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This is to certify that the

dissertation entitled

Organogenesis and Somatic Embryogenesis in
Tissue Cultures of Apple(Malus domestica Borkh.)

 

presented by

JANG RYOL LIU

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in HortiCU1tUY‘e

(.

 

Major professor

Date Dec. 1, 1981

 

MS U is an Ajfirmatiw Action/Equal Opportunity Institution 0~ 12771

 

 

 

bV1ESI_J RETURNING MATERIALS:
Place in book drop to

LJBRABJES remove this checkout from

.——. your record. FINES will

 

 

 

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stamped below.

 

 

 

ORGANOGENESIS AND SOMATIC EMBRYOGENESIS
IN TISSUE CULTURES OF APPLE

(MALQS pgugsllcg BORKH.)

BY

JANG RYOL LIU

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Horticulture

1981

ABSTRACT

ORGANOGENESIS AND SOMATIC EMBRYOGENESIS
IN TISSUE CULTURES OF APPLE

(MALflfi MEST A BORKH.)
BY

JANG RYOL LIU
Leaf, cotyledon, and hypocotyl explants were obtained
from 2- to 3-week-old open-pollinated seedlings of 'Golden

dg estiga Borkh.)

Delicious' (GD) apple (MaIu§

germinated in vitro in the light. Explants were placed

 

on a basal medium (BM), consisting of Murashige and Skoog
(MS) inorganic salts, BS vitamins, sucrose and agar,
supplemented with 6-benzylaminopurine (BAP) and
a-naphthaleneacetic acid (NAA), and were maintained at
2§D¢ 2°C in the light (cool white fluorescent lamps;
1.5 wnéz; 16 h per day) or in the dark. Leaf and cotyledon
explants cultured in the dark for the initial 3 weeks, then
transferred to light, produced 5- to 20-fold more
adventitious shoots than did those cultured:h1the light.
However, light did not significantly'influence the number
of adventitious shoots formed on hypocotyl explants. A
10-day dark treatment significantly increased the number
of adventitious snoots formed on leat‘explants regardless
of subsequent exposure to light. Five-min daily exposures
of leaf explants to red light (651 nm) suppressed
adventitious shoot formation by 80%; exposure to far-red

light (729 nm) immediately following the red light counter-

acted the effect of the latter.

JANG RYOL LIU

Light inhibited callus formation on leaf and cotyledon

explants but not on hypocotyl explants. Callus derived

from leaf explants from seedlings, immature fruit halves,

and immature embryos of GD was cultured on BM supplemented
with BAP alone or BAP plus NAA.

Globular to heart-shaped somatic embryos were induced
on leaf explants from seedlings cultured on BM supplemented
with BAP and NAA. The somatic embryos were observed 3 to
4 weeks after culture in the dark but not in the light.
On excision and transfer to BM with or without growth
regulators, most of the embryos did not undergo further
development, but 6 attained the cotyledonary stage. Two
of these developed into intact bipolar structures with root

and shoot apices.

an My Iflarentz

ACKNOWLEDGMENTS

I wish to express my sincere appreciation to my co—
advisors, Drs. K.C. Sink and F.G. Dennis, Jr. for their
valuable criticism, suggestions and encouragement during
the course of this research; also my committee members:
Drs. R.F. Carlson, J.W. Hanover, E.J. K103 and R.L. Perry.

A special note of thanks is given to Mr. C.B. chg
for his willing aid in the photobiology experiment.

I am gratefully indebted to the Department of
Horticulture for my research assistantship.

I wish to return my best thanks to my parents for their
steadfast love and prayers to God for my health and

happiness.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES
LIST OF FIGURES
INTRODUCTION -------------------------------------------- 1
LITERATURE REVIEW ....................................... 2
I. Introduction ................................... 2

A. Historical review of plant tissue culture ----2
B. Current status of tissue culture in woody

plants --------------------------------------- A

II. Woody plant organogenesis in yitro ------------- 6

A. Explants ------------------------------------ 10

B. Hormonal control ---------------------------- 11

III. Woody plant somatic embryogenesis in yitrg ----12

A. Explants ------------------------------------ 13

B. Hormonal control ---------------------------- 16

IV. Effects of light on morphogenesis lg yitrg ----- 18

A. Light intensity ----------------------------- 19

B. Photoperiod --------------------------------- 19

C. Spectral quality ---------------------------- 20

V. Epilogue -------------------------------------- 20

Section I
PLANT REGENERATION FROM APPLE (MALUS DQMESILQA
BORKH.) CALLUS, LEAF, COTYLEDON,
AND HYPOCOTYL EXPLANTS

Abstract ------------------------------------------------ 23
Introduction -------------------------------------------- 25
Materials and methods ----------------------------------- 25
Results ------------------------------------------------- 33
Discussion ------------------------------------------- ----51
References ---------------------------------------------- 56

iv

Page
SECTION II
SOMATIC EMBRYO FORMATION ON CULTURED LEAF

EXPLANTS OF APPLE (MALUS DOMESTICA
BORKH.) SEEDLINGS

Summary ---------------—---—------—5 ..................... 59
Introduction -------------------------------------------- 60
Materials and methods ----------------------------------- 60
Results ------------------------------------------------- 61
Discussion .............................................. 53
References .............................................. 59
SUMMARY AND CONCLUSIONS ----------------------------------- 71
BIBLIOGRAPHY .............................................. 7n

LIST OF TABLES

Table Page

LITERATURE REVIEW

1. Instances of adventitious shoot formation
in ELLEQ for woody species ........................ 7

2. Instances of asexual embryogenesis
in yitrg for woody plants other than
the Rutaceae ..................................... 1n

SECTION I

1. Components of media used for induction of callus,

adventitious shoots, and roots in apple

explants (mg/l). --------------------------------- 26
2. Analysis of variance for effects of light, BAP,

and NAA on adventitious shoot induction on leaf,

cotyledon, and hypocotyl explants from seedlings.-37
3. Red/far-red light effects on numbers of

adventitious buds and shoots formed on leaf

explants from seedlings. ------------------------- A3

vi

LIST OF FIGURES

Figure Page

SECTION I

1. (a) Etiolated adventitious shoots formed on leaf
explant in the dark. (b) Elongated adventitious
shoots on leaf explant. (c) Elongated adventitious
shoots on cotyledon explant. (d) Elongated
adventitious shoots on hypocotyl explants. (e)
Roots formed on adventitious shoots. (f) Plants
regenerated from explants from seedlings. -------- 3A

2. Effects of light, BAP, and NAA on adventitious
shoot induction on (a) leaf explants, (b)
cotyledon explants, and (c) hypocotyl explants
from seedlings ----------------------------------- 38

3. Effects of 10 day intervals in light and dark
on adventitious shoot induction on leaf explants
from seedlings ----------------------------------- 40

A. Effects of IAA, IBA, and NAA on root induction
on adventitious shoots from leaf, cotyledon, and
hypocotyl explants: (a) shoots derived from leaf
explants; (b) shoots derived from cotyledon
explants; (c) shoots derived from hypocotyl
explants ----------------------------------------- A5

5. (a) Shoot primordia (arrows) regenerated from
callus derived from leaf explants from seedlings.
(b) An elongated shoot from callus derived from
leaf explants from seedlings. (0) Same as 'b'
with elongated axillary shoots. (d) A plant
regenerated from callus derived from leaf
explants from seedlings. (e) Regenerated roots
from callus derived from leaf explants from
seedlings. (f) A shoot regenerated from callus
derived from receptacle and ovary tissues from
immature fruits. (g) A shoot regenerated from

callus derived from immature embryos. (h)

Adventitious shoots formed on mature leaf

explants. ---------------------------------------- A8
6. Effects of BAP and NAA on adventitious shoot

formation on mature leaf explants ---------------- 52

vii

Figure Page
SECTION II

(a) Globular to heart-shaped somatic embryos
(arrows) on a leaf section from an apple
seedling. (b) Heart-shaped somatic embryos.

(c) Globular somatic embryos after transfer to
BM. (d) A somatic embryo with fused cotyledons
and a radicle (arrow). (e) A somatic embryo
with 5 cotyledons. (f) A pair of leaf primordia
(arrow) on a somatic embryo clustered with the
neighboring somatic embryos. (g) A somatic
embryo with developing primary root, hypocotyl
and with developing shoot. (h) Same as 'g' with
developing shoot. (1) A somatic embryo with
developing primary root, hypocotyl and cotyledons.

(3) Same as 'i' with numerous roots. (k) Same as
'j' with multiple shoots.(l) Plantlets from
somatic embryos. ................................. 66

viii

Guidance Committee:

The thesis is organized in journal style in accordance
with departmental and university requirements. Two sections

were prepared following the journal article formats of

 

Blani_9elli_lia§ue_Qrsan_gulture and Plant Science
LELLQLS, respectively.

~ix

Introduction

In general, plant regeneration in yitro from single

 

cells or tissues of woody species is rare, and limited to
embryonic or juvenile sources. The biochemical differences
between cells which differentiate readily and those which
do not are still obscure. In practice, effective protocols
for plant regeneration as well as the biochemistry of dif-
ferentiation must be studied in order to understand
morphogenesis.

In apple spp. organogenesis in yitgg has not been
demonstrated; although induction of asexual embryos from
uninucleate microspores and nucellar explants is possible,
none of the embryos has developed into a plantlet.

The aim of this study was to assess the morphogenetic
potential of explants from apple (Mglus QQQQSLIQQ
Borkh.) in litgg, emphasizing effects of light and
growth regulators. Very young seedlings (2 to 3 weeks old)
germinated in xitro, immature fruits, and immature
embryos were used because they were assumed to have high
morphogenetic potentials. The system developed for explants
from seedlings was also applied to leaf explants from mature
'Golden Delicious' trees. Leaf explants from seedlings
showed relatively high potential for formation of organs

and somatic embryos which in turn developed into plantlets.

