface of the enlarging callus.
Since from the time of callus formation to the time an
organized shoot appeared many cells have divided, it is
virtually impossible to determine from the histological
observations made herein whether the cells of the entire new
shoot are the derivatives of a single epidermal cell of the
petiole or are the derivatives of a number of epidermal
cells.

Adventitious shoots from irradiated leaf-petiole cut-
tings of B. x hiemalis Fotsch have been mainly non-chimeric.
Several factors may be involved in ensuring non-chimerism
when a number of cell types are dividing. First, the cell
to give rise to the cells of the adventitious shoot may be
"chosen". Second, diplontic selection may be occurring
during callus and protrusion development. And third,

diplontic selection may be occurring after the meristem has

33

34

organized and before the shoot emerges above ground. If
diplontic selection is occurring to a lesser degree during
the early stages of development because certain cells are
"chosen", then a wide mutation spectrum including semi-
lethals would be expected at low irradiation doses. If
diplontic selection is occurring at the early stages of
development, then semi-lethals, if desired, may be obtained

only at high doses of irradiation.

BIBLIOGRAPHY

BIBLIOGRAPHY

Arends, J. C., 1970. Somatic chromosome number in 'Elatior'—
Begonias. Meded. Landb. Hogesch. Wageningen 70(20):
1-18.

Broertjes, C., 1968. Mutation breeding of vegetatively
propagated crops. Fifth Congress of the European
Association for Research on Plant Breeding, Milano:
139-165.

Broertjes, C., 1972. Use in plant breeding of acute,
chronic or fractionated doses of X-rays or fast neu-
trons, as illustrated with leaves of Saintpaulia.
Thesis Wageningen; Agric. Res. Rep. 776, 74 pp.

 

Broertjes, C., and L. Leffring, 1972. Mutation breeding of
Kalanchoé. Euphytica 21:415-423.

Doorenbos, J., 1973. Breeding 'Elatior'-Begonia. Acta
Horticulturae 31:127-132.

Doorenbos, J., and J. J. Karper, 1975. X-ray induced muta-
tions in Begonia x hiemalis. Euphytica 24:13-19.

Harris, G. P. and E. M. H. Hart, 1964. Regeneration from
leaf squares of Peperomia sandersii A. Dc: a relation-
ship between rooting and budding. Ann. Bot. New Series
28:509-525.

Hartsema, A. M., 1926. Anatomische und Experimentelle Unter-
suchungen fiber das Auftreten von Neubildungen an
Bléttern von Begonia Rex. Recueil Trav. Bot. Néerland
23:305-361.

 

McVeigh, I., 1938. Regeneration in Crassula multicava.
American Journal of Botany 25:7-11.

 

Mikkelsen, J. C., J. Ryan, and M. J. Constantin, 1975.
Mutation breeding of Rieger's Elatior Begonias.
American Horticulturalist 54:18-21.

35

36

Naylor, E. and B. Johnson, 1937. A histological study of
vegetative reproduction in Saintpaulia ionantha.
American Journal of Botany 24:673-678.

 

Sass, J. E., 1971. Botanical Microtechnique (Iowa State U.
Press, Ames, Iowa).

Sparrow, A. H., R. C. Sparrow, and L. A. Schairer, 1960.
The use of x-rays to induce somatic mutations in
Saintpaulia. African Violet Magazine:32-37.

Walker, R. I., 1940. Regeneration in the scale leaf of
Lilium candidum and L. longiflorum. American Journal
of Botany 27:114-117.

Yarbrough, J. A., 1936. Regeneration in foliage leaf of
Sedum. American Journal of Botany 23:303-307.

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