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AN ANATOMECAL STUDY OF THE OREGIN AND
DEVELOPMEN? OF ‘E‘HE 3ULBLETS 3N THE
SCALE LEAVES OF LEW BULBS

 

TH .

Yhesis for the» Degree of M. S.

MICHIGAN STA?E UNIVERSITY

Brenda Frames Goddenfi
"3 962

LIBRARY

Michigan State
University

 

AN ANATOEMCAL STUDY OF THE ORIGIN AND DEVE LSPi‘viVLINT OF

THE BULBLET S ON THE SCALE LEAVES 01"" LILY BULBS

by

BRENDA FRANCES GODDEZN

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

1)

MASTER OF SCIENC.

Department of Hortigulturc

I 962

ACKNOWLEDGEMENTS

The author is sincerely indebted to Dr. Donald P. Vi'atson for his
guidance throughout the course of study. Appreciation is due to Dr.
Charles Hammer of the Department of Horticulture and Dr. Leo Mcricle
of the Department of Botany for serving on the author's committee.

The author also wishes to express her obligations to the Garden
Club of America for providing the financial assistance which has
made possible her stay in the United States of America during the

period of study.

ii

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS.................. ii
CONTENTS........................ iii
LISTOFILLUSTRATIONS ................. iv
INTRODUCTION...................... 1
REVIEV:’0P‘LITERATURE................. 1
MATERIALS........................ 4
PROCEDURE..................

.00... 5

RESULTS......................... 6
EXTERNALMORPHOLOGY....... .. ....... 6
Bulbs.....................
ScaleLeaves................... 6
Bulbletdevelopment................ 7
INTERNALMORPHOLOGY............... 7
ScaleLeaves................... 7
Periderm and Callus Formation . . . . . . . . . .. 9
BulbletDevelopment................ 11
CONCLUSION....................... 16
SUMMARY.............

0 O O O O O O O O O O O 18

BIBLIOGRAPI‘IY I O O O O O O O O O O O O O O O O O O C 0 0 U 19

, iii

LIST OF ILLUSTRATIONS

Figure

l a. b. and c. mature bulbs (xi/2): d. e. and f. scale
leaves and bulblets following six weeks of incubation
(x 2/3). a and d. cv. Bellingham; b and e. cv.
Olympic;candf. cv. Fiesta. . . . . . . . . . .

2 Drawing of a longitudinal section of the base of a scale
leaf immediately following detachment from the parent
scale (x 75). a. adaxial epidermis; b, vascular tissue;
c, stem tissue; d. mesophyll of leaf scale; e, abaxial
surface.....................

3 Diagrammatic representation of stages of development
of a bulblet. a. visible protuberance; b. formation
of first leaf initials; c. later stage of leaf initials;
d.developedbulblet................

4 Stages of deveIOpment of a bulblet. a. visible protuber-
ance (x 45); b, formation of first leaf initials (x45);
0. later stage of leaf initials (x 45): d. developed bulb-
let (x13). 1. initiation of root primordium; 2. peri~
derm; 3. callus; 4. vascular connection; 5. root;
6.0uterbulbletscale. . . . . . . . . . . . . . .

5 Enlargement of vascular connection of Figure (1. 4. (x 70) ..

iv

10

12

13

15

INTRODUCTION

Scale propagation in the genus £393.19 has been an accepted procedure
for bath the commercial and amateur grower tor more than half a century
(Wallace 1879. Griffiths 1930. Emsweller 1957, Rockwell 331. 19531).

Most rapid regeneration in the form of bulblets has occurred on scale leaves
detached from the parent bulb following incubation of the scales in a moist

medium at a temperature of 70 degrees F.
REVIEW OF LITERATURE

Priestley and Swingle (1929). in a comprehensive anatomical review of
vegetative propagation. maintained that it was necessary to distinguish be-
tween the further deveIOpment of a dormant, but already organized. meristem
and the initiation of such a meristem as the result of developmental changes
caused by mechanical isolation of a portion of the plant. They indicated that
each species was distinctive in its behavior and required individual study
before its performance as a self propagating unit could be determined.