LITERATURE REVIEW

I. Introduction

Plant tissue culture, or the study of plant
protoplasts, cells, tissues, or organs onlmnndent medium
under aseptic conditions, serves many different purposes
in plant science. Because excised plant parts can be
cultured in vitae without whole plant influence the
technique allows the study of dedifferentiation and
morphogenesis of selected plant parts. Tissue culture also
permits rapid clonal propagation of selected genotypes or
of pathogen-free, especially virus-free, plants. Tissue
culture promises to provide new genotypes through genetb:
engineering or somatic hybridization. This review deals
primarily with the status, problems, and potentialities
of organogenesis and somatic embryogenesis in tissue
cultures of woody plants.

A. Historical Review of Plant Tissue Culture

The technique of culturing isolated plant cells was
pioneered by Haberlandt as early as 1902 (A0). Although
he failed to induce cell division, he did provide the
concept of cell totipotency, the potential of a single cell
to develop into an intact plant. Many years passed before
excised plant tissues were grown for potentially unlimited

2

3

periods in liggg, as White (127) demonstrated by the
culture of root segments of tomato in liquid medium in 193A.

In 1939 Gautheret (38) and Nobécourt (79)
independently succeeded in indefinite culture of callus
derived from carrot root segments by adding indole-3-acetic
acid (IAA) to the medium. In the same year White (125)
reported unlimited growth of callus from young stem tissue
of a hybrid Nicotiana (N. glauca x N. lahgsdorffii) on the
same medium that he had developed for growing excised root
segments of tomato, but solidified with 0.5% agar.
Furthermore, he (126) induced shoot differentiation from
the callus by transferring it to liquid medium.

In 1956 Miller et al. (68) both discovered and
synthesized the first cytokinin, 6-furfurylaminopurine
(kinetin). This discovery led them to propose the important
concepts of chemical control and manipulation of
organogenesis, whereby quantitative interactions between
cytokinins and auxins regulate shoot and root initiation
(101). In 1957 Skoog and Miller (101) demonstrated that
high ratios of kinetin to IAA induced shoots, while the
reverse ratio induced roots in tobacco stem segments and
callus.

In 1958 Reinert (93) and Steward et al. (109)
independently succeeded in inducing somatic embryos from
- suspension-cultured carrot cells. This remarkable work
was the dream of Haberlandt (40) who wrote, "... I believe,

in conclusion, that Iham not making too bold a predicthmu

u
if I point.tx> the possibility that, in this way, one could
successfully cultivate artificial embryos from vegetative
cells."1

In 1960 Morel (72) developed shoot tip cultures for
clonal propagation and recovery of virus-free plants. IHis
new technique has since been successfully applied to
numerous economically important crops.

In 1960 Cocking (26) pioneered the isolation of plant
protoplasts by dissolving the cell wall of tomato roots
with enzymes, which opened the new era of somatic cell
genetics. The breeding barriers between different plant
species may theoretically be overcome by fusing their
protoplasts. Carlson et al4 (18) first produced a somatic
hybrid plant by means of protoplast fusion between N.
glauca and N. langsdorffii in 1972.

‘B. Current Status of Tissue Culture in WoOdy Plants

Considerable success has been achieved incnuturing
dicotyledons, especially those of the Solanaceae. However,
monocotyledons and woody plants including both angiosperms
and gymnosperms have been far less amenable. Many
investigators (e.g. 12, 31) have reported many more
difficulties with these plants in inducing both cell
<division and organogenesis by using current tissue culture
techniques.

Nevertheless, in the last two decades, some remarkable

 

1English translation from (57).

5
demonstrations of embryogenesis and organogenesis have been
reported for woody plants.

Somatic embryogenesis from nucellar callus
(pseudobulbil) of Qggggs was first reported by Maheshwari
and Ranga Swamy (6A) in 1958. Later; both Singh (98) and
Sabharwal (9A) induced somatic embryos from nucelli and
ovules of polyembryonic species of Citrus cultured in
,ggggg. Other investigators succeeded in inducing somatic
embryos directly from nucelli of moncembryonic species of
Citrus containing either fertilized (87) or unfertilized
(70) ovules. Furthermore, in 1975 Vardi et al. (120)
demonstrated somatic embryogenesis of Qitggg from
protoplasts isolated from unfertilized ovular callus.

Besides gigggs and related genera, somatic
embryogenesis has been demonstrated directly from explants
or via callus in a few woody angiosperms. In 1979 Eichholtz
(3A) induced somatic embryos from excised nucelli of
NaINs. From callus of Qgfifeg, derived from soft
internodes of young orthotropic shoots, somatic embryos
were induced by Staritsky (108) in 1970. Various seedling
tissues including shoot tip, leaf, cotyledon, hypocotyl
and root tip have been cultured to study their potential
for organogenesis. In 1974 Mehra and Mehra (65) obtained
shoots from subcultured callus derived from figgggs leaf,
cotyledon, and embryo sections.

As for gymnosperms, Ball (6) first succeeded in

inducing shoots from callus derived from green shoots of

6

a Segggia burl in 1950. Konar and Oberoi (56) observed
somatic embryogenesis in cotyledonary cultures of 519;;
in 1965. Shoots were also induced from subcultured callus
of fisegdgtsgga by Cheng (23) in 1975 and by Winton and
Verhagen (132) in 1977. Induction of adventitious buds
for mass clonal propagation ig liggg of various seedling
tissues of conifers has been reported (e.g., 17, 105).

To date, most demonstrations of embryogenesis or
organogenesis in woody plants have involved embryonic or
juvenile tissues. Few papers have reported success with
mature tissues. Basic studies on the physiological and
biochemical changes occurring during phase transition may

reduce the difficulties presently encountered with woody

plants Lg vitro.

II. Woody Plant Organogenesis IQ Niggg

Cells and tissues of woody species can be induced
to form a variety of organs such as roots, shoots and
flowers in yitng. The initiation and subsequent
development of these organs is termed organogenesis, and
involves two distinct phases: dedifferentiation of the
initial explant followed by redifferentiation into organs
demewn.

In this chapter, a review of caulogenesis or
adventitous shoot formation for woody species is presented.
Table 1 provides a summary of'adventitious shoot formation

observed i3 vitro for woody species.

 

7

Table 1. Instances of adventitious shoot formation ig_vitro for woody

 

 

 

 

 

 

species
*
Species Explant Subculture Reference
of callus
Angiosperm
Nessie 59s, Shoot tip + 100
Betula pendula Stem + A8
Broussone kazinoki Hypocotyl - 81
Cisrss grandis Leaf + 20
Stem + 20
sinensis Leaf + 20
Stem + 20
ngfea arabica Leaf - A5
Elaeis guineehsis Leaf + 51
Root + 51
Eucalyptus alba ’§ Hypocotyl + 5A
citriodora Cotyledon - 60
Lignotuber + ~ A
Hedera helix -- + 53
Cotyledon - 7
Hypocotyl - 7
Nelss sylvestris Shoot tip - 1
MQ£Q§.élD§. Leaf - 82
Beagles. W Leaf + 15
canesc Shoot tip + .19
x canescens Shoot tip + 10
(E. deltgides x.£. nigra)
euroamericana Shoot tip + 19
Stem + 19
nigra
var. typica Shoot tip + 19
Stem + 19
var. italica Stem + 122

simonii x.£. nigra
Anther + 5

Table 1 (cont'd)

 

 

 

Species Explant Subculture Reference
of callus
tremula Stem + 19
ggemula (An) Stem + 129
themuleides (3n) Stem + 130, 131
W Anther' + 5
Erunus amygdalis Cotyledon + 65
Embryo + 65
Leaf + 65
Eutrsjiva rgxburghii Endosperm + 107
Ross sp. (hybrid) Stem - 50
Ssstalum alsum Hypocotyl - 89
Ulmus americana Hypocotyl + 32
Nessigigg,sngsstifolium Cotyledon - 78
Hypocotyl - 78
Gymnosperm
Abies balsamea Shoot tip - 12
Biots scientalis Upper part of - 115
seedling (UPS)
CnprQmeria japgnics Hypocotyl - A9
9mm grim UPS - 115
macrocsrps UPS - 115
sempegyiregs UPS - 115
Legig dahurica Leaf - 71
Pices abies (1n) Endosperm + 12
glauca Hypocotyl - 16
sitchensis Embryo - 123
Shoot tip - 123
Pinus contorta Embryo - 123
elliottii Cotyledon - 28
Embryo - 10A
palgeris Cotyledon - 28
Embryo - 10A

Table 1 (cont'd).

 

 

Species Explant Subculture Reference
of callus

radiata Embryo - 90
rigida Cotyledon - 28
sssigisgs_ Cotyledon - 28
simple Embryo - 69
taeds Cotyledon - 28, 66
Hypocotyl - 66
virginiana Cotyledon - 13, 28
Esesdgtsuga menziesii Cotyledon - 2A, 132
+ 132
Embryo - 21, 102, 132
Segggis_gigsgtea Cotyledon - 28
Hypocotyl - 28
sempervirens Stem + 6
Thsja ogcidentalis UPS - 115
plicata Cotyledon - 27
Shoot tip - 27
Tsuga heterophylla Cotyledon - 22

 

i
+: Induction of shoots from subcultured callus.

Induction of shoots directly from the initial explant

or through a minor callus attached to the explant.

10
A. Explants

Successful organogenesis for many woody plants
requires the use of juvenile explants (e.g., 60, 89, 113).
Explants such as cotyledons and hypocotyls excised from
embryos or seedlings are common tissue sources (Table 1).
Why explants taken from the adult phase seldom undergo
organogenesis is yiigg still remains obscure.
Differences between juvenile and mature forms are maintained
jxlcuilture. For example, tissue specific characteristics
were maintained in callus cultures derived from both
juvenile and adult phases of fléflfifié spp.vnth monthly
subculture for over two years (112).

In addition, the organ from which explants are excised
and the presence of other organs may determine response.
Hypocotyl.explants from excised embryos of N. heiii
had lower potential for shoot formation than cotyledons,
but a higher potential for root formation, while whole
embryos and excised organs from germinated seedlings
produced only callus (7).

Sometimes, different sites on the same organ have
variable potentials for organogenesis. Explants from the
proximal portion of Nggss gigs leaves exhibited
increased potential for caulogenesis compared with those
from the distal portion (82).

Explants also show polarity in regard to
organogenesis. The potential for shoot and root formation

on hypocotyl explants of N, helii resided entirely in

11
the proximal and distal end, respectively (7). A polarized
gradient of phytohormones might be involved in such
regeneration patterns (121).

Organogenesis may occur either directly from the
explant or through an intermediary callus. However, the
induction of organs directly from explants is more frequent
and constant (7A).

An extensive period of callus culture, especially
through increased numbers of subcultures, reduces
morphogenetic potential. Such loss of the potential to
differentiate has been explained by accumulated aneuploidy,
chromosome aberration, hormonal habituation, and
heterochromatization as well as spontaneous mutation and
selection during extensive culture (39).

B. Hormonal Control

The concept of auxin-cytokinin balance in regulation
of organogenesis (101) has been confirmed in a number of
species (75). However, the molecular basis of the
auxin-cytokinin interaction is still unknown.

In many conifers, the induction of adventitious shoots
from excised mature embryos does not require exogenous
cytokinin (e.g., 90, 132). Leaf explants from a few species
of figsgss rootstocks produced adventitious shoots on a
medium supplemented with 6-benzylaminopurine alone in our
laboratory (J.R. Liu and M.A. Palenick, unpublished). If
the concept of auxin-cytokinin balance for organogenesis

is valid, such exceptions might be explained by high levels

12

of endogenous auxin. Evidence exists that exogenous
application of cytokinin may induce auxin biosynthesis (52)
or auxin application may induce cytokinin biosynthesis (35,
133). Therefore, the concept of auxin-cytokinin balance
in regulation of organogenesis should be understood at the
endogenous hormonal levels of the organ-forming loci.

Besides auxin and cytokinin, other hormones play a
minor role in organogenesis. Although gibberellins (GAs)
are generally without effect in inducing organogenesis is
yiigg, GA3 stimulates shoot initiation from callus of
some herbaceous plants such as Chrysanthemum (33) and
AEEQIQQRSIS (76). Similarly, abscisic acid (ABA)
stimulates adventitious shoot formation on sweet potato
root tuber tissues (135) and protoplast-derived potato
callus (97), and adventitious root formation on hypocotyl

explants of Phaseolus coccineus (2).

III. Woody Plant Somatic Embryogenesis lg Niigg

All cells of higher plants potentially are capable
of expressing the developmental pattern of zygotic
embryogenesis. The process of embryo initiation and
development from cells that are not products of gametic
fusion is termed somatic, asexual, or adventive
embryogenesis (117). However, somatic embryogenesis cannot
be a synonym for asexual or adventive embryogenesis; the
term somatic embryogenesis should be restricted to somatic

cells, while the terms asexual or adventive embryogenesis

13
may be used to refer to the general case including embryo-
genesis from unfertilized gametic cells such as microspores.

The decisive feature of an embryo is bipolarity, with
root and shoot apices integrated into one axis at the
earliest developmental stage (A1).

Induction of somatic embryos is signs in woody
plants has been limited primarily to the Rutaceae or citrus
family in which many species produce nucellar embryos is
sils (1A). Table 2 lists the woody plants other than
the Rutaceae in which asexual embryogenesis has been
demonstrated is sisss. Note, however, that intact
plantlet formation has not been demonstrated in all cases.

A. Explants

In the Rutaceae, the nucellus has been the primary
explant (117, 1A). The successful induction of somatic
embryos directly from nucellus explants from‘monoembryonic
citrus spp. (88) has stimulated other investigators to
examine whether nucellus explants from other monoembryonic
fruit trees could give rise to somatic embryos. Success
has been achieved in Nsiss sssessiss (3A) and Esssss
hensiea (110).

In addition to the nucellus, other organs related
to megaspore production” such as the placenta, integument,
ovule, and ovary could be alternative sources of explants.
Through an intermediary callus, inner integument or ovule
explants produce somatic embryos in Cssiss (29),

Easiownis (8A) and Vitis (73).

1A

Table 2. Instances of asexual embryogenesis is vitrp for woody plants
other than the Rutaceae

 

 

 

*
Species Explant Callus Reference
intervention
Angiosperm
Assssips,hippppastanss Anther - 85
Carica papaya Petiole + 29
Ovule +/- 62
Coffea arapica Anther + 96
Leaf + 106
Stem + 108
Corylus avellana Zygotic embryo + 86
assess helix Stem + 8
Hevea brasiliensis Anther + 25
Stem + 128
Ilex aguifplium Zygotic embryo +/- A6, A7
cornuta Zygotic embryo +/- A6
Hypocotyl + A7
QDQQQ Hypocotyl + ' A7
Ligpidambsp styraciflua Hypocotyl + 103
Nsiss,gomestips Anther - 67
Microspores - 59, 67
Nucellus - 3A
We tomatoes. Ovule + 84
Ptunts torsioa Nucellus - 110
Ovule - 111
Ssstalum aipum + 61
Vitis vinifera Ovule + 73
sp. Stem + 58
Gymnosperm
Abiss baisames Shoot tip - 11
Riots. ot‘ientalis Cotyledon - 56

15
Table 2 (cont'd)

 

 

 

Species Explant Callus Reference
intervention

,Einus.toooa. Shoot tip + 30

Psspsptssgs,meszissii Shoot tip + 30

s

+: asexual embryogenesis through an intermediary callus.
-: asexual embryogenesis directly from the initial explant.

16

Tissues from young seedlings also give rise to somatic
embryos. Embryos were induced directly from hypocotyl and
cotyledon explants in Blgté.(56), or through intermediary
callus formation in Eisss (103). Induced somatic embryos
may produce accessory, or smaller additional embryos,
without forming an intermediary callus (e.g., 103, 58).

Haploid embryos have been induced via culture of
anthers or microspores. When asexual embryosenwaihduced
directly from uninucleate microspores, they are usually
haploid, but if callus formation occurs, they may be
haploid, diploid or polyploid (67, 8A, 96, 101).

B. Hormonal Control

In general, somatic embryos can be induced in most
embryonic tissues on a basal medium consisting of a balanced
inorganic mixture, vitamins and sucrose, as demonstrated
for nucellus explants from Cissss and related genera
(117), Malta (3A) and Ilex. spp. (A6, A7). Somatic
embryo initiation and development from nucellus explants
of polyembryonic Ciisss sssississs were depressed
markedly by adding 2,A-dichlorophenoxyacetic acid (2,A-D),
ABA, and ethephon, and slightly inhibited by IAA,
kinetin, and GA3. Citpss ovules excised from young fruits
of the monoembryonic type contained IAA, ABA and GA3 levels
several fold higher than those found incmnfles of the
polyembryonic type (119).

However, when callus formation precedes embryo

initiation, auxins, especially 2,A-D, may have stimulatory

17
effects on callus induction and subsequent embryo
initiation. Preculture with 2,A-D, followed by transfer
to a medium without 2,A-D, leads to somatic embryogenesis
in a few herbaceous plants such as stsss spp. (e.g.,
AA, 92). Likewise, callus induced on.a medium with 2,A—D
gave rise to somatic embryos of Csiiss and lisis after
transfer to a medium with a weaker auxin such as
-naphthaleneacetic acid (58, 106). On the other hand,
1.0 mg/l 2,A-D did not inhibit the induction of somatic
embryos from zygotic embryo callus of Cosylps, although
it prevented further development into plantlets (86).
Cytokinins are not essential for somatic embryogenesis
is yipss and are sometimes inhibitory as previously
mentioned. However, in combination with auxin, a low level
of cytokinin stimulated somatic embryo initiation in
nucellar callus of Nisis (73)- Even higher ratios of
cytokinin to auxin were required to induce embryogenic
callus in leaf explants of Cpffes arabica (106).’
Although GAs generally depress somatic embryo

initiation, GA stimulates root emergence in somatic embryos

3
of a few plants such as Citrus (55). Both cytokinin and
GAs were required for the development of somatic embryos
of yisis into plantlets (73).

ABA suppressed developmental abnormalities, such as
malformed cotyledons and accessory embryos, in somatic

embryos of Cssss sssyi, a herbaceous species, while

zeatin with or without GA increased the frequency of such

3

18

abnormalities (3).

IV. Effects of Light on Morphogenesis is Nisss

In plant tissue cultures, light is essential for
phytomorphogenesis but not for growth. Because
carbohydrates provided in the basal medium supply a readily

available energy source, plants is vitro are considered

 

heterotrophic. However, photosynthesis is limited in
chlorophyll-containing plant parts cultured is yisss
despite relatively high light intensities.

Light may control various aspects of phytomorpho-
genesis, including shoot and root initiation and possibly
somatic embryogenesis. Light was necessary for shoot
induction in Nisssisss tabapsm cv. 'Wisconsin 38' callus
(12A), but inorganic and organic additives in the basal
medium could substitute for the light requirement (116).
A tobacco hYbrid (N. glauca x N. lanssoonitii)
callus produced shoots in the dark, but not under high light
intensity (101). Light was also essential for root
development in Heiissssss tissues, provided auxin was
omitted (37). High light intensity stimulated induction
of somatic embryos from Nipptiana callus (A2).

Light as an environmental parameter of tissue culture
must be considered in terms of its components, such as light

intensity, photoperiod and spectral quality.

19
A. Light Intensity
Most tissue culture laboratories adopt lux as the
unit for light intensity. However, the term expresses
luminous intensity, that is, luminous flux area density,
which is not relevant to plant science research. The use
of this term is therefore not recommended (9). Many

’1 as the

publications on plant tissue culture use pEm’ZS
energy unit for light intensity. However,:U;is not part
of the international system of units (SI) (83) and should
not be used (9). A proper energy unit suggested by photo-
biologists is Wm'z, which is the incident radiant flux area
density, defined in SI as the irradiance (83).

When Sylvania Gro-Lux lamps were employed to provide
a 16 h daily exposure, Murashige (75) determined that
1,000 lux could be used for many herbaceous species for
Stage I (period for establishment of‘explants), and 1,000
to 3,000 lux for Stage II (period for multiplication of
propagula). With General Electric cool white lamps 3,000
to 10,000 lux was optimal for Stage III (period for rooting
and acclimation before transfer to soil).

B. Photoperiod

Because the total radiant energy of specified spectral
quality to which the culture is exposed could determine
the rate of growth and differentiation, the optimal

photoperiod may depend upon the light intensity used (75).