Later anatomical investigations (Clamp i934. McVeigh 1938. Naylor 1932.
Naylor and Johnson 1937, Walker 1940. Yarbrough 1932) described the origin
of regeneration within selected species and clarified this distinction between
dormant or reassumed meristematic activity as was proposed by Priestly and

Swingle.

Dormant meristems have been shown in Kalanchoe and Bryophyllum.

 

Clamp (1934) showed residual primary meristems derived from the original
apical meristem in Kalanchoe tubit’lora. Similarly, entire dormant meri-

stems are said to be present in Bryophyilum calycinum (Naylor 1932.

 

Yarbrough 1932).

In contrast to this type of origin the mature cells in some other genera
have been shown to reassume meristematic activity. A new individual
arose exogenously by the divisions of a group of mature epidermal cells
in Crassula multiclava (McVeigh 1938). In Sain aulla ionantha the origin
of the shoot was similar. It arose exOgenously from epidermal cells
whereas the root, in contrast. arose endOgenously from parenchymatous
tissue (Naylor and johnson 1937).

In Lilium candidum and Lilium Longiflorum the origin of regeneration

 

 

was entirely endogenous with bOth root and sheet arising as a result of

divisions of parenchyma (iialker 1940).

 

With the exception of Bryophyllum and £11193}? however. vascular
connections were developed between the parent leaf and the new individual.
In 1.3129 the vascular system of the young bulblet was indicated to be en-
tirely independent of the parent bulb scale and at no time was any vascular
connection observed (Vlalker 1940).

A phenomenum of Lilium is that regeneration only occurred after de-

tachment of the scale from the parent bulb. Hence regeneration might be
associated with wound healing. Priestly and “offender: (I922) formulated
the following three point sequence of wound healing: "1) The wounded
parenchyma surface becomes blocked by a deposit of suberin or cutin formed
in the presence of air; 2) sap accumulates at the parenchymatous surface;
3) phellogen activity develops amidst this parenchyma and gives rise to
callus tissue. In every case the essential antecedent to meristem forma-
tion is the blocking of the cut surface.”

For the purpose of the present investigation it became necessary to
make a clear distinction between callus tissue and periderm in relation
to wound healing. Lauer and Krantz (1957) introduced a new interpretation

following their study on wounding of Solanum tuberosum. They proposed

 

that callus tissue wasproduced by an increase in cell number and that the
resultant cells were capable of continued expansion and the production of
buds. Periderm was suggested to be the result of re-diiferentiation of
existing cells without division and without the capacity to produce buds.
Further differences were: the relatively slow formation of callus when
compared with the formation of wound periderm; callus formed only in
non-dormant tubers. whereas periderm deveIOped in bath dormant and non-

cbmant tubers: and callus developed only after bud removal.

MATERIALS

The following three cultivars‘: Olympic hybrids. Fiesta hybrids. and
Bellingham hybrids. were selected because of their different. yet repre-
sentative. external morphology of bulbs within the genus Lilium. The

Olympic hybrids were selected forms of Lilium leucanthuxg centifolium.

 

Fiesta hybrids were developed by a combination of Lilium dauricum

 

 

 

seedling and Lilium dayidii together with Lilium amabile and Lilium
amabile luteum. Bellingham hybrids have resulted from hybridization

of the native North American species (De Graft 1951).

‘Supplied by the courtesy of Jan de Graff. Gresham. Oregon.

PROCEDURE

Eight outer scales were removed from bulbs of each of the three
cultivars. The detached scales were dusted with a fungicide (Captan) to
prevent basal scale rot and were laid horizontally in damp peat in wooden
flats lined with polyethylene to retain the moisture. ' The bulb scales
were incubated in darkness in moist peat at 70 degrees F for six
weeks.

’ Three sample scales were collected at random at intervals of three
days. examined for external features. cut into small portions and pre-
pared for microscopic examination. The material was killed in a formalin-
acetic acid - alcohol (5:5:90). dehydrated with ethyl alcohol. embedded in
paraffin. sectioned 10 micra in thickness. and stained with safrannin
and analin blue or with safrannin and fast green Uohansen: Plant Micro-
tecbnique). Sections were cut primarily in a direction parallel to the
longitudinal aids and approximately two hundred and fifty preparations

were mounted permanently in Canada Balsam.