In addition, plants for which morphogenesis is affected

20
by photoperiod is yiys may require the same

photoperiodic cycle is vitro (75).

 

C. Spectral Quality

The spectral quality of the light significantly
controls phytomorphogenesis not only is yiys but also
is yiisp. Shoot induction from Nissiisss callus was
enhanced by blue light, while red light was equivalent mo
darkness (95, 12A). In lettuce cotyledon cultures, red
light enhanced shoot induction, a phytochome-mediated
response (53). In Hsiisnthss tuber sections, red light
(near 660 nm) was most effective in initiation of roots
(95). Red light was necessary to stimulate haploid
embryogenesis in Nicotiana microspores on a sucrose-free
medium (80). In contrast, short exposures to far-red
illumination enhanced asexual embryogenesis in Nsssss
carpts callus (77). In addition, near ultraviolet light
(371 nm) either stimulated or inhibited shoot induction
from Nisssisss callus depending upon light intensity,
indicating that flavonoids may have mediated the response

by acting as photoreceptors (95).

V. Epilogue

Phytomorphogenesis is a complex physiological process
controlled by a number of'intrinsic and extrinsic factors.
One or a few chemicals are not sufficient to substitute
for organ-specific morphogens (36, 39), such as rhizocaline,

caulocaline and florigen, which denote the conceptual

21

hormones that control morphogenesis. Because woody species
live for many years, their physiological status changes
continuously with ontogeny and seasonal fluctuations.
Therefore, the explant source is much more critical for
the induction of organs ss ssys in woody plants than
in herbaceous plants. For example, the callus from juvenile
stems of fisssss seiii forms shoots and roots, while
the callus from mature stems forms somatic embryos (8).

Why do some tissues have a higher potential to
differentiate is yisss than others? Wolpert (13A)
postulated that concentration gradients of morphogens exist
within organisms and that the developmental fate of any
given cell is determined by its position within these
gradients. Halperin (A3) has suggested that cultured cells
may retain epigenetic factors characteristic of their
differentiated functions in the tissue from which they were
excised and he attributed lack of regenerative competence
of some tissues to incomplete dedifferentiation at the
biochemical level.

What triggers cells to undergo organogenesis or
somatic embryogenesis remains obscure. Street (11A)
postulated that if polarity develops within a group of
meristematic cells as a whole, unipolar growth or an organ
primordium forms, but if the polarity develops within a
single cell, an embryo will form.

In general, woody species are less suitable to studies

of the physiology and biochemistry of morphogenesis than

22
herbaceous species due to the low frequency of
differentiation is yisss. However, the nucellus in
many species of the Rutaceae retains.a high potential flu?
embryo formation and is often used for the study of somatic
embryogenesis. The study of juvenility also benefits from
tissue culture as shown in H. ssiix.

Because of the high economic value of many woody
species, the manipulation of organogenesis and asexual
embryogenesis will be emphasized more in the next decade.
Adventitious bud formation following irradiation of leaf
explants enables us to select desirable mutants for research
and applied purposes. Somatic embryos are attractive to
pomologists because they serve as true-to-type seeds.
Haploid plants arising from androgenesis will help tree
breeders save time and labor. In addition, both genetic
engineering and somatic hybridization through protoplast
fusion will be realities for use in woody plant tissue

cultures.

SECTION I
PLANT REGENERATION FROM APPLE (MALUS DQNESILCA
BORKH.) CALLUS, LEAF, COTYLEDON,

AND HYPOCOTYL EXPLANTS

PLANT REGENERATION FROM APPLE (MALQ§ DQMESIICA
BORKH.) CALLUS, LEAF, COTYLEDON,
AND HYPOCOTYL EXPLANTS

Abstract

Leaf, cotyledon, and hypocotyl explants were obtained
from 2- to 3-week-old open-pollinated seedlings of 'Golden
Delicious' (GD) apple (Nsiss ssssssiss Borkh.) germi-
nated is yisss in the light. Explants were placed on
a basal medium (BM), consisting of Murashige and Skoog (MS)
inorganic salts, B5 vitamins, sucrose and agar, supplemented
with 6-benzylaminopurine (BAP) and a-naphthaleneacetic acid
(NAA), and were maintained at 25CE;2OC in the light (cool
white fluorescent lamps; 1.5 Wm'2; 16 h per day) or in the
dark. Leaf and cotyledon explants cultured in the dark
for the initial 3 weeks, then transferred to light, produced
5- to 20-fold more adventitious shoots than those cultured
in the light. However, light did not significantly
influence the number of adventitious shoots formed on
hypocotyl explants. A 10—day dark treatment significantly
increased the number of adventitious shoots formed on leaf
explants regardless of subsequent exposure to light.

Five-min daily exposures of leaf explants to red light

(651 nm) suppressed adventitious shoot formation by 80%;

23

2A
exposure to far-red light (729 nm) immediately following
the red light counteracted the effect of the latter.

Excised adventitious shoots were rooted on a medium
consisting of MS macronutrient salts at 1/3 concentration,
micronutrient salts, i-ihositol, thiamine HCL, sucrose and
agar with or without indole-3-acetic acid (IAA), indole-3-
butyric acid (IBA), or NAA under a light intensity of

5.0 wm'2

(16 h per day). Auxin concentration strongly
influenced the morphology of the roots induced.

Leaf, cotyledon, and hypocotyl explants were cultured
on Schenk and Hildebrandt (SH) medium supplemented with
2,A-dichlorophenoxyacetic acid (2,A-D), p-chlorophenoxy-
acetic acid (CPA) and kinetin in the light or in the dark.
Light inhibited callus formation on leaf and cotyledon
explants but not on hypocotyl explants.

Callus derived from leaf explants from seedlings,
immature fruit halves, and immature embryos of GD was
cultured on BM supplemented with BAP alone or BAP plus NAA.
Shoots and roots formed on callus in the dark but not in
the light; the frequency of shoot regeneration was 5% or
less.

Leaf explants from mature GD trees also produced
adventitious shoots in the dark on BM supplemented with
BAP and NAA.

Key words: Plant tissue culture --- Apple --- Plant

regeneration --- Adventitious shoot --- Phytochrome

 

25

Introduction

Morphogenesis is yisss has been demonstrated in
a number of woody species (6,12). However, among fruit
trees, only in the Rutaceae has asexual embryogenesis been
studied in detail (19). Asexual embryogenesis has been
reported in a few other species such as Cesiss (8),
Malta (2,11). Phunus (20) and Iitis (7,13) and
caulogenesis or adventitious shoot formation of fruit trees

only in Citrus (1), Morus (16) and Erunus (10).

 

This paper describes plant regeneration from various
parts of open-pollinated seedlings and mature trees of GD

apple.

Materials and Methods

Bisss_ssiesisis, Mature apple fruits of CD were
collected from trees at the Horticulture Research Center,
Michigan State University, E. Lansing, Michigan. lknmmht
twigs were excised from the trees in March and April.
Immature fruits approximately 2 cm in diameter were
collected in June.

Hedia_and_oultu£e_xessels. Deionized double-
distilled water was used for preparing all stock solutions
and media. The pH of all media was adjusted to 5.8 with

0.1 and 1.0 N NaOH before autoclaving at 1.A6 kg/cm2

for
15 min at 121°C. Unless otherwise indicated, the BM
consisted of Murashige and Skoog (MS) inorganic salts (15),

B5 vitamins (3), 30 g/l sucrose and 8 g/l agar (Table 1).

26

Table 1. Components of media used for induction of callus,
adventitious shoots, and roots in apple explants
(mg/l). Sucrose (30 g/l) and agar (8 g/l) were
added to all media and pH was adjusted to 5.8.

 

 

Components Basal mediuma Medium Rb SH mediumC
NHuNO3 1650.0 550.0 -
NHAHZPOA - - 300.0
KNO3 1900.0 633.3 2500.0
MgSOu-7H20 370.0 123.3 A00.0
MnSOu-HZO - - 10.0
MnSOu-AHZO 22.3 22.3 -
ZnSOu-AHZO 8.6 8.6 -
ZnSOu-7H20 - - 1.0
CuSOu-SHZO 0.025 0.025 0.2
CaC12-2H20 A00.0 166.7 200.0
KI 0.83 0.83 .0
CoC12-6H20 0.025 0.025 0.1
KH2P0Ll 170.0 56.67 -
H3B03 6.2 6.2 5.0
Na2M00u'2H20 0.25 0.25 0.1
FeSOu-7H20 27.8 27.8 15.0
NaZEDTA 37.3 37.3 20.0
i-Inositol 100.0 100.0 1000.0
Nicotinic acid 1.0 - 5.0
Pyridoxine-HCL 1.0 - 0.5
Thiamine-HCL 10.0 0.A 5.0

 

aMurashige and Skoog

inorganic salts (15) plus B5 vitamins (3).

Murashige and Skoog macronutrient salts (15) used at 1/3

concentration found in basal medium.

CSchenk and Hildebrandt (13).

27
The agar was rinsed 3 times with double-distilled water
before use.

The growth regulators, such as IAA, IBA, and (i)-
sis, issss abscisic acid (ABA) (Burdick & Jackson
Laboratories, Inc.), were sterilized separately through
0.22 m filters (MilliporeR) and added aseptically to media
which had been cooled to A50 to 500 C after autoclaving.

The culture vessels used were 60 ml glass bottles,
100 x 80 mm glass Petri dishes, and 60 x 15 or
100 x 15 mm plastic Petri dishes, which.contained 25, 80,
12 or 25 ml of medium, respectively. All Petri dishes

R strips.

were sealed with Parafilm
Exoetimehtal_oonoitions. The standard light
treatment consisted of 16 h daily exposure to approximately
1.5 Wm-2 of cool white fluorescent lamps (General Electric),
the dark treatment of placement in cardboard boxes or
wrapping with aluminum foil. Where other light intensities
are specifically indicated, the same source and length1of
illumination per day were used. Red/far-red light was
generated from incandescent bulbs (Bell and Howell, CPAO)
for slide cube projectors. Red and far-red light were
obtained using interference filters (Baird Atomic) of
651 nm and 729 nm, respectively, with a half band width of 9

to 11 nm. The intensity of red light was 1.15 WmIZ; and

that of far-red light 0.A6 Wm'z. Unless otherwise

indicated, cultures were maintained at 2501 2°C.

28

Etoohimental-oesisn and statistical analxsis.
Unless otherwise indicated, a completely randomized design
was used. The numbers of adventitious buds and shoots were
counted with the aid of a dissecting microscope, and the
data subjected to analysis of variance. Where appropriate,
logarithmic transformation of the data and mean separation
by Duncan's Multiple Range Test (DMRT) were used.

ssyentiiious shoot issuction on leafs cotyledoni
sss_sypssssyi_eiplants from seedlings. The seeds were
removed from the mature fruits after they had been in cold
storage at least 10 weeks at 5); 2°C. Embryos were
excised aseptically from the seeds and placed on BM in 100
x 80 mm Petri dishes. Each Petri dish.contained 10 to 12
embryos. The embryos germinated in a few days under

2 light intensity at 25°: 3°C.

approximately 2.8 Wm'
Two- to 3-week-cld seedlings derived from these embryos
were used as the source of explants for the tissue culture
experiments.

Expanded leaves and cotyledons were excised, and their
margins, including axillary buds and petioles, were
removed. Approximately 1 x 1 cm leaf and 1 x 0.8 cm
cotyledon sections were prepared. In additnnh 1 cm-long
hypocotyl segments were prepared from immediately below
the cotyledonary node.

Leaf explants were cultured in 60 ml bottles, using

2 explants from one seedling in each.vessel. The abaxial

surface of leaf explants was firmly pressed into contact

29

with the medium. Two cotyledon explants from one seedling
were cultured in the same way. Each hypocotyl explant was
placed horizontally on medium in 60 ml bottles.

Three levels of BAP (1.0, 3.0, and 10.0 mg/l) and
5 levels of NAA (0.0, 0.1, 0.3, 1.0, and 3.0 mg/l) were
tested in a factorial arrangement. Half of the cultures
were kept in the light and half in the dark. Dark-grown
cultures were transferred to the light after 3 weeks and
response was determined A weeks after beginning the

experiment.

E:toots_ot_loneth_o£_ihitial-lisht_ano_oank

r n n ven 'tious shoot f rma ' e f e l
:jrom seedlings. During 30 days of culture, leaf explants

were cultured in continuous darkness, continuous light,
or in alternating 10-day periods of'light and darkness on
BM supplemented with 3.0 mg/l BAP and 0.3 mg/l NAA in
60 ml bottles.

Ei£§9t§-92_5:mlfl_flgill_W

'11 ., .q - o-~ :h0o fo ma 1., . l-.f :.-,.n - _ om

ssesiisgs. Two leaf explants were placed on BM
supplemented with 3.0 mg/l BAP NAA in 60 x 15 mm Petri
dishes. Cultures were exposed daily to 5 min of red,
far-red, or red followed immediately by far-red light, or
were left in the dark.

Ropt ispugtipn pp asyentitipus shoots deriyss_isss
.QLRLEHLS flpps seedlings. Adventitious shoots were cultured
on BM supplemented with 0.3 mg/l BAP in 60 ml bottles for

30
2 to 6 weeks. Shoot tips 2 to 3 cm long were then excised
and transferred to rooting medium (medium R; Table 1) in
60 ml bottles. Concentrations of 0.011x31.0 mg/l of IAA,
IBA, and NAA were evaluated for root induction. Rooting
response was evaluated after exposure to approximately

5.0 WEI-2

light intensity for 3 weeks. The percentage of
tips which rooted, the number of roots, and their morphology
were recorded.
Eiteot-ot-lisht-ano-oa2k_tr.oatments_on_oallts
'n i n n la fro li . Each leaf, cotyledon,
or hypocotyl explant was placed on SH medium (Table 1)
supplemented with 0.5 mg/l 2,A-D, 2.0 mg/l CPA and 0.1 mg/l
kinetin (callus culture medium) in 60 x 15 mm Petri dishes.
The abaxial surface of leaf and cotyledon explants was
gently scored with a scalpel blade to stimulate callus
formation. 0f the two leaf and cotyledon explants taken
from each seedling, one was cultured in the light and the
other in the dark, using a randomized complete block design
with seedlings as blocks. Hypocotyl explants were cultured
in the same way but in a completely randomized design.
The total fresh weights of induced callus and explant were
measured 6 weeks after culture.
n a r rom l ri
r nt f ' . Callus derived from leaf,
cotyledon, and hypocotyl explants was subcultured at least
twice in the dark on the callus culture medium, then each

piece of callus of approximately A00 mg was transferred

31
to 60 ml bottles containing BM supplemented with four
levels of BAP ( 1.0, 3.0, 6.0, and 10.0 mg/l ) and 5 levels
of NAA (0.0, 0.1, 0.3, 1.0, and 3.0 mg/l) in a factorial
arrangement. Half the cultures were held in the light and
half in the dark. Also, BAP alone at 0.0, 0.1, and
3.0 mg/l was tested. The numbers of'shoots arising after
A, 6, and 8 weeks were recorded.

In addition, callus derived from leaf explants was
cultured on BM supplemented with 1.0 mg/l BAP. Four levels
of ABA (0.0, 0.1, 0.3, and 1.0 mg/l), adenine sulfate
(0, A0, 80, and 160 mg/l), NaHZPOu-HBO (0, 50, 100, and
200 mg/l), and L-tyrosine (0, 50, 100, and 200 mg/l) were
tested separately for their effects on shoot regeneration.

Resononation_o£_shoots-fnou_oallus_oenite_d_f_tom

exolants_£r_om_imoatune_ituits . I mm a t u r e f r u i t s we r- e

surface-sterilized and rinsed as previously described.
Fruit halves, embryos, and nucellar tissues were cultured
on the callus culture medium in 60 ml bottles in the dark.
Six weeks after culture, induced callus was excised from
the explants and thereafter subcultured at intervals of
A to 6 weeks in 100 x 15 mm Petri dishes on the same medium.

Pieces of callus of approximately A00 mg which had
been subcultured at least twice were placed on media in
60 ml bottles. The test described previously for callus
derived from explants from seedlings (BAP x NAA x
light/dark) was conducted to determine treatment effects

upon shoot regeneration.

32
dven ' ° ho t indu tic l af x nt fr

ssisrs tree . Dormant twigs were excised and placed in
water at 25° 1: f C under diffuse light in the laboratory.
After growth began, the shoots were surface-sterilized in
a 10% CloroxR solution for 10 min and.subsequently rinsed
3 times with sterile water. The shoot tips were excised
and their expanded leaves removed. Shoot-tip explants
approximately 1 cm long were placed on BM supplemented with
3.0 mg/l BAP in 100 x 15 mm Petri dishes. Other
environmental conditions were the same as those used for
embryo culture. Three weeks after culture, actively growing
shoots were selected. Each was transferred to a 60 ml glass
bottle containing BM supplemented with 3.0 mg/l BAP and
0.1 mg/l NAA. Proliferating axillary shoots were
subcultured on the same medium every 3 weeks.

Leaves approximately 1.5 cm long and ,1 cm wide were
excised fiwnn the cultured shoots and cut transversely into
strips approximately 2 mm wide after removing the petioles.
Five to 6 strips from one leaf were placed on BM in 601ml
bottles. Two levels of BAP (1.0 and 3.0 mg/l) and 3 levels
of NAA (0.1, ()g3, and 1.0 mg/l) were tested in a factorial
arrangement. All cultures were kept in the dark for 3

weeks, then transferred to light for an additional week.

33

Results

onentitious_shoot_ino_______uction on .l.a_l_o_o_xe f t logos...
and hypocotyl explants from seedlings. Two weeks after
culture, adventitious buds and leafy shoots were initiated
on leaf, cotyledon, and hypocotyl explants. The number
of shoots increased during the additional weeks of culture.
Shoots induced in the dark were etiolated (Figure 1a), but
resumed chlorophyll biosynthesis within a few days after
transfer to the light. Sections of callus with adventitious
shoots were transferred to BM supplemented with 0.3 mg/l
BAP alone to induce elongation (Figure 1b, c, d).

Approximately 65% of the adventitious buds were
initiated on midribs and cut edges in the proximal zone
of leaf explants and on cut edges in the proximal area of
cotyledon explants, but some were ramdomly distributed<m1
both the adaxial and abaxial surfaces. The buds and shoots
induced on hypocotyl explants occurred randomly both on
the cut edges and on the:surface. Because no intermediary
callus stage was observed under a dissecting microscope,
they appeared to arise directly from the epidermal layer.
Shoots occasionally emerged through the cracked epidermal
layer of cotyledon explants; they may have originated from
the subepidermal layer.

Ten mg/l BAP was supraoptimal for adventitious shoot
formation and these data were omitted in analysis of
variance. Leaf explants cultured in the dark occasionally

gave rise to somatic embryos on BM supplemented with

Figure 1.

3A

(a) Etiolated adventitious shoots formed on a
leaf explant in the dark. (b) Elongated
adventitious shoots on a leaf explant. (c)
Elongated adventitious shoots on a cotyledon
explant. (d) Elongated adventitious shoots on
hypocotyl explants. (e) Roots formed on

adventitious shoots: (from left) i) thread-like

roots; ii) roots with fibrous hairs; iii)
thickened roots; iv) roots from basal callus.
(f) Plants regenerated from explants from
seedlings. Each horizontal bar equals

1.0 cm length except for that in (f) which
represents 10.0 cm.

3S

 

Figure l

36
10.0 mg/l BAP and 3.0 mg/l NAA (9). Adventitious roots
as well as shoots were often produced on leaf and cotyledon
explants.

The induction of adventitious buds and shoots from
leaf and cotyledon explants was suppressed (pk0.01) by light
(Table 2; Figure 2a, b). The mean number of buds and shoots
produced per leaf explant on BM supplemented with 1.0 mg/l
BAP and 0.3 to 1.0 mg/l NAA was 3.5 or more in the dark
vs. less than 0.2 in the light (Figure 2a). Likewise, an
average of more than 5.0 buds and shoots arose on each
cotyledon explant on BM supplemented with 1.0 mg/l BAP and
0.3 mg/l NAA in the dark vs. less than 1.0 bud and shoot
per explant in the light. Suppression of adventitous shoot
induction on both leaf and cotyledon explants was evident
on BM supplemented with 1.0 to 3.0 mg/l BAP and 0.3 to 3.0
mg/l NAA (Figure 2a, b).