R ESULTS

EXTERNAL Mom-101,003

B; 113 8:

 

The bulbs of the genus M have a characteristic structure of
spirally arranged. overlapping. fleshy scale—leaves arising upon a con-
densed disc like stem. Bulbs of Olympic and Fiesta hybrids were of simi-
lar appearance and were from 8 to 10 cm s. in diameter. The Olympic
bulb was a deep red color and the Fiesta bulb was normally white with
some red pigment when exposed to light (a. b. Fig. l). The Bellingham
hybrids possessed two or more distinct bulbs arising from a rhizomatous
stem base characteristic of the wild species from which they are derived
(c. Fig. 1).

Scale leaves:

 

The outer scales of the Olympic hybrids averaged 6 cms. in length
and 3 cms. in width. They had a sessile base and were elliptically con-
cave toward the inside. tapering to an acute apex. The scales of Fiesta
showed greater variation in shape. had a similar sessile base but an
acuminulate tip. Parallel longitudinal venation was evident in some scales.
Outer scales of the Bellingham hybrids were two-jointed. the distal section
with a tapering shape comparable to the Olympic hybrid except for an aver-

age length of 3 to 4 cm s. and width of l. 5 cm s.

 

 

Bulblet develogment:

 

Bulblet development was found to be exactly similar in all three culti-
vars. The production of a proliferated callus over the wound surface was
observed externally within 5 days. By 10 days distinct protuberances be-
came evident on the callused surface and with leaf differentiation these
protuberances became distinguishable as bulblets (Fig. 3'). Throughout
the incubation period the parent scale leaf remained firm and fleshy.

Bulblet development occurred predominantly. though not exclusively.
on the adaxial surface (d. e. f. Fig. 1). Fiesta hybrids exhibited some
capacity to prochtce bulblets on the margins of the scale leaf (f. Fig. 1).
When veins were visible to the naked eye it was evident that bulblets were
contiguous to the bases of these veins.

It was observed that new bulblets became visible over a period of
six weeks so that. in the final collection. bulblets were at all stages of
growth and development. or the three chums. the Bellingham hybrids
showed the greatest regularity in the time of deveIOpment.

After six weeks the average number of bulblets on each scale of the

different cultivars was: Olympic i. 9. Fiesta 3. D. and Bellingham l. 7.

INTERNAL MORPHOLOGY

 

Scale leaves:

 

The somewhat crescent shaped scales were surrounded by an irregular

 

Fig. 1. a, b. and c. mature bulbs (x 1/2); do e. and f, scale leaves and
bulblets following six weeks of incubation (x 2/3).
a and (1, cv. Bellingham; b and e. cv. Olympic; c and f, cv. Fiesta.

layer of epidermal cells. Undifferentiated parenchymatous mesophyll.
heavily deposited with starch grains. filled the scale. Inconspicuous
vascular bundles. frequently difficult to locate in the transverse view.
were in no definite pattern. These bundles were small with xylem lying
toward the adaxial. and phloem toward the abaxlal surface.

Visible in longitudinal section in some scales examined immediately
after detachment from the parent bulb was a distinctly separate zone of
tissue adjacent to the wound surface (9. Fig. 2). The cells in this region
were conspicuously rounded. were ulth heavier cell walls. and were
without starch. In Other scales this zone was absent and the cells were
undifferentiated. Further study of several specially prepared sections
confirmed the fact that the loose cells of this sons were merely a part of
the stem tissue torn off with the scale and not a specialized separation
layer of an abscission zone as might have been concluded.

Periderm and callus forfimation:

 

The wounded cells collapsed soon after detachment of the scale. The
onset of periderm formation was indicated by an accumulation of suberin
and globular material around the damaged cells and around the walls of
cells contiguous to the wounded surface. From this surface cell destruc-
tion progressed distally with increased deposits of suberin and with vacuola-
tion of the cells.