In contrast, light suppression of adventitious shoot
formation on hypocotyl explants was not statistically
significant (Table 2; Figure 2c). Explants on BM with BAP
alone at either 1.0 or 3.0 mg/l formed no buds or shoots
except for one cotyledon explant which produced one shoot.

Eftoots_ot_lensth_o£_initial_lisht_ano_oa£k
tr atm s a v titi us sho t formation on leaf e lants
isss_ssssiisgs. Leaf explants cultured in the dark for
the first 10 days produced significantly more adventitious

buds and shoots than did those cultured in the light (Figure

37

Table 2. Analysis of variance for effects of light, BAP,
and NAA on adventitious shoot induction on leaf,
cotyledon, and hypocotyl explants from seedlings.

 

a 6
Source of Degrees of Mean-square ’

variance freedom Leaf Cotyledon Hypocotyl

 

Blocks 1° 0.25 0.03 0.01
no
Treatments 19 0.10 N.S 0.12 0.02 N.S.
i! in
Light (L) 1 0.72 0.93 0.12 N.S.
BAP (B) 1 0.02 N.S. 0.02 N.S 0.00 N.S.
**
NAA (N) A 0.1A N.S. 0.2A 0.03 N.S.
L x B 1 0.01 N.S. 0.02 N.S 0.01 N.S.
L x N A 0.08 N.S. 0.07 N.S 0.01 N.S.
B x N A 0.01 N.S. 0.02 N.S 0.02 N.S.
L x B x N A 0.06 N.S. 0.02 N.S 0.01 N.S.
Experimental 19 0.05 0.0A 0.03
error
d

Sampling error 80/120

 

p

aThe data were log-transformed.
* *

bSignificant at 1% ( ), 5% ( ), or nonsignificant (N.S.).

CTwo independent experiments used as blocks.

dEighty degrees of freedom for leaf and cotyledon explants
and 120 for hypocotyl explants.

Figure 2.

38

Effects of light, BAP, and NAA on adventitious
shoot induction on (a) leaf explants, (b)
cotyledon explants, and (c) hypocotyl explants
from seedlings. Each point represents the mean
of 6 replicates of 2 explants each (a, b) or

of 8 replicates of one explant each (c).

MEAN NUMBER OF BUDS AND SHOOTS PER EXPLANT

O

0

Figure 2

O

 

 

.0
O
0
.906. .o.
’ o
o’ 0 ‘
o’ .0. 0%
. |---8>“g—O—.O IL .4 H
s—‘H:::::::::::;

 

b con
0 nght

LEDON
O D a rk

 

— 1.0 mg/l BAP
III-III 3.0

P ._

1. .1-

O
\
coco-n ~.. --__8..
P ORA—O O" \O ..

—H/ i i . .

 

   

 

 

 

I

~

I
.-

 

O \x'" .
b. . .lu... .
- 011 homnIflOhO .4 r—O’ ...
-‘-1,' 1 1 - . fi. Iii L
0.0 0.1 0.3 1.0 3.0 0.0 0.1 0.3 1.0 3,0

NAA (mg/l)

 

Figure 3.

A0

Effects of 10 day intervals in light and dark

on adventitious shoot induction on leaf explants
from seedlings. Each vertical bar represents

the mean for 10 replicates of 2 explants each.

F value for comparison of regime initiated in
light vs. regime initiated in dark is significant
at 0.05 level.

MEAN NUMBER OF 8008 AND SHOOTS

PER EXPLANT

41

 

 

 

E] 10 DAY INTERVAL m LIGHT (L)
,- DARK (D)

 

 

 

 

Figure 3

LLL LLD LDD DLL DDL 000

 

A2

3). This promotion occurred regardless of subsequent

exposure to light (Figure 3).

Effscts of 5-min daily egppsures tp redlfar-red light

on adventitious shoos_issssiiss on leaf ex lants_§sss
seedlings. Five-min daily exposures of leaf explants to
red light suppressed adventitious shoot formation by 80%,
while far-red light did not have a significant effect (Table
3). However, exposure to far-red immediately after red

nullified the inhibitory effect of the latter.

Bppt isssgtios cs adyentitipus shppts geriysg fpps
sspissts frps sesdlisgs. Four different root morphology

responses occurred following auxin treatments (Figure 1e):
(i) thread-like roots at low auxin concentrations (0.00
to 0.03 mg/l IAA or 0.00 to 0.01 mg/l IBA), (ii) primary
with numerous secondary fibrous roots at low auxin concen-
1qration (0.03 to 0.30 mg/l IAA, 0.01 mg/l to 0.03 mg/l IBA,
(or 0.00 to 0.01 mg/l NAA), (iii) thickened roots at high
aaixin concentrations (0.30 to 1.00 mg/l IAA, 0.10 mg/l IBA,
or~ 0.03 mg/l NAA), and (iv) roots originating from callus
at. the basal portion of shoots (1.00 mg/l IAA, 0.30 mg/l
IBA, or 0.10 mg/l NAA). The first two types were typical
<3f’ the roots on seedlings from excised embryos germinated
.iti yiiss. Thickened roots and roots from callus did
END t appear to be as functional as the first two types.
Trlerefore, only the numbers of the first two types were

CO unted .

Adventitious shoots derived from leaf, cotyledon,

“3

Table 3. Red/far-red light effects on numbers of adven-
titious buds and shoots formed on leaf explants
from seedlings.

 

 

 

Treatmenta
Dark R FR R + FR
Mean number of buds 1.0Ac 0.2B 0.5AB 1.3A

and shoots per explant

 

aR: 1.15 Wm‘z 651 nm light for 5 min per day.

FR: 0.A6 Wm-2 729 nm light for 5 min per day.
bR + FR: R immediately followed by FR.

Mean for 20 replicates of 2 explants each.
Mean separation by DMRT at the 5% level.

AA
and hypocotyl explants exhibited similar rooting responses
to auxin (Figure A). Very few roots were produced without
auxin. All 3 auxins stimulated rooting at anconcentra-
tions, but became inhibiting as the concentration increased.
IAA was inbibitory at 1.00, IBA at 0.10, and NAA at
0.03 mg/l. At optimum concentrations of auxin approximately

65% of the cultures produced typical roots.

 

induptips ps explants from_ssssiisgs, White to yellowish

friable callus was induced on leaf, cotyledon, and hypocotyl
explants. The fresh weight of callus formed on leaf and
cotyledon explants in the dark was approximately twice that
formed in the light, but callus formation on hypocotyl
explants was not significantly affected by light (Table
A).

ngeneration of shoots and roots from callus derived
thom-lea£_exolants_ihom-seedlihss. Six weeks after
culture, two callus cultures of 50 derived from leaf
explants from seedlings regenerated 3 shoots each on BM
supplemented with 1.0 mg/l BAP (Figure 5a). Callus derived
from cotyledon or hypocotyl explants did not produce
shoots. Cultures which regenerated shoots had been held
in the dark. The callus which produced shoots was brown,
friable and mostly necrotic at the time when shoots
emerged.

Regenerated shoots were transferred to BM supplemented

with 3.0 mg/l BAP and 0.3 mg/l NAA in 60 ml bottles. 0n

Figure A.

A5

Effects of IAA, IBA, and NAA on root induction
on adventitious shoots from leaf, cotyledon,

and hypocotyl explants: (a) shoots derived from
leaf explants; (b) shoots derived from cotyledon
explants; (c) shoots derived from hypocotyl
explants. Each point represents the mean
response of four replicate shoots. Vertical
bars indicate the standard error.

46

 

    
   

 

 

 

 

 

 

 

 

0 Control
O—O IAA
l------l IBA "
A——~A NAA
'5
° F ~03
I 1
m .
n:
m
a.
{’3 .
O
O
a:
u.
o
E
m e.
E L
a .
z
z
<
u:
a q
0.00 0.01 0.03 0.10 0.30 1.00

AUXIN (mg/l)

Figure 4

Table A.

A7

Effect of light on fresh weight of callus formed
on explants from seedlings after 6 weeks of
culture.

 

Source of

Fresh weight of callus
per explant (mg)a

 

 

explant

Light Dark
Leaf 3253b 812A
Cotyledon 37OB 698A
Hypocotyl 321A 38AA

 

3Mean for

10, A0, and 20 replicates, for leaf, cotyledon, and

hypocotyl explants, respectively.
Mean separation within sources by DMRT at the 5% level.

Figure 5.

A8

(a) Shoot primordia (arrows) regenerated from
callus derived from leaf explants from seedlings
(b) An elongated shoot from callus derived from
leaf explants from seedlings. (c) Same as 'b'
with elongated axillary shoots. (d) A plant
regenerated from callus derived from leaf
explants from seedlings. (e) Regenerated roots
from callus derived from leaf explants from
seedlings. (f) A shoot regenerated from callus
derived from receptaCle and ovary tissues from
immature fruits. (g) A shoot regenerated from
callus derived from immature embryos. (h)
Adventitious shoots formed on mature leaf
explants; the picture was taken 2 weeks after
transfer to light. -Each horizontal bar equals
1.0 cm except for that in (a) which represents
1.0 mm.

 

Figures

50

exposure to light, they turned green and the axillary
branches elongated (Figure 5b, 0). After 3 weeks a shoot
regenerated from leaf callus produced thread-like roots
on medium R supplemented with 0.01 mg/l IAAIHKNH'a light
intensity of approximately 5.0 mm2 (Figure 5d). Adventi-
tious roots occurred sporadically on leaf callus on some
media, including BM without growth regulators, and this
callus was also necrotic (Figure 5e).

Additives such as ABA, adenine sulfate,
NaH2 POWH 0, and L-tyrosine did not increase the frequency
of shoot regeneration from callus derived from leaf explants
when cultured on BM supplemented with 1.0 mg/l BAP.

BQEQQttétlgfl_9_fl-§399t§_£22fl_QBLLE§_QW
espissps_§sss_issature frsits. Among 20 calli derived
from receptacle and ovary tissues of immature fruits cul-
tured on BM supplemented with 1.0 mg/l BAP and 0.3 mg/l
NAA, only one produced a single shoot (Figure 5f). Also,
one of 20 derived from immature embryos cultured on BM sup-
plemented with 6.0 mg/l BAP and 3.0 mg/l NAA produced two
shoots (Figure 5g). All shoots developed 8 weeks after
culture in the dark but not in the light. The callus giving
rise to shoots resembled the leaf-derived callus which
regenerated shoots in being brown, friable and mostly
necrotic. Callus derived from nucellar tissues regenerated
no shoots.

Asyentitipss shppp insssiips on lesf sisisnts fppm
satsss tsses. Adventitious buds and shoots were induced

51
on leaf explants from mature trees (Figure 5h). Approxi-
mately 90% of them formed on strips obtained from the
proximal region of the leaf. The greatest numbers of shoots
developed from explants cultured on BM supplemented with

3.0 mg/l BAP and 0.1 mg/l NAA (Figure 6).

Discussion

Light suppressed formation of adventitious shoots
and callus on leaf and cotyledon explants but not on
hypocotyl explants. Shoot regeneration from callus occurred
only in the dark. In work previously reported leaf explants
gave rise to somatic embryos when cultured in the dark but
not in the light (9) and somatic embryos developed from
nucellar explants of GD apple only in the dark (2).
However, the same mechanism(s) may not be involved in.all
these responses.

An initial dark treatment (10 days) was critical for
adventitious shoot formation on leaf explants. (hflturing
explants in the light for 10 days inhibited shoot formation
even though cultures were subsequently transferred to
darkness. Therefore, changes induced by the light were
irreversible. Similarly, adventitious bud formation is
yiisp in RIDES oontorta was enhanced by a 12-day dark
treatment of cultures prior to exposure to high light
intensity (21).

Red light suppressed adventitious shoot formation

on leaf explants, but the effect was eliminated by

52

Figure 6. Effects of BAP and NAA on adventitious shoot
formation on mature leaf explants. Each point
represents the mean of eight replicates.
Vertical bars indicate the standard error.

53

 

 

I
F ‘ ' ‘

'0
g — 1.0 mg/l BAP
a 2b IIIIIII 3.0
g o

h
9 3

fl
. -
O a

o
h 9..
2 ; 1-
E a. "s.
a .o.
c 0 :.

H .9.
r: O ’._
fl 0 'o...
‘ .: WI...

 

 

-// .

0.1 0.3 “I .0
NAA (mg/I)

Figure 6

5A

subsequent exposure to far-red light. Therefore,
adventitious shoot formation is evidently under some degree
of phytochrome control. Cool white fluorescent lamps
provide considerably more red than far-red radiation (A).
Therefore, suppression of adventitious shoot formation by
the light treatment may be due to irradiation in the red
region of the spectrum provided by the cool white
fluorescent lamps.

In contrast, 5-min daily exposures of lettuce
cotyledon cultures from dark-grown seedlings to red light
(660 on) enhanced both adventitious shoot and callus
formation and far-red light nullified the red light effect
(5). Furthermore, lettuce cotyledon cultures produced
significantly more adventitious shoots in the light (broad-
band emitting fluorescent lamps; 16 h per day) than in the
dark (5).

Light may suppress both callus and adventitious shoot
formation in apple by increasing the activity of an
inhibitor(s) which limits both dedifferentiation and
differentiation of tissue.

The concentration of auxins strongly influenced the
morphology of roots induced on adventitious shoots.
Abnormalities were most common with NAA and least with IAA,
inversely paralleling their rates of metabolism. Few
investigators have described the morphology of roots induced
on shoots is yissp.

A low frequency (5% or less) of shoot regeneration

55

from callus was obtained in this study. Additives which
have enhanced adventitious shoot formation in other tissues,
such as ABA (23), adenine sulfate (12), Na% Pg-HZO (1A),
and L-tyrosine (18), did not stimulate regeneration. 'Hua
low frequency does not appear to be due solely to increased
passages of subcultures, because in preliminary studies
callus excised from the explant did not show an appreciably
higher potential for regeneration. A.low frequency (A no
8%) of shoot regeneration was reported for almond callus
derived from various parts of embryos and seedlings (10).

Caulogenetic or rhizogenetic calli were necrotic when
organ formation was first observed. Similar observations
apply in somatic embryogenesis in callus cultures of
'Seyval' grape (7). Krul and Worley (7) have suggested
that developing embryos may secrete substances which enhance
lysis in neighboring cells or that superficial cells may
lyse in response to external conditions, releasing products
which enhance embryo production. However, whether
caulogenesis and rhizogenesis in apple callus was the cause
or the result of cell lysis is not known.

The morphogenetic potentials of mature woody tissues
are presumably extremely low (6, 22). However, in this
study, leaf explants from mature trees produced a relatively
good yield of adventitious shoots under the same system

developed for explants from seedlings.

56

REFERENCES

1.

10.

11.

12.

13.

1A.

Chaturvedi HC, Mitra GC (197A) Clonal propagation of
citrus from somatic callus cultures. HortScience
9:118-120

Eichholtz DA, Robitaille HA, Hasegawa PM (1979)
Adventive embryony in apple. HortScience 1A:699-700.

Gamborg 0L, Miller RA, Ojima K (1968) Nutrient
requirements of suspension cultures of soybean root
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Jagger J (1977) Phototechnology and biological exper-
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Kadkade P, Seibert M (1977) Phytochrome-regulated
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Karnosky DF (1981) Potential for forest tree
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Krul WR, Worley JF (1977) Formation of adventitious
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Litz RE, Conover RA (1981) Is vitrp polyembryony
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Liu JR, Sink KC, Dennis FG Jr (1982) Somatic embryo
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Mehra A, Mehra PN (197A) Organogenesis and plantlet
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Miller C0 Skoog F (1953) Chemical control of bud for-
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Mullins MG, Srinivasan C (1976) Somatic embryos and
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Murashige T (197A) Plant propagation through tissue
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17.

18.

19.

20.

21.

22.

23.

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, Skoog F (1962) A revised medium for rapid
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Physiol Plant 15:A73-A97.

 

Oka S, Ohyama K (1981) is vitro initiation of adven-
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of benzyladenine in leaf tissues of mulberry (Morus
aips). Can J Bot 59:68-7A.

 

Schenk RU, Hildebrandt AC (1972) Medium and techniques
for induction and growth of monocotyledonous and dicot-
yledonous plant cell cultures. Can J Bot 50:199-20A.

Skoog F, Miller C0 (1957) Chemical regulation of growth
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Symp Soc Exp Biol 11:118-130.

Spiegel-Roy P, Kochba J, Dagan B (1980) Embryogenesis

in citrus tissue cultures. In: Flechter A, ed, Advances
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Berlin: Springer-Verlang, p 27-A8

Stiles HD, Biggs RH (1973) Embryoids from peach explants.
HortScience 8:A0. (Abstr)

Webb KJ, Street HE (1977) Morphogenesis is vitrp of
Eisss and Pipes. Acta Hortic 78:259-269.

Winton LL (1978) Morphogenesis in clonal propagation

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Yamaguchi T, Nakajima T (1972) Effect of abscisic acid
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sweet potato. Proc Crop Sci Soc Jpn A1:531-532.

SECTION II
SOMATIC EMBRYO FORMATION ON CULTURED LEAF
EXPLANTS OF APPLE (MALUS DOMESTICA

 

BORKH.) SEEDLINGS

SOMATIC EMBRYO FORMATION ON CULTURED LEAF

EXPLANTS OF APPLE (MALUS DOMESTICA

 

BORKH.) SEEDLINGS

SUMMARY
Globular to heart-shaped somatic embryos were induced
<mn leaf explants from open-pollinated seedlings of 'Golden

Delicious' apple (Malus Domestica Borkh.) cultured on

 

a basal medium (BM), consisting of Murashige and Skoog
inorganic salts, BS vitamins, 30 g/l sucrose and 8 g/l agar,
supplemented with 10.0 mg/l 6-benzylaminopurine (BAP) amd
3.0 mg/l a-naphthaleneacetic acid (NAA). The somatic
embryos were observed 3 to A weeks after culture in the
dark but not in the light (cool white fluorescent lamps;
1.5 Wm'e; 16 h per day). On excision and transfer to BM
with or without growth regulators (3.0 mg/l BAP plus
0.3 mg/l NAA), most of'the embryos did not undergo further
development but 6 attained the cotyledonary stage. Two

of theSe developed into intact bipolar structures with root

and shoot apices.

 

Abbreviations: BAP, 6-benzylaminopurine; BM, basal

medium; NAA, a-naphthaleneacetic acid.

59

60
INTRODUCTION
Induction of somatic embryos of woody plants is
yiiss has been limited primarily to the Rutaceae, many

species of which give rise to nucellar embryos is viv

IO

(1). Somatic embryos of Cissss sissssis have been
derived from cultured protoplasts (2). Asexual
embryogenesis is yiiss has also been reported for other
fruit trees or vines including Naiss (3-S), fissnus (6,

7) and Vitis (8, 9).

 

Globular 32- and 6A-celled embryos were obtained from
uninucleate microspores of 'Jonathan' apple (5), and 2-
celled apple pollen grains developed into cotyledonary
embryos (A). Somatic embryos with differentiated cotyledons
and axes were induced in cultured micropylar halves of the
nucellus of 'Golden Delicious' apple (3).

This paper describes the developmental stages of
somatic embryos arising on cultured leaf explants from

open-pollinated seedlings of 'Golden Delicious' apple.

MATERIALS AND METHODS

Leaf explants were prepared from 2- to 3-week-old
open-pollinated 'Golden Delicious' seedlings grown
aseptically as previously described (10). Two leaf
explants, 1 x 1 cm each, were placed abaxial surface down
into 60 x 15 mm Petri dishes containing 12 ml of a basal
medium (BM) supplemented with 10.0 mg/l BAP and 3.0 mg/l

NAA. The BM consisted of Murashige and Skoog inorganic

61

salts (11), B5 vitamins (12), 30 g/l sucrose and 8 g/l
agar. The pH of all media was adjusted to 5.8 with 0.1
and 1.0 N NaOH. All media were autoclaved at
1.A6 kg/cm2 for 15 min before use. The Petri dishes were
sealed with ParafilmR and placed under a light intensity
of approximately 1.5 Wm'2 from cool white fluorescent lamps
(General Electric) on a 16-h photoperiod or in the dark
at 25°: 2°C.