After three days nuclear divisions were exceedingly prolific in the

10

 

 

 

 

 

 

 

 

 

 

 

Fig. 2. Drawing of a longitudinal section of the base of a scale leaf

immediately following detachment from the parent scale (x 75).

a, adaxial epidermis; b. vascular tissue; c. stem tissue;

d, mesophyll of leaf scale; e. abaxial surface.

ll

parenchyma cells distal to the newly formed periderm. The earlier divi-
sions were predominantly in a direction parallel to the wound surface.

In this manner. a large volume of callus tissue was developed adjacent

to the dead cells of the periderm crushing and subordinating this tissue
and sealing the damaged area (2. 3. d. Fig. 4.).

Bulblet deveknament;

 

Stages of a developing bulblet are illustrated in Fig. 3.

Regions of bulblet initiation became distinguishable by the division
and nuclear enlargement of a group of sub-epidermal cells. These divi-
sions could easily be distinguished from those of callus development by
their localization and the direction of division being predominantly parallel
to the epidermis. This increase in cell number was accompanied by anti-
clinal divisions of the epidermal cells and a protuberance was formed on
the surface of the scale (a. Fig. 4).

Later divisions occurred in all planes giving increase in size of the
bulblet primordium. At this stage the first indications of differentiation
of vascular tissue were observed occurring within existing cells of the
parenchyma of the parent scale or in the cells of the callus tissue. A
scalariform cell wall thickening characteristic of xylem elements appeared
on those cells lying between the vascular strand of the parent scale and the
developing primordium. There was some elongation of these cells (Fig. 5).

By a differential rate of cell division at the surface of the bulblet

12

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Fig. 3. Diagrammatic representation of stages of development of a bulblet.
a. visible protuberance; b, formation of first leaf initials; 0, later stage of
leaf initials; d, developed bulblet.

13

 

 

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primordium alternate leaf initials arose (b. Fig. 4). These increased
rapidly in size to surround and enclose a central meristem portion which
continued to give rise to successive leaf primordia.

The first indication of a root primordium was the formation of a
densely stained group of meristematic cells within the bulbet protuberance
below either the apical meristem or the leaf primordium (1. c. Fig. 4).

A definite root apex developed and emergence was exactly similar to nor-
mal adventitious root development. At no time was rooting of the parent
scale observed. Roots which externally appeared to have arisen from the
bulb scale were shown to have their origin within the newly formed bulbet.
Vascular tissue formed from the root meristem connected with that dif-
ferentiated between the bulblet and the parent scale.

A bulblet after six weeks from the time of detachment of the scale
possessed leaf scales and leaf initials and showed continuing development
of leaf primordia (as illustrated in 6. d. Fig. 4). Vascular strands were
forming in the larger scales. The central meristem had assumed the
characteristics of a typical apical meristem exhibiting layers of both tunica
and corpus. Two or three well developed roots were present (5. d. Fig. 4).
Connecting vascular parenchyma between the parent scale and bulblet re-
mained visible but further vascular differentiation appeared to have ceased

(4.d. Fig. 4; Fig. 5).

15

 

(x 70).

Fig. 5. Enlargement of vascular connection of Figure d, 4.

CONCLUSION

Wound healing at the broken surface of the detached scale followed
the sequence proposed by Priestley and Vs'offenden (1922) of blocking of
the cut surface and subsequent meristematic activity which gave rise to
a callus tissue.

The present observations on Lilium have illustrated a distinction

 

between callus and periderm tissue as was presented by Lauer and Krantz
(1957). Their assumption that callus was an increase in cell number.

whereas periderm was the re-difierentiation of existing cells. has been

 

substantiated and adopted. Lauer and Krantz’ conclusion that callus
formed only in non-dormant tubers and only after bud removal has not
been verified.

The development of bulblets behind the sound surface was shown to
be exactly similar in all three cultivars examined. It has been suggested
therefore that the pattern of development described is consistent through-
Out the genus. '

The bulblet arose on the leaf scale by simultaneous divisions of both
epidermal and sub-epidermal cells and was am therefore entirely endo-
genous as has been stated by Walker (1940). This type of development

of a new individual from reassumed meristematic activity in mature cells

16

17

was closely comparable. though not identical. to that shown by McVeigh

(1938), Naylor and Johnson (1937) in Crassula and Saintpaulia. This

 

type of origin in Lilium contrasted with that shown in Kalanchoe and

Bryophyll-um (Clamp 1934. Naylor 1932. Yarbrough 1932) in lacking

 

residual meristems.