Three to A weeks after culture, the explants were
observed periodically under a dissecting microscope for
somatic embryo formation. The induced somatic embryos were
excised at various developmental stages and transferred
to BM without growth regulators under the same light
intensity. When further development ceased, the embryos
were transferred to BM supplemented with 3.0 mg/l BAP and

0.3 mg/l NAA.

RESULTS

Somatic embryos, approximately 1 mm in diameter at
the globular to heart-shaped stage, appeared on the adaxial
surface of leaf explants cultured in the dark for 3 to A
weeks (Fig. 1a, b) but did not occur in the light. The
number of embryos varied from 0 to 10 per explant.
Adventitious shoots developed simultaneously with the
somatic embryos on the leaf explants. Somatic embryos.at
the globular to early heart-shaped stage had a more glossy

surface than the other nodules which occurred on the leaf

62
surface (Fig. 1a).

Embryos which remained on the leaf surface for more
than two months did not undergo further development and
eventually died. Approximately A0 were removed with a
scalpel at the early developmental stages and placed on
BM without growth regulators, most embryos enlarged slightly
and lost their glossy appearance, but did not undergo
further development (Fig. 1c). Six developed to the
cotyledonary stage (Fig. 1d, e). One embryo at this stage
had a pair of fused cotyledons and a prominent radicle
(Fig. 1e), a second developed 5 cotyledons (Fig. 1e), and
a third produced a pair of leaf primordia, bypassing the
cotyledonary stage (Fig. 1f). Four of the 6 embryos ceased
development, even after transfer to BM supplemented with
3.0 mg/l BAP and 0.3 mg/l NAA, and eventually died.

Two of the embryos produced primary roots and
hypocotyls 1 to 3 days after transfer to BM without growth
regulators (Fig. 1g, 1). One had a pair of cotyledons which
were partially fused with the hypocotyl (Fig. 1g), the other
had a pair of poorly developed cotyledons fully fused with
the hypocotyl (Fig. 11). A few days later, a pair of leaf
primordia and numerous roots emerged (Fig. 1h, j). The
plantlets did not develop further after an additional 2
weeks of culture, and therefore were transferred to BM
supplemented with 3.0 mg/l BAP and 0.3 mg/l NAA to stimulate
further development. One week thereafter,tflmw'developed

multiple leafy shoots (Fig. 1k). To stimulate further

63
elongation, they were transferred to 60 ml glass bottles
containing 25 ml of BM supplemented with 0.3 mg/l BAP.
Two weeks after transferg normal growth had begun with one
shoot dominant in each plantlet (Fig. 11). However, the

roots reverted to callus type growth.

DISCUSSION

The decisive feature of'ah.embryo is its bipolarity;
it has a root and a shoot apex integrated into one axis
at the earliest developmental stage (13). In this study,
two somatic embryos which eventually developed into
plantlets exhibited bipolarity on BM without growth
regulators. In many species somatic embryos at advanced
developmental stages usually require a simple medium lacking
growth regulators to initiate both roots and shoots, as
do zygotic embryos (8, 1A, 15).

All cotyledons developing from the somatic embryos
in this study had certain abnormalities; they were fused
with each other, multiple, or partially or wholly fused
*with the hypocotyl axis. Many investigators have observed
fused (16, 17) or multiple cotyledons (15, 18, 19) in
somatic embryos.

Whether the somatic embryos which did not develop
further were dormant (19), or disorganized and thus
incapable of development to an advanced stage, has not been
established.

Somatic embryos were induced in the dark but not in

6A

the light. In a parallel study, light suppressed the
induction of adventitious shoots on leaf explants from apple
seedlings (10); 5-min daily exposures to red light
(651 nm) were also effective whereas exposure to far-red
light (729 nm) immediately following the red light
counteracted the effect of the latter. Therefore, red light
supplied by the fluorescent lamps may have suppressed the
induction of somatic embryos, possibly by a phytochrome-
mediated mechanism. Eichholtz e; si. (1979) reported
that somatic embryos arising from micropylar halves of the
nucellus of apple were induced only in the dark (3). In
addition, in Nsssss ssspss callus, short exposure to
far-red illumination enhanced somatic embryogenesis (20).

Histological studies on the somatic embryos were
not conducted. Microscopic observation revealed that the
somatic embryos did not emerge through the cracked epidermal
layer, and no intermediary callus stage was observed;
therefore they may have arisen directly from the epidermal
layer, as reported for fissssssiss (21) and DQNQES (22).

In this study, both somatic embryos and adventitious
shoots developed simultaneously on the leaf explants. If
both are derived from the epidermal cells, investigation
of the factors which determine whether the cells become
embryos or meristems would be of interest.

In preliminary studies, somatic embryos were not found
on BM supplemented with 10.0 mg/l BAP plus less than

1.0 mg/l NAA. Therefore, certain combinations of cytokinin

65
and auxin appear to be critical for induction.

Most reports of somatic embryogenesis is yisss
in woody plants have dealt with embryogenic tissues or
callus derived from them, for example, ovule, nucellus,
or integument (1). This study differs in that somatic
embryogenesis occurred in leaf explants. Inukn'identical
culture conditions, cotyledon or hypocotyl explants from
apple seedlings did not yield somatic embryos. This
observation suggests that somatic embryos might be initiated
directly fkwnn isolated leaf mesophyll protoplasts of apple

seedlings as is the case in tomato (23).

Figure

1.

66

(a): Globular to heart-shaped somatic embryos
(arrows) on a leaf section from an apple
seedling. (b): Heart-shaped somatic embryos.
(c): Globular somatic embryos after transfer
to BM. (d): A somatic embryo with fused
cotyledons and a radicle (arrow). (e): A
somatic embryo with 5 cotyledons. (t9: A pair
of leaf primordia (arrow) on a somatic embryo
clustered with the neighboring somatic embryos.
(g): A somatic embryo with deveIOping primary
root, hypocotyl and cotyledons (arrows). (h):
Same as 'g' with developing shoot. (1): A
somatic embryo with developing primary root,
hypocotyl and cotyledons. (j): Same as 'i'
with numerous roots. (k): Same as 'j' with
multiple shoots. (l): Plantlets from somatic
embryos. Each horizontal bar equals 1.0 mm
except for that in (l) which represents 1.0 cm.

67

 

Figure l

68

 

Figure l (CONIId)

69

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P.S. Rao and S. Narayanaswami, Physiol. Plant., 27
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20.

21.

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23.

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W. Newcomb and D.F. Wetherell, Bot.

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Gaz., 131 (

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Appl.

Genet.

1970)

59

SUMMARY AND CONCLUSIONS

SUMMARY AND CONCLUSIONS

Leaf, cotyledon, and hypocotyl explants obtained from
2- to 3-week-old 'Golden Delicious' (GD) apple (Nsiss
sssssiiss Borkh.) seedlings were (cultured to assess
morphogenetic potential on a basal medium (BM), consisting
of Murashige and Skoog inorganic salts, BS vitamins, sucrose
and agar, supplemented with 6-benzylaminopurine and
a-naphthaleneacetic acid (NAA) at 25°; 2°C in the light
(cool white fluorescent lamps; 1.5 Wm'z; 16 h per day) or
in the dark.

Light suppressed formation of adventitious shoots and
callus on leaf and cotyledon explants but not on hypocotyl
explants. Shoot regeneration from callus derived from leaf
explants occurred only in the dark. Leaf explants gave
rise to somatic embryos when cultured in the dark but not
in the light.

An initial dark treatment (10 days) was critical for
adventitious shoot formation on leaf explants. Culturing
explants in the light for 10 days inhibited shoot formation
even though cultures were subsequently transferred to
darkness. Therefore, changes induced by the light were
irreversible.

Red light (651 nm) suppressed adventitious shoot
formation on leaf explants, but the effect was eliminated
by Subsequent exposure to far-red light (729 nm). There-

fore, adventitious shoot formation is evidently under some

71

 

72

degree of phytochrome control. Suppresshnicfi‘adven-
titious shoot formation and somatic embryogenesis may be
due to red light provided by the cool white fluorescent
lamps. Light may suppress both callus and adventitious
shoot formation in apple by increasing the activity of an
inhibitor(s) which limits both dedifferentiation and
differentiation of tissue.

The concentration of auxin strongly influenced the
morphology of roots induced on adventitious shoots.
Abnormalities were most common with NAA and least with
indole-3-acetic acid, inversely paralleling their rates
of metabolism.

A low frequency (5% or less) of shoot regeneration
from callus derived from leaf explants, receptacle and ovary
tissues, and immature embryos was obtained in this study.
The low frequency does not appear to be due solely to
increased passages of subcultures.

Caulogenetic or rhizogenetic calli were necrotic when
organ formation was first observed. Developing shoots or
roots may secrete substances which enhance lysis in
neighboring cells or superficial cells may lyse in response
to external conditions, releasing products which enhance
morphogenesis. However, whether caulogenesis and
rhizogenesis in apple callus were the causes or the results
of cell lysis is not known.

Leaf explants from mature GD trees also produced

relatively good yields of adventitious shoots under the

73
same system developed for explants from seedlings.

In addition, leaf explants from seedlings gave rise
to somatic embryos at the globular te>heart-shaped stage,
0n excision and transfer to BM with or without growth
regulators, most of the embryos did not undergo further
development, but 5% of them attained the cotyledonary
stage. Two developed.ixnx0 intact bipolar structures with
root and shoot apices. Microscopic observation revealed
that tdne somatic embryos did not emerge through the cracked
epidermal layer, and no intermediary callus stage was
observed; therefore, they may have arisen directly from
the epidermal layer. Under identical culture conditions,
cotyledon or hypocotyl explants from apple seedlings did
rust yield somatic embryos. This observation suggests that
embryos might be initiated directly from isolated leaf

mesophyll protoplasts of apple seedlings.

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