The dependence of the bulblet for food material from the parent
scale during its early stages of initiation has been assumed. Vascular
connections arose at the onset of bulblet development by the differentiation
of parenchyma or callus cells lying between the vascular strands of the
parent scale and the bulblet primordia. External observations have
shown the tendency for bulblet development to occur at the base of
veins. In spite of the fact that Va‘alker (1940) had not observed vascular

connections in Lilium lanai: florum and Lilium candirhzm. they were

 

present in Olympic. Fiesta and Bellingham hybrids.
Roots appeared as a subsequent development as the bulblet enlarged.
In every case roots arose from the tissue of the bulblet and their origin

could not be traced from parent tissue, as had been suggested in Saint-

 

paulia (Naylor and Johnson 1937) and Lilium (ll-talker 1940).

SUMMARY

The external morphOIOgy of the bulbs of three cultivars (Olympic.
Fiesta. and Belling-ham hybrids). of the genus M has been described.
Scales detached from these bulbs were incubated in moist peat at 70
degrees F to promote bulblet development. Sample scales were taken
at three-day intervals over a period of six weeks. and prepared for
microscopic examination.

The origin of the bulblet has been shown to be derived by the simul-
taneous division of epidermal and sub-epidermal cells following periderm
and callus formation on the wound surface. Prior to root development
vascular connections developed between the bulblet primordium and the
vascular tissue of the parent scale by the differentiation of existing
parenchyma or of cells of the callus tissue.

The initiation of new bulblets occurred throughout the six week

period of examination. .

18

BIBLIOGRAPHY

CLAMP. G. 1934. Leaf development and vegetative propagation in
Kalanchoe tubiflora. Proc. Bot. Soc. Edinburgh 31: 327-338.

 

De GRAFF, J. 1951. The New Book of Lilies. M. Barrows and Co.
Inc.. New York.

EMSWELLER. S. L. 1937. Frapagation of Lilies. North America
Lily Soc. Yearbook 10: 7-18.

GRIFFITHS. D. 1930. The Production of Lily Bulbs. United States
Department of Agriculture Circ. 102.

LAUER. F. I. and KRANTZ. F. A. 1957. Formation of buds from
callus tissue in potato. Amer. Potato Jour. 34: 158-164.

PRIESTLEY. J. H. and Sh-‘INGLE. C. F. 1929. Vegetative pmpa-
gation from the standpoint of plant anatomy. United States Depart-
ment of Agriculture Tech. Bul. 151.

. and liOF F ENDEN. L. M. 1922. Physiologcal
Studies in Plant Anatomy 5: Causal Factors in Cork Formation.
New PhytOIOgist 21: 252-268.

 

ROCKRELL F0 F0. GRAYSON. E0 Co. and MRAFF. I. 1961.
The Complete molt of Lilies. Doubleday and Co. Inc.

McVElGH. I. 1933. Regeneration in Crassula multiclava. Amer.
101113 at 25: 7'11.

 

NAYLOR. E. 1932. The Morphology of Regeneration in Bryophyllum
calycinum. Amer. Jour. Bot. 19: 32-4-0.

 

and JOHNSON. BETTY. 1937. A HistolOgical Study of
Vegetative Reproduction in Saintpaulia ionantha. Amer. Jour. But.
24: 673-678.

 

 

l9

20

WALKER. R. I. 1940. Regeneration in the scale leaf of Lilium candidum
and Lilium longiflorum. Amer. Jour. Bot. 27: 114-117.

 

 

WALLACE. Dr. 1879. Notes on Lilies and their Culture. Colchester.
England. '

YARBROUGH. J. A. 1932. Anatomical and Developmental Studies of the
foliar embryos of Bryophyllum calycinum. Amer. Jour. lbt. 19:
443-453. ‘ '_"

 

ROGM USE ONLY.